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Ingegneria metabolica “smart”
Strategie di attivazione parallela
Come si ottiene un aumento di flusso?
Aumentando S
Sottraendo P
Aumentando enzima
Causano aumenti locali che faticano a
propagarsi lungo la via (dampening)
Aumentando attività
Esaminiamo alcuni esempi di aumenti di flusso in vivo
* In lievito nello switch tra fermentazione a respirazione (DeRisi, 1997)
* Nel seme durante la mobilizzazione delle riserve lipidiche (Rylott, 2001)
* Sintesi dei lipidi durante l’embriogenesi di Arabidopsis (O’Hara, 2002)
* Altri esempi (vedi Fell)
Diauxic shift in yeast
Exploring the Metabolic and
Genetic Control of Gene
Expression on a Genomic
Scale (DeRisi et al., 1997)
Quali sono i geni che vengono
attivati e quali vengono
disattivati nella transizione da
fermentazione a respirazione?
 Microarray con tutti i geni
di lievito ibridato con mRNA a
vari tempi di crescita
Rosso = Aumento
Verde = Diminuzione
Seguiamo i trascritti nel tempo
Passando da fermentazione a respirazione
cosa cambia nel metabolismo?
4.9
PYK1
 Variazione
 Gene interessato
Rosso = Aumento
Verde = Diminuzione
Molti geni sono regolati in modo simile
Variazione coordinata di molti geni
E’ possibile classificare i geni in base alla regolazione:  6 classi
Lipid mobilization
in Arabidopsis
germinating seeds
Schematic representation of the pathways involved in storage lipid mobilization in
oilseeds: 1, ACX; 2, multifuctional protein; 3, thiolase; 4, MS; 5, ICL; 6, PEPck.
Northern analysis
Rylott EL, Hooks MA, Graham IA. (2001) Co-ordinate regulation of genes involved
in storage lipid mobilization in Arabidopsis thaliana. Biochem Soc Trans. 29:283-7.
(A) Stages of seedling development (B)
Northern blot analysis of gene expression
from 0 to 8 days after imbibition
Enzimi coinvolti
ACC
Malonyl-CoA transacilasi
KAS III, II & I
FAS - Acido grasso sintasi
Lipid synthesis during embryogenesis
O'Hara, P., et al. Plant Physiol. 2002;129:310-320
3-oxoacyl-ACP reductase (KR)
biotin carboxylase (BC)
acyl-ACP thioesterase (TE)
enoyl-ACP reductase (ENR)
acyl-carrier protein (ACP)
FAS Components Exhibit Constant mRNA Ratios
Abbondanza relativa dei trascritti
It was demonstrated recently that mRNAs encoding the four subunits of heteromeric (ACCase) acetylCoA carboxylase accumulate at a constant molar ratio throughout silique development in Arabidopsis. The
ratios were found to be CAC1:CAC2:CAC3:(accD-A & accD-B) = 0.14:1.0:0.17:0.06 (Ke et al., 2000)
Via del triptofano in lievito
Solo la simultanea espressione di molti (tutti) i geni
causa un ΔJ paragonabile al ΔEi (ΔJ ≃ CJ x ΔEi )
Evidenze sperimentali
Reguloni!
La concentrazione dei metaboliti varia molto meno del flusso
* Rate limiting step concept: more misguided than even MCA initially suggested
* Agire su un solo punto è poco efficace e potrebbe essere deleterio
Il metodo universale mantiene costanti le concentrazioni dei metaboliti [Si]
 evita effetti negativi dovuti all’aumento o alla riduzione di [Si]
Referenze
Referenze ai lavori sugli aumenti naturali in vivo Vedi anche Fell ultimo cap
* DeRisi JL, Iyer VR, Brown PO. DeRisi JL, Iyer VR, Brown PO. (1997) Exploring the metabolic and
genetic control of gene expression on a genomic scale. Science. 278:680-6.
