Download Flavour compounds in fungi

FACULTY OF SCIENCE
UNIVERSITY OF COPENHAGEN
PhD thesis
Davide Ravasio
Flavour compounds in fungi
Flavour analysis in ascomycetes and the contribution of the Ehrlich
pathway to flavour production in Saccharomyces cerevisiae and
Ashbya gossypii
Academic advisor: Prof. Steen Holmberg, Department of Biology, University of Copenhagen.
Co-supervisor: Prof. Jürgen Wendland, Yeast Genetics Group, Carlsberg Laboratory
Submitted: 01/10/14
“There is nothing like looking, if you want to find something. You certainly usually find
something, if you look, but it is not always quite the something you were after.”
― J.R.R. Tolkien, The Hobbit
Institutnavn:
Natur- og Biovidenskabelige Fakultet
Name of department:
Department of Biology
Author:
Davide Ravasio
Titel:
Flavour-forbindelser i svampe. Flavour-analyse i
ascomyceter og bidrag fra Ehrlich biosyntesevejen til
smagsproduktion i Saccharomyces cerevisiae og Ashbya
gossypii
Title:
Flavour compounds in fungi. Flavour analysis in
ascomycetes and the contribution of the Ehrlich pathway
to flavour production in Saccharomyces cerevisiae and
Ashbya gossypii
Academic advisor:
Prof. Steen Holmberg, Prof. Jürgen Wendland
Submitted:
01/10/14
Table of contents
Preface ................................................................................................................................................ 1
List of Papers ..................................................................................................................................... 2
Summary ............................................................................................................................................ 3
Resumé ............................................................................................................................................... 5
Acknowledgments .............................................................................................................................. 7
Abbreviations ................................................................................................................................... 10
1.
Introduction .............................................................................................................................. 11
1.1
The Fungi Kingdom ........................................................................................................... 11
1.2
Ascomycota: The Saccharomycotina clade ...................................................................... 12
1.3
Saccharomyces cerevisiae: the model organism ............................................................ 15
1.4
Yeast carbon metabolism ................................................................................................. 18
1.5
The Eremothecium genus: Ashbya gossypii and Eremothecium cymbalariae ............ 20
Ashbya gossypii ....................................................................................................................... 21
Eremothecium cymbalariae .................................................................................................... 23
1.6
Fungal system and their contribution to industrial processes ........................................ 23
1.7
Aroma and flavour definition, chemical type .................................................................. 24
1.8
Flavour additives and natural flavours ............................................................................ 26
1.9
Bioflavour production (Ehrlich pathway, FFAs and lactate) .......................................... 29
Ehrlich pathway ....................................................................................................................... 30
Fatty acids as substrates for flavour formation ....................................................................... 33
Metabolism of lactate and citrate ............................................................................................ 34
1.10
Biological properties of quorum sensing molecules and VOCs as signaling molecules . 35
1.11
Biotechnological application of fungal VOCs .................................................................. 37
1.12
Fungal VOC collection and detection ............................................................................... 38
VOC collection .......................................................................................................................... 38
VOC separation ........................................................................................................................ 39
VOC detection........................................................................................................................... 39
2.
Aim ............................................................................................................................................40
3.
Objectives and state-of-the-art ................................................................................................ 41
3.1
Functional analysis of the ARO gene family in S.cerevisiae and A. gossypii ................. 41
3.2
Analysis of the different volatile profiles of A. gossypii and E. cymbalariae is correlated
to their genetic backgrounds. ...................................................................................................... 43
3.3
4.
5.
Flavour molecules produced in the Saccharomyces clade. ............................................. 44
Discussion ................................................................................................................................. 44
4.1
Reporter assay for ARO genes in S. cerevisiae and A. gossypii ...................................... 44
4.2
Use of a lacZ- reporter assay to correlate reporter gene activity with flavour production
........................................................................................................................................... 46
4.3
General flavour differences between A. gossypii and E. cymbalariae ........................... 47
4.4
VOCs produced in the Saccharomyces clade ................................................................... 49
Conclusions and Outlook ......................................................................................................... 53
References ........................................................................................................................................ 55
Paper 1 .............................................................................................................................................. 64
Paper 2 ............................................................................................................................................. 65
Paper 3 ............................................................................................................................................. 66
Appendix .......................................................................................................................................... 67
Preface
This thesis “Flavour compounds in fungi: Flavour analysis in ascomycetes and the contribution
of the Ehrlich pathway to flavour production in Saccharomyces cerevisiae and Ashbya gossypii”
represents an overview of my PhD internship carried out at the Carlsberg Laboratory in
Denmark. This project was supervised by Prof. Jürgen Wendland, head of the Yeast Genetics
Group in the Carlsberg Laboratory and Prof. Steen Holmberg at the Department of Biology,
University of Copenhagen. My thesis was funded by the European Union Marie Curie Initial
Training Network, Cornucopia.
1
List of Papers
I.
Davide Ravasio, Andrea Walther, Kajetan Trost, Urska Vrhovsek and
Jürgen Wendland (2014). An indirect assay for volatile compound production in
yeast strains. Scientific report 4: 3707.
II.
Davide Ravasio, Jürgen Wendland and Andrea Walther (2014). Major
contribution of the Ehrlich pathway for 2-phenylethanol/rose flavour production in
Ashbya gossypii. FEMS Yeast Res. doi: 10.1111/1567-1364.12172.
III.
Davide Ravasio, Silvia Carlin, Teun Boekhout, Urska Vrhovsek, Jürgen
Wendland and Andrea Walther (2014). A survey of flavor production among
non-conventional yeasts. Manuscript.
2
Summary
Fungi produce a variety of volatile organic compounds (VOCs) during their primary or
secondary metabolism and with a wide range of functions. The main focus of this research work
has been put on flavour molecules that are produced during fermentation processes, mainly
esters and alcohols derived from the catabolism of amino acids. These compounds are produced
by the Ehrlich pathway. The conversion of amino acids into aroma alcohols is accomplished by
three enzymatic steps: i) a transamination, ii) a decarboxylation and iii) a dehydration reaction.
The transaminase and decarboxylase enzymes are encoded by the ARO gene family which
represents a widely conserved set of genes in the Saccharomyces clade. Comparative genomic
analysis revealed conservation of these genes also in the riboflavin over producer Ashbya
gossypii, a closely related species belonging to the Eremothecium clade. ARO80 is a
transcription factor that represents the key regulator of the ARO gene family. The first part of
the thesis will unveil the ARO80-dependent regulation of the Ehrlich pathway in both
Saccharomyces cerevisiae and A. gossypii.
Promoter analyses of the ARO genes in S. cerevisiae showed that the ScARO9 promoter region
is directly regulated by the ScAro80 transcription factor. This interaction has been used to
create a lacZ-reporter system to correlate the formation of two volatile compounds, 2phenylethanol and 2-phenylethyl acetate in yeast with ARO9 expression levels. This indirect
genetic assay also provides a tool for the prediction of volatile production in other
Saccharomyces sensu stricto species. It can be used to screen a large number of strains for their
flavour production within a short time and with low costs and effort.
In Ashbya single mutations in the ARO genes led to a strong reduction in volatile production,
especially in the amount of isoamyl alcohol and 2-phenylethanol. In contrast, the
overexpression of the transcriptional regulator AgARO80 did only increase the level of isoamyl
alcohol but did not enhance the 2-phenylethanol yield. Promoter analyses of the ARO genes in
A.
gossypii
identified
both
ARO8
and
ARO10
to
be
activated
by
Aro80.
In this study we further analyzed the aroma profile of another Eremothecium species,
Eremothecium cymbalariae. This species lacks most of the ARO genes involved in amino acid
catabolism. The only ARO gene present in E. cymbalariae is a homolog of the A. gossypii
ARO8a, which is a non-syntenic homolog of ARO8 in yeast. We compared the VOC profiles of
both species in order to investigate the consequences of their different gene set up on their
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flavour profiles. Here we found that in contrast to A. gossypii E. cymbalariae does not produce
2-phenylethanol and 2-phenylacetate.
The last part of this thesis presents the initial characterization of twenty non-conventional
yeasts (NCY) and their potential application in fermentative processes. These strains have been
selected as they have been previously isolated from various fermented food sources. This
selection of strains was used in fermentations with the aim of identifying new interesting flavour
producers. Fermentation profiles, volatile analyses, off-flavour identification and resistance to
osmotic/oxidative stress have been addressed to highlight new candidates to use for industrial
applications. This resulted in the identification of Wickerhamomyces anomalus and Pichia
kluyveri as high producers of esters fruity compounds, which contribute to enhance the
complexity of wine and beer product. In addition the strain Debaromyces subglobosus showed
high yields of aldehydes and fruity ketones, which constitute active aroma compounds in dry
cured ham.
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Resumé
Svampe producerer en række flygtige organiske forbindelser (VOC, Volative Organic
Compounds) under deres primær eller sekundær metabolisme og med en bred vifte af
funktioner. Hovedfokus i dette forskningsarbejde er på flavour molekyler, der produceres under
gæringsprocesser, primært estere og alkoholer fra aminosyrers catabolisme. Disse forbindelser
dannes under Ehrlich reaktionsvejen. Omdannelsen af aminosyrer til alkoholer opnås ved tre
enzymatiske trin: i) transaminering, ii) decarboxylering og iii) dehydreringsreaktion.
Transaminase- og decarboxylase enzymer kodes af ARO-genfamilien, som repræsenterer et
bredt konserveret sæt af gener i Saccharomyces cladus. Komparativ genomisk analyse viste
konservering af disse gener også i riboflavin-overproduceren Ashbya gossypii, en nært
beslægtet art, der tilhører Eremothecium cladus. ARO80 er en transkriptionsfaktor, der
repræsenterer nøgleregulatoren i ARO-genfamilien. Første del af afhandlingen viser ARO80afhængig regulering af Ehrlich-biosyntesevejen i både Saccharomyces cerevisiae og A. gossypii.
Promotor analyse af ARO-generne i S. cerevisiae viste, at ScARO9 promotor-regionen er direkte
reguleret af ScAro80 transskriptionsfaktoren. Denne interaktion er blevet brugt til at konstruere
et lacZ-reporter-system for at korrelere dannelsen af to flygtige forbindelser, 2-phenylethanol og
2-phenylethylacetat i gær med ekspressionsniveauet af ARO9. Dette indirekte genetiske assay er
også et redskab til at forudsige produktionen af flygtige forbindelser i andre Saccharomyces
sensu stricto arter. Det kan anvendes til på kort tid og lave omkostninger at screene et stort
antal stammer for deres flavourproduktion.
I Ashbya, førte enkeltmutationer i ARO gene rned til en kraftig reduktion i produktion af flygtige
forbindelser, især i mængden af isoamylalkohol og 2-phenylethanol. I modsætning hertil førte
overekspression af den transkriptionelle regulator AgARO80 kun til øget
isoamylalkoholdannelse men ikke forøget 2-phenylethanol udbytte. Promotoranalyser af ARO
generne i A. gossypii viste at både ARO8 og ARO10 aktiveres af Aro80.
I denne undersøgelse analyserede vi yderligere aromaprofilen i en anden Eremothecium art,
Eremothecium cymbalariae. Denne art mangler de fleste af ARO generne, involveret i
aminosyre catabolisme. Det eneste ARO-gen, der findes i E. cymbalariae er en homolog af A.
gossypii ARO8a, som er et ikke-syntenisk homolog af ARO8 i gær. Vi sammenlignede VOCprofilerne i begge arter for at undersøge konsekvenserne af deres forskellige gen-set up på deres
smagsprofiler. Her fandt vi, at A. gossypii i modsætning til E. cymbalariae ikke producerer 2phenylethanol og 2-phenylacetat.
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Den sidste del af denne afhandling præsenterer den indledende karakterisering af tyve ikkekonventionelle gærtyper (NCY, Non-Conventional Yeasts) samt deres potentielle anvendelse i
gæringsprocesser. Disse stammer er valgt, fordi de tidligere er blevet isoleret fra forskellige
fermenterede fødevarer. Formålet var at identificere nye interessante flavour-producenter.
Gæringsprofiler, analyse af volatile forbindelser, off-flvour bestemmelse og modstandsdygtighed
mod osmotisk/oxidativ stress er blevet undersøgt for at adressere nye kandidater til brug for
industriel anvendelse. Dette resulterede i identifikationen af Wickerhamomyces anomalus og
Pichia kluyveri som høj-producenter af frugtagtige esterforbindelser. Disse kan bidrage til at
øge kompleksiteten af vin og øl produkter. Desuden viste Debaromyces subglobosus høje
udbytter af aldehyder og frugtagtige ketoner, der udgør aktive aromastoffer i tørret skinke.
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Acknowledgments
Sometimes what you need it is a chance to prove yourself and have the courage to make it
happen. My PhD experience and this thesis would not be possible without all the support I
received to carry out this astonishing scientific opportunity.
First, I would like to give my honest and profound gratitude to Prof. Jürgen Wendland to
offer me this position. Without his acceptance and thoughtful guidance this PhD would not have
been possible. You bet on me even though you could have chosen many other more qualified
students. I just want to let you know that your inputs, ideas, supervision and support became
important for the success of this PhD project.
I am indebted to my academic supervisor Prof. Steen Holmberg because his supervision
during my PhD and kindness to examine this thesis. My deepest thanks to the opponents of my
PhD defense which have spent time to read this thesis and evaluate my work.
I am particularly grateful also to Andrea. We have done so many things together than I do not
know where to start. You helped me to settled down and get around here in Copenhagen. You
taught me almost all the techniques I acquired during my PhD. We shared funny moments
together during conferences and extra work activities. Our LONG discussions were extremely
useful to improve my skills and delineate well organized experiments. I have so many things to
be thankful that one page would not be enough. I guess my biggest satisfaction was to introduce
you the GC/MS technique. That time you were the student and I was the teacher. This turned
around situation made me understand all the progress I made thanks to you and Jürgen.
The next person I would like to give my gratitude is Prof. Laura Popolo, my previous
supervisor at the University of Milan. She trained and offered me a wonderful year experience in
her lab during my master thesis. The research work with C. albicans opened my future
development, clarified my interests and opened the way to my career decision. You told me
about the Cornucopia project and you convinced me to send the application. From that moment
on my life changed completely and I will never stop to thank you for encouraging me to take this
first step.
Klaus, you are like the German Terminator but with less muscles and HUGE brain. Every time
Google did not have an answer for me you had one. You are an unlimited source of useful
information. If we connected your brain to the server we could also survive without internet
connection. Thanks also for bringing up funny stories and topics during our lunches. You have
the attitude to have always the right word to say, turning each situation in a pleasant moment.
Lisa, I can be the Superman in the lab but you are definitely the Supermom in the daily life! You
managed to get a great success during your PhD even though you had a husband and a small
daughter to take care of. Apparently all the early mornings did not bother you at all. On the
contrary you looked always resolute and full of motivation. Keep going with this attitude and I
wish you a bright future, wherever that will be!
7
I have to give my special thanks also to “my girls” Ana and Jevgenia. I have started this PhD
path with you two in the office. I will never forget our discussions and the funny moments we
shared together. Ana thanks for your IT help. If Neo in Matrix was the chosen one you can be
our Neo here at Carlsberg. I will not be surprised if one day I will see a plug behind your skull!
Jevgenia, “tusind tak” for supporting my/our research every day. I do not know how many
selection plates and YPD media I used that were made by you. You made our daily experience in
the lab easier, taking care of the needs of everyone in the group. The word “multitasking” fits
perfectly to you! 
Therese, thanks for the good time in the lab as well as being an inspiration for starting my
thesis writing. The experience in Asilomar was the greatest conference ever thanks to you, Ana
and Andrea. We had a lot of fun and I will never forget the pleasant and joyful atmosphere you
helped to create.
Claudia, you do not talk too much but when you show your qualities you are among the best. In
our last team building event you brought out your innate talent with the bowling. By keeping a
low profile and using a questionable technique you defeated everyone in the group (except
Jürgen of course). Thank you too for the good company and the nice discussion we had from
time to time.
Klara, you were the last one who joined our group but in these few months you shared your
honest beer enthusiasm with us. Thanks for helping us with the organization of the FungiBrain
workshop. You have been a very irreplaceable resource to coordinate all the activities and to
accommodate the students. I wish you all the best with your PhD project and do not stop to use
passion in your work!
Claes, thanks a lot with your help with the Danish summary. With my current Danish skills I
could have written maximum 2 lines, full of grammar mistakes most likely.
Big thanks to Dr. Urska Vrhovsek and her team for introducing me to the GC/MS technique
and for hosting me during my stay at the Fondazione Edmund Mach, Research and Innovation
Centre (Italy). Your exceptional and high qualified support was extremely important for the
success of this PhD thesis.
Special thanks also to the entire Carlsberg laboratory for providing a high quality research
and exciting work environment. The last three years have been memorable and they will always
represent an example of genuine and high scientific research work.
I am also deep indebted with the Cornucopia consortium, with all the PIs and organizers of
the network. You have done an incredible work with me and all the other PhD students. You
have found the right mixture of good scientific spirit and social activities to make this journey
precious. A special gratitude to Prof. Jure Piskur who was the coordinator and main
organizer of the EU-project. Your scientific mood will always be with us as well as the funny
moments you promoted.
A particular note also to all the PhD (and non) fellows in the Cornucopia consortium. Anna,
Jiří, Nerve, Honeylet, Md, Alicia, Amparo, Ida, Raquel, Benjamin, Dorota and Jana
8
you helped to make these three years’ experience brilliant and extraordinary. We shared worries,
doubts, happiness, stress, connections, interests, information and much more. Thanks to all of
you for making every Cornucopia workshop a pleasant event.
Lucia, ci siamo conosciuti all’inizio di questa esperienza. La tua permanenza doveva essere
breve ma il destino ha voluto che restassi e condividessimo preoccupazioni e gioie. A dire il vero
abbiamo pure finito con il condividere la lavatrice ma questa è tutta un’altra storia. Grazie di
essere sempre stata un’ottima amica nonostante i miei continui scherni. Sappi che sei stata di
grande aiuto in tutto, anche se a volte posso non averlo dimostrato come dovevo. Spero
vivamente che tutti i tuoi sogni si realizzino e che in un modo o nell’altro tu riesca a capire che
non sei proprio “brava” come pensi nelle attività manuali! :-P
Solfa, Andrea, Sara e Conzi che dire... voi siete senz’altro i numeri 1. Mi avete assistito e
supportato fin dal principio di questo viaggio. Mi siete stati accanto nelle lunga distanza ma
soprattutto nei miei ritorni a casa. Senza di voi restare qui sarebbe stato impossibile. I bei
trascorsi passati insieme mi sono stati di conforto negli ultimi anni, aiutandomi a superare
momenti “bui” che avrebbero potuto farmi vacillare. Restate come siete, vi voglio bene.
Frank, io e te siamo cresciuti insieme e abbiamo condiviso tante di quelle cose che manco me le
ricordo tutte. Comunque per dovere di cronaca io me ne ricordo più di te di sicuro! Ad un certo
punto le nostre strade si sono divise...ma è più forte di noi, non esiste che ci perdiamo di vista.
Finchè al cinema ci saranno film inutili da vedere noi dobbiamo andarci. Siamo come Frodo e
Sam, non esiste che ci separino. Grazie di essere come sei e volerti bene mi sembra quasi
scontato dopo tanti anni!
Papà, mamma, sorellina, nonne, Piero e bodolino. Nessuno più di voi ha supportato
questa insolita esperienza. Non so neanche da che parte cominciare con i ringraziamenti. Siete le
colonne portanti della mia vita, quindi PhD o meno voi avrete sempre il mio amore e la mia
gratitudine. Mi siete mancati moltissimo in questi anni e Dio solo sa quanto vi avrei voluto qui
accanto a me. Niente di tutto ciò che sono sarebbe stato possibile senza il vostro aiuto. Grazie di
T-U-T-T-O!!!!!!
9
Abbreviations
ADP
Adenosine diphosphate
ATP
Adenosine triphosphate
bp
Base pair
ChIP
Chromatin Immunoprecipitation
DNA
Deoxyribonucleic acid
FAD
Flavin adenine dinucleotide
FFA
Free fatty acid
FID
Flame ionization detector
GC-MS
Gas chromatography–mass spectrometry
kb
Kilo base pair
NAD
Nicotinamide adenine dinucleotide
NADP
Nicotinamide adenine dinucleotide phosphate
NCY
Non-conventional yeast
PCA
Principal component analysis
PCR
Polymerase chain reaction
pFA
plasmid for Functional Analysis
pKa
Logaritmic acid dissociation constant
PUFA
Polyunsaturated fatty acid
RNA
Ribonucleic acid
SPME
Solid-phase microextraction
TCA
Tricarboxylic acid cycle
TCD
Thermal conductivity detector
TF
Trascriptional factor
VOC
Volatile organic compund
X-Gal
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
10
1.
Introduction
1.1
The Fungi Kingdom
Fungi are eukaryotic, heterotrophic organisms, encompassing both single-celled yeasts and
multi-cellular filamentous fungi. Their existence is strictly linked to the decomposition of
organic material. Many fungal species can survive in oligotrophic environments, scavenging
nutrients from the substrate which they colonize [1]. With over 900 million years of evolution
the Fungal Kingdom shows an enormous evolutionary and biological diversity [2] (Figure 1).
Mycologists have classified fungi into four groups according to their sexual reproduction and
molecular structure: chytridiomycetes, zygomycetes, ascomycetes and basidiomycetes.
chytridiomycetes occupy a wide range of natural habitats and today represent the most ancient
phylum of fungi present on earth [3]. Unlike the others, chytridiomycetes produce zoospores,
motile spores propelled by a flagellum which allows them to move towards light or chemical
stimuli [4]. Basidiomycetes contain about 30,000 described species and are best known for the
production of large fruiting bodies. Most of the fungal body is made of the mycelium, which
constitutes the vegetative part of the fungus. The fruiting bodies, the mushrooms, are the results
of sexual reproduction and are normally the visible part of the fungus. zygomycetes, which
encompass most of the organisms we define as “molds”, typically grow inside their food. They
can reproduce sexually and asexually, through a process that is light governed. Their rapid
asexual reproduction involves the formation of sporangia and sporangiospores. The presence of
sporangia and unseptate hyphae represents a common feature in this class of fungi [5]. The
ascomycota is the most studied phylum, and accounts for approximately 75% of all described
fungi. Their defining feature is the ascus, a sexual structure in which non-motile ascospores are
produced. However, asexual reproduction is the most common form of propagation, responsible
for the rapid spread of these fungi into new areas or the conquest of short-lived [6]. The phylum
harbors very diverse organisms and includes a number of industrially relevant fungi (i.e.
Saccharomyces cerevisiae, Penicillium chrysogenum) but also plant pathogens (i.e.
Magnaporthe grisea, Cryphonecrita parasitica, Fusarium oxysporum) and human pathogens
(i.e. Candida albicans, Aspergillus fumigatus, Coccidioides immitis). Ascomycetes have gained
great importance especially as producers of antibiotics and in food production (bread, beer,
wine, cheese, etc.).
11
Figure 1. Phylogeny of the fungal kingdom. The diamonds represent evolutionary branch points and
indicate approximate time points. The colored lines define the major fungal groups [7].
1.2
Ascomycota: The Saccharomycotina clade
The ascomycota, or sac fungi, are the largest monophyletic phylum of Fungi, with over 64,000
species [8]. Molecular phylogenetic analyses of nuclear and mitochondrial ribosomal RNA
divided them into three main groups: 1) Schizosaccharomyces and Protomyces; 2) ‘filamentous
fungi’ (Pezizomycotina); and 3) budding yeast (Saccharomycotina) [7]. This last group
comprises yeasts and is home to the most commonly known fungi. Numerous studies have
examined the phylogenetic relationships among yeasts of the ‘Saccharomyces complex’ (Figure
2). Multigene sequence analysis resolved 75 species into 14 clades [9].
12
Figure 2. Phylogenetic tree resolving species of the ‘Saccharomyces complex’ into 14 clades [9].
S. cerevisiae (Clade 1) is part of the Saccharomyces sensu stricto complex, which includes S.
paradoxus, S. mikatae, S. cariocanus, S. kudriavzevii, S. arbicola, S. eubayanus and S.
uvarum. Among them stable interspecies hybrids are common and widely used in wine, cider,
and beer industry. Yeast hybrids share multiple origins, harboring chimeric allopolyploid
genomes with different ratios of their parental genomes. The hybridization event may have
taken place in the production environments during the domestication of the strain under manmade selection conditions or could occur in nature (Figure 3) [10] [11].
13
S. cerevisiae
S. cerevisiae x
S. kudriavzevii
S. paradoxus
S. mikatae
S. pastorianus
S. kudriavzevii
S. carlsbergensis
S. arboricola
S. eubayanus
S. bayanus
S. uvarum
Figure 3. Saccharomyces sensu stricto complex and hybrids [12]
The genetic constitution of these hybrids offers the advantage to acquire physiological properties
from both parents, generating strains better suited for extreme environmental or production
conditions. Yeast breeding and selection processes have been used to improve industrial strains ,
e.g. by generating strains with novel flavours, cold adapted hybrids with higher fermentation
rates or osmotolerance [13] [14]. The first Saccharomyces interspecies hybrid identified was the
lager brewing yeast S. pastorianus (S. carlsbergensis). Initially named Unterhefe No. 1 by Emil
Chr. Hansen the hybrid has been used in beer production since 1883. The introduction of this
hybrid as pure strain in beer industry revolutionized the production process completely,
stabilizing and improving fermentation performance [15]. Later, other yeast hybrids have been
discovered and characterized. S. cerevisiae × S. kudriavzevii hybrids are widely used in wine
and brewing production [16] [17]. S. cerevisiae × S. bayanus yeasts often appear in wine, cider
and brewing production. They favor to preserve the S. bayanus-like genome and reduce the
S. cerevisiae part [18]. Furthermore, S. cerevisiae × S. paradoxus natural and artificial crosses
have been documented [19] [20]. In the natural isolates introgression occurred and rare progeny
was found due to post zygotic isolation [21]. In conclusion, Saccharomyces yeasts and their
hybrids represent key resources in different industrial processes. Their existence inspired new
molecular genetic studies, offering new insight into the evolution of genomic variation and
genomic architecture.
14
This study will focus not only on representatives of clade 1 but also on other clades: clade 13 with
Hanseniaspora as representative
genus; clade 9 with
Torulaspora; clade 7 with
Zygosaccharomyces species and clade 12 with Eremothecium species. The Eremothecium genus
contains attractive study models and industrially important species, which will be described
below in more detail (Paragraph 1.4). Among the Saccharomycotina I will also consider other
species such as Candida, Kazachstania, Pichia, Wickerhamomyces and Kluyveromyces, which
are not represented in Figure 2.
1.3
Saccharomyces cerevisiae: the model organism
Saccharomyces cerevisiae with its intrinsic ability to ferment sugar into carbon dioxide and
ethanol has gained a central role in food industry in being a leavening agent for bread and a
producer of alcoholic beverages [22]. The use of fermentation, particularly for beverages, has
existed since the Neolithic era. The oldest archeological evidence has been documented dating
from 7000–6600 B.C. in Jiahu, China (Figure 4) [23]. Even without any deeper knowledge
about the process, early human societies could use the combined leavening action of yeasts and
bacteria (i.e. sourdough). Around the 19th century bakers had started to use the yeasts from beer
brewers by collecting them after wort fermentation [24]. As consequence of that the bread
started to have a sweet note, lacking the sourness acquired by co-fermenting with Lactobacilli.
In the late 1700 Antoine Lavoisier was the first who tried to describe the fermentation process
identifying the raw material necessary for the process: sugar, water, and “ferment” paste [25]. At
that time the fermentative process was considered a simple chemical reaction where the “living
material” had only a secondary role. In the second half of the 19th century, Louis Pasteur proved
that alcoholic fermentation was the result of a microbial process (Figure 4) [25]. In 1857 Pasteur
showed that lactic acid fermentation is also caused by living organisms and some years later he
defined fermentation as a “form of life without air” (“Pasteur effect”). By the early 1900s still no
one could exactly explain the biochemistry of the process until the German chemist Eduard
Buechner could ferment a sugar solution by using the first “yeast-extract” ever made. He proved
that “cellular machineries” inside microorganisms are able to catalyze all the chemical reactions
that occur inside the cell [26]. Enzymatic studies followed and scientists began to analyze and
purify the components of cell-free extract. Eduard Buchner detected the active components of
the cells extract, coined them as “zymase” in 1897 (Figure 4). Some years later Arthur Harden
and William Young could distinguish two fractions of yeast extract: i) A high molecular weight
and heat sensitive fraction, which contained mainly enzymes; and ii) a low molecular not heat
15
sensitive fraction, which was enriched in cofactors (i.e. NAD, NADH, NADP,ATP, ADP, etc.). A
major breakthrough in enzyme research was then reached after the 1940s when Otto Meyerhof
and Luis Leloir resolved details about the glycolytic pathway, showing the complexity of an
enzymatic pathways for the first time [27] [28].
Another important milestone in the history of fermentation was the definition of the Crabtree
effect by the English biochemist Herbert Grace Crabtree [29]. This effect can be described as the
production of ethanol in aerobic condition instead of the production of biomass via the
tricarboxylic acid cycle (TCA). This mechanism clearly denies the “Pasteur effect” described
above. At high glucose concentrations and in the presence of oxygen, S. cerevisiae and other
Crabtree positive yeasts first consume the glucose and then start consuming the by-product
ethanol when the glucose is depleted. This mechanism is now known to be controlled by glucose
repressing respiratory enzymes (e.g. Mig1), which control sugar metabolism and glucose
transport activity [30].
Several authors speculated that the origin of the fermentation mechanism and the relative
molecular controls are linked with the origin of modern plants with fruits, at the end of the
Cretaceous age, more than 125 million years ago [31]. In this environment the competition for
fruit sugars started among the microbial communities. The Crabtree effect could have been a
strategic solution for yeast to outcompete bacteria. By sacrificing biomass, the production of
ethanol and the increased glucose uptake would have given yeasts an advantage to secure the
carbon source and inhibit the environment from microbial competitors [32].
16
Figure 4. Timeline showing the major discoveries which have contributed to define the fermentation
process.
In the 1930s advances in microbiology and biochemistry made it possible to obtain the first
mutated yeast by physical and chemical treatment. The easy propagation of yeast cells and
manipulation of the genome combined with the simple cellular physiology made S. cerevisiae a
model system. Sequencing technologies provided the possibilities to sequence entire genomes,
unveiling the comprehensive biological information contained therein. In this genomic era
S. cerevisiae was the first eukaryotic species to be completely sequenced [33]. With this
knowledge about the genetic material questions about gene regulation, structure-function
relationships of proteins and chromosome structure could be answered [34]. Moreover, the
finding that 40% of yeast proteins share part of their primary amino acid sequence with the
corresponding human protein indicate that genes and functions are widely conserved in certain
processes [35]. For instance, human and yeast HMG-CoA reductase proteins are 66% identical.
They are responsible for starting the steroid biosynthesis in both species. Even though yeast
does not produce cholesterol the yeast proteins can function in the human cholesterol
biosynthesis pathway [36].
The yeast receptor Ste2 is involved in the recognition of α-mating factor. A similar protein is
found in human called β-adrenergic receptor which functions as a regulator of the blood
17
pressure [36]. Overall many secretary proteins, heat-shock proteins, G-proteins and
transcriptional factors of yeast show homologs in the human genome and have similar function
in both species. This relationship can be used as a powerful tool to unveil the function of
unknown sequences of human and other higher eukaryotic genes [37].
Even though S. cerevisiae has played a central role as a model organism as far back as the early
1900s it is just one representative of the entire yeast community. Most of these species were
described in the last 20 years (i.e. sensu stricto species), but the yeast variety is enormous and
new species are discovered continuously [38].
Therefore, studying new species represent a unique opportunity to open and create new
research directions, intended to understand more about biological processes or to unveil new
industrial applications.
1.4
Yeast carbon metabolism
A tightly regulated carbon metabolism is a fundamental requirement for in the fermentation
process. Yeast can thrive on different carbon sources but glucose and fructose are favored over
others [39]. In yeasts the glucose uptake occurs by facilitated diffusion through the plasma
membrane by hexose transporters. These plasma membrane proteins belong to the HXT family
where each of them exhibits different glucose affinity depending on the glucose concentration.
Their expression is tightly regulated by glucose sensor proteins placed in the plasma membrane.
Due to these sensors the expression of the optimal hexose transporters is achieved for the
respective
concentration
of
glucose
available
outside
the
cell
[40]
[41].
When yeast is grown on carbon sources other than glucose (e.g. maltose) the sugar is imported
into the cell by a proton symport mechanism. This transport requires the establishment of an
electrochemical gradient of protons which can only be achieved by depletion of internal ATP
[42] [43].
The first step of the glycolytic pathway is the conversion of glucose into glucose-6-phosphate by
hexokinases (Hxk1/Hxk2) or the glucokinase Glk1 (Figure 5; 1). This step is the first irreversible
step of the glycolysis and results in the retention of glucose inside the cell. In particular, Hxk2 is
highly expressed during the growth phase when fermentable carbon sources (i.e. glucose,
fructose…) are provided. Hk1 and Glk1 on the other hand are de-repressed when a nonfermentable carbon source (i.e. ethanol, glycerol…) is present [39].
18
After phosphorylation a series of chemical reactions lead to the formation of pyruvate [44]. Once
pyruvate is formed it can be fed either into the respiratory or fermentative pathways (Figure 5;
2). In the respiratory pathway, pyruvate enters the mitochondria where it is converted into
acetyl CoA by the mitochondrial pyruvate dehydrogenase enzyme complex [45].
The acetyl CoA is then further oxidized in the tricarboxylic acid cycle (TCA cycle) where it
generates reducing power (i.e. NADH and FADH) necessary for the final production of ATP
(Figure 5; 3). Since TCA intermediates are also used as building blocks to generate biomass,
several anaplerotic reactions are available to ensure a proper flux in the TCA cycle. An example
of an anaplerotic reaction is the enzymatic conversion of pyruvate into oxaloacetate, which can
directly enter the mitochondria [44] (Figure 5; 4).
In fermentative conditions the pyruvate is decarboxylated into acetaldehyde and - depending on
the redox status of the cell - it can follow two fates. Acetaldehyde can be reduced to ethanol by
the enzyme alcohol dehydrogenase [46] or oxidized to acetate by aldehyde dehydrogenase [47]
(Figure 5; 5 and 6). If we look at the energetic yields, respiration is far more efficient.
Fermentation generates only a net of 2 ATP per molecule of glucose, whereas respiration
produces 38 ATP in S. cerevisiae. On the other hand, fermentation does not utilize an
electrochemical gradient to form ATP, which simplifies the overall redox balance of the cell.