* O'Hara P, Slabas AR, Fawcett T. (2002) Fatty acid and lipid biosynthetic genes are expressed at
constant molar ratios but different absolute levels during embryogenesis. Plant Physiol. 129:310-20
* Rylott EL, Hooks MA, Graham IA. (2001) Co-ordinate regulation of genes involved in storage lipid
mobilization in Arabidopsis thaliana. Biochem Soc Trans. 29:283-7.
* Niederberger P, Prasad R, Miozzari G, Kacser H. (1992) A strategy for increasing an in vivo flux by genetic
manipulations. The tryptophan system of yeast. Biochem J. 287:473-9.
* Zhao J, Last RL.(1996) Coordinate regulation of the tryptophan biosynthetic pathway and indolic
phytoalexin accumulation in Arabidopsis. Plant Cell. 8:2235-44.
* Eastmond PJ, Rawsthorne S. (2000) Ccoordinate changes in carbon partitioning and plastidial
metabolism during the development of oilseed rape embryos. Plant Physiol. 122:767-74
•Universal method: Kacser and Acerenza (1993) A universal method for achieving increases in
metabolite production Eur J. of Biochemistry 216:361-367
•Lütke-Eversloh T, Stephanopoulos G. (2008) Combinatorial pathway analysis for improved L-tyrosine
production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene
overexpression. Metab Eng. 10:69-77.
Ingegneria metabolica “in batch”
(6)
Espressione di fattori di trascrizione che regolano
positivamente gli enzimi della via metabolica
S
+
TF
+A
* Terpenoid Indole Alkaloyd (TIA)
* via dei flavonoidi cere, glucinolati...
+
B
+
C
Usando i fattori di trascrizione probabilmente si
mantengono le “giuste proporzioni tra gli enzimi
CAVEAT: ci sono limiti a questa strategia?
P
Certo, alcuni enzimi come già molto abbondanti
(es. quelli del calvin o glicolitici)
Fig. 1. Biosynthesis of TIAs in C. roseus.
Solid arrows indicate single enzymatic
conversions, whereas dashed arrows indicate
multiple enzymatic conversions.
Numerosi enzimi della via sono stati identificati
e clonati. Esiste un fattore di trascrizione capace
di attivarli tutti insieme?
Abbreviations of enzymes:
AS, anthranilate synthase; DXS, D-1-deoxyxylulose 5phosphate synthase; G10H, geraniol 10-hydroxylase; CPR,
cytochrome P450-reductase; TDC, tryptophan
decarboxylase; STR, strictosidine synthase;
SGD,strictosidine b-D-glucosidase; D4H,
esacetoxyvindoline 4-hydroxylase; and DAT, acetyl-CoA:4O-deacetylvindoline 4-O-acetyltransferase.
Genes regulated by ORCA3 are underlined.
T-DNA activation tagging
Struttura del T-DNA
Punto di inserzione del
T-DNA nel genoma
ORF attivata
dall’inserzione
Linea cellulare selezionata con inibitori delle TDC. L’inserzione
del T-DNA porta ad un aumento del flusso nella via
Molti altri geni della stessa via sono indotti nella linea cellulare
Il metabolismo secondario:
Flavonoidi, Antociani e
Lignina
Genes encoding all
enzymes indicated in
red are clock-controlled
Myb transcription
factor PAP1
I geni in rosso sono implicati
nella biosintesi dei
fenilpropanoidi e sono
controllati dal ritmo circadiano
Alcuni geni sembrano essere regolati in maniera molto simile
dal punto di vista temporale. Può essere segno di un controllo
comune mediato cioè dallo stesso fattore di trascrizione?
Activation tagging
Il mutante pap1-D presenta una colorazione rossa (carattere
dominante) e accumula antocianine (una classe di flavonoidi)
Molti geni della via dei fenilpropanoidi (e
sue diramazioni: flavonoidi, antocianine)
sono espressi maggiormente nel mutante.