Moreover to counterbalance this low ATP yield yeasts increase the glycolytic flux by up
regulating glucose transporters. This mechanism ensures a rapid conversion to pyruvate and
final fermentation products, producing enough ATP to allow cell growth [48].
19
Figure 5. Schematic overview of the carbon metabolic pathways in yeasts.
1.5
The Eremothecium genus: Ashbya gossypii and
Eremothecium cymbalariae
The Eremothecium genus was instated by Antonino Borzi (1852-1921) with the description of
Eremothecium cymbalariae in 1888 [49]. The genus has been assigned to clade 12 (Figure 2) of
the ‘Saccharomyces complex’ [9] and shows interspecific divergence [50]. It contains seven
members: Eremothecium gossypii (better known as Ashbya gossypii; [51]; Eremothecium
cymbalariae [49]; Nematospora/Eremothecium coryli [52] [53]; Nematospora/Holleya/Erem
othecium sinecaudum [52], [54]; Eremothecium ashbyi [55]; Ashbya aceri [56].
20
Ashbya gossypii
Ashbya gossypii is a filamentous fungus, originally isolated from cotton as a pathogen causing
stigmatomycosis. The fungus uses insects of the Pyrrohocoridae family as vectors [51]. With
today’s use of insecticides fungal infections via insect vectors are less problematic. A. gossypii is
mainly used in industry for the production of riboflavin (vitamin B2) [57] [58]. Several studies
have elucidated the molecular pathways involved in riboflavin production [59] and aimed at
improving the riboflavin yield by utilizing different substrates [60] [61] [62] [63]. The life cycle
of A. gossypii starts with the germination of needle-shaped spore and the formation of a
spherical germ cell (Figure 6). This stage is characterized by DNA replication, accompanied by
an isotropic growth phase. After a switch from isotropic to polar growth a hyphal tip is formed
that grows to a juvenile mycelium by lateral branching. The hyphae are compartmentalized into
multinucleated cells whose borders are separated by chitin-rich septa. About 20-24 hours post
germination hyphal maturation results in the formation of a mature mycelium, which is
characterized by an accelerated growth speed and dichotomous branches at the hyphal tips.
Upon nutrient limitation, initiated in older segments of a mycelium, hyphae generate sporangia
which contain needle shaped spores. The life cycle ends with the formation of 26-30 µm long
spores, clumping together via terminal filaments [57].Due to the biotechnological interest in
riboflavin production molecular analyses were already started in the 1990s. Molecular studies
revealed the identification of an efficient homologous recombination system in A. gossypii [64].
Based on that, efficient gene targeting methods for molecular manipulation have been
successfully developed by the Philippsen group [65].
In 2004 the A. gossypii complete genome sequence was published. Its annotation revealed a
large degree of synteny with the genome of the close relative S. cerevisiae [66]. About 95% of all
the 4718 protein-coding genes in A. gossypii have a homologue in yeast, indicating that the basic
cellular functions and morphogenetic machineries are well conserved [57]. However, the
Eremothecium and Saccharomyces genera diverged more than 100 million years ago before the
whole genome duplication occurred in S. cerevisiae. This duplication event created about 5000
twin ORFs in the duplicated S. cerevisiae genome, raising the possibility to diverge the function
of the duplicates. A. gossypii with its 8.8 million base pairs evolved to a very compact genome
caused by the reduction of intergenic regions [50].
21
Figure 6. A. gossypii life cycle. Isotropic growth phase of the germinated spore (a) is followed by the
generation of the first germ tube (b). The formation of the second germ tube is opposite to the first (c).
The growing hyphae start the lateral branching pattern (d) before they mature and switch from lateral to
dichotomous tip branching with an increased growth speed (from 10 µm/h to 200 µm/h) (e). In older
hyphae sporangia formation and hyphal fragmentation occur, and sporulation and breakdown of the
ascus walls takes place (f) [57]. The picture in the background of the life cycle shows a juvenile mycelium
of A. gossypii stained with Calcofluor white. Septal sites and hyphal cell walls contain chitin and are,
therefore, highlighted with the dye.
Additionally, it could be shown that the average length of introns (107 bp) is less than half of the
average intron size in S. cerevisiae (244 bp) [56]. The ease of genetic manipulation combined
with the available genome sequence and industrial interest of the strain made A. gossypii an
attractive model for studying the formation and maintenance of filamentous growth and a
model for comparative genomics and evolutionary studies [57].
22
Eremothecium cymbalariae
Eremothecium cymbalariae is a close relative of A. gossypii, which was recently sequenced and
annotated. Its genome consists of 9,7 million bp encoding for 4712 [50].Both fungi share about
97%of homologous genes. Nevertheless, there are significant differences in their genomes. A.
gossypii has only 7 chromosomes whereas E. cymbalariae has 8. The high GC content of 52% in
A. gossypii was not found in E. cymbalariae. With 40% GC content it is more similar to that of
S. cerevisiae (38.3%) [50]. As for A. gossypii, Eremothecium cymbalariae is a homothallic
fungus. [67] In contrast to A. gossypii the spores are produced in aerial sporangia. Additionally,
E. cymbalariae is not over producing riboflavin like A. gossypii [50] (Figure 7).
Figure 7. Juvenile mycelium of A. gossypii (A) and E. cymbalariae (B). In the upper right corner are
displayed the different spores shape of A. gossypii and E. cymbalariae.
1.6
Fungal system and their contribution to industrial processes
In the history of mankind fungi have been involved in many processes such as the production of
fermented food and drinks. The use of fungi in spontaneous fermentations led to an increase in
the shelf life of the food products and to the production of fermented beverages. The
consumption of alcoholic drinks may have started as a religious procedure but became very
popular and was a way of avoiding water-borne diseases [68]. Later, biotechnological advances
provided new ways to use fungi as cell factories [69]. Discoveries and the development of
molecular biology provided new insights into biological pathways offering wide applications in
the food, chemical, cosmetic and pharmaceutical sector [70]. Nowadays, biotechnological
applications are generally preferred over chemical processes. Among others the main reasons
are that chemical syntheses often involve environmentally unfriendly production processes
coupled with an undesirable racemic mixture of compounds [71]. Advantages of bio-production
23
are the reduced material and energy consumption, increased use of renewable and
biodegradable materials, reduced waste generation and the production of more environmentally
friendly stereospecific products [72]. Table 1 contains a list of some common industrial
application of fungi.
1.7
Aroma and flavour definition, chemical type
The word “aroma” derives from Latin and was coined in the 13 century to define “sweet odors”.
Also the Greek word “ἄρωμα“describes any “seasonal spice or sweet herb”. “Flavour” on the
other hand, derived from the vulgar Latin word flator , literally "that who blows”, and the old
French word flaour "smell, odor" (http://www.etymonline.com/). Today flavours are defined as
a complex combination of the olfactory, gustatory and trigeminal sensations perceived during
tasting (according to the International Organization for Standardization). Nevertheless, the term
"flavour" is used to describe different things. Sensory experts use flavour to describe the
combination of taste and smell. Flavour chemists typically mean a single aroma, while chefs
tend to use it not only on the food per se, but also for its overall presentation on the plate. In
many cases the flavouring agents have a defined molecular structure, making food attractive and
eating or drinking a pleasure. Flavour substances can be either volatile or non-volatile. The
latter consists mainly of carbohydrates, amino acids and fruit acids, and includes taste
substances only. The volatile part includes both taste and odor substances, and contains
alcohols, esters, acids, aldehydes, amines and sulfur and nitrogen compounds (Table 2). When
we compare the relative sensitivity of our senses we realize the big difference between our smell
and taste sensitivities. The sense of smell is approximately 10.000 fold more sensitive than our
sense of taste [73]. Furthermore, without an appropriate volatile bouquet our food would taste
very alike. Hence studying the vast variety of volatile constituents of food and fermented
products is of great importance.
24
Application
List of compounds
Organism
Description
Food, drinks and fodder
Alcohols,
pyrazines,
ketones
Saccharomyces sp., Lactobacillus sp.,
Brettanomyces sp., Zygosaccharomyces
sp., Hanseniospora sp. and Candida sp.
Bread, beer, wine, cheese are just a sample of the fermented
products produced by yeast fermentation.
Metabolic products
esters,
acids,
aldehydes
and
B group vitamins
Ashbya gossypii , Eremothecium ashbyi,
Brewer’s yeast
Antibiotics: Cephalosporin C,
Penicillin, Griseofulvin
Penicillium notatum, Cephalosporium
sp., Penicillium griseofulvum
ImmunoCyclosporin A
suppressives:
Tolypocladium inflatum
Statins: Lovastatin, Mevastatin
Aspergillus terreus, Penicillium citrinum
Polyunsaturated fatty acids
(PUFAs)
Morteriella
circinelloides
Antitumor: Taxol
Taxomyces andreanae
Pigments
Blakeslea trispora, Monascus purpureus
Microbial leaching
Citrate, Succinate,
Oxalacetate
Biological control of pathogen
Naphthalene,
Nitrosoamide
Environment protection
Depolymerases
Oxalate,
Citronellal,
isabellina,
Fungal secondary metabolites are extremely important to our
health and nutrition. More and more metabolites are produced
by fungal species, some of them modified and optimized by
genetic engineering (for istance organic acids, vitamins,
antibiotics, steroids). In this category we also find production of
enzymes and recombinant polypeptides (insulin, hirudin, statins,
taxol) .
Mucor
Mucor sp., Paecilomyces sp., Penicillium
sp., Trichoderma sp., Cladosporium sp.,
Alternaria and Apergillus sp.
Metal-containing ores are percolated with the microrganism. The
metal is then solubilized by the fungal metabolism and then
extracted or precipitated.
Phoma sp., Muscodor sp., Rhizopus sp.,
Fusarium sp.
In oder to reduce the need for chemical compounds is now
possible to prepare a mixture of fungal species to control insect
pests.
Apergillus sp., Pestalotiopsis sp., Mucor
sp.
Gentically modied fungi have been shown to be able to degrade
plastic materials and highly toxic hydrocarbon species
Table 1. Sample list of the more common biotechnological application of fungi.
25
1.8
Flavour additives and natural flavours
Adding substances to food for conservation, flavouring, or appearance is not a modern-day
invention [74]. Before the first refrigeration system was invented, salts were used to preserve
meats and fish. Sugar was added to preserve fruits and cloves were placed in hams to inhibit the
growth of bacteria [75]. Ancient cultures added sulfites to preserve wine and spices and
colorings were used to enhance flavours of foods [74]. Today, there are thousands of food
additives found in foods.
Functional group
Alcohol
-OH
Source
Example
Smell
Plants
Geraniol, linalool
Menthol
Fresh, floral
Mint
Aroma-active alcohols > c3
Sweet or pungent
Fat
Milk products
Diacetyl
Like butter
Cheese
Formic acid
Capric acid
Pungent
Like goats’ milk
Ester, lactone
Solvent (these chemicals
are used as solvents)
Ethyl acetate
Glue
-COOR
Fruit
Methyl/ethyl butyrate
Pineapple
Amyl/butyl acetate
Pentyl butyrate
2-isobutyl-3methoxypyrazine
Banana
Apricot
Aldehyde; ketone
-CHO; >C=O
Acid (C1-C12)
-COOH
Pyrazine
aromatic =N-
Roasted,
fermented foods
S-compounds: aliphatic,
aromatic
Vegetables
Phenols (mono-, poly-)
Smoked food
cooked,
Earth, spice, green pepper
2-acetyl-tetrahydro-pyridine
Diallyldisulphide
1,2-dithiolane-4-carboxylic
acid
Guaiacol
Cresol
Popcorn
Garlic
Asparagus
Wood smoke
Tarry
Table 2. List of the most common volatile compounds and relative perception descriptors
(www.scienceinschool.org).
The increasing demand for high quality food products made a radical change in food production
and technology necessary. Most of the global market is now dominated by processed food which
requires food additives to reestablish flavour and taste characteristics. A food additive is defined
as “any substance the intended use of which results (…), in its becoming a component or
otherwise affecting the characteristics of any food” [76]. Direct food additives are added
26
intentionally to a food for a specific purpose like the sweetener aspartame, sugar or salt which
are used to give taste and texture to the products. Indirect food additives are those substances
which become part of the food product during the packaging, storage and handling [77]. Direct
food additives have different functions: they improve the texture appearance, keep freshness,
increase the nutritional contents and improve flavour and taste of the products [78] (Table 3).
Due to the increasing number of food additives, the European Economic Community (EEC)
established code names to identify food additives. All food additives used in the European Union
are identified by an E-number and each food additive is assigned a unique number. The
numbering scheme follows precise rules which place the food additive in the respective category
(for more details look at the European commission website: http://ec.europa.eu/food/food/fAE
F/additives/lists_authorised_fA_en.htm).
Typical
flavour
additives
are
fruit
flavours,
sweeteners, cheesy flavours, buttery or roasted flavours, dosed in either natural or artificial
concentrations. The difference between them is not at the chemical level. The natural process
and the chemical synthesis lead to the same chemical compound. The difference between
natural and chemical flavours lies in the source of the flavour. The so called “natural” flavours”
are substance obtained by appropriate physical, enzymatic or microbiological processes from
material of vegetable, animal or microbiological origin [76]. Food manufacturers often use
natural flavours simply because the term "natural" appears healthier to consumers. The result of
this trend was a remarkable increase in natural flavour production, reaching a market of $3.5
billion
in
2011
and
a
predicted
(http://www.marketsandmarkets.com).
27
market
of
$5
billion
in
2017
Types of food
additivies
Function
Examples
Fruit sauces and jellies, beverages,
baked goods, cured meats, oils and
margarines, cereals, dressings,
snack foods, fruits and vegetables
Beverages,
baked
goods,
confections,
table-top
sugar,
substitutes, many processed foods
Names Found on Product Labels
Ascorbic acid, citric acid, sodium benzoate, calcium propionate,
sodium erythorbate, sodium nitrite, calcium sorbate, potassium
sorbate, BHA, BHT, EDTA, tocopherols (Vitamin E)
Preservative
Prevent food spoilage from bacteria, molds, fungi, or
yeast
Sweeteners
Add sweetness with or without the extra calories
Color Additives
Offset color loss due to exposure to light, air,
temperature
extremes,
moisture
and
storage
conditions; correct and enhance natural variations in
color
Many processed foods, (candies,
snack foods margarine, cheese, soft
drinks))
FD&C Blue Nos. 1 and 2, FD&C Green No. 3, FD&C Red Nos. 3 and 40,
FD&C Yellow Nos. 5 and 6, Orange B, Citrus Red No. 2, annatto
extract, beta-carotene, grape skin extract, cochineal extract or carmine,
paprika oleoresin, caramel color, fruit and vegetable juices, saffron
Flavours and Spices
Add specific flavours (natural and synthetic)
Pudding and pie fillings, gelatin
dessert mixes, cake mixes, salad
dressings, candies, soft drinks, ice
cream
Natural flavouring, artificial flavour, and spices
Flavour Enhancers
Enhance flavours already present in foods (without
providing their own separate flavour)
Many processed foods
Fat Replacers
Provide expected texture and a creamy "mouth-feel" in
reduced-fat foods
Nutrients
Replace vitamins and minerals lost in processing
(enrichment), add nutrients that may be lacking in the
diet (fortification)
Emulsifiers
Allow smooth mixing of ingredients, prevent separation
Stabilizers
Thickeners
and
Produce uniform texture, improve "mouth-feel"
Baked goods, dressings, frozen
desserts, confections, cake and
dessert mixes, dairy products
Flour,
breads,
cereals,
rice,
macaroni, margarine, salt, milk,
fruit beverages, energy bars,
Salad dressings, peanut butter,
chocolate,
margarine,
frozen
desserts
Frozen desserts, dairy products,
cakes, pudding and gelatin mixes,
dressings, jams and jellies, sauces
Beverages,
frozen
desserts,
chocolate, low acid canned foods,
baking powder
Sucrose (sugar), glucose, fructose, sorbitol, mannitol, corn syrup, high
fructose corn syrup, saccharin, aspartame, sucralose
Monosodium glutamate (MSG), hydrolyzed soy protein, autolyzed
yeast extract, disodium guanylate or inosinate
Olestra, cellulose gel, carrageenan, polydextrose, modified food starch,
microparticulated egg white protein, guar gum, xanthan gum, whey
protein concentrate
Thiamine hydrochloride, riboflavin (Vitamin B2), niacin, niacin amide,
folate or folic acid, beta carotene, potassium iodide, iron or ferrous
sulfate
Soy lecithin, mono- and diglycerides, egg yolks, polysorbates, sorbitan
monostearate
Gelatin, pectin, guar gum, carrageenan, xanthan gum, whey
pH control agents and
acidulants
Control acidity and alkalinity, prevent spoilage
Leavening Agents
Promote rising of baked goods
Breads and other baked goods
Anti-caking agents
Keep powdered foods free-flowing, prevent moisture
absorption
Humectants
Retain moisture
Salt, baking powder, confectioner's
sugar
Shredded coconut, marshmallows,
soft candies, confections
Yeast Nutrients
Dough strengtheners
and conditioners
Firming agents
Enzyme preparations
Promote growth of yeast
Breads and other baked goods
Calcium sulfate, ammonium phosphate
Produce more stable dough
Breads and other baked goods
Ammonium sulfate, azodicarbonamide, L-cysteine
Maintain crispness and firmness
Modify proteins, polysaccharides and fats
Gases
Serve as propellant, aerate, or create carbonation
Processed fruits and vegetables
Cheese, dairy products, meat
Oil cooking spray, whipped cream,
carbonated beverages
Calcium chloride, calcium lactate
Enzymes, lactase, papain, rennet, chymosin
Carbon dioxide, nitrous oxide
Table 3. Summary list of the most common food ingredients (http://www.fda.gov)
28
Lactic acid, citric acid, ammonium hydroxide, sodium carbonate
Baking soda, monocalcium phosphate, calcium carbonate
Calcium silicate, iron ammonium citrate, silicon dioxide
Glycerin, sorbitol
1.9
Bioflavour production (Ehrlich pathway, FFAs and lactate)
Up to date, plants and animals represent the main source of bioflavours. Nevertheless, these
compounds are often produced in low concentration, making extraction, purification and
formulation too expensive for the market. Therefore, microbial processes represent a promising
frontier, offering de novo flavour synthesis or bioconversion of natural precursors [79]. The
ability to produce flavour compounds can be applied in two different ways: i) in situ flavour
generation, where the compounds become an integral part of food or beverage (i.e. cheese,
yogurt, beer, wine) and ii) specifically designed processes where the obtained aroma compounds
are isolated and used later as ‘natural’ additives in food production [80]. Several fungi are able
to produce various mixtures of so-called gas-phase or generally named volatile organic
compounds (VOCs). Diverse definitions of the term VOCs are in use, depending on laws and
government agencies. Biologically generated VOCs can be defined as any organic chemical that
has a high vapor pressure at room temperature. Fungal VOCs are mainly derived from
secondary metabolism pathways. Due to their small sizes they can be very difficult to detect.
Furthermore, VOC profiles are species or strain dependent and can vary according to different
parameters such as temperature, micronutrients, oxygen, redox status of the cell, pressure,
incubation time, carbon and nitrogen sources and others environmental factors [81]. At the
beginning of the 20th century, researchers clarified some of the major biochemical pathways
involved in flavour formation and unveiled relationships between a specific microorganism and
a desirable mix of flavours [79]. Despite that, flavour yields are often quite low and the limited
knowledge about the biochemical pathways and the regulation of key enzymes still represents a
bottleneck for novel technologies.
In the following paragraphs I provide a brief description of the three main molecular pathways
involved in the flavour synthesis in fungi (Figure 8), with emphasis on the catabolism of amino
acids and related events in yeast.
29
Figure 8. Schematic representation of the three main pathways responsible for the flavour formation in
yeast (modified from [82]).
Ehrlich pathway
In 1907 the German biochemist Felix Ehrlich (1877-1942) proposed that amino acids were split
by a “hydrating” enzyme activity to form the corresponding fusel alcohols, along with carbon
dioxide and ammonia [83]. Following work by Lampitt [84], Yamada [85], and [86] [87] could
show that the higher alcohols produced by yeast are derived from amino acid catabolism. This
pathway, which later was named after its discoverer, Ehrlich, utilizes valine, leucine, isoleucine,
methionine, phenylalanine, tyrosine and tryptophan to generate higher volatiles with distinctive
flavours (Table 4).
30
Amino
acid
a-Keto acid
Fusel aldehyde
Fusel alcohol
Fusel acetate
Leu
4-Methyl-2-oxopentanoate
3-Methylbutanal
Isoamyl alcohol
Isoamyl acetate
Val
3-Methyl-2-oxobutanoate
2-Methylpropanal
Isobutanol
Isobutyl acetate
Ile
3-Methyl-2-oxopentanoate
2-Methylbutanal
2-Methylbutanol
Ethyl pentanoate
Phe
3-Phenyl-2-oxopropanoate
2-Phenylethanal
2-Phenyl ethanol
2-Phenylacetate
Tyr
3-(4-Hydroxyphenyl
2-oxopropanoate
2-(4Hydroxyphenyl)
ethanal
2-(4Hydroxyphenyl)
ethanol
2-(4Hydroxyphenyl)
ethanoate
Trp
3-(Indol-3-yl)-2oxopropanoate
2-(Indol-3-yl)ethanal
Tryptophol
2-(Indol-3-yl)
ethanoate
Met
4-Methylthio-2oxobutanoate
Methional
Methionol
3-(Methylthio)
propanoate
Table 4: The Ehrlich pathway intermediates (modified from [88]).
The catabolism of the amino acids is characterized by three enzymatic steps: including a
transaminase, a decarboxylase and an aldehyde dehydrogenase reaction (Figure 9). Although
this pathway has been discovered a century ago little is known about the metabolic and genetic
regulation of each step. Furthermore, it is still unclear why these secondary metabolites are
produced since yeast could use the amino acids for its protein synthesis instead. Several
publications consider the higher volatile production as a way to remove toxic compounds from
the cell, to help maintaining the NADH/NAD+ ratio and redox balance [89] or to balance the
cells nitrogen sources [90] [91]. Furthermore, volatiles are also known to be important signal
molecules used as attractants to insect which are then used as vectors for dispersal [92].
31
Figure 9: The Ehrlich pathway. The three enzymatic reactions responsible for converting aromatic and
branched-chain amino acids into aromatic compounds (modified from [88]). Respective genes of
S. cerevisiae are listed aside for each enzymatic reaction.
Increasing demand for natural flavour compounds, such as 2-phenylethanol, isoamyl alcohol
and their respective esters has led to a new interest in research to investigate this pathway in
order to control the flavour production in fermented products.
The first transamination step of the catabolic degradation of amino acids via the Ehrlich
pathway is controlled by four genes, namely BAT1, BAT2, ARO8 and ARO9. BAT1 and BAT2
encode mitochondrial and cytosolic amino acid aminotransferases, respectively. The expression
of the mitochondrial enzyme Bat1 is higher in the exponential phase of growth whereas the
cytosolic Bat2 is preferably expressed during stationary phase [93]. Recent studies
demonstrated the fundamental importance of BAT2 during flavour production. Overexpression
and deletion of this gene resulted in a drastic increase or reduction of the level of higher volatile
compounds, respectively [94]. The two additional aminotransferases ARO8 and ARO9 are not
specifically expressed in distinct growth phases only. However, little is known about the
regulation of ARO8 or ARO9 [88]. The second reaction of the Ehrlich pathway is a
decarboxylation step. This step can be performed by five thiamine diphosphate (TPP)dependent decarboxylases, namely Pdc1, Pdc5, Pdc6, Aro10, and Thi3. Presumably, each
32
decarboxylase has an α-keto acid specificity but to date there are no convincing results available.
Only Aro10 seems to have a broad activity towards α-keto acids [88].
The third step of the Ehrlich pathway is a reduction/oxidation. This step is strictly dependent on
the growth condition. Under anaerobic condition yeast cells have excessive production of
NADH. The Ehrlich pathway may then guarantee an efficient NAD(P)+ regeneration. The
cellular redox status is most likely the major factor that controls the ratio of fusel acid or fusel
alcohol production. In the S. cerevisae genome six aldehyde dehydrogenase and 16 alcohol
dehydrogenase genes are found. Unfortunately, glucose-limiting chemostats with phenylalanine,
methionine, or leucine as the sole nitrogen source did not show conclusive results of their
regulation since these enzymes strongly overlap in their function [95].
Transcriptional and post-transcriptional regulation of the Ehrlich pathway genes is poorly
understood. ChIP–chip experiments (chromatin immuno-precipitation on a microarray)
revealed that ARO80 is the main transcription factor (TF) that regulates the expression of the
ARO9, ARO10 and ARO80 itself [96]. Other approaches e.g. in vivo studies confirmed this
regulation pattern [97]. Aro80 belongs to the Zn2Cys6 family of transcription factors known to
bind palindromic elements. Recent literature reveals several ARO80 binding sequences which
are all different and unique binding site [96] [98] [99]. Although the DNA binding motive is
unclear all studies agree on the Aro80-dependent expression of ARO10 and ARO9.
Once volatile compounds are made they are transported out of the cell. The export of higher
alcohols might occur by simple membrane diffusion, although the ATP-dependent transporter
Pdr12 seems to be involved in the export of weak organic acids [100].
Fatty acids as substrates for flavour formation
Free fatty acids (FFAs) are considered important precursors for different varieties of volatile
compounds, especially in the flavour development of cheese. Lipolysis is an important
biochemical step in cheese ripening. In particular the process is essential in Italian cheese
varieties and blue mold cheeses. Indeed, milk-fat contains high concentration of short and
intermediate triglycerides which then can become free after hydrolysis. Lipases in cheese can
originate from several sources: milk, starter bacteria or fungi but also lipase preparations.
Lactococcus sp. has a weak lipolytic capability but if present in high numbers can be responsible
for the liberation of high levels of FFAs. In contrast, Penicillium sp. exhibits high levels of
33
lipolytic activity and is responsible for the distinctive lipolysis in mold ripened cheeses [82]. As
shown in Figure 10 FFAs are the precursors for a number of flavour molecules.
Figure 10. General pathways for the catabolism of FFAs (modified from [82]).
Floral and fruity lactones are the results of a spontaneous intramolecular esterification of
hydroxyl fatty acids. For instance, γ -and δ -lactones contribute to a peculiar peach note in
Cheddar cheese. FFAs can also interact with alcohols to yield long chain esters. In Emmenthal
cheese fourteen different esters have been found and in the Italian Parmiaggiano Reggiano up to
38 esters, suggesting their importance in the final product [82].
Metabolism of lactate and citrate
Lactose is the starting fermenting substrate and the major carbon sources in food industry
applications. For example, the dairy processing starts with certain starter bacteria (Leuconostoc
sp., Lactococcus sp., Clostridium sp.) able to metabolize the lactose into a racemic mixture or Lor D-lactate (Figure 8). The lactate is then utilized by bacteria to produce mainly propionate,
acetate and CO2. Acetate and propionate contribute to the flavour of the cheese whereas carbon
dioxide is important for the “eye” formation in the cheese, the typical holes found in Emmenthal
and Swiss-type cheeses [82]. Camembert and Brie are the results of mixed-fermentation of
bacteria and fungi. Initially lactic acid bacteria release lactate which is rapidly metabolized by
fungi such as Debaryomyces hansenii, Geotrichum candidum, Penicillium camemberti [101].
The role of the fungi is to convert the lactate into CO2 and water, and therewith to increase the
pH at the cheese surface. Deacidification is an important step during cheese production, because
it allows growth of coryneform bacteria involved in the ripening and flavour maturation of many
34
cheeses. Furthermore, the alkaline pH on the surface makes the final maturation step possible
that is carried out by Penicillium camemberti in Camembert cheese. In this last part the amino
acids released from the casein are converted into NH3 and fruity esters [82].
As mentioned earlier citrate is a key intermediate in the TCA cycle but it is also one of the
constituents of milk (ca. 8 mmol∙L-1). Further, citrate is present in fruit juices, vegetables and is
generally added as a preservative to foods. In nature, only a limited number of bacteria are able
to ferment citrate [101]. Generally, it is not used as a carbon source but it is metabolized
together with lactose and other carbon compounds. The products of the citrate metabolism are
4-carbon compounds, diacetyl, acetate, acetoin and 2,3-butanediol. Diacetyl is well studied and
usually produced only in small amounts (1-10 µg∙mL-1 in milk) [82]. In cheeses and yogurts it has
a very positive effect on the aroma profile because it gives the typical buttery note to the final
products. In contrast, diacetyl is considered an off-flavour in lager beer production when it
exceeds a concentration higher than 0,15 ppm [102].
1.10 Biological properties of quorum sensing molecules and VOCs as
signaling molecules
Biofilm formation and morphological development are controlled by the exchange of chemical
compounds, better known as quorum-sensing signals. When a quorum sensing molecule reaches
a critical concentration, the entire population of a species reacts by activating specific target
genes. For instance, yeasts grown in the presence of isoamyl alcohol, one of the major alcohols
produced during beer/wine fermentation, induce pseudohyphal growth [103] [104]. The effect is
not limited to this alcohol alone, since 2-phenylethanol, another compounds derived from the
amino acids catabolism, seems to affect morphogenesis as well and induces invasive growth
[105]. The same two compounds are produced in low concentration also in Candida albicans
and Candida dubliniensis. In these species they participate in the dynamics of biofilm formation
[106]. However tyrosol and farnesol are the main extracellular molecule which controls mycelia
development in C. albicans. Tyrosol stimulates germ tube formation, whereas farnesol act as an
inhibitor of mycelia development [107] [108]. When both signals are present the farnesol is
dominant over tyrosol [109].
Further, volatiles and several flavouring molecules are known to be used as biological signals for
communication.
Many
fungal-insect,
fungal-fungal,
fungal-bacterial
and
fungal-plant
interactions have been discovered in a broad range of disciplines. For practical reasons very few
35
studies have been done using gas phases, assuming that the physical state of the compounds
only matters the critical dosage necessary to trigger an effect. Furthermore VOCs produced by a
given fungal species can raise different responses, depending on the environmental context. The
classical example is the ubiquitous fungal volatile 1-octen-3-ol. In Aspergillus nidulans this C-8
compounds acts as inhibitor of conidiation when it reaches high concentrations. It is a selfcontrol system to steer the asexual reproduction. The inhibitory effect is reversible since
conidiospores could normally germinate when the 1-octen-3-ol was removed [110]. When
Arabidospis thaliana is exposed to 1-octen-3-ol the plant’s defense mechanisms become active
[111] blocking the expansion of the pathogen, Botrytis cinerea, on infected leaves. In insects, the
C-8 alcohols production by wood-decay fungi stimulate the reproduction of the conifer feeding
bark beetle, and inhibit the growth of their natural predators [112].
Furthermore, the R enantiomer of the 1-octen-3-ol is commercially known as “roctenol” and is
widely used as insect attractant [113] [114]. Even though fungal volatiles are generally perceived
as attracting agents for insects, they can also act as repellents. Oviposition of female houseflies is
blocked in substrates infected by Fusarium, Phoma, Rhizopus, and other fungi. The VOCs
responsible for this reaction are dimethyl disulfide, phenylacetaldehyde, 2-penylethanol,
citronellal, and norphytone [115]. Bacillus nematocida is an interesting example of bacterial
predation against nematodes. It produces the worm attracting, volatile, organic compounds
benzaldehyde and 2-heptanone. The secretion of these bacteria attracts the nematodes and once
in the nematode guts they produce proteases leading the host to death [116]. Moreover, several
fungi are known to establish symbiotic interactions with plants as mycorrhizae. Gas phase
molecules can easily diffuse in cavity of the soil, reach far distances and produce intermediate
molecules to promote interspecies interactions. This mutualistic relationship between fungi and
plants is dynamic and controlled by different stimuli such as nitrogen to phosphate ratio,
abundance in the soil, soil moisture, etc. Advantages of such mycorrhizal interactions are an
increased
nutrient
uptake
and
resistance
to
pathogen
attacks
[117].
In conclusion, these small metabolites produced by a variety of organisms are responsible for a
number of biological activities and interactions. Many of them are still poorly understood and
need to be investigated. Presumably, technological advances and further research will elucidate
more of these chemical interactions, offering new prospective for applied disciplines.
36
1.11
Biotechnological application of fungal VOCs
Beside their role in biological activities VOCs have also an enormous potential in
biotechnological applications. In agriculture they can be used as biocontrol compounds,
reducing fungicides applied on crop plants. Precisely designed mixtures of volatiles are able to
inhibit plant pathogens [118]. The advantage of using small volatile compounds lies in their
ability to travel long distances and infiltrate in the soil pores. For instance, the non-pathogenic
Fusarium oxysporum MSA35 lives in association with a consortium of ectosymbiotic bacteria.
This association is able to suppress Fusarium wilt of pathogenic F. oxysporum. It has been
shown that the volatiles emitted by the fungus-bacteria consortium are able to modify the
mycelia surface of another pathogenic Fusarium isolates [119]. Another example is Muscodor
albus which is able to inhibit the growth of the broccoli pathogen Rhizoctonia solani and the
pathogen Phytophthora capsici, which cause root rot in bell pepper [120]. Mixture of volatiles
from M. albus can also be used in non-agricultural applications, for instance in controlling and
significantly reducing common building molds [121]. Also, it has been discovered that a number
of antifungal volatiles are active against several human pathogens [122] [123], and this may offer
an additional alternative to fight against drug resistant strains.
Recent evidence suggests that single VOCs are generally not effective [124] [125]. Organic
volatile mixtures are more predominant making it necessary to decrypt this chemical complex to
achieve the antifungal activity observed in vivo. The analysis of these VOC mixtures could also
become useful in medical areas to identify bacterial, viral and fungal infections in the lungs
[126]. Characteristic volatile mixture can serve as biomarkers to assess human infections by
using rapid and non-invasive techniques [127]. VOC emissions can also be used to mimic flower
essences to attract pollinating insects and facilitate pollen transfer [128]. By using mixtures of
volatiles that are normally produced by fungi to attract and promote their dispersion nature,
scents can be created to increase yields of fruit and seed crops [129]. Botanophila flies, which
are the common vectors for the endophytic fungal genus Epichloë, are attracted by a volatile
sesquiterpenoid alcohol, and a methyl ester emitted by the fungus [130]. Additionally, fungal
VOCs can be applied for their insecticidal activity. An example is naphthalene produced by
Muscodor vitigenus, an endophytic fungus which colonizes the Amazonian plant Paullinia
paullinioides. This compound was initially used in “moth balls” due to its effective functions as
insect repellent [131]. In the last years VOCs have also been considered as biofuel precursors or
additives. Many organic volatiles derived from saprotrophic fungi, including alcohols, alkanes,
alkenes, esters, ketones, sesquiterpens, propylene, ethane, are similar to biofuel target
compounds [132] [133] [134]. But only very little information is available concerning the
37
pathways involved. New studies including metabolomic and transcriptomic data are now trying
to correlate and clarify which genes and enzymes are involved in the production of these
hydrocarbon compounds [135].
1.12
Fungal VOC collection and detection
VOC collection
The intrinsic small nature of VOCs and their low concentrations make them very difficult to
characterize and study. In addition, fungi produce very complicated mixtures of volatiles which
require powerful separation technologies to unravel this complicated composition of organic
compounds. Depending on the application and the chemical nature of the volatile of interest,
different sample preparation, separation, identification, and quantification methods can be
applied. At present, gas chromatography-mass spectrometry (GC-MS) is the traditional
technology applied because it combines efficient separation methods with highly sensitive
detection capabilities and precise quantification (up to part-per-billion/trillion) (Figure 11).
Sampling and trapping of the VOCs is the initial critical step of the analysis. Each sampling
method has its own advantages and disadvantages, the choice should be made according to
chemical nature of the analyte(s) and the concentration in the gas mixture. The goal is to
prepare an extract that is representative for the original sample. Chemical instability, variation
in volatility and matrix composition are the main factors which have to be considered in this
process. In general the extraction methods can be separated into two classes: adsorbing and
solvent extraction methods. In adsorbing techniques a fiber/membrane is used which traps and
collects volatile compounds present in the headspace. No sample preparation is required for the
headspace sampling. It can be done either for liquid or solid samples. Besides, gradients of
temperature and an inert gas flow can be applied to improve the sampling of the headspace.
Various fibers are available on the market coated with different selective properties, depending
on the polar character of the analyte(s). Among the different absorbing materials are charcoal
filters, Tenax and Super Q fibers are the most frequently used. In all cases the volatile
compounds released from the sample are trapped and concentrated into the absorbing material.
Later thermal desorption is applied to release the organic compounds into the GC-MS system.
Recently, trapping procedures using the solid phase micro-extraction (SPME) fiber technique
have become a popular method in several contexts [136]. The technique combines extraction,
concentration and introduction of the volatiles into one automated step, offering the advantage
to directly profile living fungal cultures [137]. Steam distillation extraction (SD) and liquid38
liquid extraction offer the advantage to extract volatiles that show reduced volatility [138].
Therewith they get separated from the aqueous phase containing proteins and carbohydrates
which are not wanted in the GC-MS system. Though, the choice of the organic solvents (i.e.
dichloromethane, hexane, pentane) can affect the final VOC profile, excluding highly volatile or
early eluting compounds [139]. Already in 1995 Larsen and Frisvad showed that different
profiles of fungal VOCs can be obtained by applying varying collection techniques.
VOC separation
After the extraction step the separation of the volatiles occurs in the GC system. The gaseous
compounds fly through a thin column, pushed by an unreactive gas (usually nitrogen or
hydrogen). The process is also called ‘mobile-phase’. The chemical components of the VOC
mixture have different progression rates into the column (‘stationary phase’), depending on the
strength of adsorption. Compounds which have a low affinity with the column are eluted first
and vice versa chemicals which establish more interactions with the solid phase are eluted last.
The separation process can be optimized by modifying different parameters such as polarity of
the column, temperature, pressure of the mobile phase and amount of samples run through the
column.
VOC detection
Once the separation is completed the organic compounds are received and captured by a
detector. The flame ionization detector (FID) and the thermal conductivity detector (TCD) are
the most common and robust detectors used in combination with GC systems. They can detect a
wide range of compounds with a sensitivity of approximately 0.5ng/µL for carbon compounds.
Nowadays most of the GC systems are coupled with a mass spectrometer (MS) which acts as the
detector. In the MS the compounds eluting from the column are bombarded with high-energy
electrons breaking them into charged fragments. These small fragments are separated by their
mass to charge ratios (m/z) in the analyzer and produce a spectral pattern unique to the
compound. Compounds can then be recognized by using a library of mass spectra.
The resulting output of the detector is a chromatogram, based on peaks and areas. The retention
time taken from the tip of the peak can be used to identify the compounds. The area below the
peak is proportional to the amount of analyte in the sample. A mathematical integration of the
peak and a calibration curve then allow a quantification of each compound of interest.
39
Figure 11. Schematic representation of a GC-MS system.
2.
Aim
This PhD thesis project is part of the EU Marie Curie Initial Training Network Cornucopia. The
general task of the program has been to make use of the yeast biodiversity and find new
applications for non-conventional species. The goal of this thesis is to give more insight into the
molecular pathways, in particular into the Ehrlich pathway, which drives flavour production in
fungi.
The Ehrlich pathway is a linear and well conserved pathway that converts amino acids into
either fusel acids or fusel alcohols. In yeast this secondary metabolism is one of the major routes
that contribute to the aroma profile of fermented products. One of the goals of this study was to
examine the main genes and their corresponding proteins that are involved in the Ehrlich
pathway. In particular, I focussed on the ARO gene family of Saccharomyces cerevisiae. As a
result I gained insight into the transcriptional regulation of the ARO genes. This knowledge
enabled me to develop a tool that allows the prediction of volatile production in Saccharomyces
sensu stricto species that can be used to screen a large number of strains within a short time and
low effort and investment.
40
I further analysed the function of the ARO gene family in Ashbya gossypii, a very aromatically
smelling Eremothecium species. In particular, I focused my attention on the key transcription
factor of the pathway, ARO80. I could show that the deletion of each of the ARO genes except
for ARO8a affects the flavour profile of the fungus. Furthermore, I investigated the volatile
compound profiles of the two closely related Eremothecium species, A. gossypii and
E. cymbalariae. The purpose was to determine differences in flavour production and relate
these profiles to the gene set of the species.
Finally, 53 novel flavour producers strains selected within the Saccharomyces clade have been
investigated to study their potential for industrial fermentation. For this purpose a high
throughput screening of non-conventional yeasts was performed to test fermentation
performance and flavour characteristics of these non-Saccharomyces yeasts.
3.
3.1
Objectives and state-of-the-art
Functional analysis of the ARO gene family in S.cerevisiae and A.
gossypii
During the last years several studies have been started to elucidate the genetic regulation of the
Ehrlich pathway, however, not with focus on the ARO gene family. ARO8 and ARO9 were
initially isolated by the complementation of the phenylalanine/tyrosine auxotrophy [140], and
were defined as the aromatic amino acid aminotransferases I and II, respectively [141]. Aro8 is
responsible for the phenylalanine and tyrosine biosynthesis and is constitutively expressed.
Aro9p, on the other hand, is more a catabolic enzyme and is involved in the tryptophan
degradation [142] [140]. However the double mutant aro8/aro9 can be partially complemented
by one of the two, suggesting broad substrate specificity and reversibility of the transaminase
reaction [140]. The irreversible decarboxylation step was prematurely attributed to the pyruvate
decarboxylases, PDC1, PDC5 and PDC6 [143]. Only years later, after combining genetic,
physiological, and biochemical approaches, Aro10p was identified as the major decarboxylases
of the Ehrlich pathway, showing an overlapping substrate specificity when phenylalanine,
leucine, or methionine was used as a nitrogen source [144] [145].
The transcriptional regulation of the ARO genes is mediated by two factors: the amino acids
availability and the quality of the nitrogen source. The amino acid activation is controlled by the
transcription factor Aro80 [141] [146], whereas the response to nitrogen is governed by nitrogen
41
catabolite repression (NCR) conveyed by the GATA factors Gat1 and Gln3. Optimal conditions
for the ARO9 and ARO10 induction are therefore the presence of inducers, aromatic amino acids
and the absence of an optimal nitrogen sources like ammonia or glutamine [146]. Only recently,
the interplay of GATA activators and the transcription factor Aro80 has been explained in
respect of the amino acids catabolism. As shown by Kyusung Lee and Ji-Sook Hahn Aro80 is
constitutively bound to its target promoters and is necessary to recruit Gat1 and Gln3 to the
Aro80 target promoters. Nevertheless, the binding of the GATA factors is independent of the
Aro80 activity and they do not have any influence on the binding of Aro80. Figure 12 shows the
proposed transcriptional activation model of ARO9 and ARO10.
Figure 12. ARO9 and ARO10 transcriptional control by Aro80 and GATA activators (modified from
[146]).
Although studies have been done to elucidate the interplay between Aro80 and the GATA
factors, still little is known about the precise binding site of Aro80 to the target promoter
regions. Several binding sites have been proposed which make the establishment of a consensus
sequence difficult [97] [99] [96] [88] [98]. All authors agreed that the binding should be in
proximity of the CCG triplets, the typical recognition site for the Zn2Cys6 transcription factor
family. In Paper 1 we identified promoter regions of several genes which were more responsive
to ARO80 over expression. We co-transformed a plasmid containing ARO80 under control of
the A. gossypii TEF-promoter and plasmids containing ARO8, ARO9, ARO10 and ARO80
promoter-StlacZ reporter gene fusions into S. cerevisiae. We measured the β-galactosidase
activity and identified the promoter regions which were most responsive to the ARO80
expression level. Based on the assumption that high expression levels of the ARO80
transcription factor results in high activation of the Ehrlich pathway we used the ARO9
promoter to correlate reporter gene activity and flavour formation in yeast. To determine the
42
applicability of the ARo9-StlacZ system as a screening tool we transformed various strains with
this ARO9p-StlacZ reporter gene construct. Then we measured the β-galactosidase activities and
compared them with the corresponding flavour profiles. The same approach has been used to
study the genetic regulation of the conserved ARO genes in A. gossypii. We tested the Aro80
dependent expression of the AgARO genes by applying the same set up described above
(Figures 9, Paper 2 and our unpublished data).
3.2
Analysis of the different volatile profiles of A. gossypii and
E. cymbalariae is correlated to their genetic backgrounds.
The contribution of S. cerevisiae to the aromatic profile of food and beverages is well
documented [147]. However the fungal biodiversity has not been explored to its full potential. In
Paper 2 we studied the potential use of A. gossypii for flavour production, employing the GCMS technique to explore the complete aroma profile. Comparative genomic analysis has shown a
conservation of the key genes of the Ehrlich pathway in A. gossypii, especially the family of the
ARO genes. As shown in the Table 5, A. gossypii does not contain the ARO9 gene but we found a
duplication of ARO8. The two ARO8 paralogs were named ARO8a (AGR167w, non-syntenic
homolog of S. cerevisiae ARO8) and ARO8b (AFR548w, syntenic homolog of S. cerevisiae
ARO8).
Species
Transaminase
Decarboxylase
S. cerevisiae
YGL202W (ARO8)
YDR380W (ARO10)
YHR137W (ARO9)
YLR044C (PDC1)
YHR208W (BAT1)
YLR134W (PDC5)
YJR148W (BAT2)
YGR087C (PDC6)
AGR167w (ARO8a)
ACR211W (ARO10)
AFR548c (ARO8b)
ACL134C (PDC1)
ACL072C (BAT1)
AAL073W (PDC6)
Ecym_7228 (ARO8a)
Ecym_2369 (PDC1)
Ecym_7477 (BAT1)
Ecym_3186 (PDC6)
A. gossypii
E. cymbalariae
Table 5. Genes contributing to the Ehrlich pathway.
43
To address the metabolic role of the ARO genes for the Ehrlich pathway in A. gossypii, we
generated the respective deletion mutants and investigated the overall impact on flavour
production. In addition, we also analysed the VOC formation of the closely related specie
E. cymbalariae, which does not show the fruity flavour characteristics of A. gossypii. In line
with that, E. cymbalariae possess an even more limited set of genes that encode for enzymes of
the Ehrlich pathway (Table 5). The same gene setup was observed in L. waltii, where only the
transaminase; ARO8a and ARO8b, are present (see Paper 2 for more details). Here, the
objective was to present a comparative flavour profile of A. gossypii and E. cymbalariae, in
order to extend our knowledge of the Ehrlich pathway and its role in the production of natural
flavouring molecules.
3.3
Flavour molecules produced in the Saccharomyces clade.
A wide selection of non-conventional yeasts (NCY) has been studied in this thesis. The list
contained more than 50 strains encompassing a wide variety of species within the
Saccharomyces clade. Compared to previous works our approach was not limited to certain
geographical regions [148]. On the contrary, our selection included strains isolated worldwide
from different natural sources (i.e. meat, cereals, fruits, cocoa beans, olive, cheese, etc.). The
strain repertoire was also based on the previous identification of these strains in fermentation
processes [149]. The complete list of strains is shown in Table 1 (Paper 3). All strains were
tested on plate assays for their response to stresses (i.e. ethanol, osmolarity and temperature)
but also for the production of off-flavours (H2S and phenolic compounds). Furthermore, all
strains were tested in fermentation condition and the produced volatiles were sampled by
headspace solid-phase microextraction (SPME) and analyzed by gas chromatography–mass
spectroscopy (GC–MS). Data mining and statistical analysis software were employed to cluster
and highlight species related flavours.
4.
Discussion
4.1
Reporter assay for ARO genes in S. cerevisiae and A. gossypii
The genetic regulation of the Ehrlich pathway has gained increasing interest in the last years,
with respect to the critical role in fermenting processes. Particular attention was paid to the
44
ARO genes family, due to the broad substrate activities of the encoding enzymes. The
transcriptional control of these genes depends at least in part on the transcription factor Aro80.
Recent studies have proposed the molecular mechanism of this network [146]. However, despite
computational predictions and chromatin immuno-precipitation (ChIP) experiments, it was not
clearly shown which genes were more responsive to increased ARO80 expression. In addition,
previously there was no information available on the regulation of ARO genes in A. gossypii.
The ß-galactosidase assay presented in this thesis (see also Paper 1 and Paper 2 for detailed
assay information) investigated the potential of AgAro80/ScAro80 to activate target promoter
regions (Figure 13).
AgARO80
ScARO80
AgARO80
900
1800
800
1600
700
1400
Miller Units
Miller Units
1000
600
500
400
300
1200
1000
800
600
200
400
100
200
0
ScARO8
ScARO9
ScARO10
ScARO80
0
ScARO80
AgARO8a
AgARO8b
AgARO10
AgARO80
Figure 13. A ß-galactosidase reporter assay was used to examine target promoters of AgAro80 and
ScAro80 and to quantify the activation of these promoters. Plasmids containing AgTEF1p driven
AgARO80 or ScARO80 sequence and plasmids with an ARO gene promoter StlacZ constructs were cotransformed into S. cerevisiae. StlacZ served as a reporter to quantify the activation of the target
promoter.
In A. gossypii, Aro80 positively regulates the expression of all ARO promoters tested. The
highest activation was found for the AgARO8a promoter. In S. cerevisiae, ScAro80
predominantly activates the ScARO9 promoter, but shows very low activation of other ARO gene
promoters. It was described in the literature that ScAro8o does not regulate the expression of
ScARO8 [97]. Our results could confirm this finding (see Figure 13 upper diagram). However, in
contrast to the computational results and ChIP experiments, we could not observe a strong
activation of the ScARO10 promoter region. Surprisingly, we could observe a higher activation
of ARO10p by AgAro80 than by ScAro80. The functional analysis of the AgARO deletion
mutants showed no growth phenotype of the mutants but a substantial reduction in the
production of the rose flavour 2-phenylethanol (Paper 2). Interestingly, and unlike
45
S. cerevisiae, neither the double mutant AgARO8a/b nor the single mutants showed any amino
acid auxotrophies. This may imply a different regulation in the amino acids biosynthetic
pathway or an additional enzyme which can provide such an activity in A. gossypii.
4.2
Use of a lacZ- reporter assay to correlate reporter gene activity with
flavour production
Flavours are small volatile organic compounds produced during yeast fermentation. Their
natural presence in the gas-phase has been used to collect, separate and analyze them by
modern GC-MS technologies. Even though high-throughput fermentation screening systems
have been improved enormously over the last years, the aroma profiling still requires a lot of
manual and analytical work. In addition the fermentation process needs days to be completed,
preventing the possibility of fast screening with low material and labour costs. In Paper 1 we
discussed an indirect genetic method to identify flavour active strains, exploring their genetic
potential to produce high amounts of aromas for an industrial interest. ScAro80 represents one
of the key transcription factors involved in the regulation of the Ehrlich pathway in yeast. Based
on the results of the ß-galactosidase assay, we can conclude that the ARO80 expression level
directly influences the regulation of the ARO9 promoter. Further experiments found a
correlation between the β-galactosidase activity of ARO9 and the amount of β-phenylacetate and
β-phenylethanol produced during fermentations. This finding was recently supported by other
studies which have used ARO9 and ARO80 overexpression to improve significantly the
biocatalytic production of β-phenylethanol [150]. Thus, this newly developed fast tool can
conveniently streamline the identification of yeast strains with potential flavour characteristics.
It is designed to be applied for high throughput screening of yeast collections. However, the
genetic tool builds on the conservation of the transcriptional regulatory circuits found in
S. cerevisiae. Thus, one limitation of the assay is the range of species or hybrids in which the use
of ScARO80 provides reliable correlations with flavour production Nevertheless, new screening
tools can be developed based on our genetic tool using other species’ ARO80 target genes.
Furthermore, the genetic tool can be used to screen a large variety of S. cerevisiae strains or
hybrids for the flavour production as well as large numbers of progeny of these strains.
46
4.3
General flavour differences between A. gossypii and E. cymbalariae
In this study we present the aroma profile analysis of two closely related filamentous
ascomycetes, A. gossypii and E. cymbalariae (Paper 2). We used the volatile profiling analysis
to provide a quick access to the metabolites they produced. Specifically, we focused on all the
VOCs linked to the Ehrlich pathway in which particularly aromatic and branched chain amino
acids get catabolized into higher flavour compounds, also known as fusel products.
In general, the amounts of branched-chain aldehydes are rather high in both fungal species,
whereas the production of aromatic volatiles was found to be specific for A. gossypii. Comparing
the gene set linked to the Ehrlich pathway we observed that E. cymbalariae lacks two important
genes that are known to be involved in catabolism of aromatic amino acids, namely ARO9 and
ARO80 [151]. A. gossypii lacks only the homolog of ARO9 but contains two paralogs of ARO8.
We could show that the transcription factor AgAro80 can activate the transcription of both
ARO8 genes as well as ARO10 in A. gossypii (Figures 13). Although A. gossypii lacks the
homologous ScARO9 gene, we concluded that one of the ARO8 genes may function as a
transaminase in the catabolism of phenylalanine. Though, this has to be confirmed by future
experiments.
Branched-chain aldehydes such as 3-methyl butanal, 2-methyl butanal and 2-methyl propanal
are derived from branched chain amino acid degradation. These compounds are key flavour
metabolites [152]. Mainly perceived as malty or chocolate-like they are usually formed during
the fermentation of many food related substrates, especially during the fermentation of cocoa
beans. The high reactivity of the aldehyde carbonyl group makes this class of compounds very
easy to be reduced to the respective alcohols or oxidized to the corresponding acids. A high flux
in the leucine biosynthesis pathway of both fungi might explain the high levels of these
intermediates. Having an exaggerated leucine production could maintain the Ehrlich pathway
catabolism of leucine and therefore keep a high level of intermediates at all times. A genetic
evidence for a high production and flux in the leucine synthesis pathway might support this
hypothesis. In A. gossypii a duplication of the LEU4 (AFL229w) gene has been found (ADL015c,
non syntenic homolog of ScLEU4, Figure 14).
47
Figure 14. Gene synteny of the LEU4 loci in S. cerevisiae, A. gossypii and E. cymbalariae. The red stars
indicate positions of tRNA in A. gossypii.
LEU4 encodes a α-isopropylmalate synthase, responsible for the first step in the leucine
biosynthesis pathway. In S. cerevisiae LEU4 has a paralog, LEU9, which arose from the whole
genome duplication. Functional analyses in conjunction with non-fermentable carbon sources
demonstrated that only LEU4 is required to maintain normal growth rates of S. cerevisiae in the
absence of leucine [153] LEU9 plays an auxiliary role in yeast since about 80% of total αisopropylmalate synthase activity in wild-type cells is provided by LEU4 [154] The second LEU4
gene in A. gossypii (ADL015c) is not in synteny to the S. cerevisiae LEU4 or LEU9 gene and
therefore arose by a unique duplication event in A. gossypii (Figure 14). In E. cymbalariae no
duplication of the LEU4 gene occurred. The role of LEU4 in A. gossypii has been investigated by
deletion mutants. The single LEU4 deletions did not show any growth delay or difference in the
VOCs production. We failed to delete both LEU4 genes of A. gossypii concluding that this
double deletion is lethal even in media with excessive amounts of leucine. These results suggest
a different regulation of the leucine biosynthesis pathway in A. gossypii.
3-methyl butanal is a key intermediate in the isoamyl alcohol biosynthesis. By continuous
feeding of the pathway with this leucine derived intermediate the fungus can then overproduce
the respective fusel alcohol. However, the reason why the Eremothecium species produce high
yields of this particular alcohol is still unknown. In S. cerevisiae isoamyl alcohol together with 248
phenyl ethanol, and tryptophol can induce pseudohyphal growth and stimulate flocculation
[104] [105]. In A. gossypii the production of fruity flavours could provide a benefit in its
ecological niche to attract spore-transmitting insects for dispersal to reach new sites of infection.
Further experiments are necessary to test this hypothesis.
4.4
VOCs produced in the Saccharomyces clade
Non-conventional yeasts (NCY) represent a new frontier for industrial applications. In the last
years a lot of studies have explored the yeast biodiversity looking for new traits that can make a
breakthrough in biotechnological processes. In this study we focused our attention on novel
flavour producers within the Saccharomyces clade. Initial results showed that yeast strains
which belong to the same species exhibited similar VOCs pattern (Paper 3). During
fermentation we collected various parameters (i.e. fermentation kinetics, ethanol content, pH of
the media and sugar content) which allowed us to make a rational selection of the NCY we
wanted to continue with. This approach streamlined the strain selection to 18 representative
NCY, which were then further tested for specific traits and flavour profiles.
Comparing the fermentation profiles we noticed that almost all the NCY finished fermentation
after four days and showed a typical sigmoidal growth curve-like pattern of the CO2 release.
The final Plato at the end of all fermentations was in a range between 3 and 4. It is not yet
known why yeast strains stop fermenting before all fermentable sugar is consumed [155]
Accumulation of toxic intermediates, oxidative stress, lacking of micronutrients and deplete of
all the fermentable sugars could be possible explanations for an incomplete sugar fermentation.
The only exception we noticed was in fermentation of Wickerhamomyces anomalus. The strain
did not show a lag-phase of CO2 release but a linear CO2 curve instead, which ended after eleven
days. During the fermentation of W. anomalus we observed a water-insoluble layer emerging on
top of the fermentation cylinder. This might explain the slower and constant CO2 release. The
formation of the film layer has been reported as a typical phenomenon in stored wines.
Apparently the residual amount of oxygen in the headspace of the fermenters triggers the
formation of a biofilm. The main volatiles produced by this film are acetic acid, acetaldehyde
and acetate esters [156].
49
Nevertheless, Wickerhamomyces anomalus and Pichia kluyveri generated the highest amount
of CO2 at the end of the fermentation process (up to 2.8 g/40ml media). On the other hand, they
produced the lowest amount of ethanol with 6 and 4% v/v, respectively.
The strain Debaromyces subglobosus showed an opposite fermentation profile. It was able to
produce 8% v/v ethanol and utilize most of the sugar available in the media. However, it
released the lowest CO2 content (1, 3 g/40ml media). In all the other fermentations we noticed a
reasonable relation between ethanol production and sugar consumption.
Ethanol is not the only product of the fermentation process. Volatiles also contribute to CO2
production. Our data showed a clear correlation between the fermentation performance of
Wickerhamomyces anomalus and Pichia kluyveri species and the amount of volatiles produced.
The highest CO2 release was coupled with the extremely high production of esters in these
strains. In particular, the strains produced large amounts of ethyl acetate, isoamyl acetate and
phenyl acetate. Together with the esters high yields of acetic acid were registered. Both ethanol
and acetic acid are generated from the common intermediate acetaldehyde by the aldehyde
dehydrogenase, reducing one molecule of NAD(P)+. Ethanol and acetate can both inhibit yeast
growth if present at high concentrations. Ethanol generally diffuses out from cell and just a very
low concentration is retained inside. Acetic acid accumulation inside the cell is toxic.
It can generate high turgor pressure and trigger the formation of free oxygen radicals and
consequently oxidative stress [157]. Moreover, the passive diffusion through the plasma
membrane is very limited due to the largest dissociated form of the acid since the intracellular
pH is higher than 4.75, which is the pKa of the acetic acid. Therefore, the cell needs to either
pump it out by ATP-hydrolyzing transporter systems (ABC transporters) or convert the acetate
into a less toxic compound. The first option of detoxification implies the use of ATP, which is
quite limited in fermentative conditions [158]. In the second case an enzymatic reaction is
required. Ethyl acetate can be synthetized by two different pathways in which both require
ethanol as substrate. Alcohol acetyl transferases can synthetize ethyl acetate by a reaction that
involve acetyl co-enzyme A as a cofactor [159]. Generally, this pathway is involved in the
production of isoamyl acetate, using isoamyl alcohol instead of ethanol as a substrate for the
reaction. The other enzymatic way for the production of ethyl acetate is by esterase activity. This
reaction that is a reverse hydrolysis uses the acetate and ethanol to produce ethyl acetate (Figure
15).
50
Figure 15. Enzymatic routes for the production of ethyl acetate.
W. anomalus differs from S. cerevisiae by producing most of acetate esters by the inverse
esterase reaction [160]. More recent studies from Rojas et al. (2002) [161] confirmed the high
ester synthase activity in W. anomalus. The authors argue that the natural overproduction ethyl
acetate could be related with the anti-fungal activity of this compound. Along with other NCY,
W. anomalus could have developed the ability to accumulate damaging compounds to
outcompete S. cerevisiae or other microorganism for nutrients, oxygen and space. For this
reason, W. anomalus has been studied as a bio control agent, especially against molds. The
inhibitory effect against Penicillium roqueforti has been shown to be related to the combined
effect of ethanol and ethyl acetate [162].
Esters are also important in food production due to the organoleptic properties that they imply.
A balance among them is important to give the typical bouquet to specific product. The high
ester production of W. anomalus, P. kluyveri and Z. mellis has been reported in other works and
it is known as a common feature for NCY. Together with their lower fermentation abilities the
uncontrolled production of esters is the main reason that has prevented their use as starter
culture in mixed fermentation. Despite that, recent studies have reevaluated the application of
these strains, applying them in different production processes. For instance, Wickerhamomyces
sp. are commonly used in the production of soya-sauce, where the high amount of esters,
especially ethyl acetate have shown to give the strong and sweet/caramel like aroma to the
product [163]. Pichia kluyveri has been extensively tested as multistarter culture for cocoa,
tequila and wine fermentation. It has been shown that controlled mixed fermentation of Pichia
kluyveri and Kluyveromyces marxianus enhanced the sensory quality of the cocoa beans,
offering a valid alternative to the standard spontaneous fermentation used in cocoa production
[164]. Moreover the strains have been investigated for industrial application of tequila mixed
51
fermentation. Compare to standard S. cerevisiae fermentations, P. kluyveri and K. marxianus
mixtures were more efficient in alcohol and ester production [165]. P. kluyveri strains are also
commercialized by Chr. Hansen in products like Viniflora® FrootZen™. The strains are used to
start the alcoholic fermentation of must until they reach an alcohol content of 4%. Then a
standard S. cerevisiae strain is added to complete the process. Again, the advantage of the
second strain is to add additional fruity flavours to the wine resulting in a higher complexity of
the wine (for more details look at http://www.pros.co.nz/PDF/PDS/FROOTZEN%20PDS.pdf).
Debaromyces subglobosus showed another exceptional profile of volatiles. Characterized by
delayed fermentation the strain was yet able to produce large amount of aldehydes. In literature
Debaromyces sp. are described as strains with weak fermentation properties [166] but a notable
halotolerance [167]. We could confirm this high tolerance in our studies as well by growing
D. subglobosus in SD media with 4% NaCl (Paper 3, data not shown). This natural salt
tolerance of the genus has been used for production processes such as the synthesis of dairy
products such as cheese and fermentation of meat products [166].
Fruity ketones are important chemical compounds in cheese ripening as well. In line with that,
the largest amount of pyranone was detected in D. subglobosus. In contrast, we detected
extremely low levels of esters by GC-MS. Van den Tempel and Jacobsen (2000) [168] found
high esterase activity in different isolates of D. hansenii which could explain this low amount of
esters. In meat production Debaryomyces sp. have been reported to generate volatile
compounds during the fermentation of dry-fermented sausages. In particular, several species
inhibit the formation of lipid oxidation compounds and generate ethyl esters [169].
Also, branch-chained aldehydes are critical components for the maturation of meat products,
giving nutty, cheesy and salty notes to the food products [152]. Generally these aldehydes are
produced by heat-induced processes (Strecker degradation) but small amounts of aldehydes are
also produced enzymatically during food fermentation. D. subglobosus shows the highest
production of aldehydes among our yeast selection. As one of them 3-methyl butanal, an
intermediate in leucine degradation, is produced in higher amount in fermentations with
D. subglobosus and Z. mellis. 3-methyl butanal is a common aldehyde produced in cured-hams
by lactic acid bacteria and Micrococcaceae [170]. Since the strains produced detectable amounts
of 3-methyl butanal but also other compounds derived from Strecker degradation, i.e. 5hydroxymethylfurfural, furfural, 5-methylfurfural they might have a possible application in
meat processing.
In summary we did an initial characterization of 20 NCYs by correlating their fermentation
performances with their aroma profiles. In this first screening D. subglobosus, W. anomalus, P.
52
kluyveri and Z. mellis displayed unique fermentation and volatile profiles that may be used for
supply of additional flavours, e.g. in mixed fermentations.
5.
Conclusions and Outlook
Flavours and volatile compounds have a growing market potential, especially for food and
beverages or fragrance industries. Fungi are able to produce various mixtures of VOCs and thus
provide various opportunities to generate desirable natural flavours. The Ehrlich pathway is one
of the main routes involved in the synthesis of volatile aroma compounds, especially during
yeast fermentations of beverages. Previous observations identified the ARO gene family as a
target set of genes with a strong impact on the activity of the Ehrlich pathway. Comparative
genetic analysis showed a clear conservation of this gene family in the Saccharomyces clade. In
our experiments we focused on the key transcriptional factor, ARO80. By using a lacZ-reporter
system we quantified the Aro80-dependent expression of key ARO genes in both S. cerevisiae
and A. gossypii. Expression of ARO-genes is also known to depend on other transcriptional
regulators, e.g. the general biosynthetic amino acids regulator GCN4, which contributes to the
complex regulation of ARO genes. At present, the regulation of ARO-genes has only been
studied on a transcriptional level. Further studies are needed to elucidate if there also is a
regulation of ARO genes on a post-transcriptional stage.
In our promoter analyses we found that the ScARO9 expression is induced by Aro80. This
prompted us to generate a ScARO9- lacZ reporter system. This indicated that the expression
level of ARO9 correlates with the formation of two aroma alcohol products, 2-phenylethanol and
2-phenylethyl acetate. We went on to transfer this reporter tool into other Sacchamyces sensu
stricto species. With these studies we could confirm the correlation of ARO9 gene expression
with the production of 2-phenylethanol and 2-phenylethyl acetate. This tool opens the way for
high throughput screenings of entire strain collections or the analysis of progeny pools using
microtiter plate assays. This inexpensive indirect assay offers new opportunities for the isolation
of candidate strains with increased flavour production..
Our data with A. gossypii showed that AgARO8a/b and AgARO10 are regulated by AgAro80.
Deletion of almost all the ARO genes resulted in a severe decrease in flavour production,
especially in 2-phenylethanol and isoamyl alcohol. However, overexpression of ARO80 resulted
53
specifically increased the production of isoamyl alcohol but not of 2-phenylethanol. Experiments
in S. cerevisiae showed an increased flavour production on media with poor nitrogen sources.
These growth conditions could be tested in A. gossypii as well to further increase the production
of flavour components. Additionally, spiking of media with amino acids could induce flavour
product formation. Further experiments will be necessary to identify the underlying causes of
natural over production of isoamyl alcohol in A. gossypii. We have begun to analyze the leucine
biosynthesis pathway of A. gossypii. Also, the biological function for the production of the high
levels of this alcohol should be addressed. Recent studies showed that 2-phenylethanol and
isoamyl alcohol emitted by the fungus Aureobasidium pullulans are involved in the attraction of
eusocial wasps [92]. Similar experiments could be used in A. gossypii to test the ability to attract
spore-transmitting insects on which A. gossypii depends for its dispersal.
The close relative of A. gossypii, Eremothecium cymbalariae, produced a very distinct flavour
profile, particularly with regards to its high production of ethyl acetate. The biological function
of this is also unknown. Ethyl acetate could represent a strategy to avoid high concentrations of
acetic acid in a similar mechanism as described for W. anomalus (see paragraph 4.4). Studies
could be done to address the enzymatic activity of esterases and alcohol acetyl transferase in
E. cymbalariae.
In collaboration with the EU consortium Cornucopia, which is focusing on the identification of
novel potentials of NCY, we characterized a selection of 18 different species that are presented in
this thesis. In this characterization we recorded their fermentation performance followed by a
flavour profiling. The volatile aroma profiles of W. anomalus, P. kluyveri and Z. mellis and
D. subglobosus were unique from all other strains. These four species will be characterized
further in various fermentation conditions, i.e. different carbon and nitrogen sources,
temperature and pH to explore their potential in food production. Additionally, these strains
might be favorable in mixed-fermentations.
By using NCY strains in different co-fermentation regimes new flavour mixes may be generated
and to be used in new kinds of beverages.
54
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63
Paper 1
Paper 1
This paper investigated the regulatory network of the ARO gene family in S. cerevisiae. The ARO
gene set expresses enzymes that belong to the Ehrlich pathway, producing flavoured alcohols
and esters by the amino acid catabolism. ScARO80 is a Zn2Cys6 transcription factor and
activator of the pathway. The study investigated the ARO80-dependent expression of the ARO
target promoter regions. ScARO9 expression is strongly regulated by ScAro80. This established
ScARO9 as a potential reporter to correlate the gene activation and flavour formation in yeast.
To determine the applicability of the ARO9-lacZ system as a screening tool we transformed
various strains with the ARO9p-lacZ plasmid. GC-MS results confirmed the ability of the lacZsystem to predict the yield of two flavour compounds, 2-phenylethanol and 2phenylacetate.