Il mutante pap1-D presenta una maggiore
attività enzimatica e più lignina.
La sovraespressione di Pap1 o
Pap2 in Tabacco o Arabidopsis
porta ad un’intensa pigmentazione
Come identificare i fattori implicati
nella trascrizione di vie metaboliche
mutanti classici (indotti o spontanei)  gene
activation tagging o sovraespressione
Coregolazione  elementi comuni in cis  elementi comuni in
trans (?)  identificazione del fattore tramite One-hybryd
Identificazione….
Attenzione: i fattori di trascrizione sono enzimi (?) e spesso agiscono in
sinergia
Immagini cortesia del prof. C. Martin
Lobe
Tube
Geni regolatori in
Anthyrrinum majus
Diversi geni della via
sono down-regulated
nel mutante delila ma
solo nella zona con
ridotta pigmentazione
Tobacco crosses: 35S:Del x 35S:Ros1
Piante di Arabidopsis che sovraesprimono uno solo
dei due fattori non mostrano accumulo. Quando sono
coespressi l’aumento di flusso è notevole.
Immagini cortesia del prof. C. Martin
Sinergismo
!
Rosea1 + Delila can give 100-fold + activation and
anthocyanin levels of up to 10 mg/g fwt. They can also
increase flux through pathway branches 2.5-fold.
Other regulatory combinations are not so potent
Immagini cortesia del prof. C. Martin
Fattori di trascrizione coinvolti nella
regolazione del metabolismo in pianta
Broun P. (2004) Transcription factors as tools for metabolic engineering in plants. Curr Opin Plant Biol. 7:202-9.
Altri esempi:
- Cernac et al. (2006) The WRI1 gene encodes an AP2/EREBP transcription factor involved in the
control of metabolism, particularly glycolysis, in the developing seeds. Plant Physiology 141:745757.
- Xie et al. (2006) Metabolic engineering of proanthocyanidins through co-expression of
anthocyanidin reductase and the PAP1 MYB transcription factor. Plant J. 45:895-907.
- Metabolismo degli olii in foglia: Santos Mendoza et al., (2005) FEBS Lett. 579:4666-4670. LEAFY
COTYLEDON 2
- Kannangara et al. (2007) The transcription factor WIN1/SHN1 regulates Cutin biosynthesis in
Arabidopsis thaliana. Plant Cell. 2007 Apr;19(4):1278-94.
- Aharoni et al. (2004) The SHINE clade of AP2 domain transcription factors activates wax
biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in
Arabidopsis. Plant Cell. 16:2463-80.
- Baud and Lepiniec (2009) Regulation of de novo fatty acid synthesis in maturing oilseeds of
Arabidopsis, Plant Physiol. Biochem. 47:448–455.
- Ruuska et al. (2002) Contrapuntal networks of gene expression during Arabidopsis seed filling, Plant
Cell 14:1191–1206.
- Shen et al. (2010) Expression of ZmLEC1 and ZmWRI1 increases seed oil production in
maize, Plant Physiol. 153:980–987.
- Pouvreau et al. (2011) Duplicate maize Wrinkled1 transcription factors activate target genes
involved in seed oil biosynthesis, Plant Physiol. 156:674–686.
- Zhang et al. (2002) Similarity of expression patterns of knotted1 and ZmLEC1 during somatic and
zygotic embryogenesis in maize (Zea mays L.), Planta 215:191–194.
- Maeo et al. (2009) An AP2-type transcription factor, WRINKLED1, of Arabidopsis
thaliana binds to the AW-box sequence conserved among proximal upstream regions of genes
involved in fatty acid synthesis, Plant J. 60:476–487.
WIN1: wax inducer (biosintesi delle cere)
Broun P, Poindexter P, Osborne E, Jiang C-Z, Riechmann JL: WIN1, a
transcriptional activator of epidermal wax accumulation in Arabidopsis.