64
OPEN
SUBJECT AREAS:
MICROBIOLOGY
TECHNIQUES
FUNGAL GENETICS
Received
9 August 2013
Accepted
6 December 2013
Published
15 January 2014
Correspondence and
requests for materials
should be addressed to
J.W. (juergen.
wendland@
carlsberglab.dk)
An indirect assay for volatile compound
production in yeast strains
Davide Ravasio1, Andrea Walther1, Kajetan Trost2, Urska Vrhovsek2 & Jürgen Wendland1
1
Carlsberg Laboratory; Yeast Genetics Gamle Carlsberg Vej 10 DK-1799 Copenhagen V, Denmark, 2Fondazione Edmund Mach
Research and Innovation Centre Food Quality and Nutrition Department Via E.Mach 1, I-38010 S.Michele all’Adige, Italy.
Traditional flavor analysis relies on gas chromatography coupled to mass spectrometry (GC-MS) methods.
Here we describe an indirect method coupling volatile compound formation to an ARO9-promoter-LacZ
reporter gene. The resulting b-galactosidase activity correlated well with headspace solid phase micro
extraction (HS/SPME) GC-MS data, particularly with respect to the formation of rose flavor. This tool
enables large-scale screening of yeast strains and their progeny to identify the most flavor active strains.
T
he organoleptic perception of beer depends mainly on substances produced by yeast during the fermentation
process. Flavor active substances are volatile compounds and include higher alcohols, esters, and fatty acids.
In the wine industry attempts are made to increase flavour compounds by either simultaneous or sequential
co-fermentations using either different yeast strains, i.e Saccharomyces cerevisiae with a non-Saccharomyces yeast,
or mixing bacterial strains, e.g. Oenococcus oeni, with wine yeasts5,8,14,17. Research interest in natural flavors
produced by yeasts has gained increasing interest, particularly focusing on isoamyl alcohol (banana flavor)
and b-phenylethanol (flowery, rose flavor). Both compounds are produced during amino acid catabolism in
yeast9. The Ehrlich pathway, a linear pathway requiring three enzymatic activities, is responsible for converting
aromatic amino acids (phenylalanine, tyrosine, and tryptophan), branched-chain amino acids (leucine, isoleucine, and valine) and methionine into higher alcohols. The regulation of the Ehrlich pathway depends at least in
part on the Zn2Cys6 transcription factor Aro80, which regulates ARO9 and ARO10 in a nitrogen source dependent manner (Fig. 1A)12. One of the key bottle necks in flavor research is the requirement of chemical analytical
tools to measure volatile compounds produced during fermentation, which is generally done using HS/SPME
extraction methods coupled to GC-MS2,15. This method, however, is time consuming, requires additional quantitation as well as prior lab scale fermentations and sample preparations, which are often difficult to optimize for
high throughput screening.
In order to identify a promoter that is most responsive to ARO80 overexpression, we co-transformed ARO80
under the control of the Ashbya gossypii TEF-promoter with plasmids containing ARO8, ARO9, ARO10, and
ARO80 promoter-lacZ reporter gene fusions into S. cerevisiae (Fig. 1B). To investigate whether expression of the
reporter genes was actually Aro80-dependent we quantified b-galactosidase activity in strains bearing the endogenous ARO80, an ARO80 deletion, or the ARO80 overexpression construct (Fig. 1C). This established the ARO9
as a potential reporter for a strain’s flavor production.
To correlate ARO9 reporter gene activity with flavor formation we first determined its activity in a set of strains
with S. cerevisiae background expressing ARO80 at wild type levels. This included the laboratory strain CENPK,
two hybrid lager yeast strains, collectively known as S. pastorianus as well as a Bordeaux wine yeast. For
comparison we used these strains in bench-top fermentation assays and at the end of fermentation volatiles were
extracted by HS/SPME and analyzed via GC-MS (Tab. S1). For the comparison of volatile compound formation
with b-galactosidase activity we focused our attention to phenylalanine catabolites (rose flavor). This showed that
b-galactosidase activity of the ARO9-lacZ reporter correlated well with the amount of b-phenylacetate and bphenylethanol produced by these strains (Fig. 2).
To determine the applicability of this tool beyond S. cerevisiae we used the ARO9-reporter with strains from the
Saccharomyces sensu stricto complex including S. bayanus, S. cariocanus, S. eubayanus, S. kudriavzevii, S. mikatae, S.
paradoxus, and S. uvarum (Fig. 2). The flavor profiles show that there is a great variability in volatile formation
between these strains (Tab. S1). This variability is also reflected in the b-galactosidase activity in these strains indicating
that high b-galactosidase activity pairs with increased flavor production. A correlation curve was analyzed comparing
b-galactosidase activity with the combined flavor values for 2-phenyl ethanol and 2-phenyl acetate (Fig. 2C). This took
into account that Aro9 enzymatic activity is upstream of 2-phenyl ethanol and 2-phenyl acetate production.
SCIENTIFIC REPORTS | 4 : 3707 | DOI: 10.1038/srep03707
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Figure 1 | Identification of a reporter gene for Ehrlich Pathway activity. (A) Amino acids (branched-chain amino acids, leucine, isoleucine, and valine,
aromatic amino acids, phenylalanine, tyrosine, and tryptphan, or methionine) are converted in the Ehrlich pathway to fusel alcohol or fusel acids in a
three step process. The genes encoding enzymes that catalyze single steps are indicated. Oxidation of aldehydes to fusel acids is done by aldehyde
dehydrogenases (e.g. ALD1). Reduction of aldehydes to fusel alcohols is done by alcohol dehydrogenases (e.g. ADH1). Transcriptional regulation by
Aro80 and co-factor requirement is indicated. (B) Plasmids carrying the ARO80 overexpression and one of the ARO-promoter-lacZ reporter gene
constructs were co-transformed into S. cerevisiae (BY4741). (C) Quantitative b-galactosidase assay with strains bearing the indicated ARO-promoter-lacZ
constructs in strains in which ScARO80 was either overexpressed or deleted, or contained the wildtype ARO80.
Fermented beverages contain only small amounts of volatile compounds; yet, these are of paramount importance for the flavor profile
and organoleptic perception of a beverage19,20. Changes in brewing
technology, e.g. introduction of high-gravity brewing, can drastically
alter the flavor composition - in this case - by resulting in an increase
in the amount of acetate esters. Consumer preference is towards all
natural flavors and unique flavor signatures10. Based on this nonGMO preference, three main roads are currently followed to improve
flavor content of beverages: (i) choice of the starter culture, (ii) mixed
fermentations using different yeast species or a combination of yeast
and bacterial species, and (iii) selection of strains high in volatile
compound formation via yeast breeding approaches1,5,6,22.
For example, yeasts belonging to the genera Hanseniaspora and
Pichia are good producers of acetate esters, whereas mixed fermentations with S. cerevisiae and Lachancea thermotolerans increased the
level of b-phenylethanol4,21. Furthermore, mixed fermentations,
including S. cerevisiae and a bacterial strain e.g. Oenococcus oeni,
promise to provide novel flavor variations17.
With the highly advanced gene function analyses in S. cerevisiae
the genetic repertoire involved in volatile compound formation has
SCIENTIFIC REPORTS | 4 : 3707 | DOI: 10.1038/srep03707
been elucidated to a great extent18. The Ehrlich pathway plays a
central role in aromatic and branched-chain amino acid catabolism
resulting in the conversion of amino acids to aroma compounds9.
Several studies have described an increase in flavor production by
selecting for yeast strains resistant to fluoro-amino acids. An
increased production of isoamyl alcohol, for example, can be
achieved by selecting mutants resistant to trifluoroleucine3. In such
strains a mutation of D578Y in the LEU4 gene releases feedback
inhibition and initiates increased production of leucine and its catabolites16. Using a genetic approach it was shown that overexpression
of the alcohol acetyl transferases ATF1 and ATF2 substantially
increased the production of isoamyl acetate20.
The indirect assay described in this study converts Ehrlich
pathway activity into a reporter gene readout that can be quantified as b-galactosidase activity. We base the tool on the ARO9
promoter as the ARO8 promoter was not responsive to Aro80 and
has been shown to be under general control13. With this method
we can preferably assay rose flavor. Apparently, however, this
reporter is not discriminatory towards branched chain amino
acids (Tab. S1).
2
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Figure 2 | Comparison of b-galactosidase activity with volatile compound formation. (A) Assay with either the indicated S. cerevisiae strains (A) or with
Saccharomyces sensu stricto strains (B). Upper panels depict b-galactosidase activity based on the ARO9p-lacZ reporter construct. Lower panels show bphenylethanol and b-phenylacetate volatile compounds. Note: Fermentation with the wine strain in (A), was done in YPD due to its lack of MAL-genes.
The low amount of flavor produced by S. mikatae, S. cariocanus, and S. cerevisiae in (B) is due to their inability to end-ferment granulated malt used in
these fermentations. Correlation of -galactosidase activity and the combined yield of phenylalanine catabolites are shown in (C).
Our tool is fast and convenient and can be adapted for use with
high throughput microtiter plate assays in yeast7,11. Thus this indirect
flavor assay system is inexpensive and allows screening of large libraries of yeast strains as well as F1/F2 populations of interbred
strains. This will lead to the rapid identification of strains with potentially improved flavor characteristics compared to the parental
strains. Additionally, different growth regimes can lead to altered
flavor production. This allows the implementation of changes in
oxygen supply and use of different nitrogen sources.
1. Arroyo-Lopez, F. N. et al. Yeasts in table olive processing: desirable or spoilage
microorganisms? Int J Food Microbiol 160, 42–49 (2012).
2. Canuti, V. et al. Headspace solid-phase microextraction-gas chromatographymass spectrometry for profiling free volatile compounds in Cabernet Sauvignon
grapes and wines. J Chromatogr A 1216, 3012–3022 (2009).
3. Casalone, E. et al. Genetic and biochemical characterization of Saccharomyces
cerevisiae mutants resistant to trifluoroleucine. Res Microbiol 148, 613–623
(1997).
4. Comitini, F. et al. Selected non-Saccharomyces wine yeasts in controlled
multistarter fermentations with Saccharomyces cerevisiae. Food Microbiol 28,
873–882 (2011).
5. Domizio, P. et al. Outlining a future for non-Saccharomyces yeasts: selection of
putative spoilage wine strains to be used in association with Saccharomyces
cerevisiae for grape juice fermentation. Int J Food Microbiol 147, 170–180 (2011).
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Saccharomyces cerevisiae improves stress resistance and fermentation
performance. Yeast 29, 343–355 (2012).
7. Gietz, R. D. & Schiestl, R. H. Microtiter plate transformation using the LiAc/SS
carrier DNA/PEG method. Nat Protoc 2, 5–8 (2007).
8. Gobbi, M. et al. Lachancea thermotolerans and Saccharomyces cerevisiae in
simultaneous and sequential co-fermentation: a strategy to enhance acidity and
improve the overall quality of wine. Food Microbiol 33, 271–281 (2013).
9. Hazelwood, L. A., Daran, J. M., van Maris, A. J., Pronk, J. T. & Dickinson, J. R. The
Ehrlich pathway for fusel alcohol production: a century of research on
Saccharomyces cerevisiae metabolism. Appl Environ Microbiol 74, 2259–2266
(2008).
10. Hugenholtz, J. Traditional biotechnology for new foods and beverages. Curr Opin
Biotechnol 24, 155–159 (2013).
11. Ichikawa, K. & Eki, T. A novel yeast-based reporter assay system for the sensitive
detection of genotoxic agents mediated by a DNA damage-inducible LexA-GAL4
protein. J Biochem 139, 105–112 (2006).
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12. Iraqui, I., Vissers, S., Andre, B. & Urrestarazu, A. Transcriptional induction by
aromatic amino acids in Saccharomyces cerevisiae. Mol Cell Biol 19, 3360–3371
(1999).
13. Iraqui, I., Vissers, S., Cartiaux, M. & Urrestarazu, A. Characterisation of
Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic
aminotransferases I and II reveals a new aminotransferase subfamily. Mol Gen
Genet 257, 238–248 (1998).
14. Kim, D. H., Hong, Y. A. & Park, H. D. Co-fermentation of grape must by
Issatchenkia orientalis and Saccharomyces cerevisiae reduces the malic acid
content in wine. Biotechnol Lett 30, 1633–1638 (2008).
15. Mauriello, G., Capece, A., D’Auria, M., Garde-Cerdan, T. & Romano, P. SPMEGC method as a tool to differentiate VOC profiles in Saccharomyces cerevisiae
wine yeasts. Food Microbiol 26, 246–252 (2009).
16. Oba, T., Nomiyama, S., Hirakawa, H., Tashiro, K. & Kuhara, S. Asp578 in LEU4p
is one of the key residues for leucine feedback inhibition release in sake yeast.
Biosci Biotechnol Biochem 69, 1270–1273 (2005).
17. Rossouw, D., Du Toit, M. & Bauer, F. F. The impact of co-inoculation with
Oenococcus oeni on the trancriptome of Saccharomyces cerevisiae and on the
flavour-active metabolite profiles during fermentation in synthetic must. Food
Microbiol 29, 121–131 (2012).
18. Styger, G., Jacobson, D., Prior, B. A. & Bauer, F. F. Genetic analysis of the
metabolic pathways responsible for aroma metabolite production by
Saccharomyces cerevisiae. Appl Microbiol Biotechnol 97, 4429–4442 (2013).
19. Verstrepen, K. J. et al. Flavor-active esters: adding fruitiness to beer. J Biosci Bioeng
96 (2003).
20. Verstrepen, K. J. et al. Expression levels of the yeast alcohol acetyltransferase genes
ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile
esters. Appl Environ Microbiol 69, 5228–5237 (2003).
21. Viana, F., Gil, J. V., Genoves, S., Valles, S. & Manzanares, P. Rational selection of
non-Saccharomyces wine yeasts for mixed starters based on ester formation and
enological traits. Food Microbiol 25, 778–785 (2008).
22. Zott, K. et al. The grape must non-Saccharomyces microbial community: impact
on volatile thiol release. Int J Food Microbiol 151, 210–215 (2011).
Acknowledgments
This research was supported in part by the European Union Marie Curie Initial Training
Network Cornucopia (http://www.yeast-cornucopia.se/).
Author contributions
D.R. carried out the molecular experiments; D.R. and K.T. carried out flavor measurements;
A.W., J.W.W. and U.V. designed the experiments, A.W. and D.R. prepared the figures,
J.W.W. wrote the main manuscript text; all authors reviewed the manuscript.
3
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Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
SCIENTIFIC REPORTS | 4 : 3707 | DOI: 10.1038/srep03707
How to cite this article: Ravasio, D., Walther, A., Trost, K., Vrhovsek, U. & Wendland, J. An
indirect assay for volatile compound production in yeast strains. Sci. Rep. 4, 3707;
DOI:10.1038/srep03707 (2014).
This work is licensed under a Creative Commons AttributionNonCommercial-NoDerivs 3.0 Unported license. To view a copy of this license,
visit http://creativecommons.org/licenses/by-nc-nd/3.0
4
Paper 2
Paper 2
The riboflavin over producer Ashbya gossypii produces significant amounts of fruity flavours
compare to the close relative Eremothecium cymbalariae. Comparative genomic analysis
showed that E. cymbalariae lacks most of the Ehrlich pathway genes, especially members of
conserved ARO gene family. In A. gossypii mutation in the ARO genes led to a strong reduction
in volatile production compare to the wild-type. On the other hand AgAro80 over expression
produced significantly higher yields of VOCs, especially of isoamyl alcohol. Furthermore, in the
study we compared the kinetic volatile profiles of the two Eremothecium species. The volatile
compound analysis showed that both Eremothecium species produce large amounts of isoamyl
alcohol while E. cymbalariae, lacking the major components of the ARO family, does not
produce 2-phenylethanol.
65
RESEARCH ARTICLE
Major contribution of the Ehrlich pathway for 2-phenylethanol/
rose flavor production in Ashbya gossypii
€ rgen Wendland & Andrea Walther
Davide Ravasio, Ju
Carlsberg Laboratory, Yeast Genetics, Copenhagen V, Denmark
€rgen Wendland,
Correspondence: Ju
Carlsberg Laboratory, Yeast Biology, Gamle
Carlsberg Vej 10, DK-1799 Copenhagen V,
Denmark. Tel.: +45 3327 5230;
fax: +45 3327 4708;
e-mail: [email protected]
Received 13 May 2014; accepted 3 June
2014.
DOI: 10.1111/1567-1364.12172
Editor: Terrance Cooper
Keywords
ARO80; VOC; volatile flavour compounds;
biodiversity.
Abstract
Aroma alcohols of fermented food and beverages are derived from fungal
amino acids catabolism via the Ehrlich pathway. This linear pathway consists
of three enzymatic reactions to form fusel alcohols. Regulation of some of the
enzymes occurs on the transcriptional level via Aro80. The riboflavin overproducer Ashbya gossypii produces strong fruity flavours in contrast to its much
less aromatic relative Eremothecium cymbalariae. Genome comparisons indicated that A. gossypii harbors genes for aromatic amino acid catabolism
(ARO8a, ARO8b, ARO10, and ARO80) while E. cymbalariae only encodes
ARO8a and thus lacks major components of aromatic amino acid catabolism.
Volatile compound (VOC) analysis showed that both Eremothecium species
produce large amounts of isoamyl alcohol while A. gossypii also produces high
levels of 2-phenylethanol. Deletion of the A. gossypii ARO-genes did not confer
any growth deficiencies. However, A. gossypii ARO-mutants (except Agaro8a)
were strongly impaired in aroma production, particularly in the production of
the rose flavour 2-phenylethanol. Conversely, overexpression of ARO80 via the
AgTEF1 promoter resulted in 50% increase in VOC production. Together these
data indicate that A. gossypii is a very potent flavour producer and that
amongst the non-Saccharomyces biodiversity strains can be identified that could
provide positive sensory properties to fermented beverages.
YEAST RESEARCH
Introduction
Fermented beverages contain a variety of flavour active
compounds that convey a fruity aroma, which is very
prominent in beer and sake (Verstrepen et al., 2003; Kitagaki & Kitamoto, 2013). Volatile esters represent the most
important group. Amongst them 2-phenylethanol, isoamyl
alcohol, and ethyl caproate convey rose/flower, banana/fruity, and apple aromas, respectively. In lager beer high
amounts of isoamyl alcohol can be present while other
esters tend to contribute only minor amounts to the overall
flavour bouquet. Alterations in beer flavour profile have
been experienced when changing the production conditions. The amount of higher alcohols increases when using
oxygenated wort or higher fermentation temperatures
(Valero et al., 2001). Additionally, high-gravity brewing
(providing increased amounts of sugars) results in
increased production of acetate esters (Saerens et al., 2008).
The biochemistry of volatile compound formation is
based on amino acid catabolism. The corresponding
FEMS Yeast Res && (2014) 1–12
pathway was already described in 1907 by the German
biochemist Felix Ehrlich, (1907). The pathway consists of
three reactions: the initial transamination of an amino
acid, a decarboxylation and a subsequent alcohol dehydrogenase reaction (Fig. 1; for review see Hazelwood
et al., 2008). In Saccharomyces cerevisiae several genes and
gene families can perform each reaction. The transamination can be carried out by Aro8/Aro9 and the
branched-chain amino acid transaminases Bat1/Bat2. The
decarboxylation occurs through the Ehrlich pathway gene
ARO10 or by the pyruvate decarboxylases Pdc1/Pdc5/
Pdc6. This will convert an amino acid first into an a-keto
acid and then into an aldehyde. In S. cerevisiae these aldehydes are then converted either to aroma alcohols or
aroma acids via either alcohol dehydrogenases (encoded
by the ADH-gene family) or aldehyde dehydrogenases
(encoded by the ALD-gene family), respectively. Genetic
studies of these genes and others, including for example
the alcohol acetyl transferase genes ATF1/ATF2, contributed greatly to the understanding of the molecular basis of
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
2
D. Ravasio et al.
2-oxoglutarate
Amino acid
Glutamate
CO2
α-keto acid
O
NAD(P)H, H+
Aldehyde
O
R
O
R
OH
OH
O
O
O
Transaminase
Acetate
R
CH
OH
NH2
CoA
Alcohol
R
R
Acyl CoA
NAD(P)+
Decarboxylase
Dehydrogenase
R1
Acetyltransferase
Amino acid
α-Keto acid
Fusel aldehyde
Fusel alcohol
Fusel acetate
Leu
4-Methyl-2-oxopentanoate
3-Methylbutanal*
Isoamyl alcohol*
Isoamyl acetate**
Val
3-Methyl-2-oxobutanoate
2-Methylpropanal*
Isobutanol*
Isobutyl acetate**
Ile
3-Methyl-2-oxopentanoate
2-Methylbutanal*
2-Methylbutanol
Ethyl pentanoate
Phe
3-Phenyl-2-oxopropanoate
2-Phenylethanal
2-Phenyl ethanol*
2-Phenylacetate**
* Compounds detected in A. gossypii and E. cymbalariae; ** Compound detected in E. cymbalariae only
flavour production in S. cerevisiae (Iraqui et al., 1998;
Dickinson et al., 2003; Verstrepen et al., 2003; Vuralhan
et al., 2005; Lilly et al., 2006; Ravasio et al., 2014).
The need for all-natural flavour production may guide
the selection of suitable lager or wine yeasts. Alternatively,
other yeast strains, collectively termed non-Saccharomyces
yeasts may be considered as flavour producers (Domizio
et al., 2011). This yeast biodiversity has not been evaluated to its full extent. Yeast species derived from several
genera, including Dekkera, Hanseniaspora, Lachancea,
Pichia, Saccharomycodes, Zygosaccharomyces, have been
shown to contribute to the flavour profile of fermented
beverages in interesting new ways either in sequential fermentation or when used as co-inoculants (Viana et al.,
2008; Ciani et al., 2010; Gobbi et al., 2013).
Ashbya gossypii, a known and industrially exploited overproducer of riboflavin/vitamin B2, is a filamentous ascomycete within the Saccharomycetaceae. Ashbya gossypii is a
protoploid, pre-whole genome duplication species with a
small genome of just 8.7 Mb + rDNA-repeats (Dietrich
et al., 2004). Molecular studies have centered on increasing
riboflavin productivity (Stahmann et al., 2000; Jimenez
et al., 2008; Park et al., 2011). With the identification of
highly efficient homologous recombination gene function
analyses were initiated studying polar hyphal growth and
nuclear dynamics in A. gossypii (for review see Wendland &
Walther, 2005; Schmitz & Philippsen, 2011). Comparative
genomics revealed marked differences between A. gossypii
and its close relative, the filamentous fungus Eremothecium
cymbalariae. These include genome size (8.7 Mb vs.
9.7 Mb), chromosome number (7 vs. 8), degree of synteny
with the yeast ancestor (higher in E. cymbalariae), and
GC-content (52% vs. 40%; Wendland & Walther, 2011).
Furthermore, we noticed decisive differences in growth
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Fig. 1. Ehrlich pathway conversion of amino
acids into fusel alcohols and esters. The
relevant co-factors are indicated for each
reaction. Specific molecular groups are
highlighted and enzymatic functions for each
reaction are provided. For the amino acids
processed in Ehrlich pathway reactions the
intermediates are noted. Several of these
substances were detected in Eremothecium
spec. as indicated.
pattern, colony morphology and flavour production
between both species.
The aim of this report was to study the potential of
A. gossypii for flavour production and to compare it with
its relatives E. cymbalariae and S. cerevisiae. For that purpose we targeted the Ehrlich pathway ARO-genes for
deletion in A. gossypii and employed gas chromatography
methods to measure the volatile aroma compound formation in these strains. Our results reveal the potential use
of A. gossypii for natural flavour production in beverages,
which provides further support for screening the nonSaccharomyces biodiversity for novel strains to be used in
the food and beverage industry.
Materials and methods
Strains and media
Strains generated and used in this study are shown in
Table 1. Yeast strain CEN.PK2 and WS34/70 were grown
in YPD (1% yeast extract, 2% peptone, 2% glucose) at
30 °C. Ashbya gossypii and E. cymbalariae were grown in
AFM (1% yeast extract, 2% caseine peptone, 2% glucose)
at 30 °C. Antibiotic substances (G418 or ClonNAT) were
added using a final concentrations of up to 200 lg mL1
for the selection of transformants. Plates were incubated
for 7 days at 30 °C prior to photography. For the growth
assays diameter of the mycelium was measured daily. Biological triplicates were performed. For sporulation, an
overnight culture of A. gossypii was further incubated in
minimal medium (1.7 g L1 YNB w/o ammonium sulphate and w/o amino acids, 0.69 g L1 CSM, 20 g L1
glucose, 1 g L1 asparagine, and 1 g L1 myo-inositol)
for up to 5 days.
FEMS Yeast Res && (2014) 1–12
3
Ehrlich pathway in Ashbya gossypii
Table 1. Strains used and generated in this study
Strain
Collection number
Genotype
Source
ATCC 10895 A. gossypii
Eremothecium cymbalariae
Saccharomyces pastorianus
CEN.PK2
A. gossypii
A. gossypii
A. gossypii
A. gossypii
A. gossypii
A. gossypii
71
C64
C44
C598
C692
C694
C703
C632
C696
C787
leu2
Wild-type
Weihenstephan Lager Yeast 34/70
Prototrophic strain, (CEN.PK113-7d x CEN.PK113-1A)
Δagr167w (Δaro8a)
Δafr548w (Δaro8b)
Δagr167w/afr548w (Δaroa/b)
Δacr211w (Δaro10)
Δadr199c (Δaro80)
ADR199C XL (ARO80XL)
Lab Strain
DBVPG 7215
Lab Strain
Lab strain
This study
This study
This study
This study
This study
This study
Transformation
Transformation of A. gossypii was done by electroporation as described previously (Wendland et al., 2000).
PCR-based cassettes were amplified from pFA-GEN3/
pFA-SAT1 plasmids (see Table 2) using gene specific S1
and S2 primers. For the deletion of ARO80 a disruption
cassette was cloned containing a 2 kb ORF-fragment
using the primer G1-AgARO80 (#6117) and G4-AgARO80 (#6118). An internal 1.1 kb EcoRV-NdeI fragment was replaced by kanMX. The ARO80 disruption
cassette was cleaved from the vector backbone with KpnI
and SpeI and contained flanking homology regions of
0.45 and 0.6 kb at the 50 and 30 ends, respectively.
To create a chromosomal ARO80 overexpression strain
an integration cassette was generated. The ARO80-ORFterminator fragment was amplified using the primer
A1-AgARO80 (#6170) and A4-AgARO80 (#6171). The
resulting 3.2 kb fragment was then cloned downstream
of the ScTEF1-promoter of the pFA-GEN3-ScTEF1p
vector via XhoI/SacII. The AgARO80-promoter served as
50 -flanking homology region. This fragment was amplified
by PCR (primers #6287-#6288) and after a PvuII/MscI
cleavage cloned into the unique PvuII site upstream of
the GEN3 marker gene in pFA-GEN3-ScTEF1p-AgARO80, resulting in pFA-AgARO80p-GEN3-ScTEF1p-AgARO80. Two NcoI sites within the flanking homolog
regions were used to release the overexpression cassette,
which provided flanking homology regions of 0.26 and
2.7 kb at the 50 and 30 ends, respectively.
Table 2. Plasmids used and generated in this study
Plasmid
Description
Source
#121
#550
pFA-KanMX6
pFA-SAT1
#C886
#C791
#C886
pFA-AgARO80p-GEN3-ScTEF1p-AgARO80
pSK-AgARO80::kanMX
pFA-AgARO80pGEN3-ScTEF1p-AgARO80
Philippsen lab
Schaub et al.
(2006)
This study
This study
This study
FEMS Yeast Res && (2014) 1–12
Primary heterokaryotic transformants were sporulated and
homokaryotic mutant mycelia were generated from uninucleate and haploid spores. For each desired genetic manipulation, two independent transformants were generated.
All primers are listed in Table 3. Primers were obtained
from Integrated DNA Technologies (IDT, Leuven, Belgium). Diagnostic PCR was used for verification of correct
integration of a disruption cassette and concomitant
absence of the target gene in homokaryotic deletion strains.
Fermentation conditions
Tall tube cylinders containing 200 mL medium were used
for fermentation. As fermentation broth YPD (with 15% glucose) was used for CEN.PK2 and WS34/70 at 20 °C, respectively. The starting cell density was set to OD600 at 0.2. Tall
tubes containing magnetic stirrers were placed on stirrer
pads run at 190 r.p.m. Fermentation progress was monitored over 5 days measuring CO2 loss and reduction of sugar
content using a DMA 35 Anton Paar densitometer, which
determines medium gravity in °Plato. The fermentation was
considered finished when the sugar content did not drop
further. Fermentation supernatants were decanted and used
for GC/MS, GC/flame ionization detector (FID) analysis. All
fermentations were carried out in biological duplicates.
Eremothecium growth conditions for flavor
profiling
Strains were pre-cultured o/n in AFM. One millilitre of
each pre-culture was used to inoculate a 150 mL AFM
culture in a 250 mL baffled flasks. At specific time points
50 mL samples were collected. In order to preserve the
volatiles and stop cell metabolism samples were kept at
20 °C until analysis.
Analytical methods – GC/MS, GC/FID
Two different extraction methods were used in this study.
For the comparison of A. gossypii mutants with yeast
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
4
D. Ravasio et al.
Table 3. Oligos used in this study
Oligo
Name
Sequence 50 -to-30 *
#6129
#6130
#6109
#6110
#6111
#6112
#6127
#6128
#6113
#6114
#6115
#6116
#6121
#6122
#6123
#6124
#6125
#6126
#6297
#6171
#6117
#6118
#6193
#6305
#1202
#1198
#6717
#6718
S1-AGR167w (ARO8a)
S2- AGR167w (ARO8a)
G1-AGR167w
G4-AGR167w
I1-AGR167w
I2-AGR167w
S1-AFR548w (ARO8b)
S2-AFR548w (ARO8b)
G1-AFR548w
G4-AFR548w
I1-AFR548w
I2-AFR548w
S1-ACR211w (ARO10)
S2-ACR211w (ARO10)
G1-ACR211w
G4-ACR211w
I1-ACR211w
I2-ACR211w
50 -ADR199c (KpnI)
30 -ADR199c (XbaI)
G1-ADR199c
G4-ADR199c
I1-ADR199c
I2-ADR199c
G2-KanMX6
G3-KanMX6
G2-SAT1
G3-SAT1
TAAGTCAGCAAGATCGCTGGGCGCTAAGGTAGATAACGACAAGAGgaagcttcgtacgctgcaggtc
GCACCACGAGGCAGGGCAGGTGACTGGAGGCTAGTATTTTATGGActgatatcatcgatgaattcgag
CTCGGAACCGGGTCAGTTC
GACCTTGGAAGTGGACTCC
CAGCACACGGAGAAGTTCCAC
CCACGAGCGTGCTGCCCATC
CCGCACAGTGGCTCCCGCAGGGCGCTTCTTTGGCTGAGCGCTCCGgaagcttcgtacgctgcaggtc
AAGTCTTGGCCACAAGTGGCAATCGAAGTCGGCACCTATAAGTGActgatatcatcgatgaattcgag
CTCGTCAAAGCTGGCTTACTG
CTGGAGACGCTCTCCTCGG
GACGTGCTGGCGACGGTCG
GGAGAACGAGTCCAGACGC
AACAGGGGTAGGAGGTTTACAGGAGGTAAGCGAGCGGCACGAGACgaagcttcgtacgctgcaggtc
CCCCTGCATGTCTTCTGTTGCTCGTCTGCGGAGTAGCTACGCAGCctgatatcatcgatgaattcgag
GTGGCTATTCGTGGGCTGG
GCGTAGAACCAGCTCTCTTC
GGCTCTCAATGGCGTGGCAG
CGCATAGTATTGCAGGCGTGC
AGAATAggtaccGTCCCAGTGCTTTTGTGACCGTC
ATAAGAtctagaGTACCAGCTCCATAGTCCATGGTAATC
CTCATCACTTGTGTGGAGCC
GCCGTAGAGGATGCGCTC
GTCGAATCGCGGTTCTCCG
CTGAACTCCGGCTCATCGC
GCGTTTCCCTGCTCGCAGGTC
CGCCTCGACATCATCTGCCC
GCAATAAATCTTGGTGAGAACAGC
GCGGCATTGACCTCTTCACG
*Restriction sites are in lower case.
strains, a solvent extraction with carbon disulphide (CS2)
was used. The samples were stirred for 30 min, centrifuged and 2 lL of the liquid organic phase was injected
into the gas chromatograph (Agilent 6890 GC). 1-octanol
was used as an internal standard. Detection of volatiles
was performed using a FID. The volatiles were separated
on a DBWAX capillary column (30 m 9 0.32 mm 9
0.25 lm). For comparison of Eremothecium strains volatile compounds were measured using biological and technical duplicates for dynamic headspace sampling. As an
internal standard 4 methyl-1 pentanol was added to each
sample. All samples were equilibrated to 37 °C in a water
bath and then purged with hydrogen (100 mL min1) for
20 min while stirring at 200 r.p.m. The volatile compounds were collected on Tenax-TA traps (250 mg, mesh
size 60/80, density 0.37 g mL1, Buchem bv, Apeldoorn,
The Netherlands) at 37 °C. To eliminate excess water
traps were purged by hydrogen flux of 100 mL min1 for
10 min. The trapped volatiles were desorbed using an
automatic thermal desorption unit (ATD 400, Perkin
Elmer, Norwalk, CT) and were automatically transferred
to a gas chromatograph-mass spectrometer (789OA GC
system, 5975C VL MSD, Agilent technologies, Santa
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Clara, CA). Separation of volatiles was carried out on a
ZB-Wax capillary column (Zebron capillary, 30 m 9
0.25 mm 9 0.5 lm). The mass spectra were recorded in
electronic impact mode at 70 eV in a mass/charge ratio
from 15 to 300 m/z. Data analysis was performed
using the software program MSDCHEMSTATION (Version
E.02.02.1431, Agilent Technologies). Identification of
compounds was based on comparison with a mass spectral database (Nist 1.0.0.23). One characteristic quantifier
ion and two to three qualifier ions were selected for each
compound. The peak area of the quantifier ion was used
for quantification.
Results
Comparison of flavour production between
A. gossypii and S. cerevisiae
The amino acid catabolism of branched-chain amino acids
(Leu, Val, Ile) and phenylalanine via the Ehrlich pathway
generates a-keto acid and fusel aldehyde intermediates
that are converted to fusel alcohol, which can be esterified
into fusel acetates (Fig. 1). Ashbya gossypii produces a
FEMS Yeast Res && (2014) 1–12
5
Ehrlich pathway in Ashbya gossypii
characteristic highly fruity flavour when grown either on
plates or in liquid culture. For a comparison of flavour profiles via GC/FID measurements of volatile compounds we
used A. gossypii grown in AFM for 5 days, the S. cerevisiae
strain CEN.PK2, and the Weihenstephan lager yeast production strain grown in YPD (15% glucose; Fig. 2). All strains
were incubated up to 5 days in either oxidative (A. gossypii)
or fermentative conditions (yeast strains). To increase the
volatile production the sugar content of the media was
increased to 15% glucose. Since the growth of A. gossypii
was significantly reduced in media with sugar content higher
than 2% we incubated this stain in standard AFM medium.
This showed that A. gossypii was superior in 2-phenylethanol production compared to these yeast strains.
Comparison of flavour production between
Eremothecium species
Volatile compound formation has not been analysed so far
in Eremothecium species. To monitor any change in VOCs
formation over time we measured the volatiles produced by
liquid cultures at day three, four, and five representing medium to late stages of growth in both Eremothecium species
(Tables 4 and 5). Within this comparison between A. gossypii and E. cymbalariae we identified only a marginal production of 2-phenylethanol production in E. cymbalariae.
In contrast E. cymbalariae had low levels of isobutylacetate
which were absent in A. gossypii (Fig. 3a). Both species
showed a strong production of isoamyl alcohol (Fig. 3b).
Production of both isoamyl alcohol and 2-phenylethanol in
A. gossypii increased over the assayed period and were
500
50
WS34/70
450
CEN.PK2
Ag
45
40
350
35
300
30
250
25
200
20
150
15
100
10
50
5
mg L–1
mg L–1
400
0
0
Iso butanol
Isoamyl
alcohol
2-phenyl
ethanol
Isoamyl
acetate
2-phenyl
ethyl acetate
Fig. 2. Comparison of flavor compounds between Ashbya gossypii (Ag)
and yeast strains. Gas chromatography was employed to measure the
indicated fusel alcohols or esters. Strains: Weihenstephan lager yeast
(WS34/70) and Saccharomyces cerevisiae laboratory strain CEN.PK2. For
culture and growth conditions see ‘Materials and methods’.
FEMS Yeast Res && (2014) 1–12
highest at day 5. Eremothecium cymbalariae, on the other
hand, produced isoamyl acetate and 2-phenyl acetate but
only in the initial period of growth and these compounds
were significantly reduced at day 4 and 5. Thus our analysis
indicates that A. gossypii produces strong notes of banana
and rose flavour, that is flavour alcohols, while E. cymbalariae produces higher amounts of esters. Specifically, E. cymbalariae generated high amounts of ethyl acetate and ethyl
propionate, which at moderate concentration impart pear
and pineapple like flavours, respectively (Table 5).
Components of the Ehrlich pathway in
A. gossypii
We noted previously that E. cymbalariae lacks most components of the Ehrlich pathway which are, however, present in A. gossypii (Wendland & Walther, 2011). Here we
provide a more detailed analysis of the Ehrlich pathway
gene set in comparison to other yeasts and the compiled
yeast ancestral genome prior to the whole genome duplication (Gordon et al., 2009). In contrast to S. cerevisiae
and the yeast ancestor pre-duplication yeast species may
harbour two ARO8 paralogs, here termed ARO8a and
ARO8b for A. gossypii, Kluyveromyces lactis and Lachancea
waltii (Table 6). The E. cymbalariae genome encodes
ARO8a but lacks ARO8b, ARO9, ARO10, as well as the
transcriptional regulator ARO80. Comparison of Aroproteins indicated that the transaminases Aro8a, Aro8b
and Aro9 are sufficiently divergent to cluster into separate
groups (Fig. 4). Interestingly, the amino acid sequence
identity between Aro8a and Aro8b paralogs of one species
is lower (in the three species evaluated on average 48.1%)
than that of Aro8a or Aro8b proteins of different species:
on average Aro8a proteins show about 55% amino acid
sequence identity, Aro8b proteins about 61.0%. Eremothecium species are distinguished from other yeasts by their
lack of ARO9.
Deletion of AgARO-genes does not confer
growth phenotypes
Via PCR-based gene targeting we generated A. gossypii single deletion mutants lacking either ARO8a, ARO8b, ARO10,
or ARO80 as well as a double mutant with deletions of
ARO8a and ARO8b. Growth comparisons of these mutants
with the parental strain showed no radial growth delay
(Fig. 5).
VOC analysis of A. gossypii Ehrlich pathway
mutants
The A. gossypii ARO-mutant strains with deletions in
Ehrlich pathway components were analysed for their
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
6
D. Ravasio et al.
Table 4. Ashbya gossypii VOCs produced over 3 days
3rd day, ppm
Alcohols
1 Butanol
2 Phenylethanol
Benzylalcohol
Isoamyl alcohol
Isobutanol
Propanol
Esters
2 Phenylacetate
Ethyl acetate
Ethyl phenylacetate
Isoamylacetate
S-methylthioacetate
Aldehydes
2-Methyl-2 butenal
(E-E) 2,4-Hexadienal
2-Butenal
Acetaldehyde
3-Methyl benzaldehyde
Benzaldehyde
Benzeneacetaldehyde
Butanal
2-Methyl-1 butanal
2-Methyl-1 propanal
3-Methyl-1 butanal
5 Methyl-2-phenyl hexanal
Ketons
2 Pentanone
2-Butanone
c-Decalactone
Acids
Acetic acid
Ethyl benzoate
Ethyl butanoate
Ethyl caproate
Ethyl isobutyrate
Ethyl propionate
Valeric acid
Aromatic compounds
2,5-Dymethyl-pyrazine
2-Ethyl-5 methyl-pyrazyne
2-Ethyl-6-methyl-pyrazyne
3-Ethyl-2,5-dimethyl-pyrazine
3-Phenylfuran
3-Phenylpentane
1-3-Bis-1,1-dymethylethyl-benzene
Others
E-1-Hydroxy-1,3butadiene
Hexane
5th day, ppm
4.46
4.00
0.11
459.53
36.52
10.87
(0.27)
(0.56)
(0.08)
(11.27)
(2.47)
(0.66)
6.05
32.86
0.16
754.01
74.16
5.79
(0.49)
(6.85)
(0.03)
(23.27)
(4.50)
(0.59)
0.36
90.82
0.16
850.24
51.63
/
(0.10)
(12.42)
(0.04)
(37.03)
(2.95)
0.03
173.46
0.07
0.41
3.32
(0.00)
(13.02)
(0.01)
(0.04)
(0.37)
0.39
194.17
0.12
3.85
0.26
(0.13)
(2.59)
(0.01)
(1.46)
(0.02)
1.99
70.45
0.18
2.57
0.22
(0.30)
(2.39)
(0.01)
(1.04)
(0.02)
3.50
0.15
73.01
0.71
(0.05)
(0.17)
(2.40)
(0.30)
0.50 (0.23)
/
3.02
0.69
0.57
0.53
1.89
/
0.22
1.90
0.34
49.09
4.31
71.29
(0.03)
(1.28)
(0.09)
(8.19)
(0.57)
(5.21)
/
(1.91)
(0.06)
(0.06)
(0.02)
(0.19)
/
0.43 (0.07)
/
/
0.69 (0.05)
/
1.00 (0.13)
7.68 (1.10)
/
38.40 (5.25)
3.26 (1.03)
24.09 (0.41)
55.31
3.31
57.37
0.13
/
0.73 (0.02)
5.47 (0.36)
2.09 (1.51)
0.15
0.23
2.23
0.26
0.38
4.47
2.36
(0.05)
(0.05)
(0.04)
(0.00)
(0.37)
(0.54)
(1.94)
2.42
0.12
0.10
0.16
0.09
(0.13)
(0.08)
(0.03)
(0.02)
(0.01)
1.17 (0.07)
4.69 (0.35)
2.72 (0.73)
/
(0.45)
(0.44)
(2.30)
(0.06)
/
/
0.22 (0.01)
7.78 (0.13)
1.22 (0.63)
0.12 (0.09)
0.89 (0.14)
0.79 (0.49)
0.31 (0.27)
0.68 (0.38)
/
1.00 (0.04)
3.24 (0.37)
14.23 (1.09)
0.24 (0.02)
2.16 (0.14)
2.47
0.20
0.63
0.25
0.08
0.86
0.08
aroma alcohol profile using GC/FID (Fig. 6a). We found
a striking effect of deletions of ARO8b, ARO10, and
ARO80 in the severe reduction in 2-phenylethanol formation in A. gossypii. Additionally, the amount of isoamly
alcohol was decreased in these strains to about 80% of
the wild type level. Deletion of Agaro8a did not result in
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
4th day, ppm
(0.08)
(0.01)
(0.09)
(0.02)
0.00
(0.05)
(0.06)
0.11 (0.12)
0.66 (0.18)
/
/
3.11 (0.64)
7.29 (0.85)
2.52
0.22
0.94
0.21
0.05
(0.01)
(0.01)
(0.15)
(0.01)
(0.01)
/
0.06 (0.02)
/
0.37 (0.10)
reduction of isoamyl ethanol or 2-phenylethanol production and the aro8a/aro8b double mutant exhibited the
aro8b phenotype with respect to these flavours. Thus we
conclude that the Ehrlich pathway in A. gossypii plays a
major role in 2-phenylethanol production and also contributes to isoamyl alcohol formation.
FEMS Yeast Res && (2014) 1–12
7
Ehrlich pathway in Ashbya gossypii
Table 5. Eremothecium cymbalariae VOCs produced over 3 days
3rd day, ppm
Alcohols
1-Butanol
1-Propanol
2 Phenylethanol
3-Methyl-3-Buten-1-ol
Benzylalcohol
Citronellol
Ethanol
Isoamyl alcohol
Isobutanol
Phenol
Esters
2 Phenylacetate
2-Furanmethanol acetate
Amylacetate
Isoamylacetate
Isobutylacetate
Ethyl acetate
Heptyl acetate
Propylacetate
Benzylacetate
Aldehydes
2-Butenal
2-ethyl-trans-2-butenal
2-Methyl propanal
2-Methyl-2-butenal
2-Methyl-butanal
2-Mehtyl-benzaldehyde
3-Methyl-butanal
Acetaldehyde
Benzaldehyde
Butanal
Caproaldehyde
Ketons
2 Butanone
2,3-Butanedione
Acetophenone
c-Decalactone
3-Hydroxy-2-butanone
Acids
Amyl-propionate
Ethyl Butyrate
Ethyl Isobutyrate
Ethyl propionate
Propyl propionate
Valeric acid
Acetic acid
Aromatic compounds
2,5-Dihydro-furan
3-Ethyl-2,5-dimethyl-pyrazyne
2,5-Dymethyl-pyrazine
2-Ethyl-5-methyl-pyrazyne
3-Phenylfuran
Benzonitrile
FEMS Yeast Res && (2014) 1–12
2.50 (0.02)
/
0.26 (0.06)
/
0.04 (0.00)
/
0.22
275.51
8.32
0.09
(0.03)
(2.28)
(0.92)
(0.01)
6.78
0.11
0.13
367.37
2.37
941.94
0.09
13.63
1.49
(0.11)
(0.01)
(0.00)
(8.69)
(0.09)
(41.37)
(0.02)
(1.08)
(0.06)
4th day, ppm
7.31
0.83
0.29
0.30
0.04
0.06
0.22
468.72
22.15
0.10
(0.36)
(0.15)
(0.01)
(0.02)
(0.00)
(0.00)
(0.07)
(8.72)
(2.00)
(0.00)
31.60 (1.75)
/
42.15 (2.37)
0.75 (0.03)
0.21 (0.01)
(2.41)
(0.38)
(11.86)
0.01 (0.01)
(0.58)
(0.00)
(28.03)
(1.35)
(0.04)
(0.06)
(0.25)
(0.92)
(0.01)
(0.90)
(0.05)
(0.01)
(0.07)
0.00 (0.00)
108.03
2.94
1.20
13.18
10.58
0.04
35.93
0.76
0.53
0.87
/
(0.30)
(3.42)
(0.07)
(0.11)
(0.19)
(4.66)
(0.01)
(4.59)
(0.04)
(0.04)
/
(0.00)
(0.17)
(0.35)
(0.01)
(1.60)
(1.32)
3.31
4.11
0.16
9.41
14.56
(0.63)
(0.48)
(0.03)
(2.74)
(0.59)
5.61
4.00
0.13
11.26
11.77
(2.03)
(0.43)
(0.00)
(0.67)
(0.83)
5.69 (0.67)
1.05 (0.03)
(1.94)
(0.01)
(0.04)
(0.02)
4.20
2.70
0.64
119.46
0.00
7.87
0.31
(0.45)
(0.12)
(0.08)
(3.26)
(0.00)
(1.70)
(0.03)
1.42
0.76
0.19
66.70
(0.02)
(0.01)
(0.01)
(0.25)
(0.04)
(0.00)
(0.00)
(0.04)
1.63
0.17
2.28
0.14
0.06
0.08
(0.04)
(0.04)
(0.17)
(0.02)
(0.01)
(0.02)
1.30
14.24
0.12
5.19
11.94
(0.64)
(0.13)
(0.05)
(0.01)
(0.00)
(0.00)
(0.02)
(19.73)
(2.39)
(0.01)
7.13
0.47
507.13
/
2.12
/
49.04
0.63
1.14
7.80
47.71
0.12
93.29
0.75
0.58
0.34 (0.12)
/
/
15.23
1.68
720.65
/
2.73
/
/
0.05 (0.00)
16.34
2.68
0.31
0.35
0.04
0.05
0.17
526.42
42.60
0.06
0.05 (0.02)
/
/
/
/
/
5th day, ppm
/
65.65
0.31
2.49
0.25
/
/
2.44
0.16
0.06
0.08
/
1.23 (0.06)
0.19 (0.03)
4.00
0.15
2.40
0.15
0.04
0.05
(0.07)
(0.01)
(0.06)
(0.00)
(0.00)
(0.00)
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
8
D. Ravasio et al.
Table 5. Continued
3rd day, ppm
Others
(E)1-Hydroxy-1,3-butadiene
2,4,5-trimethyl-3-oxazoline
2-Isopropylbut-2-enal
Hexane
4th day, ppm
/
/
/
0.70
1.04
0.40
0.22
0.22 (0.01)
(0.09)
(0.03)
(0.03)
(0.03)
5th day, ppm
0.53
1.24
0.33
0.49
(0.09)
(0.05)
(0.01)
(0.09)
ppm expressed as I.S
(a)
10
100
8
80
6
60
4
40
2
20
0
10
4
5
E. cymbalariae
5
0
3
Ashbya
0
3
4
day
3
5
4
2-Methylpropanal
Isobutanol
Isobutyl acetate
Phe
2-Phenylethanal
2-Phenylethanol
2-Phenylacetate
ppm expressed as I.S
Val
100
10
80
8
60
6
40
4
20
2
5
0
0
3
4
5
3
4
5
day
ppm expressed as I.S
(b)
100
Ashbya
60
40
20
0
3
ppm expressed as I.S
E. cymbalariae
80
4
day
5
Ile
2-Methylbutanal
2-Methylbutanol
Ethyl pentanoate
Leu
3-Methylbutanal
Isoamyl alcohol
Isoamyl acetate
100
1000
1000
800
600
400
200
0
80
60
40
20
0
3
4
5
800
600
400
200
0
3
4
day
5
3
4
5
Fig. 3. Time course analysis of flavor
production in Ashbya gossypii and
Eremothecium cymbalariae. Strains were
cultured for 5 days and samples were
analyzed between days 3–5. Volatile
compounds were analyzed via GC/MS. Ehrlich
pathway products for the amino acids valine,
phenylalanine, isoleucine and leucine were
quantified. Compounds written in grey are
intermediates of the respective amino acid
degradation but were not detected.
Table 6. ARO-gene content in different fungal species
Transaminase
Species*
ARO8a
ARO8b
ARO9
Decarboxylase
ARO10
Transcription factor
ARO80
Ancestor
S. cerevisiae
K. lactis
L. waltii
A. gossypii
E. cymbalariae
Absent
Absent
A04906
17462
AGR167W
Ecym_7228
Anc_3.511
YGL202W
F10021
18982
AFR548C
Absent
Anc_2.102
YHR137w
D11088
14582
Absent
Absent
Anc_5.456
YDR380W
E02707
Absent
ACR211W
Absent
Anc_5.525
YDR421W
A01804
Absent
ADR199C
Absent
*Species depicted were: Saccharomyces cerevisiae, Kluyveromyces lactis, Lachancea waltii, Ashbya gossypii, Eremothecium cymbalariae.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Yeast Res && (2014) 1–12
9
Ehrlich pathway in Ashbya gossypii
EcymAro8a
AgAro8a
LwAro8a
KlAro8a
KlAro8b
LwAro8b
ScAro8b
AgAro8b
LwAro9
KlAro9
ScAro9
KlAro80
AgAro80
ScAro80
AgAro10
KlAro10
ScAro10
Fig. 4. Phylogenetic tree of transaminase and decarboxylase genes.
Aro-proteins of Ashbya gossypii (Ag), Eremothecium cymbalariae
(Ecym), Lachancea waltii (Lw), and Kluyveromyces lactis (Kl) were
analyzed using DNASTAR MEGALIGN software.
Overexpression of AgARO80 enhances flavour
production in A. gossypii
Since the deletion of ARO80 showed a clear negative
effect on flavour production we went on to analyse if
overexpression of ARO80 could further increase flavour
levels. To this end the chromosomal AgARO80 gene was
placed under the control of the strong constitutive TEF1
promoter from S. cerevisiae (see ‘Materials and methods’).
Resulting transformants were compared to the parental
wild type strain analysed under the same conditions as
Fig. 5. Functional analysis of Ashbya gossypii
ARO-genes. Radial colony growth of the
indicated strains inoculated at the center of
each petri plate was monitored over 8 days.
Growth was either at either 30 °C or 37 °C.
Mycelia were grown on AFM plates and
photographs were taken at the end of the
growth period.
FEMS Yeast Res && (2014) 1–12
Ag aro8a
before (Fig. 6b). It became apparent that ARO80 overexpression resulted in a 2.5 fold increase of isoamyl alcohol
and isobutanol levels derived from branched-chain amino
acid catabolism, whereas the levels of 2-phenylethanol
were basically unchanged, which could be attributable to
the limited uptake of phenylalanine from the medium.
Discussion
Non-conventional yeasts (NCY) harbour a great potential
as novel production strains, for example the production
of L-lactic acid by Candida sonorensis, or the production
of another highly useful chemical building block, Dxylonic acid, by Pichia kudriavzevii, for the production of
virus like particles in Kluyveromyces lactis or for the production of all-natural flavours by a wide variety of NCYs
(Goretti et al., 2013; Ilmen et al., 2013; Toivari et al.,
2013). The scope of this biodiversity within the Saccharomycetaceae has but just begun to be exploited and will
provide a rich source for biotechnological research in the
future (Domizio et al., 2011). This group of yeasts is very
attractive based on their ease of cultivation and molecular
genetic tractability combined with small genome sizes
allowing for rapid determination of draft genome
sequences and the functional analysis of target genes.
In this study we have analysed the flavour profile of
two closely related Eremothecium species, which are both
filamentous fungi belonging to clade 12 of the Saccharomycetes (Kurtzman & Robnett, 2003). Both species differ
on the genomic level and morphologically, for example
regarding extensive aerial mycelium formation in E. cymbalariae compared to synnemata formation in A. gossypii
and also in the shape of their spores (Wendland &
Ag aro8b
Ag aro8a/b
Ag aro10
Ag aro80
Ag wt
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
10
D. Ravasio et al.
wt
aro8a
aro8b
aro8a-b
aro10
aro80
(a) 600
ppm (mg L–1)
500
400
300
200
100
0
Isobutanol
Isoamyl alcohol
wt
(b) 600
2-Phenyl ethanol
TEF1p-ARO80
ppm (mg L–1)
500
400
300
200
100
0
Isobutanol
Isoamyl alcohol
2-Phenyl Ethanol
Fig. 6. Volatile compound formation in Ashbya gossypii aro-mutants.
(a) the indicated mutant strains were grown as described in
‘Materials and methods’ and fusel alcohols of the valine, isoleucine,
and phenylalanine were assayed via GC/FID. (b) Overexpressionof
the ARO80 transcription factor enhances isoamyl formation in
A. gossypii.
Walther, 2005). The purpose of this study was to determine differences in flavour production between these
Eremothecium species and relate these to the different
genetic makeup of ARO-genes. Our flavor profiling
results revealed a preference regarding flavour alcohol
production in A. gossypii vs. ester production in E. cymbalariae. Higher ester production in E. cymbalariae
resembles that found in lager yeast strains fermenting
high gravity wort with high concentrations of sugar
(Verstrepen et al., 2003). Considering the slow growth of
E. cymbalariae even the relatively low amounts of 2% glucose in our experiments may have triggered an excess
amount of pyruvate formation resulting in increased acetate ester formation. In line with this we have observed
larger amounts of ethyl acetate and other acetate esters in
E. cymbalariae compared to the faster growing A. gossypii
(see Tables 4 and 5). Ashbya gossypii formed very large
amounts of both isoamyl alcohol and 2-phenylethanol.
From S. cerevisiae it is known that 2-phenylethanol formation is restricted to the growth phase (Stark et al.,
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
2002). Ashbya gossypii generated the highest amounts of
2-phenylethanol among the species we tested in our
experiments, which makes it a very efficient natural producer of this flavour. Our deletion analyses showed that
Ehrlich pathway genes are of central importance for the
production 2-phenylethanol. The microbial production of
2-phenylethanol has recently received considerable attention as an all-natural flavour in the food and cosmetics
industry. Due to the toxic effect of large amounts of
2-phenylethanol (>2.5 g L1) in situ removal strategies
using organic solvents have been developed to avoid a
decrease in growth rate (Stark et al., 2003; Eshkol et al.,
2009; Achmon et al., 2011; Hua & Xu, 2011).
The production of more fruity notes in beverages could
benefit from for example co-fermentations using S. cerevisiae and another non-conventional yeast strain. Such
co-fermentations have gained increased popularity in the
wine industry (Ciani et al., 2010). Using a lacZ-based
assay system we recently evaluated the flavour profiles of
S. cerevisiae and its closely related species (Ravasio et al.,
2014). In this study we could show that overexpression of
ARO80 has a profound impact on flavour production.
Thus a more broadly applicable assay for non-conventional yeasts could help linking the expression level of
Ehrlich pathway genes to the flavour production capabilities of a previously uncharacterized isolate.
Ashbya gossyppi is well-known for its riboflavin overproduction. Recent studies hypothesized that flavindependent detoxification of plant compounds may open
new niches for insects (Sehlmeyer et al., 2010; Dietrich
et al., 2013). Involving Ashbya in such a plant-insect system required adaptation to an insect environment to sustain viability in milkweed bugs (e.g. during hibernation
periods) and overproduction of riboflavin to detoxify
toxic alkaloids produced for example by oleander. These
environmental stresses may have resulted in alteration
and streamlining of the Ashbya genome compared to
E. cymbalariae. On the other hand, with such a niche
found, reinforcing the relationship of Ashbya with certain
insects used for spreading of Ashbya could have occurred
by using volatile compounds such as 2-phenylethanol as
attractant. Our study opens new aspects of linking genome evolution to both biotic and abiotic environmental
challenges.
Acknowledgements
This research was supported in part by the European
Union Marie Curie Initial Training Network Cornucopia.
Sequence data for Ashbya gossypii was obtained from the
Ashbya Genome Database website at http://agd.vital-it.ch/
index.html. We thank Dr Mikael Agerlin Petersen, University of Copenhagen for help with VOC measurements.
FEMS Yeast Res && (2014) 1–12
Ehrlich pathway in Ashbya gossypii
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Paper 3
Paper 3
Non-conventional yeasts (NCY) constitute a rich and accessible resource for new traits that
can find applications for new biotechnological processes. In this manuscript we investigated a
selection of 18 NCYs, encompassing species that represent the entire Saccharomyces clade. The
strains have been tested in fermentation condition and fermentation parameters were collected.
In addition the strains have been evaluated under stress condition (i.e. osmotic and oxidative
stress) and for the production of off-flavours by several plate assays. Even though each species
displayed unique characteristic, the volatile profile analysis highlighted four extreme flavour
producers: W. anomalus, P. kluyveri and Z. melli showed very high ester production; in
contrast D. subglobosus produced high levels of fruity ketones and aldehydes.
66
third group of five strains produced higher
A survey of flavor
production among
non-conventional
yeasts
ethanol concentrations than the reference
strain and the fourth group consisting of two
strains is characterized by either highest or
no
ketone
production.
We
present
a
comprehensive overview of these strains
identifying those specialized for volatile
alcohol and ester formation or ethanol
Davide Ravasio, Silvia Carlin, Teun
Boekhout, Urska Vrhovsek, Jürgen
Wendland and Andrea Walther
production, respectively.
Introduction
Beer is one of the most widely consumed
alcoholic beverages in the world. In 2003 the
Abstract
worldwide beer production reached around
Fungi produce a variety of volatile organic
1.82 billion hectoliters and increased to a
compounds (VOCs) during their primary and
volume of 1.97 billion hectoliters in 2013 (©
secondary metabolism. In particular, in the
Statista 2014).
beverage industry these volatiles contribute
to the flavor and aroma profile of the final
product.
Herein,
we
evaluated
the
fermentation ability and aroma profiles of
non-conventional yeasts (NCY) that have
been isolated from various food sources. A
total of 60 strains have been analyzed with
regard to their fermentation and flavor
profile.
After
representative
a
primary
strains
were
screen
18
selected
for
further strain characterizations based on
principal component analysis as well as their
fermentation performances.
Beer production is divided into two main beer
styles: Ale and Lager beers. Generally, ale is
produced by top fermenting yeasts and
typically at higher temperatures of 15-30 °C.
This normally results in faster fermentation.
Ales are known for their fruity aromas which
are regarded as a distinctive character of top
fermenting beers.
The word “Lager” actually derives from the
German word “lagern”, which means “to
store”. Lager beers are produced by bottom
fermenting yeasts typically at temperatures
below 15 °C. The aroma of lager beers is more
These 18 strains have been further divided
neutral compared to Ale type beers since they
into four groups: Group 1 consists of six
contain lower amounts of fruity flavors.
strains
with
exceptionally
high
flavor
production, while group 2 includes five
strains with very low flavor production. A
Top fermenting and bottom fermenting
yeasts
are
two
distinct
Saccharomyces
species. Top fermenting yeasts typically
belong to the species S. cerevisiae. At the end
contribute to the beer by a solvent-like aroma
of fermentation these yeasts rise to the
which gives a warm mouth feel. The latter two
surface of the fermenter creating a thick cell
are prevalent for their sweet/rose and
layer. Bottom fermenting yeasts are called
fruity/banana like aroma, respectively [3].
S. carlsbergensis or S. pastorianus. These
yeasts
are
different
interspecies
parental
hybrids
strains
that
of
can
two
be
subdivided into two subgroups namely Saaz
type strains and Frohberg type strains, which
derived from 2 independent hybridization
events [1].
Esters
that
fermentation
are
produced
contribute
during
mainly
to
beer
the
fruitiness of the product. They can be divided
into two main groups. The first group of
acetate esters consists of fruity esters such as
ethyl acetate (solvent like, fruity), isoamyl
acetate (banana) and 2-phenyl acetate (rose).
spontaneous
Ethyl or medium-chain fatty acid esters such
fermentation of wort is typical for Belgian
as ethyl hexanoate, ethyl octanoate and ethyl
Lambic sour beers. Here, the fermentation
decanoate belong to the second group of
process is not initiated through inoculation
esters. They give a fruity apple or wine like
with specific yeasts. The wort is filled in a
flavor to the beer [4].
Beer
production
by
shallow open vessel that allows microbial
intrusion of bacteria and wild yeasts [2]
The main aldehyde present in beer is
acetaldehyde. It can be formed of ethanol
Beer is a very complex product consisting of
when oxygen is present [5]. Other aldehydes
both volatile and non-volatile components
that contribute to the beer aroma are divided
that form the final aroma. The contribution of
into
yeasts to the final flavor bouquet is dependent
aldehydes of Maillard reaction and aldehydes
on the beer style. In ale beers the contribution
of fatty acid oxidation. Strecker aldehydes
of the yeast strain to the final aroma is
derive from the degradation of leucine,
considered to be around 30 % whereas in
isoleucine and phenylalanine during wort
lager beers the contribution is only 15 %. The
boiling and aging. Maillard aldehydes consist
volatile compounds produced by the yeast
of various heterocyclic compounds which
strain belong to different chemical groups.
develop due to the reaction of sugars and
The most prominent and dominating flavor
amino acids. The most abundant Maillard
compounds
aldehyde in beer is furfural [6]. One of the
are
higher
alcohols,
esters,
aldehydes, ketones and organic acids.
Among the higher alcohols n-propanol, isobutanol, 2-phenylethanol and isoamyl alcohol
are the
most abundant alcohols. They
three
groups:
Strecker
aldehydes,
predominant aldehydes of the fatty acid
oxidation is trans-2-nonenal (T2N). It creates
a stale papery taste that is generally released
during storage of beer.
Further, ketones contribute an important
recent years SPME has been widely used as a
aroma to the beer. In particular, diacetyl (2,3-
solvent-free
buanedione) can influence the flavor of beer.
analysis of volatiles in beverages.
In
brewing
process
it
is
considered
undesirable due to its unpleasant buttery
flavor and its low taste threshold. Diacetyl
can be produced during the biosynthesis
pathway
of
valine
and
isoleucine.
Acetolactate, an intermediate of the valine
biosynthesis pathway can be oxidized to
diacetyl preferably under high fermentation
temperatures. At the end of fermentation and
during beer maturation diacetyl can be
reabsorbed by the yeast cells and converted to
2,3-butanediol via the intermediate acetoin.
Both acetoin and 2,3-butanediol have a much
higher taste threshold than diacetyl [7].
Short-chain
organic
acids
can
extraction
method
for
the
Lately, non-conventional yeasts (NCY) or
non-Saccharomyces
yeasts
importance
fermented
for
have
gained
alcoholic
beverages. They produce various mixtures of
volatile compounds and therewith contribute
to the aroma profile of beverages. [8].
However,
these
strains
have
not
been
characterized in great detail. A vast number
of non-conventional yeast strains has been
isolated from various food sources and
collected in a strain collection database such
as CBS (Centraalbureau voor Schimmelcultures, Utrecht, Netherlands). In this study
we aimed at covering a broad spectrum of
greatly
species isolated from different substrates like
influence the final beer flavor. They can
berries, fruits, cheese, fruit flies or even soil
reduce the pH during fermentation and
and spanning a broad evolutionary distance
therewith give a sour taste to the beer.
within the Saccharomycetes.
Medium-chain fatty acids such as caproic,
caprylic and capric acid can add a rancid
Material and Methods
goaty flavour to the beer. Since they can
Strains and media
influence the foam stability of the beer they
Strains used in this study are shown in Table
are undesirable compounds.
1. Prior to spotting assays yeast strains were
Given the low concentration and volatile
nature of these aroma compounds gas
chromatography
coupled
to
mass
spectrometry (GC–MS) offers an optimal
technique to analyze the flavor profile of beer.
Coupled to this, several sampling methods
such as solid-phase micro extraction (SPME),
steam distillation extraction (SD) and liquidliquid extraction have been developed. In the
grown in YPD (1 % yeast extract, 2 % peptone,
2 % glucose) at room temperature o/n. Then,
the cells were harvested by centrifugation,
washed in sterile deionized water and finally
diluted to an OD600 of 0.1. Dilution series in
steps of 1:10 were then spotted onto the
plates. For growth tests on granmalt medium
(150 g/l malt granules, 5 g/l yeast extract, 2%
agar), plates were incubated for 2 days at
room temperature prior to photography. YPD
Analytical methods GC/MS
plates were incubated at 10, 20 and 37°C for
Volatile
up to 4 days to assess the strains’ thermo
analyzed by using a Thermo Scientific TSQ
tolerance. SD based medium (20 g/L glucose,
Quantum GC Triple Quadropole GC-MS. 2-
6.7 g/l YNB w/o amino acids but with
octanol was added as an internal standard to
ammonium
with
each sample. The standard was chosen as a
addition of cinnamic acid (100 µg/l) was used
compound known not to be present in the
to test the strains ability to produce phenolic
fermentation samples. 2.5 ml of sample were
off-flavors. BiGGY agar plates (Bismuth
prepared in a 20 ml vial added with
Glucose Glycine Yeast Agar, 1 g/l yeast
appropriate amounts of sodium chloride,
extract, 10 g/l glycine, 10 g/l glucose, 13 g/l
50µl NaN3 0,1%, 25 µl of the internal
agar, 3 g/l sodium sulphite, 5 g/l bismuth
standard and ascorbic acid. All samples were
ammonium citrate) were prepared according
incubated for 10 min at 50°C. The volatile
to the manufacturer’s instructions (Fluka).
compounds were collected and separated on a
These plates were then incubated at 20°C for
Divinylbenzene/Carboxen/Polydimethylsilox
up to 4 days.
ane fiber (DVB-CAR-PDMS) for an extraction
sulphate,
20 g/l
agar)
compounds
were
detected
and
time of 40 min. A Solgel-wax column, 30
Fermentation conditions
Fermentation was performed in 50 ml tubes
filled with 40 ml YPD (16°Plato) at 20°C.
m/I.D 0.25 mm/Film 0.25 μm, was used for
all analyses.
Each fermentation was started with a strain
The oven was kept at 40 °C for 4 min then
density of OD600 = 0.2. The stirring was set at
increased by 6 °C/min to 250 °C and kept at
300 rpm using a triangular magnetic stirrer.
the final temperature for 5 min. The injector
Fermentation performance was monitored up
and interface temperatures were kept at 250
to 14 days by daily measurement of the CO2
°C as well. Helium was used as the carrier gas
release. The sugar content was measured by a
with a flow rate of 1.2 ml/min. The time for
DMA 35 Anton Paar densitometer (medium
thermal desorption of analytes was 4 min.
gravity in °P). The Plato measurements were
The MS detector was operated in full scan
taken at the beginning and at the end of each
mode at 70 eV with a scan range from 35 to
fermentation process. The fermentation was
350 m/z.
defined as finished when the CO2 loss was not
increasing anymore and the Plato value did
not reduce further for 2 days. A supernatant
of 35 ml was then used for GC-MS analysis.
All
fermentations
biological duplicates.
were
carried
out
in
Data analysis was performed using the
software
ThermoXcalibur
(Version
2.2
SP1.48, Thermo scientific). Identification of
compounds was based on comparison with a
mass spectral database (NIST version 2.0).