Proc Natl Acad Sci USA 2004, 101(13):4706-11
Activation of wax production in Arabidopsis plants that
overexpress WIN1, an ERF-type transcription factor, and
concurrent induction of wax pathway genes. Morphological
phenotype of (a) a control (wt) and (b) 35S::WIN1 plants. Note the
glossy appearance of 35S::WIN1-overexpressing leaves. Scanning
electron microscope (SEM) images of (c) control and (d)
35S::WIN1 leaf surfaces: WIN1 overexpressors produce wax
crystals, which are absent from control leaves. (Magnification:
3000x.) Stomatal cells are shown at the centre of the images. (e)
Northern analysis of the expression of wax pathway genes in
35S::WIN1 and control plants: KCS1, which encodes a putative
fatty acid elongase, and CER1, encoding a putative fatty acid
decarbonylase, are induced in 35S::WIN1 plants.
Northern and microarray analyses of 35S::WIN1 plants indicated that several genes that are implicated in wax
biosynthesis, such as ECERIFERUM1 (CER1) and 3-KETOACYL-COA SYNTHASE1 (KCS1), were upregulated
in the WIN1-overexpressors
wt
b and c are representative
of medium, and high
levels of leaf glossiness
35S::WIN1
35S::WIN1
Total fatty acids per seed for the untransformed mutant (wri1) and wild type (WT) (a),
and transgenic lines in the wri1 background (b) or the wild type background (c).
Fatty acid composition
Lipid and fatty acid compositions,
after LEC2:GR induction in leaves
Lipid composition.
Transcriptional regulation of triacylglycerol biosynthesis
in maturing seeds of Arabidopsis thaliana
LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3 (ABI3), and
FUSCA3 (FUS3) arenormally expressed predominantly in seeds, can induce the
deposition of seed oil in vegetative tissues when ectopically activated in seedlings.
Family
TF Name
Summary of Role in Seed Oil Deposition
B3
domain;
AFL
Clade
ABSCISIC ACID INSENSITIVE3
(ABI3),
LEAFY COTYLEDON2 (LEC2),
FUSCA3 (FUS3)
Master regulators of embryogenesis and seed maturation;
mutation/overexpression often associated with pleiotropic effects; direct and
indirect regulation of suites of genes involved in carbohydrate and lipid
metabolism, including fatty acid synthesis, triacylglycerol assembly and packaging
HAP3/C
BP
LEAFY COTYLEDON1 (LEC1),
LEC1-LIKE (L1L)
Subunits of CCAAT binding proteins; capable of working independently of CBP;
master regulators of embryogenesis and seed maturation; direct and indirect
regulation of genes involved in carbohydrate and lipid metabolism
AP2
WRINKLED1 (WRI1)
Direct target of master regulators having more specific role towards seed oil
biosynthesis; mutants dramatically reduced in seed oil content and wrinkled
appearance; direct and indirect regulation of carbohydrate and lipid metabolism
genes, particularly plastidial fatty acid synthesis
Dof
GmDof4 GmDof11
Transgenic expression yields higher seed oil levels; direct and indirect regulation of
lipid metabolism genes; possible negative regulators of seed storage proteins
CHD3
PICKLE (PKL)
Putative chromatin remodeling factor; represses master regulator genes at
germination; associated with the repressive chromatin mark H3K27me3
PRC2
FERTILIZATION INDEPENDENT
ENDOSPERM (FIE),
SWINGER (SWN),
EMBRYONIC FLOWER2 (EMF2)
Components of Polycomb Repressive Complex 2 that catalyze deposition of
H3K27me3; repressors of seed maturation genes in vegetative tissues
B3
domain;
HSI2
Clade
HIGH-LEVEL EXPRESSION
OF SUCROSE INDUCIBLE
GENE2 (HSI2)/VAL1,
HSI2-LIKE1 (HSIL1/VAL2),
HSL2/VAL3
Act redundantly to repress AFL Clade genes and other positive regulators of seed
maturation during germination and in seedlings; possible chromatin remodeling
activities
AP2
APETALA2 (AP2)
Negative regulator of seed size, possibly via carbohydrate metabolism in the seed
coat; effects on seed oil deposition likely indirect
HD-ZIP
GLABRA2 (GL2)
Negative regulator of oil content; loss of seed mucilage proposed to make more C
available for fatty acid synthesis
Zhong and Ye (2009) Transcriptional regulation of
lignin biosynthesis. Plant Signal Behav. 4:1028-34.