One characteristic quantifier ion and two to
three qualifier ions were selected for each
compared to a set of brewing and wine yeast
compound. The peak area of the quantifier
strains. Each selected strain was assigned to a
ion
code based on its coordinates on a 96 well
was
used
for
quantification.
The
concentration of each volatile was expressed
as µg/l 2-octanol I.S.
plate.
As was expected, most strains harbored good
Multivariate data analysis
fermentation properties and produced final
Multivariate data analysis was performed
ethanol concentrations above 5% v/v (see
using StatSoft, Inc. STATISTICA version 8.0
below). Only 12 strains produced ethanol
(data analysis software system, 2007). A PCA
below 5 % v/v. Eight out of these 12 strains
model
data
have been unable to grow in fermentative
interpretation. The matrix contained the
conditions and were therefore repeated in
initial 60 strains considered in this study and
oxidative conditions. However, these strains
the average of the relative 62 VOCs detected.
have been discarded from our strain selection
The data were standardized to the mean of 0
due to their inability to grow under anaerobic
and unit standard deviation was scaled.
conditions. Three of the four strains that have
Eigenvalues and eigenvectors of the matrix
produced low ethanol concentrations in
were calculated and the relative plot was
fermentation
created.
Wickerhamomyces anomalus, the fourth
was
employed
to
simplify
Results and Discussion
strains
is
are
an
isolates
isolate
of
of
Pichia
membranifaciens. Out of this group of strains
Strain selection and identification of
with a low ethanol production we have
representative isolates
chosen strain F10, a Wickerhamomyces
Via collaboration of the EU-ITN Cornucopia,
anomalus strain as representative for our
focusing on non-conventional yeasts, with the
subset of strains.
CBS Fungal Strain Collection we selected 53
different species of the Saccharomyces clade
(Table 1). We prioritized strains with a proven
background from fermented liquids, fruits,
vegetables, or meat. These strains, therefore,
may have evolved superior features in
fermenting different sugars and thereby
producing ethanol or have been recognized as
contributing flavor to the samples. All strains
were fermented in 16 °Plato YPD. Then flavor
profiles were analyzed using GC-MS and
Overall
we
determined
62
different
headspace volatiles in the fermentation
samples of the 60 yeast strains tested. Key
volatile classes that were detected include
esters, alcohols aldehydes, ketones and acids.
However, the number and amount of VOCs
detected varied considerably among the
different
species.
The
complete
list
of
identified volatiles for each species is shown
in Table 2. Each volatile was grouped into the
respective chemical class. Esters were the
most prominent group of volatiles. In total 22
the 2 dimensional projection of our data. The
different esters could be identified and within
first principal component (PC1) explained
this group ethyl-esters were particularly
22.44 % of the total variability between the
dominating such as ethyl hexanoate and ethyl
fermentation
acetate, a fruity wine or apple like flavor and
associated with parameters like small ethyl
sweet pear drop flavor, respectively.
esters. PC2 explained 13.40 % of the total
Alcohols
comprised
chemical
group
the
second
produced
during
major
these
fermentations. Besides ethanol we found 14
different alcohols. Yet, only two compounds,
2-phenylalcohol, known for its rose aroma,
and isoamyl alcohol, a banana like flavor,
were produced by all the strains we analyzed.
results.
PC1
was
mainly
variability. It was more associated with
aldehydes
and
ketones.
Both
principal
components together explained 35.84 % of
the variability between the 60 fermentations.
The array of species populated all four
quadrants of the PCA plot showing the large
diversity amongst these yeasts. However,
within the dataset different species were well-
Fermentations were conducted in tall tube
segregated according to the respective genus.
cylinders with no aeration. Therefore, oxygen
For example, Wickerhamomyces and Pichia
depletion
these
sp. occupied the bottom right quadrant and
conditions. Formation of aroma alcohols is
separate clearly from the other species based
favored under oxygen limited conditions over
on their production of small fruity esters
the production of aroma acids. Thus only six
particularly ethyl acetate, ethyl hexanoate,
acids were identified in our VOC profiling
isoamyl
including acetic acid, butyric acid, decanoic
Zygosaccharomyces,
acid, hexanoic acid, isovaleric acid and
Debaryomyces sp. were found in the upper
octanoic acid. Of these acids acetic acid and
quadrants. These species had the highest
butyric acids were produced by almost all
scores for aldehydes, acids and ketones which
strains
clearly separated them from the other
occurs
and
were
rapidly
the
under
most
abundant
compounds in this chemical class.
To provide an overview of the yeast VOC
profiles, multivariate data analysis (PCA,
principal component analysis) was applied as
a statistical technique to allow visualization
and grouping of the yeast strains based on the
acetate
and
phenethyl
acetate.
Saccharomyces
and
species. The genera Candida, Lachancea,
Kazachstania,
Hanseniaspora
Meyerozyma
species
were
and
found
to
produce similar volatile profiles as lager yeast
strains based on those PCA. Therefore, these
species grouped closely together.
class and amount of volatiles. This PCA was
The loading plot (PC1 vs. PC2) represented in
based on data derived from biological
Figure 1B interprets the relationship between
duplicates of all 60 strains. Figure 1A shows
the biochemical variables (VOCs). Small
esters are well separated by the PC1.
Saccharomyces cerevisiae (E11) isolated from
Aldehydes, acids and ketones, on the other
Spanish sherry produced very high amounts
hand, cluster together as they are significantly
of fruity alcohols (isoamyl alcohol and 2-
present in Zygosaccharomyces, Saccharo-
phenyl ethanol) and acids (acetic acid and
myces and Debaryomyces species. This
butyric acid). Zygosaccharomyces mellis
analysis allowed the grouping of our strains
(G4) isolated from wine grapes showed an
in
their
overall increased production of volatiles such
production of either esters and alcohols or
as fruity alcohols and esters as well as acids,
aldehydes and ketones.
aldehydes and ketones. In contrast, five
two
major
classes
based
on
Based on our results of the PC analysis we
continued our experiments with a subset of
18 strains. The selection of these strains was
done by considering the results of the
multivariate analysis of the VOCs as well as
other factors obtained during and after the
fermentation such as ethanol production,
fermentation
speed
or
efficient
sugar
conversion. As a reference for the evaluation
of strains we used the fermentation results of
the strain Weihenstephan WS34/70 (G10).
strains, namely Geotrichum candidum (C3),
Kazachstania servazii (C9), Kluyveromyces
dobzhanskii (D4), Meyerozyma
guilliermondii
(D11)
and
Nakaseomyces
bacillisporus (E2) showed an overall reduced
volatile
production
Weihenstephan (G10).
in
comparison
However,
to
Kazach-
stania servazii (C9) produced the highest
ethanol concentration among the stains
tested. Another five strains were selected due
to
their
higher
ethanol
concentrations
compared to the reference strain namely
Six strains were selected based on their
Clavispora
extraordinary volatile profile: In Candida
fermentati (D6), Saccharomyces cerevisiae
diversa (B4) and Clavispora lusitaniae (B8)
(F2), Saccharomycodes ludwigii (F3) and
fermentations
Schwanniomyces
we
measured
very
high
lusitaniae
(B8),
occidentalis
Lachancea
(F4).
The
amounts of furfural. Candida diversa was
latter is interesting and important for its
further recognized for its high production of
amylolytic system. This fungus can degrade
ethyl hexanoate, a wine or apple like flavor,
numerous different starches completely such
and butyric acid. Pichia kluyveri (E7) and
as potato, barley or wheat starch [9].
Wickerhamomyces anomalus (F10) produced
exceptionally high amounts of fruity alcohols
and their respective esters such as isoamyl
alcohol, 2-phenylethanol and isoamyl acetate
and 2-phenyl acetate as well as ethyl
hexanoate. Further these strains produced
very
high
amounts
of
acetic
acids.
The remaining two strains were chosen
according to a unique trait found in their
volatile profiles: Hanseniaspora vineae (C7)
has been chosen since this strain produced
the highest acetoin concentration during
fermentation. Acetoin is an intermediate in
the conversion of diacetyl to 2,3-butanediol.
equal amounts of CO2 every day. During the
During fermentation yeast produces diacetyl
fermentation of this strain we observed a
that is considered a buttery off-flavor. During
water-insoluble layer and a biofilm of cells
maturation the diacetyl can be taken up and
appearing on the fermentation cylinder. This
consumed
enzymatic
suggests that this layer may have reduced the
conversion to acetoin and subsequently to
evaporation of CO2 and therewith caused the
2,3-butanediol and therewith remove the off-
constant and continuous CO2 release. In fact,
flavor from the liquid [10]. In contrast,
the fermentation liquid of Wickerhamomyces
Torulaspora microellipsoides (F7) was the
anomalus showed the lowest pH measured
only strain of our selection that did not
among all stains suggesting that the dissolved
produce any ketones in the fermentation
CO2 was contributing to the acidification of
experiment.
the liquid. The formation of such a film has
by
yeast
Fermentation
by
results
an
and
strain
characterization
These 18 strains have been processed further
through strain characterization tests on
various growth conditions and for the
production of common off-flavors that are
present in lager beer, namely H2S and
phenolic off-flavors. The results are shown in
Table 3. The table values are represented as a
heat map.
density prior to fermentation start to allow
the tracking of the fermentation speed of each
strain.
stored wines. The residual amount of oxygen
in the headspace of the fermenters then
triggers the formation of the biofilm. It has
been shown that this bio-layer produced
mainly acetic acid, acetaldehyde and acetate
esters, volatiles that have been identified in
high
concentrations
in
W.
anomalus
fermentation [11].
The most rapid fermentation as represented
All strains were adjusted to the same optical
single
been reported as a typical phenomenon in
Fermentation
rates
were
followed by measuring the CO2 release per
day (Figure 2A). As prerequisite we only
selected strains that were able to ferment the
medium within seven days. As only exception
we selected Wickerhamomyces anomalus
(F10) due to its exceptional volatile profile.
Different to the other 17 strains that showed a
sigmoidal CO2 release W. anomalus had
almost a linear fermentation curve releasing
by fast CO2 loss was observed with the beer
production strains WS34/70 (G10). However,
this strain did not cause the highest CO2
release.
Pichia
kluyveri
(E7)
finished
fermentations after four days with the highest
CO2 release. The lowest CO2 release occurred
in
the
fermentation
of
Debaryomyces
subglobosus (B11). Fermentation was finished
after four days, although only about two
thirds of the average CO2 release occurred.
Surprisingly, the remaining sugar content of
this fermentation was rather low with 3.45 °P
and the ethanol produced was high with
Growth on other carbon sources and
7.44 %.
temperatures
Since we conducted the fermentation using
glucose based medium we did not expect
drastic changes in the pH of the final liquid
due to the buffering capacity of the medium
itself. Nevertheless, we observed the lowest
pH
after
fermentation
with
Wickerhamomyces anomalus (F10; pH 4.79).
Apart from the suggested dissolution of CO2
in
the
fermented
liquid
this
strain
additionally produced the highest amounts of
acids which explain this low pH of the final
liquid. In contrast, Clavispora lusitaniae (B8)
produced the lowest amount of acids and
therewith resulted in the highest pH of 6.07.
For most of the strains we could observe a
correlation of the sugar consumption and the
produced ethanol (Figure 2B). The highest
production
low or high temperatures such as 10 °C and
37°C we setup dilution series on plates and
observed their growth after four and two days
respectively (Table 3). We found that 60 % of
the strains were able to grow on high
temperature whereas none of the strains was
showing full growth at the low temperature of
10 °C. Further we tested the ability of the
strains to grow on an alternative carbon
source such as maltose. Only two strains of
our selection showed full growth on maltose
plates namely Candida diversa (B4) and
Clavispora lusitaniae (B8).
Off-flavor formation
Ethanol production
ethanol
In order to test the strains ability to grow on
was
observed
for
Kazachstania servazii (C9) with 9.2 % v/v.
Wickerhamomyces anomalus (F10), on the
We focused our attention on the production
of off-flavors that are important factors for
the selection of strains in brewing industry
such as diacetyl, phenolic compounds like 4vinylguaiacol and hydrogen sulfide (Tables 2
and 3).
other hand, produced the lowest amount of
Diacetyl (butanedione or butane-2,3-dione), a
ethanol
highest
buttery like flavor, can be detected by GC-MS.
remaining sugar content. Hanseniaspora
Within our selected strains we found four
vineae
(E7)
strains where we could detect diacetyl. Since
produced a lower ethanol yield than the
diacetyl can be converted to 2,3-butanediol
estimation due to the sugar consumption
through
would imply. This could be connected to the
compared the acetoin production of the
increased production of acetic acids in both
strains as well. Both acetoin and 2,3-
strains.
butanediol can diffuse out of the cell, but
correlating
(C7)
and
with
Pichia
Additionally,
the
kluyveri
Pichia
kluyveri
produced vast amounts of ethyl acetate.
the
intermediate
acetoin
we
neither of them contributes to the undesired
buttery flavor [7]. Hanseniaspora vineae (C7)
produced the highest amounts of acetoin but
did not show detectable amounts of diacetyl.
a high sulfite reductase activity and produce a
[12].
detectable amount of H2S while white
Phenolic compounds such as 4-vinylguaiacol
are produced by enzymatic decarboxylation of
ferulic acid. When found in beers they are
responsible for a pungent clove-like aroma.
Key enzymes for the ferulic acid conversion
are PAD1 and FDC1. In our screening we used
cinnamic acid as a substrate to identify POF+
and POF- strains. POF+ strains express the
genes PAD1 and FDC1 and can therewith
decarboxylate cinnamic acid to styrene to
circumvent the inhibitory effects of cinnamic
acid [13]. Of the 18 strains in our study only
two showed very high production of phenolic
flavors, namely Debaryomyces subglobosus
(B11) and Meyerozyma guilliermondii (D11).
colonies produced no detectable sulfide [15].
However, this assay does not give absolute
quantitative measurements but a tendency of
sulfite reductase activity. We found three
strains with apparent low sulfite reductase
activity, namely Nakaseomyces bacillisporus
(E2), Saccharomycodes ludwigii (F3) and
Torulaspora
measure
microellipsoides
the
H2S
(F4).
formation
To
during
fermentation the lead acetate method can be
used. Here, hydrogen sulfide interacts with
the lead in the lead acetate matrix to form a
dark precipitate. This allows an estimate of
the volatile H2S in the headspace of the
fermentation [16].
However, the production of 4-vinylguaiacol
Complexity of flavor production
could not be detected by GC-MS since our
Within our selection of strains we observed
fermentation was based on YPD where ferulic
different complexities in flavor production
acid is not expected. Typically, ferulic acid
(Figure 3). The highest complexity of 47
derives from barley germination where it is
volatile compounds resulting in the largest
released from the aleurone layers as ester-
number of different flavors was produced by
linked
Debaryomyces
feruloyl
arabinoxylans.
During
subglobosus
(B11).
D.
mashing and wort boiling it is released from
subglobosus, also known as Candida famata
its bound form and found in significant
var. flareri, is a natural overproducer of
concentrations in the wort [14].
riboflavin and D-Arabitol depending on the
Hydrogen sulfide is a rotten egg flavour that
is
undesired
in
fermented
beverages.
Hydrogen sulfide is produced by yeast during
fermentation and during the maturation
phase. On BiGGY-agar the sulfite reductase
activity of a strain can be estimates since
color of colonies can be correlated with levels
of H2S production. Brown color colonies have
iron concentration of the media [17] Strain
optimization and development have reached
a riboflavin production up to 20 g/l with this
species [18] [19]. Interestingly, this strain was
also identified as the one with the lowest CO2
release during fermentation. Fermentation
was
delayed
but,
nevertheless,
finished
already after four days. Further, this strain
was standing out for its high production of
and Z. mellis produced detectable amounts of
pyranone and furfural. Pyranone was recently
3-methyl butanal as well as other compounds
identified as signaling molecules in bacterial
derived from Strecker degradation, such as 5-
communications similar to quorum sensing.
hydroxymethylfurfural,
[20]. Fruit ketones such as pyranone are
methylfurfural (Figure 4). Furfural is a
known to be important for processes like
colorless oily liquid with the odor of almonds.
cheese ripening [21]. In contrast, we found
It is further known as one of the components
very low levels of fruity esters in our GC-MS
found in vanilla. Although both strains have
analysis
fermentation.
been isolated from fruits they might have a
Already in 2000 Van den Tempel and
possible application in meat processing. Z.
Jacobsen identified high esterase activity in
mellis is already known to be involved in
various isolates of D. hansenii which may
vinegar fermentation. Together with other
cause the low concentration of esters in
species of this genus such as Z. bailii, Z.
D. subglobosus [22].
bisporus and Z. rouxii, Z. mellis belongs to a
of
D. subglobosus
Branched-chained aldehydes are favorable
furfural,
5-
group of osmotolerant yeasts [9].
components for the maturation of meat
The lowest flavor complexity was found for a
products, giving nutty, cheesy and salty notes
strain of Saccharomyces cerevisiae (E11)
to the food products [23] Usually these
isolated from Spanish sherry (Figure 3).
aldehydes
Despite
are
by
Strecker
heat-induced
process
compounds this strain was exceptionally high
converting α-amino acids into an aldehydes.
in its higher alcohol production namely in
Small amounts of these aldehydes are
butanol and isoamyl alcohol. Additionally,
produced
food
high amounts of acetic acid and butyric acid
set
as well as acetaldehyde were produced
degradation,
produced
a
enzymatically
fermentation.
D. subglobosus
during
Within
our
strain
(B11)
showed
the
most
abundant production of aldehydes (Figure 4).
One other prominent aldehyde was 3-methyl
butanal. This compound is an intermediate of
the leucine degradation pathway. It was also
found
in
high
concentrations
in
Zygosaccharomyces mellis (G4). 3-methyl
butanal
is
a
known
flavor
component
aldehyde associated with the production of
cured-hams by lactic acid bacteria and
Micrococcaceae [24] Both, D. subglobosus
the
low
number
of
31
flavor
(Figure 4).
With 36 different flavors Wickerhamomyces
anomalus (F10) is among the strains with
intermediate flavor complexity (Figure 3).
However, this strain was noticed as a
prominent producer of high amounts of
esters such as butyl acetate, ethyl acetate,
isoamyl acetate and 2-phenyl acetate. Within
the
group
of
higher
alcohols
Wickerhamomyces anomalus produced the
highest
amount
of
2-phenyl
ethanol.
Moreover, the highest yields of acetic acids
Conclusion
and acetaldehyde were found in this species
In our study we present a comprehensive
(Figure 4). As mentioned above W. anomalus
overview of the volatile compound formation
formed a water insoluble layer on top of the
potential of a set of 18 strains that were
fermentation consisting of a film that could
chosen
produce acetic acid, acetaldehyde and acetate
conventional
esters.
generalists that produce a broad variety of
from
a
selection
yeasts.
of
We
60
non-
found
both
organic volatiles but also specialist that had a
In earlier studies W. anomalus has been
isolated from a range of cereal based sources.
It has been reported from sourdoughs and
was found as the dominating yeast in
sourdough microbial ecosystems next to S.
cerevisiae. The prevalence of the fungus was
associated
with
its
osmotolerance
and
increased acid tolerance in comparison to S.
cerevisiae [25]. Further, it was shown that W.
anomalus can outgrow other yeasts and
become the dominant fungal species most
likely due to its ability to assimilate lactate
produced by lactic acid bacteria [26].
low complexity of different volatile but
produced
vast
compounds.
strains
amounts
Furthermore,
produced
of
a
certain
number
higher
of
ethanol
concentrations than the reference strain.
Among the generalists in our selection we
identified Debaryomyces subglobosus (B11).
This strain produced the highest complexity
of
volatiles
(a
total
of
47)
but
low
concentrations of esters when compared to
the reference strain Weihenstephan WS34/70
(G10). However it produced high amounts of
aldehydes and ketones.
Pichia kluyveri (E7) produced a total of 41
volatiles during our fermentations. It was
recognized by its high production of volatile
esters that reach up to fivefold concentrations
found in the reference strain (Figure 4).
Pichia kluyveri is found in 'wild ferments' of
Zygosaccharomyces mellis (G4) produced
with 44 volatiles almost as many volatiles as
D. subglobosus. However the concentration
of fruity esters and alcohols was significantly
increased when compared to the reference
strain.
wine. Chr. Hansen A/S has selected P.
kluyveri for its ability to boost fruit flavours
Wickerhamomyces
and launched products such as Frootzen™
Pichia kluyveri (E7) were noticed because of
that can be used in sequential inoculation
their enormous production of volatile esters
with standard wine yeast. In a recent study P.
but low ethanol production. In contrast,
kluyveri
Kazachstania servazii (C9) was identified as
marxianus
starter
together
with
were
presented
yeasts
fermentation [27].
for
Kluyveromyces
as
controlled
potential
cocoa
anomalus
(F10)
and
strain with the highest ethanol production
but with a reduced production of volatiles.
The yeast biodiversity holds a plethora of
strains that show useful characteristics such
as ethanol production and flavor formation.
This requires a detailed evaluation of the
initially identified favorable strains under
different
conditions
to
ascertain
their
properties and potentially promote these
strains in specific fermentation regimes.
Figures and Tables
Table 1:
Strains used in this study. Each selected strain was assigned to a code based on its coordinates on a
96 well plate.
Pos. CBS number
Taxon name
Substrate of isolation
Origin
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
C2
C3
CBS 10151
CBS 12367
CBS 4074
CBS 8058
CBS 1760
CBS 2649
CBS 6936
CBS 4373
CBS 767
CBS 2659
CBS 8139
CBS 615.84
Cured ham
Brie Régalou cheese
Grape must
Berries of Vitis coignetiae
Pickling vat with 22% brine
Grape juice
Citrus essence
Dry white wine
Norway
Apple
Italy
Netherlands
France
C4
CBS 95
C5
CBS 6783
C6
CBS 2585
Candida alimentaria
Candida alimentaria
Candida diversa
Candida kofuensis
Candida versatilis
Candida stellate
Clavispora lusitaniae
Debaryomyces fabryi
Debaryomyces hansenii
Debaryomyces subglobosus
Dekkera anomala
Geotrichum candidum
Hanseniaspora
guilliermondii
Hanseniaspora occidentalis
var. citrica
Hanseniaspora uvarum
C7
CBS 2568
C8
C9
C10
C11
D2
D3
D4
D5
D6
CBS 2352
CBS 4311
CBS 3019
CBS 2186
CBS 398
CBS 7775
CBS 8530
CBS 1557
CBS 7005
D7
CBS 3082
D8
CBS 7703
D9
CBS 5833
D10
CBS 2030
Meyerozyma guilliermondii
D11
CBS 8417
Meyerozyma guilliermondii
Brie cheese
Fermenting bottled
tomatoes
Orange juice
Sour dough
Drosophila persimilis (fruit
Hanseniaspora vineae
fly)
Hyphopichia burtonii
Pollen, carried by wild bees
Kazachstania servazii
Soil
Kazachstania spencerorum
Soil
Kazachstania transvaalensis Soil
Kazachstania unispora
Kluyveromyces aestuarii
Neotredo reynei (shipworm)
Kluyveromyces dobzhanskii Drosophila sp.
Kluyveromyces marxianus
Stracchino cheese
Lachancea fermentati
Alpechín
Drosophila pinicola (fruit
Lachancea kluyveri
fly)
Either fruit or leaf of fruit
Lachancea waltii
tree
Berries of Vitis labrusca
Metschnikowia pulcherrima
(Concord grapes)
Insect frass on Ulmus
americana (elm tree)
Brine bath in cheese factory
Japan
Japan
USA
France
Israel
South Africa
Netherlands
Italy
Portugal
Finland
South Africa
South Africa
Brazil
Canada
Italy
Spain
USA
USA
Netherlands
Pos. CBS number
Taxon name
Substrate of isolation
E2
CBS 7720
Nakaseomyces bacillisporus
E3
E4
CBS 2170
CBS 8255
Nakaseomyces delphensis
Pichia
Exudate of Quercus emoryi
(Emory oak)
Sugary deposit on dried figs
Kefyr
E5
CBS 2020
Pichia farinosa
Fermenting cacao
E6
E7
E8
E9
CBS 2057
CBS 188
CBS 5147
CBS 191
Pichia fermentans
Pichia kluyveri
Pichia kudriavzevii
Pichia membranifaciens
E10
CBS 429
Saccharomyces cerevisiae
E11
F2
F3
CBS 1250
CBS 1782
CBS 820
F4
CBS 2863
F5
CBS 6741
F6
F7
CBS 133
CBS 427
F8
CBS 248
F9
CBS 249
F10
CBS 261
F11
CBS 262
G2
CBS 4689
G3
CBS 1082
G4
G5
G6
G7
CBS 726
C1030
C1039
C746
G8
CBS 1513
G9
G10
G11
C482
WS34/70
C598
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomycodes ludwigii
Schwanniomyces
occidentalis
Schwanniomyces
polymorphus var. africanus
Torulaspora delbrueckii
Torulaspora microellipsoides
Wickerhamomyces
anomalus
Wickerhamomyces
anomalus
Wickerhamomyces
anomalus
Wickerhamomyces
anomalus
Zygosaccharomyces bailii
var. bailii
Zygosaccharomyces
bisporus
Zygosaccharomyces mellis
Saccharomyces pastorianus
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces
carlsbergensis
Saccharomyces cerevisiae
Saccharomyces pastorianus
Saccharomyces cerevisiae
Brewers yeast
Olives
Fruit juice
Wine
Fermenting must of
champagne grapes
Sherry
Super-attenuated beer
Grape must
Origin
USA
South Africa
Trinidad
and Tobago
Italy
Spain
Germany
Soil of vineyard
Spain
Soil
South Africa
Ragi
Apple juice
Indonesia
Germany
Red currants
Netherlands
Berries
Ragi
Indonesia
Beer
Grape must
Italy
Tea-beer fungus
Indonesia
Wine grapes
Brewers' yeast
Wine yeast
Brewers' yeast
Germany
Brewers' yeast
Brewers' yeast
Brewers' yeast
Laboratory strain
Table 2:
Complete list of volatiles detected by the total list of 60 strains
B2
Stdev
B3
Stdev
B4
Stdev
B5
Stdev
B6
Stdev
B7
Stdev
B8
Stdev
B9
Stdev
B10
Stdev
B11
Stdev
C2
Stdev
C3
Stdev
C4
Stdev
C5
Stdev
C6
Stdev
C7
Stdev
C8
Stdev
C9
Stdev
C10
Stdev
C11
Stdev
D2
Stdev
D3
Stdev
D4
Stdev
D5
Stdev
D6
Stdev
D7
Stdev
D8
Stdev
D9
Stdev
D10
Stdev
D11
Stdev
Alcohols
Benzyl alcohol
Butanol
Dodecanol
Fenchyl alcohol
Furaneol
Isoamyl alcohol
Propanol
2-Ethyl-1-hexanol
2-Furanmethanol
2-Methyl propanol
2-Nonanol
2-Phenyl ethanol
3 Ethoxy - 1 Propanol
3-(Methylthio)-1-propanol
1,1
0,0
8,5
2,5
0,0
50,0
0,0
0,0
0,0
0,0
0,0
239,2
0,0
1,2
0,1
1,4
0,1
0,0
0,0
0,0
0,0
0,0
0,0 301,2 63,3 452,6
0,7 13,2
5,0 13,2
2,1 14,9
0,6
2,1
0,1
0,0
0,0
0,0
0,0
0,0
0,0 11,1
6,4
0,0
11,8 20,1
8,6 342,6 26,0 706,6
0,0
0,0
0,0
0,0
0,0 15,4
0,0
3,3
1,9
4,7
0,4
7,0
0,0
0,0
0,0 52,6
8,5
0,0
0,0
0,0
0,0
6,0
1,7 42,4
0,0
0,0
0,0
0,0
0,0
0,0
25,0 317,5 103,7 445,0 108,5 373,8
0,0
0,0
0,0
0,0
0,0
0,0
0,1
0,0
0,0 48,3
3,4 28,3
0,0
0,0
27,7 69,0
6,8
8,1
0,0
0,0
0,0
0,0
62,0 206,7
3,1
0,0
0,6
4,7
0,0
0,0
2,5
0,8
0,0
0,0
66,3 154,5
0,0
0,0
1,1
0,0
0,0
13,3
1,8
0,0
0,0
1,4
0,0
0,0
0,0
0,0
0,0
15,5
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
2,1
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
110,9
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,3
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
24,5
3,5
0,0
0,0
0,8
3,1
0,0
4,2
0,6
0,0
23,9
0,0
0,0
3,4
0,0
0,0
14,2
0,7
1,8
0,2
8,7
4,5
0,0
51,2
227,2
0,0
0,0
193,1
0,0
9,4 225,6 78,8 709,2 126,6 296,5
40,5 375,8 188,7 328,6 99,3 138,4
0,0
0,0
0,0 14,0
1,0 30,7
0,0
0,0
0,0 141,1 44,7 121,4
9,7 249,2 36,9 52,5
9,8
0,0
0,0
0,0
0,0 96,1 19,0 103,7
45,6 40,1
16,5 255,4
6,3
0,0
21,0
0,0
0,0 147,9
26,7
0,0
11,4
7,0
0,0
0,0
6,3
0,0
0,1
9,7
0,5 11,7
0,5 258,8
15,1 91,2
0,4
0,0
0,0
0,0
28,8 171,9
0,0
0,0
0,0
0,0
0,0
0,0
0,2
3,9
11,0
0,6
0,1
0,1
2,7
0,0
3,1
20,3
0,0
0,0
4,9
594,4 29,5 434,7
23,8
6,4
8,4
0,0
0,0
0,0
29,2
0,8
6,5
729,5 271,4 579,8
0,0
0,0 12,4
5,3
1,8
4,6
164,7 14,0 49,6
44,1 17,4 31,1
2,9
0,3
2,3
577,4 15,5 425,0
0,0
0,0
0,0
55,4
5,4 127,5
0,5
0,0
19,2 243,1
3,4
7,4
0,0
2,5
0,1 12,1
19,5 244,8
1,1 31,6
0,0
5,3
4,1 81,4
2,4 17,8
0,3
0,0
7,9 73,7
0,0
0,0
20,6
0,0
0,0
71,6
1,3
0,2
1,7
13,6
2,4
0,4
6,1
3,5
0,0
5,2
0,0
0,0
3,5
361,0
11,2
0,0
26,3
499,2
5,7
5,4
139,9
26,2
2,2
302,6
0,0
30,8
0,1
78,9
1,7
0,0
1,1
61,6
0,2
0,3
6,7
1,2
0,2
20,1
0,0
9,6
4,1
412,9
14,0
0,0
37,2
589,9
4,5
4,9
159,4
24,4
1,9
365,9
0,0
36,8
1,1
0,0
41,3 146,2
5,8
2,9
0,0
1,6
2,6
0,0
34,8 523,1
0,7 11,5
0,5
3,7
11,2 14,4
3,9 25,0
0,1
0,0
12,9 202,9
0,0
0,0
7,8 63,9
0,0
1,6
24,0 134,1
0,4
3,0
0,2
0,0
0,0
0,0
50,1 580,8
1,5
3,3
0,9
3,6
6,7
0,0
2,5 29,7
0,0
1,2
11,8 333,7
0,0
0,0
4,7 18,1
0,4
4,9
83,7 349,7
2,0
9,2
0,0
0,0
0,0
0,0
86,3 533,5
0,9 11,5
0,2
6,0
0,0
0,0
7,4 17,9
0,4
0,0
29,9 415,2
0,0
0,0
4,6 30,8
0,2
0,0
28,1 192,2
0,3
6,0
0,0
0,0
0,0
0,0
95,3 414,8
2,4
6,9
1,6
5,5
0,0
0,0
1,1 14,6
0,0
8,5
10,0 348,0
0,0
0,0
4,7 54,4
0,0
0,0
53,5 364,6
4,9
5,2
0,0
0,0
0,0
0,0
13,2 455,2
0,5 10,2
2,0
4,4
0,0
0,0
3,0 11,5
0,9
3,1
46,7 329,7
0,0
0,0
7,5 29,6
0,0
20,3
0,0
0,0
0,0
37,4
0,7
0,4
0,0
1,3
1,4
55,5
0,0
2,5
0,0
505,8
14,5
0,0
0,0
767,1
29,0
7,2
0,0
12,5
2,0
608,6
2,6
352,6
0,0
0,0
16,0 172,6
1,3
2,2
0,0
0,0
0,0
0,0
11,8 527,3
8,7 16,1
0,6
3,8
0,0
0,0
2,9 19,3
0,1
0,0
52,0 215,2
0,8
0,0
55,2 33,2
0,2 70,6
0,0
0,0
0,7
0,0
0,0 867,7
28,3 18,4
0,0
0,0
0,0 11,8
0,0
0,9
7,6 10,5
11,0 34,9
0,0
0,0
0,0
0,0
25,6 63,0
0,0
0,0
8,7
0,0
0,0 94,9
0,6
0,0
0,2
2,0
0,1
0,2
0,0 69,0
0,0 12,2
0,0 24,8
7,1
0,0
0,0
58,6
6,3
0,0
7,0
0,0
0,9
3,4
0,0
0,0
7,4
0,0
0,0
6,3
0,0
0,8
0,0
27,3
0,2
0,4
2,1
0,0
0,0
0,0
1,6
0,0
1,1
0,0
0,2
0,0
0,0
0,0
0,0
0,0
0,9
1,9
0,1
0,5
0,2
0,0
0,0
11,5
3,7
30,0
0,9
198,2
80,8
124,0
58,6
39,7
77,0
133,7
18,8
0,0
244,4
0,0
31,4
8,8
13,8
1,4
1,5
0,0
0,0
30,0
0,2
3,0
1,9
0,0
0,1
0,6
0,4 177,0
10,0 47,8
18,4 62,9
15,4 46,8
2,1 11,6
1,3 23,0
43,6 38,4
1,2 13,3
0,0
0,0
16,8 110,4
0,0
0,0
0,8 15,6
0,4 11,2
1,8
5,0
0,1
1,8
0,2
0,9
0,0
0,0
0,0
0,0
0,4 24,2
2,0 34,8
0,0
0,0
0,1
0,0
30,7 471,4
10,4 22,2
28,2
0,0
19,8
5,9
5,3
0,2
18,6
5,1
6,2
0,7
0,6
0,0
0,0
0,0
56,4
2,3
0,0 54,3
6,5
0,0
9,1 116,9
3,2
4,2
1,3
2,5
0,4
0,5
0,0
0,0
0,0
0,0
4,7 10,4
0,2
7,4
0,0
0,0
0,0
5,4
36,3 303,1
0,1 69,4
0,0
0,0
2,2
4,7
0,2
0,7
0,6 24,5
0,2 18,3
0,0 76,2
0,0
4,9
0,2 29,8
2,2 38,3
0,0
4,9
1,1 75,2
0,3 17,3
0,1 14,6
0,0
2,9
0,0 32,0
0,0
0,0
1,4 79,0
4,2 49,2
0,0
0,0
1,4
0,3
74,6 662,8
15,6 19,2
0,0 93,8
2,1 163,0
0,4
6,9
19,5 11,7
15,8 56,2
21,7
0,0
0,1
0,0
28,2 185,7
6,8 26,3
2,8 15,3
16,5 90,5
7,1
2,0
4,9
3,1
0,3
0,2
11,7
0,0
0,0
2,1
11,4 28,4
5,5
1,3
0,0
0,0
0,0
9,7
21,1 143,7
0,0 94,7
1,6 51,5
29,5 13,1
0,7
6,4
1,4 26,3
4,6 27,7
0,0 91,4
0,0
9,6
33,8
0,0
2,1
0,0
4,6 18,6
3,8
4,7
0,1
0,0
0,2
0,9
0,0
0,6
0,0
0,0
0,2
1,7
2,6 79,5
0,1
0,0
3,5
9,7
0,7
14,7
0,4
1,2
9,4
8,4
2,5
3,3
0,0
0,0
2,6
0,1
0,0
0,2
0,0
0,0
0,4
20,6
6,1
0,0
0,0
39,5
5,1
6,6
12,6
0,5
3,3
12,7
0,0
0,0
72,5
7,7
6,9
9,9
0,0
1,9
0,0
8,4
0,0
2,2
47,1
2,0
3,4
244,9 36,3
0,0
0,0
0,0
0,0
985,2 111,3 168,8
94,6
6,5 56,6
348,0 46,9
0,0
123,1 19,5
9,2
0,0
0,0
9,8
0,0
0,0 24,2
92,1 19,7 26,4
0,0
0,0
0,0
0,0
0,0
0,0
244,3 22,9 30,9
0,0
0,0
0,0
0,0
0,0 13,8
321,6 54,2 49,8
0,0
0,0 14,3
6,0
1,1
1,2
0,0
0,0
0,9
0,0
0,0 86,3
0,6
0,0
0,0
50,6 19,7 23,4
511,2
199,9
59,4
151,6
0,0
128,9
82,9
66,1
3,2
31,5
0,0
4,7
323,9
132,4
16,9
174,6
0,0
144,3
33,2 347,3
9,1 172,7
8,7
0,0
4,6
0,0
0,0
0,0
16,4
3,9
82,3 491,4
24,3 175,9
0,0
0,0
0,0 120,6
0,0
0,0
1,1 65,8
24,1 608,1
13,0 147,4
0,0
0,0
13,5 112,6
0,0
0,0
7,4 69,0
27,5 165,1
18,1 129,2
0,0
0,0
10,7
0,0
0,0
0,0
6,3
0,0
26,0 547,4 110,9 736,7
8,8 155,2
6,6
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0 133,9
0,0
0,0
0,0
0,0
0,0
0,0
0,0 90,4
64,0 362,2
0,0 192,6
0,0
0,0
31,7 85,1
0,0
0,0
16,6 44,5
16,5 610,4 127,2 1115,2
71,9 176,2 35,2
0,0
0,0
0,0
0,0
0,0
26,6 82,0
6,0 169,7
0,0
0,0
0,0
0,0
10,0
0,0
0,0 142,0
13,3
42,5
395,1
43,5
5,6
1,6
176,5
0,0
153,3
554,1
1,1 12,2
12,6 29,8
13,3 147,5
4,8
0,0
1,5
3,2
0,7
1,9
16,8 31,0
0,0 15,5
10,7 36,5
4,1 252,9
0,7
6,9
1,1 10,6
2,0 168,9
0,0 22,5
1,8
3,9
0,7
1,1
0,8 65,0
0,2 11,5
0,9 65,0
4,9 188,7
0,6
3,1
0,2
5,2
2,4
0,1
8,7
1,2
3,7
25,0
1,0
32,2
0,4
7,6
4,3
0,0
14,8
0,4
1,8
7,7
12,3 11,9
2,0
4,2
54,1 54,6
6,1 15,8
1,4
0,6
0,5
0,6
33,3
0,0
0,0
0,0
7,9 21,9
70,2 109,7
0,5
0,5
21,1
2,6
0,7
0,1
0,0
0,0
6,9
47,0
8,4
19,4
22,2
0,0
2,5
1,0
8,9
6,8
8,3
0,0
1,4 12,6
1,8 24,8
25,9 53,5
0,0 73,9
0,7
0,0
0,1
0,0
1,3 307,6
0,4
7,3
8,7 68,3
0,0 29,9
1,6
1,3
1,1
5,8
0,0
0,0
33,9
2,3
6,2
1,8
25,6
28,6
9,2
0,0
0,0
0,0
0,0
0,0
0,0
5,5
8,6
1,9
0,2
0,0
0,0
0,0
0,0
0,0
0,0
0,0
10,6
34,9
9,6
0,0
1,3
0,7
0,0
0,0
11,9
0,0
20,0 27,2
0,0
0,0
97,9 163,0
0,0
0,0
8,2
0,0
0,0
0,0
0,3
0,9
0,0
0,0
3,9 26,7
0,0
0,0
7,5 170,4
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,4
0,0
0,0
9,2 29,8
0,0
0,0
20,9 266,2
0,0
0,0
0,0
0,0
0,0
0,0
0,0
5,8
0,0
0,0
34,6
0,0
26,3
0,0
29,2
0,0
1,6
0,0
7,6
0,0
34,3
0,0
0,0
0,0
0,6
0,0
1,4
0,0
18,1
0,0
0,0
0,0
0,4
0,0
5,1
0,0
0,0
0,0
0,0
0,0
1,0
0,0
2,6 53,8
0,0
0,0
0,0 190,6
0,0
0,0
0,0
0,0
0,0
0,0
0,1
0,5
0,0
0,0
9,8
0,0
24,4
0,0
0,0
0,0
0,2
0,0
14,4
12,4
0,0
0,0
0,0
0,0
10,8
2,2
7,5
1,5
0,0
0,0
0,0
0,0
3,3
0,0
43,1
0,0
0,0
0,0
11,3
0,0
0,0
0,0
0,7
4,4
0,4
4,2
0,0
0,2
3,8
12,2
0,2
5,8
8,1
22,6
0,4
1,6
5,2
14,2
0,0
0,0
37,9 322,1
1,6 10,0
0,0
0,0
0,0
0,0
40,4 544,3
4,5 10,3
0,4
4,4
0,0
0,0
6,6 12,8
0,0
1,6
85,3 416,5
0,0
1,1
0,9 31,3
0,0
0,0
53,2 331,7
11,0
5,6
0,0
0,0
0,0
0,0
53,9 491,1
0,6
7,8
0,2
3,8
0,0
0,0
3,6
7,2
0,0
2,7
32,7 314,6
0,5
0,0
18,4
0,0
0,0
0,0
58,3 299,3
4,0
6,1
0,0
0,0
0,0
0,0
33,1 406,7
1,3 14,6
0,2
2,9
0,0
0,0
0,3 10,3
0,5 23,1
28,5 314,2
0,0
0,0
0,0
0,0
0,0
3,4
14,4 504,4
6,6
4,4
0,0
0,0
0,0
0,0
52,2 523,9
8,7 20,6
1,2
6,4
0,0
0,0
1,6
9,7
20,9
3,9
15,8 400,2
0,0
0,0
0,0 28,3
0,6
0,0
10,7 416,7
0,3
8,5
0,0
0,0
0,0
0,0
93,6 589,6
6,0 31,7
1,7
4,9
0,0
0,0
3,6 18,8
0,6 21,5
50,7 430,8
0,0
0,0
7,4 38,4
0,0
0,0
16,7 337,5
2,4
6,5
0,0
0,0
0,0
0,0
23,7 695,7
9,1 15,4
0,2
4,6
0,0
0,0
3,1 35,9
4,5
1,4
18,9 373,1
0,0
0,0
10,7
8,0
0,0
68,4
0,2
0,0
0,0
0,3
0,1
0,5
0,0
3,1
0,0
36,8
0,0
2,6
0,0
0,0
2,4
521,2 65,8 268,2
9,8
4,4
4,9
0,0
0,0
0,0
0,0
0,0
0,0
830,0 101,6 658,9
19,6
2,9 11,4
8,0
5,2
3,7
0,0
0,0
0,0
36,5
3,2 30,4
1,2
0,1
1,0
448,5 89,4 382,1
0,0
0,0
0,0
132,7 50,4 11,8
0,2
0,0
0,0
2,6
38,1 385,0 132,0 380,4
1,5
5,5
0,9
5,2
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
66,6 597,4
2,4 581,0
1,7 13,6
1,9 21,4
0,7
2,7
0,2
4,4
0,0
0,0
0,0
0,0
3,9 13,8
3,9 23,5
0,1
1,2
0,2
1,9
8,2 533,7 25,4 431,0
0,0
0,0
0,0
0,0
5,3 45,8 15,6 12,6
0,1
2,3
87,8 303,7
1,0
5,0
0,0
0,0
0,0
0,0
19,5 449,1
2,4
7,9
0,3
4,7
0,0
0,0
1,2 13,8
0,0 16,1
8,7 329,5
0,0
0,0
0,9
0,0
0,3
3,0
14,3 288,4
1,9
5,1
0,0
0,0
0,0
0,0
28,0 477,3
0,5 12,0
0,3
4,0
0,0
0,0
1,3 11,1
2,1 10,4
1,3 312,3
0,0
0,0
0,0 19,9
0,5
4,5
40,4 385,6
2,2 11,7
0,0
0,0
0,0
0,0
23,6 502,1
0,1 12,7
0,2
7,0
0,0
0,0
0,5 11,9
1,2
2,1
27,6 383,8
0,0
0,0
2,1 40,8
1,2
86,9
4,3
0,0
0,0
64,1
1,7
3,2
0,0
2,0
0,4
59,4
0,0
7,6
1,1
0,0
0,0
2,0
8,0
0,0
1,8
1,2
2,7
7,2
0,0
0,0
5,7
0,0
1,0
10,9
1,0
0,5
0,2
3,2
0,0
3,8
4,0
7,7
0,0
19,0
9,3
3,8
8,9
2,3
7,7
6,5
0,0
0,0
38,1
0,0
0,7
15,1
0,9
3,7
0,0
0,0
0,3
4,9
7,0 13,2
9,5
0,0
0,2
0,0
11,5 102,2
6,0 35,2
27,4
0,0
1,9
0,0
3,5 10,1
6,6
5,6
14,9 94,8
0,0
0,0
0,0
0,0
23,9 246,6
0,0
0,0
0,5 12,8
22,4 40,1
0,6
6,4
0,6
0,7
0,0
0,0
0,0
0,0
0,0
0,0
6,6 31,7
1,2
0,0
0,0
37,0
17,2
0,0
0,0
1,1
1,7
2,6
0,0
0,0
31,9
0,0
1,2
8,1
5,8
0,2
0,0
0,0
0,0
6,9
8,7
0,0
0,0
2,8
5,1
44,5
22,8
1,3
0,8
8,6
0,0
0,0
32,8
0,0
12,4
3,0
1,0
1,3
0,1
36,6
0,0
3,5
14,4
5,8
0,0
0,0
0,0
0,0
25,9 181,5
31,6 36,9
0,0
0,0
36,2
6,9
0,5
2,0
4,8
0,0
7,9 40,2
0,0
0,0
0,0
0,0
80,7 89,5
0,0
0,0
0,0
0,0
62,4 23,7
0,0
6,6
1,9
3,3
0,0
0,5
31,4 20,2
0,5
3,7
2,5 64,2
1,1
0,0
0,0
32,7
6,7
0,0
0,8
0,0
0,0
10,8
0,0
0,0
7,5
0,0
0,0
0,0
1,9
1,0
0,0
0,3
1,9
9,0
52,5
0,0
0,0
497,0
36,5
0,0
34,5
1,8
0,0
44,6
0,0
0,0
181,9
0,0
0,0
299,2
0,0
9,0
0,4
451,4
2,5
66,2
0,0
5,4
0,0
0,0
0,0
0,0
44,6 183,9
1,6 29,4
0,0
0,0
5,1
4,4
0,1
2,2
0,0
0,0
4,9 70,0
0,0
0,0
0,0
0,0
27,9 90,4
0,0
0,0
0,0
0,0
19,9 24,1
0,0
7,2
0,8
2,9
0,0
0,5
91,1 19,6
0,4
3,5
18,3 49,0
0,4
0,0
0,0
9,5
1,2
0,0
0,4
0,3
0,0
61,9
0,0
0,0
12,4
0,0
0,0
1,0
0,5
0,2
0,2
2,6
0,2
13,8
1,9
0,1
0,0
15,9
4,8
0,0
0,8
0,1
0,0
7,7
0,0
0,0
1,4
0,0
0,0
8,4
0,2
0,7
0,0
4,6
0,1
1,3
4,0
6,4
0,7
57,3
2,1
83,4
8,5
4,3
1,8
28,0
12,2
0,0
34,7
0,0
4,0
12,9
1,8
1,2
0,0
1,8
0,0
2,7
53,1
13,5
0,6
562,4
88,4
117,1
133,6
5,7
26,1
70,5
5,4
0,0
210,5
0,0
105,1
127,5
7,0
12,3
0,5
48,4
0,0
23,3
4,6 12,4
1,0 17,4
0,4
0,0
67,1 116,5
2,1 17,0
14,7 70,5
19,7 63,9
0,8
2,7
6,0 24,2
17,0 45,4
4,8
0,0
0,0
0,0
21,4 134,4
0,0
0,0
10,2 105,7
29,4 46,5
3,1
0,0
3,8
1,6
0,2
0,1
8,3 97,7
0,0
0,0
3,0 17,6
3,1
3,9
0,0
34,8
3,9
13,4
9,8
0,5
5,1
23,4
0,0
0,0
15,1
0,0
29,2
9,6
0,0
0,6
0,2
2,8
0,0
3,7
24,6 874,8
12,5 133,2
0,0
0,0
7,2 99,1
0,0
0,0
0,0 109,4
70,0 225,3
8,1 109,4
0,0
0,0
35,2 37,5
0,0
0,0
34,5
0,0
5,7 375,8
35,8
0,0
0,0
0,0
5,3 54,9
0,0
0,0
0,0 57,1
30,1 1014,2
47,8 260,3
0,0
0,0
9,3 73,1
0,0
0,0
0,0
0,0
5,6 958,6
13,1 176,5
0,0
0,0
8,6 72,9
0,0
0,0
0,0
0,0
10,4
42,1
0,0
16,3
0,0
0,0
Esters
Butyl acetate
Ethyl (4E)-4-decenoate
Ethyl 2-methylbutyrate
Ethyl acetate
Ethyl butanoate
Ethyl decanoate
Ethyl dodecanoate
Ethyl heptanoate
Ethyl hexadecanoate
Ethyl hexanoate
Ethyl isobutyrate
Ethyl isovalerate
Ethyl octanoate
Ethyl propanoate
Ethyl tetradecanoate
Isoamyl acetate
Isoamyl butyrate
Isobutyl acetate
Isobutyl butanoate
Phenethyl acetate
S-methyl thioacetate
2-Methyl propanoate
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
15,8
0,0
1,8
148,7
159,7
0,0
53,5
37,2
13,7
294,5
0,0
0,0
337,0
0,0
21,5
68,0
5,7
0,0
0,9
175,9
0,6
27,5
8,6
5,9
0,0
0,0
0,4
0,0
47,0 124,0
80,4 23,3
0,0
0,0
9,4 24,9
13,9
1,5
0,9
0,0
41,3 69,4
0,0
0,0
0,0
0,0
28,6 89,7
0,0
0,0
4,1
0,0
4,4 21,2
2,0
2,5
0,0
3,3
0,4
0,4
7,5 23,1
0,1
8,9
4,6
0,0
0,1
0,0
0,7
0,0
10,9
0,0
0,0
0,9
0,0
0,0
0,0
2,3
1,3
0,0
0,0
0,4
2,4
0,0
0,2
0,0
0,0
42,0
0,0
3,3
0,0
0,0
0,1
0,5
0,0
0,0
1,1 87,8
0,0
0,0
0,0
0,0
0,1
1,8
0,0 11,9
0,0 102,7
0,0
0,0
0,6
0,0
0,1 96,9
0,0
0,0
0,0 12,1
0,0
8,1
0,2
3,9
0,0
1,5
0,0
1,2
0,0
0,0
0,0
0,0
3,2
0,0
3,5
0,0
0,0
0,0
32,3
0,0
7,5
0,0
7,4
0,0
0,0
0,0
4,0
0,0
6,6
3,9
3,7
1,1
1,4
0,0
0,0
30,4
134,3
0,0
0,0
1130,9
25,1
154,5
89,4
3,1
14,6
55,2
0,0
0,0
210,7
85,0
23,6
165,3
1,3
16,6
0,0
205,1
0,0
25,7
49,4
173,9
0,0
625,0
17,5
254,4
275,4
5,4
12,1
58,5
0,0
0,0
263,5
0,0
21,9
157,5
2,6
4,4
0,0
0,0
0,2
21,1
9,5
198,6
0,4
321,1
34,2
330,3
253,4
19,2
10,1
71,2
0,0
0,0
259,8
0,0
29,6
43,7
9,0
1,6
0,6
0,0
0,0
15,5
13,3
0,0
0,0
112,3
12,3
158,1
103,1
3,2
14,4
49,8
0,0
0,0
168,5
0,0
94,8
57,0
1,8
2,2
0,1
192,7
0,0
25,5
60,9
0,0
0,0
524,5
137,8
0,0
36,5
2,3
5,7
69,3
0,0
0,0
191,8
0,0
0,0
252,7
4,2
5,9
0,5
618,5
1,1
22,5
74,1
159,7
0,0
688,8
55,0
334,5
71,8
2,6
9,7
78,7
0,0
0,0
274,7
0,0
15,7
145,9
6,4
5,0
0,4
315,1
0,4
34,0
4,2
13,3
0,0
94,1
10,6
38,4
7,6
0,7
9,7
18,6
0,0
0,0
10,4
0,0
1,8
58,7
1,4
2,9
0,2
73,5
0,2
3,6
22,1
10,7
0,0
326,5
43,9
0,0
6,2
1,6
0,0
24,4
0,0
0,0
131,5
0,0
0,0
109,9
2,5
9,0
0,3
320,0
0,4
24,4
19,0
101,5
2,7
619,2
138,4
382,1
85,1
11,5
35,8
103,1
63,3
0,0
358,6
0,0
39,8
47,6
9,1
7,3
1,0
22,8
0,0
53,4
Acids
Acetic acid
Butyric acid
Decanoic acid
Hexanoic acid
Isovaleric acid
Octanoic acid
39,1 359,1
0,0 186,1
0,0
0,0
55,6
0,0
0,0
0,0
36,1
0,0
1,4 511,0
14,8 133,0
0,0
0,0
0,0 86,9
0,0
0,0
0,0
0,0
1,7 596,4 181,9 454,6
22,6 162,3 75,7 126,8
0,0
0,0
0,0
0,0
10,5 82,5 31,0
0,0
0,0
0,0
0,0
0,0
0,0 48,3 18,4
0,0
28,8 1038,2
1,2
0,0
0,0
0,0
0,0 107,0
0,0
0,0
0,0
0,0
85,4 976,2 292,8 317,0
0,0 355,4 78,3 178,3
0,0
0,0
0,0
0,0
4,3 109,0 35,3 69,6
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
11,3 232,8
0,0 170,4
0,0
0,0
8,4 51,4
0,0
0,0
19,8 77,8
6,3 817,1
1,7 326,1
0,0
0,0
1,7 48,3
0,0
0,0
6,0
0,0
Aldehydes
Acetaldehyde
Benzaldehyde
Furfural
Phenyl acetaldehyde
1 -Decanal
1-Nonanal
3-Methyl butanal
4-Methyl benzaldehyde
5 Methyl furfural
5-Hydroxymethylfurfural
0,5
14,0
2,3
79,4
1,1
0,0
83,2
0,0
0,0
0,0
13,1 18,9
5,1
0,0
64,5 192,7
15,5
0,0
0,0
5,7
0,0
0,0
39,7 65,3
0,0
0,0
0,0 78,0
0,0 283,5
4,9 21,8
0,0
8,7
10,9 138,8
0,0
0,0
0,0
6,4
0,0
1,1
5,7
0,0
0,0
0,0
6,7 46,0
11,4 195,5
14,2
2,7
29,8
0,0
1,3
0,0
0,0
0,0
38,3
31,7
1,9
28,5
12,8
9,3
1,3
1,0
19,9
0,0
5,4
20,7
0,0
2,3
0,0
0,0
0,0 175,4
0,0
0,0
3,7 23,4
0,0 21,3
0,0
1,3
0,0
0,0
0,1
2,8
0,0
0,0
48,8 133,2
0,0
0,0
18,9
0,0
2,4
0,0
0,3
2,7
0,0
0,0
0,6
0,0
15,9
0,0
0,0
0,0
0,3
0,0
4,1
0,0
0,0
0,0
14,6
0,0
0,6
0,0
0,3 47,3
0,0
0,0
0,0 460,3
0,0
0,0
2,0 61,2
0,0
0,0
0,1
4,2
0,0
0,0
0,7
2,5
6,4
34,7
0,3
0,2
14,8
60,7
282,8
51,6
6,3
0,0
161,5
18,1
136,4
287,5
17,0
31,0
358,2
56,0
4,7
1,7
218,0
0,0
187,5
317,9
1,7
2,2
1,7
0,0
0,3
0,2
0,0
0,0
1,0
0,0
11,4
26,6
12,4
0,0
0,0
0,0
0,0
0,0
0,0
0,0
4,9
3,3
1,4
0,0
0,0
0,0
0,0
0,0
0,0
0,0
66,9
5,5
6,4
0,0
3,7
1,8
0,0
0,0
6,3
0,0
3,9
0,8
1,0
0,0
0,6
0,4
0,0
0,0
0,8
0,0
19,2
34,1
6,9
0,0
2,5
1,1
0,0
0,0
6,1
0,0
1,9
8,3
0,6
0,0
0,6
0,3
0,0
0,0
2,0
0,0
49,7
27,9
5,1
0,0
3,2
1,5
0,0
0,0
4,9
0,0
3,9
2,0
2,5
0,0
1,9
0,7
0,0
0,0
0,5
0,0
13,7
21,8
5,0
0,0
3,3
1,7
0,0
0,0
0,0
0,0
2,1
2,2
3,0
0,0
0,6
0,1
0,0
0,0
0,0
0,0
9,5
22,9
5,5
0,0
0,0
0,0
0,0
9,3
6,7
0,0
0,1
4,1
0,5
0,0
0,0
0,0
0,0
1,5
0,3
0,0
22,1
19,6
6,3
0,0
3,2
1,3
0,0
0,0
6,5
0,0
9,7
3,2
1,7
0,0
0,1
0,3
0,0
0,0
1,4
0,0
26,3
6,5
4,6
0,0
4,6
0,0
0,0
0,0
3,9
0,0
2,1
2,2
0,6
0,0
1,6
0,0
0,0
0,0
1,6
0,0
16,2
27,7
5,1
0,0
0,0
0,0
0,0
0,0
6,3
0,0
1,2
1,5
0,8
0,0
0,0
0,0
0,0
0,0
0,9
0,0
23,0
8,4
4,3
0,0
2,4
0,0
0,0
0,0
3,5
0,0
4,9
1,5
1,1
0,0
1,2
0,0
0,0
0,0
0,0
0,0
12,5
31,3
4,3
0,0
0,0
0,0
0,0
10,5
4,5
0,0
0,9
5,1
0,3
0,0
0,0
0,0
0,0
0,8
0,5
0,0
13,9
31,9
4,4
0,0
0,0
1,8
0,0
7,0
3,8
0,0
1,6
1,9
0,2
0,0
0,0
2,0
0,0
0,2
0,1
0,0
17,9
37,3
3,9
0,0
4,5
2,4
0,0
8,7
3,3
0,0
1,9
6,0
0,8
0,0
0,8
0,4
0,0
0,6
0,2
0,0
20,9
32,1
4,2
0,0
3,4
1,7
0,0
6,9
2,9
0,0
4,6
3,8
0,1
0,0
1,1
0,1
0,0
0,6
0,1
0,0
26,5
32,7
4,0
0,0
0,0
2,4
0,0
8,2
2,8
0,0
15,8
8,8
0,2
0,0
0,0
1,0
0,0
1,1
0,5
0,0
18,8 203,0
0,0
0,0
0,0
0,0
0,0
0,0
0,6 25,4
0,0
0,0
0,0
2,2
0,0
0,9
8,0
0,0
0,0
0,0
1,9
0,0
0,5
0,2
17,9
0,0
0,0
0,0
8,5
0,0
5,4
1,5
2,7
0,0
0,0
0,0
1,7
0,0
0,2
0,0
94,6
0,0
0,0
0,0
60,5
0,0
2,7
1,8
2,1
0,0
0,0
0,0
6,5
0,0
1,3
1,0
14,5
3,1
0,0
0,0
30,8
0,0
1,6
1,0
3,1
1,0
0,0
0,0
33,8
0,0
0,1
0,3
3,7
29,2
0,0
0,0
0,0
0,0
16,2
1,4
2,8
5,1
0,0
0,0
0,0
0,0
2,4
1,1
2,8
8,0
0,0
0,0
0,0
0,0
11,3
1,7
0,7
3,5
0,0
0,0
0,0
0,0
3,7
0,7
5,6
17,6
0,0
0,0
0,0
0,0
14,8
0,7
3,4
4,1
0,0
0,0
0,0
0,0
1,8
0,0
2,3
5,2
0,0
0,0
13,8
0,0
2,5
0,4
0,4
1,1
0,0
0,0
7,9
0,0
0,2
0,1
2,7
28,2
0,0
0,0
0,0
0,0
0,0
0,5
0,4
4,7
0,0
0,0
0,0
0,0
0,0
0,1
1,8
7,3
0,0
0,0
8,4
0,0
2,1
0,3
0,1
0,4
0,0
0,0
0,2
0,0
0,1
0,0
1,0
14,7
0,0
0,0
0,0
0,0
1,2
0,3
0,0
3,0
0,0
0,0
0,0
0,0
0,1
0,0
0,9
13,6
0,0
0,0
0,0
0,0
6,2
1,9
0,1
0,6
0,0
0,0
0,0
0,0
1,7
0,2
2,8
32,2
0,0
0,0
0,0
0,0
10,8
2,9
0,2
1,3
0,0
0,0
0,0
0,0
0,4
1,0
2,9
19,1
0,0
0,0
0,0
0,0
26,8
3,5
0,9
2,6
0,0
0,0
0,0
0,0
2,6
0,2
5,4
14,8
0,0
0,0
0,0
0,0
3,4
1,4
0,9
5,3
0,0
0,0
0,0
0,0
0,3
0,1
0,6
3,7
0,2
2,9
0,0
0,4
6,8
14,6
1,0
0,4
5,6
20,9
0,3
0,4
4,0
9,5
0,4
0,8
4,3
18,6
0,4
3,1
6,6
13,7
0,4
4,0
5,7
15,1
0,4
0,5
6,6
18,8
0,7
1,5
4,9
14,3
0,5
0,6
6,2
15,4
0,2
1,5
5,8
14,7
0,1
1,4
5,9
16,1
0,6
0,2
4,8
17,4
0,6
0,6
5,3
20,2
0,8
4,4
Ketones
Acetoin
0,0
Diacetyl
0,0
Pyranone
0,0
2-Cyclopentene-1,4-dione
0,0
2-Dodecanone
12,3
2-Methyltetrahydrothiophen-3-one
0,0
2-Nonanone
4,8
2-Undecanone
0,0
0,0
0,0
0,0
0,0
0,9
0,0
0,1
0,0
0,0
0,0
0,0
0,0
21,8
0,0
0,0
0,0
0,1
0,2
0,0
2,7
4,2 42,0
0,0
0,0
5,3 319,2
0,0
0,0
0,0 40,6
0,0
0,0
0,6
6,2
0,0
0,0
Pyrazine
2,5-Dimethyl-3-ethylpyrazine
2,6-Dimethylpyrazine
0,4
4,5
0,0
0,2
8,2
29,0
1,0
0,4
6,2
20,4
7,6
20,1
0,8
3,7
8,8
21,0
0,3
0,7
0,5
4,2
0,1
0,4
9,7
23,2
0,8
1,8
8,1
21,4
1,3
3,0
6,9
34,6
0,6
0,8
5,4
16,5
E2
Stdev
E3
Stdev
E4
Stdev
E5
Stdev
E6
Stdev
E7
Stdev
E8
Stdev
E9
Stdev
E10
Stdev
E11
Stdev
F2
Stdev
F3
Stdev
F4
Stdev
F5
Stdev
F6
Stdev
F7
Stdev
F8
Stdev
F9
Stdev
F10
Stdev
F11
Stdev
G2
Stdev
G3
Stdev
G4
Stdev
G5
Stdev
G6
Stdev
G7
Stdev
G8
Stdev
G9
Stdev
G10
Stdev
G11
Stdev
Alcohols
Benzyl alcohol
Butanol
Dodecanol
Fenchyl alcohol
Furaneol
Isoamyl alcohol
Propanol
2-Ethyl-1-hexanol
2-Furanmethanol
2-Methyl propanol
2-Nonanol
2-Phenyl ethanol
3 Ethoxy - 1 Propanol
3-(Methylthio)-1-propanol
1,6
360,1
4,5
0,0
0,0
457,9
15,2
4,5
0,0
9,8
0,8
263,6
0,0
38,0
0,3
16,0
0,1
0,0
0,0
16,0
1,6
0,9
0,0
0,9
0,0
24,8
0,0
21,8
0,0
372,6
3,7
0,0
0,0
424,2
16,3
4,2
0,0
5,1
1,3
298,7
0,0
141,9
0,0
0,0
6,8 369,5
1,1
2,3
0,0
0,0
0,0
0,0
3,2 375,5
0,5
6,3
0,1
5,3
0,0
0,0
0,1 16,7
0,2
0,5
18,7 212,8
0,0
0,0
30,5 20,9
0,0
0,0
0,0
0,0
12,6 449,4 115,4 199,0
0,0
2,5
0,9
1,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
4,6 728,7 138,0 603,8
5,5 13,5
2,6
5,6
2,8
6,6
2,5
3,1
0,0
0,0
0,0
0,0
2,3 39,6
7,7 22,0
0,2
0,5
0,1 370,1
2,8 425,8
8,5 342,8
0,0
0,0
0,0
0,0
3,0 39,8
7,1
0,0
0,0
0,0
0,0
0,0
76,1 698,6 37,9 91,6
0,1
6,8
4,6
1,6
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
1,8 870,6 42,4 502,9
1,3 31,6
7,4
2,3
0,2
8,7
4,4
2,7
0,0
0,0
0,0
0,0
1,4 26,3
4,2 10,5
62,0
3,3
0,3 278,3
47,0 1178,3 645,5 328,2
0,0
0,0
0,0
0,0
0,0
0,0
0,0 32,0
8,9
26,2
0,6
281,1
56,7
0,0
12,9
1,2
4,3
44,0
0,0
0,0
98,0
0,0
8,6
43,4
2,9
1,2
0,4
26,5
1,2
12,8
2,8
1,6
0,5
24,4
8,3
0,0
4,7
0,1
1,8
5,1
0,0
0,0
4,1
0,0
0,6
15,2
0,0
0,3
0,0
4,0
0,3
2,8
21,1
109,2
0,2
281,1
84,5
0,0
22,8
5,4
4,7
102,9
0,0
0,0
229,9
0,0
15,6
102,6
8,1
0,8
0,3
322,5
1,1
12,6
4,9 56,7
20,8
3,9
0,0
0,6
45,4 106,0
15,7 21,9
0,0
0,0
3,5 27,5
0,5
0,0
0,5
0,0
0,9 32,7
0,0
0,0
0,0
0,6
11,4 44,7
0,0
0,0
3,2
0,0
35,7 20,1
1,0
2,9
0,2
1,9
0,0
0,4
11,9 15,7
0,3
4,7
2,7 24,6
9,5
6,8
1,2
6,0
0,5
1,5
17,4 262,8
3,4 65,3
0,0
0,0
9,2 34,2
0,0
1,6
0,0 10,7
4,1 66,2
0,0
0,0
0,1
4,8
4,2 66,0
0,0
0,0
0,0 10,1
2,9 45,0
0,1
5,8
2,1
4,8
0,2
0,9
1,8 25,7
0,7
5,4
0,8 33,9
1,5
4,5
0,6
47,6
10,1
0,0
6,6
0,5
1,7
13,1
0,0
1,9
26,4
0,0
2,6
5,9
1,2
1,1
0,2
5,5
1,1
5,4
0,6
0,0
1,6
5,3
0,5
0,0
0,1
0,5
1,5
1,9
0,0
0,3
7,0
0,0
0,1
3,2
0,8
0,4
0,1
1,8
0,0
12,9
257,4
121,0
0,0
34,6
0,0
0,0
4,0 308,1
24,2 136,7
0,0
0,0
9,6 33,6
0,0
0,0
0,0
0,0
43,2 299,9
23,5 125,4
0,0
0,0
14,6 77,4
0,0
0,0
0,0
0,0
11,2 466,7
35,4 170,0
0,0
0,0
22,9 73,2
0,0
0,0
0,0
0,0
61,9 194,6
26,4 120,4
0,0
0,0
14,8
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
31,4 719,3
6,2 494,1
0,6
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0 54,0
22,9 1840,0 151,4 819,3
0,1
0,0
0,0
3,9
0,5
3,0
0,7
3,3
0,0
0,0
0,0
0,0
2,6 76,5
2,3 40,5
70,6
0,8
0,5
0,9
0,3 869,2 77,4 413,2
0,0
0,0
0,0
0,0
13,6 51,4 30,4
0,0
0,0
88,7
0,0
0,0
7,4
75,1
0,1
0,1
0,0
3,5
0,0
21,1
0,0
0,0
0,0
1660,4
0,0
0,0
29,1
3702,4
66,4
5,1
0,0
192,8
0,7
562,3
0,0
66,8
0,0
0,0
14,9 383,2
0,0
1,3
0,0
0,0
3,6
0,0
46,1 565,3
7,5
0,0
0,4
4,1
0,0
0,0
28,4 20,8
0,2
2,1
41,2 222,3
0,0
0,0
7,3 38,1
0,0
1,8
78,4 397,1
0,3
0,0
0,0
0,0
0,0
0,0
95,8 645,8
0,0 26,4
2,0
3,3
0,0
0,0
0,1 16,7
0,6 20,5
29,8 456,9
0,0
0,0
10,6 39,2
0,5
0,0
21,0 596,8
0,0
7,2
0,0
0,0
0,0
0,0
21,8 444,8
5,4 15,0
0,2
5,5
0,0
0,0
1,7
2,5
2,9
5,1
72,4 561,1
0,0
0,0
14,8
0,0
0,0
0,0
0,0
0,0
48,8 437,2 67,6 461,4
3,9
2,2
0,9
1,6
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
25,1 740,0 103,4 539,0
2,3 39,2 15,4 15,7
2,0
4,5
3,9
2,8
0,0
0,0
0,0
0,0
0,9 26,8 13,3 20,9
2,0 16,1
1,1
0,0
95,9 290,7 166,2 424,7
0,0
0,0
0,0
0,0
0,0 35,8 28,3
0,0
1,2
3,0
0,1
84,6
18,8
15,1
0,9
0,1
0,1
2,4
0,0
0,0
23,4
0,0
0,0
1,7
0,0
0,6
0,1
5,6
0,3
21,3
8,2
63,7
0,1
7,3
8,5
79,8
52,4
2,5
2,5
20,4
0,0
0,0
92,7
0,0
1,1
19,8
0,0
0,2
0,1
39,9
0,0
0,2
0,0
0,0
27,2 734,0
0,1
2,1
0,0
0,0
0,0
0,0
64,1 615,5
2,2 19,8
1,0
4,5
0,0
0,0
3,0 20,6
0,0
0,0
72,4 434,5
0,0
0,0
0,0 53,9
0,0
0,0
12,0 668,4
0,1
3,2
0,0
0,0
0,0
0,0
49,5 632,2
2,8 16,8
1,6
2,6
0,0
0,0
2,6 22,4
0,0
0,0
88,1 854,3
0,0
0,0
11,7
0,0
0,0
0,0
49,1 565,9
2,1
3,1
0,0
0,0
0,0
0,0
55,3 632,8
3,6 17,7
1,7
6,8
0,0
0,0
17,8 30,6
0,0
0,0
8,4 963,7
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
2,6
76,7 755,4 167,8 1290,6 145,1 518,6
1,2
6,4
0,5
4,7
3,3
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
6,1
99,7 1081,6 52,4 2363,5 256,6 409,1
5,6 26,0
9,8 18,2
3,0 13,5
5,3
0,0
0,0
0,0
0,0
4,3
0,0
0,0
0,0
0,0
0,0
0,0
2,6 48,9 30,5 61,9
9,4 26,7
0,0
0,0
0,0
0,0
0,0 10,0
13,4 1908,8 48,1 1693,3 322,7 289,5
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0 11,6
0,0
0,0
0,0
5,0
0,4
0,0
0,0
0,0
0,0
0,0
7,7 1826,7 152,2 1189,7 129,3 396,5 14,3 822,7 373,3 476,6
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
1,5
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
1,6 56,3 31,7 33,4
7,0 10,7
6,2
0,0
0,0
0,0
34,9 1798,2 77,3 1973,2 461,9 709,7 267,2 511,7 641,5 781,6
3,4 34,0 23,1 36,8
1,6 16,9
6,9 49,7 34,9 25,3
0,1
2,8
0,8
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0 56,1 35,2 37,8
8,7
0,0
0,0
0,0
0,0
0,0
4,2 59,0 38,6 76,7 32,2 16,0
0,0 35,2 20,8 27,9
2,8
0,0
0,0 12,4
3,8
0,0
0,0
0,0
0,0
0,0
13,1 787,3 82,6 771,3 13,9 328,4 18,5 503,2 239,8 248,5
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
4,5
0,0
0,0 83,5 42,0
0,0
0,0 273,4 94,2 30,8
0,0
0,0
55,8 458,4
0,2
2,0
0,0
0,0
0,0 10,9
39,2 748,2
1,5 10,8
0,0
0,0
0,0 12,3
0,7 15,3
0,0
0,0
3,2 416,9
0,0
0,0
6,9 31,3
0,0
0,0
1,5 398,8
0,4
2,0
0,0
0,0
1,3
8,7
76,1 800,6
1,2 13,7
0,0
0,0
0,2 10,2
1,8
8,3
0,0
0,0
80,5 486,1
0,0
0,0
2,4 35,7
0,0
0,0
24,0 399,7
0,2
0,0
0,0
0,0
3,5 16,1
19,9 965,7
2,5 15,5
0,0
0,0
2,5 18,0
1,2 21,2
0,0
0,0
24,0 397,2
0,0
0,0
11,9 28,0
0,0
53,4
0,0
0,0
1,5
39,5
0,2
0,0
2,8
3,6
0,0
14,0
0,0
3,6
0,0
0,0
801,4 123,8
0,0
0,0
0,0
0,0
24,7 14,0
726,8 15,9
44,5 13,1
0,0
0,0
31,7
8,8
6,2
2,8
0,0
0,0
681,3 12,8
0,0
0,0
187,6 23,8
17,8
12,1
0,0
29,9
3,7
19,1
44,4
0,3
0,0
9,8
0,0
0,0
24,3
0,0
0,0
9,2
0,0
2,0
0,0
37,4
1,5
3,2
155,4
119,1
0,5
392,8
58,6
563,1
299,7
0,9
0,0
94,2
0,0
1,3
419,2
0,0
0,0
440,9
0,0
11,1
0,3
422,9
4,1
21,5
76,7
48,6
0,2
61,8
27,4
27,6
9,0
0,5
0,0
42,8
0,0
0,6
76,4
0,0
0,0
66,1
0,0
5,4
0,2
17,0
2,3
2,2
164,8 83,1 118,3
4,8 198,5 31,8
285,5 57,8 377,1 33,2 290,4
5,8
0,5
0,2
0,3
0,1
0,3
0,0
384,8 56,8 461,7 11,4 501,5 17,1
80,8 12,5 57,3
9,5 148,8 20,2
514,4 356,3 235,7 21,9 1222,9 34,5
250,7 83,0 73,5
9,0 545,2 24,8
1,5
0,6
2,4
0,1
1,3
0,1
6,6
1,2
8,2
0,1 21,6
0,9
71,6 33,3 46,9
0,0 177,5 22,2
0,0
0,0
0,0
0,0
0,0
0,0
1,0
0,2
0,7
0,2
0,9
0,0
416,6 53,5 754,1 33,3 1146,6 42,7
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
561,5 194,5 610,1 112,9 676,4 116,3
0,0
0,0
0,0
0,0
0,0
0,0
7,8
4,9 12,7
3,9
9,5
1,7
0,1
0,1
0,4
0,0
0,3
0,0
350,4 46,1 424,0 21,1 485,9 88,3
2,1
0,9
7,8
2,1
0,0
0,0
22,9
2,5 33,0
2,8
0,0
0,0
8,5
0,0
6,2
65,9
0,0
81,2
590,9
0,0
180,2
199,4
0,0
451,8
21,4
0,0
13,0
6,7
0,0
18,3
700,4 126,2 719,7
0,0
0,0 118,4
132,9 17,0 28,2
130,8
1,2 210,6
0,0
0,0
0,0
330,9 26,5 420,9
Esters
Butyl acetate
Ethyl (4E)-4-decenoate
Ethyl 2-methylbutyrate
Ethyl acetate
Ethyl butanoate
Ethyl decanoate
Ethyl dodecanoate
Ethyl heptanoate
Ethyl hexadecanoate
Ethyl hexanoate
Ethyl isobutyrate
Ethyl isovalerate
Ethyl octanoate
Ethyl propanoate
Ethyl tetradecanoate
Isoamyl acetate
Isoamyl butyrate
Isobutyl acetate
Isobutyl butanoate
Phenethyl acetate
S-methyl thioacetate
2-Methyl propanoate
1,9
0,0
3,3
73,7
32,4
0,0
2,4
1,0
6,7
16,4
0,0
1,2
60,0
0,0
2,8
27,6
9,7
1,6
0,9
19,0
0,8
39,2
1085,5 76,9 12,9 14,2
63,0
5,7 39,3
3,7
12,6
0,3
5,2
2,8
7329,9 645,1 571,8 491,1
458,6 24,9 27,6 23,2
487,4 32,4 64,5 40,2
190,7 80,6
4,0
1,9
17,0
5,1
0,0
0,0
131,1
8,4
0,0
0,0
238,1 73,8 10,3
1,1
0,0
0,0
8,6
2,1
22,8
5,6
1,5
0,7
1197,4 63,3 129,9
0,5
0,0
0,0
0,0
0,0
79,4
9,1
2,0
0,0
1955,4 136,5 155,6 148,2
95,2
4,4 10,1
7,3
314,9 38,1 11,5 14,5
11,5
4,7
0,7
0,5
1144,1 711,6 146,9 121,1
0,0
0,0
0,0
0,0
38,2
4,7 30,3
1,9
13,3
0,0
18,1
671,0
307,4
96,8
96,2
3,2
90,4
175,1
0,0
36,1
347,3
0,0
77,4
88,2
0,0
11,9
6,8
68,6
15,7
32,7
1,5
0,0
9,9
42,2
12,0
6,5
49,7
0,3
9,8
11,5
0,0
3,4
14,9
0,0
3,1
4,9
0,0
2,2
6,6
60,4
4,5
9,6
37,4
0,0
9,2
524,7
155,6
76,6
34,9
5,0
26,7
90,7
0,0
17,1
246,4
0,0
22,0
163,9
0,0
24,1
5,4
53,6
0,0
48,8
2,9 21,9 19,7 61,6
9,1
7,8
0,0
0,0
0,0 233,1 11,3 38,3
1,0
0,0
0,0
0,1
0,0
0,5
40,2 1121,9 121,4 245,1 11,1 515,1
32,0 158,7 54,8 10,9
2,8 90,1
28,7
0,0
0,0 858,3 26,1 218,1
7,6
0,0
0,0 399,3 45,1 32,5
0,6
0,0
0,0
1,5
0,1
2,4
1,6 23,1
3,9
8,0
1,2
3,6
6,2 77,9
3,7 61,1
1,1 40,3
0,0
0,0
0,0
0,0
0,0
0,0
7,9
0,0
0,0
0,0
0,0
0,0
17,6 84,7 22,2 528,5 151,8 411,8
0,0
0,0
0,0
0,0
0,0
0,0
5,8
0,0
0,0 12,3
5,4
0,0
22,7 101,2
7,4 224,8 14,4 20,1
0,0
0,0
0,0
0,0
0,0
0,0
14,9 21,1
2,3
6,7
4,8
2,1
1,2
1,9
0,4
0,2
0,1
0,6
6,0
0,0
0,0 341,2
9,2 37,9
0,0 33,0
1,6
0,0
0,0
1,2
2,2 85,1
8,3 21,1
2,4 66,3
20,8
1170,5
0,2
156,1
27,9
1191,2
487,7
7,4
11,3
120,1
0,0
0,0
682,7
0,0
60,4
52,9
0,0
0,5
0,2
382,4
0,0
14,4
7,2
3,4 421,1 22,4
100,1 65,8 25,9
4,7
5,9
2,6
3,7
1,4
595,6 82,3 2631,8 55,9
221,5 10,5 155,2
5,4
1033,7 595,4 169,7 16,9
382,3 294,8 71,8 81,4
8,2
9,8
6,9
2,8
13,1 10,2 19,3
6,0
128,5 30,7 127,9
0,4
40,7 30,3
0,0
0,0
9,3
4,2
7,2
1,4
881,0 190,6 417,0 127,8
0,0
0,0
0,0
0,0
50,1 54,8 23,7 12,1
44,5
9,6 602,5 26,8
0,0
0,0
0,0
0,0
4,5
0,5 126,6 37,4
1,3
0,3
5,3
3,8
0,0
0,0 384,5 102,4
0,0
0,0
0,0
0,0
48,7
3,6 14,6
3,3
13,5
497,5
0,2
498,3
71,9
1546,7
418,1
3,3
0,0
73,0
0,0
0,0
433,4
0,0
0,0
20,5
0,0
1,3
0,7
34,5
0,8
29,7
1,1
2,5
0,0
11,9
14,1
49,0
71,6
0,9
0,0
22,6
0,0
0,0
87,7
0,0
0,0
3,6
0,0
0,3
0,2
17,5
0,4
1,2
1387,1 16,7 1035,9
15,5
1,0
0,0
13,7
1,8 21,8
4627,3 32,6 6045,5
616,5 46,7 531,2
106,5 11,4 108,3
105,8
5,8 64,6
4,3
0,8
7,7
67,2
4,6 74,4
350,8 22,7 323,7
0,0
0,0
0,0
10,1
3,4
9,7
239,1 73,8 447,4
0,0
0,0
0,0
86,4 34,6 41,2
1888,9 163,1 1779,0
83,0 32,0 102,1
338,8 53,2 518,8
20,4 14,4 34,7
1227,5 97,4 1134,8
0,0
0,0
0,0
15,2
2,1 37,2
53,0
0,0
17,5
4,4
17,5
54,3
0,3
5,5
6,3
17,9
0,0
1,5
40,7
0,0
6,1
25,6
25,9
34,3
26,4
88,4
0,0
11,0
2796,6
0,0
17,8
#####
950,9
32,4
66,9
2,9
99,6
339,3
0,0
7,9
420,0
573,8
63,0
5710,3
192,5
1950,3
81,6
2493,0
0,0
29,3
70,0
0,0
9,1
153,5
230,4
7,0
1,0
0,1
6,7
152,4
0,0
1,4
56,7
18,7
5,6
56,5
1,1
31,1
13,3
195,0
0,0
1,2
1992,1
0,0
128,8
#####
1543,5
0,0
0,0
5,3
111,3
605,0
0,0
81,1
192,8
301,0
88,7
3562,4
0,0
759,8
65,3
1727,3
0,0
0,0
160,2 38,4
4,3 454,3 61,2 157,8 28,4 270,0 18,9 224,4 109,7 207,3
0,0
0,0
0,0 211,1 13,3 105,5
7,6 422,1 31,6 658,8 263,8 459,3
12,2
2,2
0,3
1,0
0,3
3,4
1,4
1,1
0,9
0,3
0,1
0,2
101,3 1390,4 271,1 4023,9 352,8 1940,9 407,2 616,1 239,4 674,7 34,1 504,7
158,6 211,2 51,3 147,9 82,8 88,1
9,7 70,0 10,3 60,7 32,9 34,5
0,0
0,0
0,0 629,4 66,9 414,8 82,0 916,5 24,9 217,0 35,4 339,1
0,0
0,0
0,0 197,9 44,4 67,0
4,7 195,6 64,4 69,6 51,7 225,0
3,4
3,7
0,6
3,8
2,5
0,8
0,2
4,6
0,1
1,8
0,0
2,0
5,9 27,1
5,1 89,9
5,0 50,5 10,5 44,5
5,2
0,0
0,0
0,0
57,7 57,6 17,1 203,3
9,9 202,6 26,4 125,6 18,5 130,2 46,6 80,1
0,0 28,0
0,7
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
14,6
2,8
0,7
0,0
0,0
4,4
1,4
0,8
0,2
0,5
0,2
0,2
14,5 184,1 37,8 480,5 22,3 447,8 137,3 996,5 62,3 725,5 279,5 498,2
90,6
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
13,9 14,2
4,7
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
59,1 79,2 10,2 773,6 39,8 488,2 179,6 777,8
5,7 914,0 462,1 556,1
0,0 13,7
6,3
0,0
0,0
0,0
0,0
5,0
2,0
0,0
0,0
0,0
84,8 13,1
1,1 86,1 49,9 53,3
3,0 37,8 16,3 21,0 13,8 28,4
3,6
4,0
0,7
4,2
2,2
1,3
0,7
0,5
0,2
0,4
0,2
0,4
113,9 31,2
4,8 188,2 17,0 262,3
8,9 484,0 16,3 529,0 187,7 400,1
0,0
0,0
0,0
1,7
0,9
0,0
0,0
4,8
1,9
0,0
0,0
2,1
0,0 74,0 10,9
0,0
0,0
0,0
0,0 23,8
5,2 49,2 32,0 16,6
Acids
Acetic acid
Butyric acid
Decanoic acid
Hexanoic acid
Isovaleric acid
Octanoic acid
5,9 1561,5
6,8
0,0
0,0
0,0
0,0 56,7
0,0
0,0
0,0 132,8
99,0 226,6
0,0 180,1
0,0
0,0
17,9 26,8
0,0
0,0
1,1 48,3
75,9 495,9
27,0 349,6
0,0
0,0
1,4 147,1
0,0
0,0
3,3 51,2
44,3 928,3 126,8 2445,9
25,2 263,6
1,6 483,0
0,0
0,0
0,0
0,0
14,3 78,9 10,7 76,0
0,0
0,0
0,0
0,0
4,2 46,9
3,5
0,0
25,0 461,2
49,4
0,0
0,0 350,1
26,3
0,0
0,0
0,0
0,0 375,2
67,8 565,6
0,0 303,8
83,0
0,0
0,0 64,2
0,0
0,0
88,7 74,9
57,9 867,3
58,1
0,0
0,0 127,5
27,1
0,0
0,0
0,0
30,5 218,9
41,6 794,6 34,4 1069,5 190,7 562,1
0,0
0,0
0,0
0,0
0,0
0,0
6,4 366,6 116,2
0,0
0,0 406,3
0,0 59,2 19,6
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
35,1 404,7 146,3 60,0 30,0 64,8
13,6 2264,4
0,0 62,6
75,6
0,0
0,0
0,0
0,0
0,0
6,1
0,0
87,9 2465,7 224,2 4941,9
7,0 183,2 30,1 135,1
0,0
0,0
0,0
0,0
0,0 63,2
7,9
0,0
0,0
0,0
0,0
0,0
0,0 34,6 48,8 103,7
57,7 3801,5 552,2 1283,7 358,8 2812,8
10,0 460,2 54,0 349,9 51,7 642,3
0,0
0,0
0,0
0,0
0,0 82,2
0,0
0,0
0,0 63,6
2,4 214,6
0,0
0,0
0,0
0,0
0,0
0,0
6,3
0,0
0,0 29,2
2,5 145,3
62,2
50,2
42,8
29,5
0,0
8,9
1425,9 264,7 537,3
233,6 16,5
0,0
105,7
9,0 152,0
153,1 13,3
0,0
0,0
0,0
0,0
217,0
9,9 377,7
79,0 1019,8 482,2 469,6
0,0
0,0
0,0
0,0
76,0 36,7 16,4 62,6
0,0 271,4 95,6 164,5
0,0
0,0
0,0
0,0
20,5 697,5 298,5 418,0
41,7
3,3
5,6
26,2
0,0
35,5
1809,9 95,5
543,9 45,9
754,8 22,4
0,0
0,0
139,5
1,2
1026,0 184,5
14,5 138,4
1,1 38,1
3,2 18,3
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
7,7
0,0
1,3
6,6
0,0
0,0
20,9
40,7
63,6
109,1
0,0
0,0
666,8
0,0
142,5
200,0
5,8 62,2
0,5 32,4
8,9 90,1
6,9 69,9
0,0
0,0
0,0
0,0
35,1 425,8
0,0
0,0
6,2 158,2
12,2 186,0
20,2
4,7
4,2
17,1
0,0
0,0
95,5
0,0
5,5
5,0
19,0
13,5
28,5
0,0
0,0
0,0
0,0
0,0
50,8
69,1
1,8
1,3
10,2
0,0
0,0
0,0
0,0
0,0
2,0
4,3
11,3
15,1
14,9
0,0
0,0
0,0
0,0
0,0
28,7
7,7
4,2
6,8
8,1
0,0
0,0
0,0
0,0
0,0
16,9
4,7
7,5
9,6
7,4
0,0
0,0
0,0
0,0
0,0
10,8
0,0
0,8 20,7
0,6 12,1
1,3 32,7
0,0 29,6
0,0
0,0
0,0
0,0
0,0 154,6
0,0
4,8
1,0 39,3
0,0 76,7
3,1
2,3
2,2
0,5
0,0
0,0
17,9
0,6
2,3
1,5
11,3
11,6
27,1
20,2
0,0
0,0
95,3
0,0
39,3
38,5
0,2
8,6
0,7 14,8
5,9 48,0
7,0 34,0
0,0
0,0
0,0
0,0
53,0 171,0
0,0
0,0
13,7 68,5
5,5 127,9
0,4
6,9
0,1 28,3
4,5 84,1
3,0 55,2
0,0
0,0
0,0
0,0
1,5 238,3
0,0
0,0
6,1 123,2
5,0 213,7
1,4
3,6
26,8
16,1
0,0
0,0
24,0
0,0
33,6
8,8
0,0 23,3
0,0
0,0
66,2 1105,9
0,8 134,1
0,0
0,0
0,0
0,0
5,1
0,0
0,0
0,0
4,2 33,1
0,0
0,0
65,1 689,7
5,9 102,4
0,0
0,0
0,0
0,0
0,0 19,1
0,0
0,0
4,9
2,8
0,0
0,0
61,7 150,8
22,8 39,5
0,0
0,0
0,0
0,0
7,2
0,0
0,0
0,0
1,8
0,0
13,4
6,3
0,0
0,0
0,0
0,0
0,0
0,0
4,4
15,8
0,0
0,0
3,4
0,0
0,0
0,0
2,6
7,1
0,0
0,0
0,1
0,0
2,0
0,0
2,4
5,6
0,0
0,0
0,0
0,0
1,1
3,5
0,0
0,0
1,9 282,5
0,5 31,4
0,0
0,0
0,0
0,0
0,0
3,1
0,0
0,0
0,3
0,0
0,0
0,0
24,1 213,8
1,0 27,2
0,0
0,0
0,0
0,0
0,7
0,0
0,0
0,0
0,0
0,0
0,0
0,0
7,6 306,4
8,9 44,1
0,0
0,0
0,0
0,0
0,0 25,3
0,0
0,0
0,0
0,0
0,0
0,0
11,1 465,5
3,7 76,2
0,0
0,0
0,0
0,0
2,9
6,1
0,0
0,0
0,0
0,0
13,5
12,4
0,0
0,0
1,2
0,0
0,8
0,2
3,4
10,3
1,4
1,6
2,5
11,1
Aldehydes
Acetaldehyde
Benzaldehyde
Furfural
Phenyl acetaldehyde
1 -Decanal
1-Nonanal
3-Methyl butanal
4-Methyl benzaldehyde
5 Methyl furfural
5-Hydroxymethylfurfural
8,1
28,9
3,2
0,0
0,0
1,0
0,0
5,1
2,2
0,0
0,3
1,0
0,4
0,0
0,0
0,1
0,0
0,1
0,6
0,0
15,9
24,5
3,1
0,0
4,0
1,4
0,0
4,1
1,4
0,0
0,9
2,2
0,1
0,0
0,1
0,1
0,0
1,3
0,4
0,0
14,8
7,7
2,8
0,0
1,9
1,6
0,0
0,0
1,3
0,0
14,2
4,7
0,6
0,0
1,1
0,8
0,0
0,0
0,6
0,0
22,7
5,0
3,9
0,0
0,0
1,4
0,0
0,0
1,7
0,0
0,1
7,9
0,0
0,0
0,0
0,0
0,1
0,1
4,1
24,0
0,0
0,0
0,0
0,0
1,4
0,3
0,2
1,7
0,0
0,0
0,0
0,0
0,0
0,0
6,2
5,6
0,0
0,0
0,0
0,0
1,4
0,5
0,3
1,8
0,0
0,0
0,0
0,0
0,4
0,1
2,3
3,3
0,0
0,0
0,0
0,0
1,5
0,6
0,1
0,2
4,3
11,3
0,1
0,5
4,5
16,3
0,7
4,6
4,0
15,0
3,7
1,2
0,3
0,0
0,0
0,9
0,0
0,0
0,3
0,0
10,8
0,0
2,1
0,0
0,0
0,0
0,0
3,7
1,2
0,0
0,9
0,0
0,5
0,0
0,0
0,0
0,0
0,2
0,2
0,0
38,9
35,3
7,5
0,0
12,6
0,0
0,0
0,0
4,1
0,0
0,5
0,0
0,4
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,2 314,5
0,2 64,3
0,0
0,0
0,0
0,0
0,0
0,0
17,1
3,5
5,5
0,0
0,0
0,0
0,0
0,0
3,9
2,1
0,1
0,6
9,7
12,7
6,3
5,1
3,8
0,0
3,9
0,0
0,0
0,0
2,5
0,0
12,8
0,0
1,8
0,0
0,9
0,0
0,0
3,9
0,8
0,0
9,6
0,0
0,3
0,0
1,2
0,0
0,0
0,0
0,0
0,0
56,2
6,3
3,6
0,0
0,0
0,0
0,0
0,0
2,2
0,0
1,9
1,0
0,9
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
1,0 320,8 100,7
1,0 43,0 35,6
1,1
0,0
0,0
0,0
0,0
0,0
3,2
0,0
4,7 36,4
8,7 103,8
4,3 10,4
0,7 16,6
0,3 36,1 12,4 14,9
0,0 82,6 16,2 33,6
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0 480,9 211,4
0,0
0,0
0,0
0,0
0,0
1,9 74,0
9,2 94,1
0,0
0,0
0,0
0,0
11,7
15,4
19,0
12,4
0,0
0,0
0,0
0,0
5,6
0,0
14,7
12,4
5,6
0,0
5,2
0,0
0,0
3,9
11,4
0,0
4,9
0,6
2,1
0,0
1,9
0,0
0,0
1,2
5,9
0,0
23,4
22,9
6,7
0,0
0,0
0,0
0,0
0,0
11,9
0,0
4,1
0,1
0,8
0,0
0,0
0,0
0,0
0,0
2,8
0,0
18,7
31,8
7,9
0,0
0,0
0,0
0,0
0,0
6,4
0,0
4,8
3,3
3,7
0,0
0,0
0,0
0,0
0,0
1,5
0,0
13,0
16,4
2,9
0,0
0,0
0,0
0,0
0,0
8,2
0,0
7,1
7,3
3,3
0,0
0,0
0,0
0,0
0,0
2,8
0,0
27,4
12,0
6,0
0,0
0,0
0,0
0,0
6,5
5,0
0,0
18,6
1,5
0,3
0,0
0,0
0,0
0,0
1,6
1,0
0,0
11,1
26,4
7,1
0,0
4,6
0,0
0,0
0,0
6,7
0,0
0,0
3,4
0,1
0,0
1,7
0,0
0,0
0,0
0,8
0,0
66,4
21,5
5,6
0,0
0,0
0,0
0,0
0,0
3,4
0,0
21,5
7,1
0,5
0,0
0,0
0,0
0,0
0,0
0,5
0,0
28,4
31,4
11,9
0,0
0,0
0,0
0,0
19,2
14,6
0,0
1,5 135,4
4,4 29,8
2,5
6,1
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
1,0
5,6
0,4
3,7
0,0
0,0
0,3
0,0
0,0
0,0
0,0 432,3
0,0 61,8
0,0
0,0
0,0
0,0
1,4
4,9
0,0
0,0
18,6
24,4
5,2
0,0
0,0
0,0
0,0
0,0
0,2
0,0
18,6
23,5
15,9
19,4
0,0
0,0
82,8
0,0
17,4
13,4
0,6
1,6
1,3
0,5
0,0
0,0
15,9
0,0
2,8
0,8
Ketones
Acetoin
0,7
Diacetyl
10,3
Pyranone
0,0
2-Cyclopentene-1,4-dione
0,0
2-Dodecanone
0,0
2-Methyltetrahydrothiophen-3-one
0,0
2-Nonanone
0,8
2-Undecanone
0,3
0,0
0,0
30,9
5,9
0,0
0,0
5,8
0,0
0,0
0,0
0,0
81,5
0,0
0,0
0,0
0,0
0,0
0,0
0,0
20,0
0,0
0,0
0,0
0,0
3,4
0,0
6,4
0,0
0,0
0,0
2,6
0,0
1,2
0,0
8,2
0,0
0,0
0,0
1,4
0,0
19,3
0,0
7,7
0,0
0,0
0,0
34,1
0,0
8,9
0,0
1,8
0,0
0,0
0,0
4,5
0,0
2,2
0,0
2,6
0,0
0,0
0,0
0,0
0,0
0,6
0,0
2,3
0,0
0,0
0,0
0,0
0,0
0,0
0,0
2,1
0,0
0,0
0,0
14,7
0,0
0,0
0,0
0,7
0,0
0,0
0,0
16,1
0,0
4,4
0,0
0,0
0,0
0,0
0,0
0,0
0,0
5,1
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
0,0
26,7
0,0
0,0
0,0
0,0
0,0
0,0
0,0
7,8
0,0
0,0
0,0
0,0
0,0
0,0
0,0
6,6
0,0
36,0
14,0
0,0
0,0
0,0
0,0
7,7
0,0
5,3
0,5
0,0
0,0
0,0
0,0
0,0
0,0
0,0
4,3
0,0
0,0
0,0
0,0
0,0
0,0
0,0
2,7
0,0
0,0
0,0
0,0
0,0
0,0
24,6
0,0
0,0
0,0
0,0
0,0
1,9
0,8
2,8
17,5
0,3
2,3
3,2
9,1
0,6
1,1
6,2
13,5
0,5
2,6
5,3
13,6
2,9
1,7
5,2
12,2
1,2
1,3
3,3
9,3
0,5
1,2
5,9
12,7
0,0
1,0
2,9
7,4
2,4
5,4
6,4
10,9
0,8
1,6
0,9
3,6
0,1
0,0
4,3
11,9
0,0
0,0
0,0
0,0
31,7 144,5
0,0 17,1
0,0
0,0
0,0
0,0
0,0
7,2
0,0
0,0
Pyrazine
2,5-Dimethyl-3-ethylpyrazine 3,7
2,6-Dimethylpyrazine
13,9
1,2
3,6
2,9
12,9
2,5
0,4
3,4
12,1
0,6
1,9
3,0
12,4
0,4
2,0
4,5
27,3
1,0
2,6
3,7
12,1
0,1
5,9
2,8
12,7
0,6
9,0
7,8
2,8
2,8
0,5
3,4
7,1
0,1
1,3
4,6
11,9
0,9
0,5
2,5
11,5
0,2
0,5
3,7
15,1
0,1
0,9
4,1
9,5
0,0
2,1
Table 2B:
List of 62 volatiles that have been detected by GC-MS measurement
Alcohols
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Benzyl alcohol
Butanol
Dodecanol
Fenchyl alcohol
Furaneol
Isoamyl alcohol
Propanol
2-Ethyl-1-hexanol
2-Furanmethanol
2-Methyl propanol
2-Nonanol
2-Phenyl ethanol
3 Ethoxy - 1 Propanol
3-(Methylthio)-1-propanol
Esters
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Butyl acetate
Ethyl (4E)-4-decenoate
Ethyl 2-methylbutyrate
Ethyl acetate
Ethyl butanoate
Ethyl decanoate
Ethyl dodecanoate
Ethyl heptanoate
Ethyl hexadecanoate
Ethyl hexanoate
Ethyl isobutyrate
Ethyl isovalerate
Ethyl octanoate
Ethyl propanoate
Ethyl tetradecanoate
Isoamyl acetate
Isoamyl butyrate
Isobutyl acetate
Isobutyl butanoate
Phenethyl acetate
S-methyl thioacetate
2-Methyl propanoate
Acids
37
38
39
40
41
42
Acetic acid
Butyric acid
Decanoic acid
Hexanoic acid
Isovaleric acid
Octanoic acid
Aldehydes
43
44
45
46
47
48
49
50
51
52
Acetaldehyde
Benzaldehyde
Furfural
Phenyl acetaldehyde
1 -Decanal
1-Nonanal
3-Methyl butanal
4-Methyl benzaldehyde
5 Methyl furfural
5-Hydroxymethylfurfural
Chetons
53
54
55
56
57
58
59
60
Acetoin
Diacetyl
Pyranone
2-Cyclopentene-1,4-dione
2-Dodecanone
2-Methyltetrahydrothiophen-3one
2-Nonanone
2-Undecanone
Pyrazines
61
62
2,5-Dimethyl-3-ethylpyrazine
2,6-Dimethylpyrazine
Table 3:
Characterization of fermentation performance. .
The table values are represented as a heat map with grey values. The legend shows the range for
each grey value for the respective condition/test.
Figure 1A:
Principal component analysis of the set of strains and their chemical compounds. The matrix is
based on the full set of 60 strains and the average of the 62 VOCs detected. Strains are presented
with their assigned coordinates (see Table 1). Species belonging to the same genus are represented
with the same color. A higher resolution of the central plot is shown as inset at the upper right
corner.
Figure 1B:
Loading plot of the principal component analysis of all components. Components were grouped
according to the chemical class, numbered and colored similarly. The complete list of volatiles is
shown in table 2B.
Figure 2A:
Fermentation profile of the sub-set of 18 strains. The CO2 release was measured daily for each
individual strain. Fermentation was followed for 11 days.
Figure 2B:
Final Sugar content and ethanol production of the subset of 18 strains. Sugar concentration was
measured in °P after 11 days of fermentation. The ethanol concentration is shown as % ethanol
(vol/vol).
Figure 3:
Volatile organic compounds produced during fermentation by the subset of 18 strains. The
compounds were grouped, numbered and colored according to their chemical class. The complete
list of compounds is shown in Table 2B.
Figure 4:
Concentration of selected volatiles in six strains that produced exceptionally high concentrations of
organic volatiles
Candida diversa (B4); Debaryomyces subglobosus (B11); Pichia kluyveri (E7); Saccharomyces
cerevisiae (E11); Wickerhamomyces anomalus (F10); Zygosaccharomyces mellis (G4);
Saccharomyces pastorianus (G10).
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Appendix
PCA based disruption cassette were used to generate the A.gossypii deletion mutants as
described by Wendland, J., et all., 2000 [65] The deletion cassettes were amplified from pFAGEN3 (C634) and pFA-SAT1(550) using S1- and S2- primers which contained 50 bp homologus
flanks to the target gene. The amplified DNA product was precipitated, purified and
transformed into A.gossypii by electroporation. Diagnostic PCR was used to verify the correct
integration of the cassette into the target gene locus. G1(upstream) and G4(downstream)
annealing primers of the marker integration site, and GEN3/SAT1 primers (G2 and G3) were
used for the verification. I1/I2 primers were used to confirm the homokaryotic deletion(HOM)
of the target gene in the A.gossypii genome. In Figure 1 there is a schematic view of the process.
Figure 1. Schematic representation of the PCA-based target protocol used in A.gossypii.
A first round of transformation was necessary to generate the heterokaryons mutants. Then the
heterokarions
were
let
sporulate
in
selective
condition
and
the
haploid
spores
macromanipulated s into selective media to generate homokaryons mutants. Two independent
mutants were generated for each gene deletion.
67
68
69
70