According to Metabolic Control Analysis, the parallel
activation (multisite modulation) of enzymes within a
biochemical pathway is the optimal strategy for changing
fluxes  retains metabolite and control homeostasis
How universal is the “universal method” in
vivo?
PDS (Phytoene Desaturase)
If a mRNA level changes, what happens to
other ones in the same metabolic pathway?
 mRNA is not equal to protein 
Two-gene
flux changes over long times
scatterplot
Use data from many different
tissues, mutants, conditions…
Pearson
correlation
coefficient
PSY (Phytoene Synthase)
A square matrix
At4g15560
At5g11380
At5g62790
At2g02500
At2g26930
At1g63970
At5g60600
At4g34350
At3g21500
1.00
0.12
0.01
0.03
0.04
0.20
0.25
0.19
0.19
At4g15560
0.12
1.00
0.35
0.75
0.66
0.65
0.66
0.73
0.73
At5g11380
0.01
0.35
1.00
0.35
0.31
0.40
0.21
0.18
0.15
At5g62790
0.03
0.75
0.35
1.00
0.69
0.70
0.78
0.78
0.68
At2g02500
0.04
0.66
0.31
0.69
1.00
0.77
0.72
0.67
0.56
At2g26930
0.20
0.65
0.40
0.70
0.77
1.00
0.80
0.67
0.60
At1g63970
0.25
0.66
0.21
0.78
0.72
0.80
1.00
0.88
0.74
At5g60600
0.19
0.73
0.18
0.78
0.67
0.67
0.88
1.00
0.77
PSY (Phytoene Synthase)
At3g21500
PSY (Phytoene Synthase)
At4g34350
0.19
0.73
0.15
0.68
0.56
0.60
0.74
0.77
1.00
From numbers to colours
Gene A
Gene B
Gene A
Essentially the same
strategy published recently
by Toufighi K, et al. (2005)
Plant J. 43:153-63
The Botany Array Resource:
e-Northerns, Expression
Angling, and promoter
analyses.
Gene B
The Red Square…
Gene
ABCDEFGHIJKLMNOPQRS
Group 1
Group 1 & 3
Group 3
are coregulated
Coregulated genes close in the list will appear as a red square
Apply the correlation analysis to the entire
“metabolic genome” (enzymes, transporters….)
B
A
B
Isoprenoid
biosynthesis
two indipendent
pathways in plants:
A cytosolic
B plastidial
Lange and Ghassemian (2003) Genome
organization in Arabidopsis thaliana: a
survey for genes involved in isoprenoid
and chlorophyll metabolism. Plant Mol
Biol. 51:925-48.
Plastidial
pathway:
Carotenoids
Phytyl
Plastoquinone
Phylloquinone
Tocopherol
Mono-terpenes
Phytochrome
Gibberellic acid
Abscissic acid.
Figure from Lange and Ghassemian (2003)
1000
5
0
0
g
e
n
e
s
1414
2000
2750
5
0
0
g
e
n
e
s
Plastidial
IPP
Cytosolyc
IPP
(meval.)
1
0
0
◄ GGPP synthases: 10 isoforms
Carotenoid
g
e
n
e
s
Chlorophyll
GA
GGPP synthase
GGPP
Prenyl group
Phytyl PP
Chlorophyll
(At3g20160)
At3g29430
At3g32040
At4g36810
(At4g38460)
At3g29430 and At3g32040
provide GGPP for…
Which GGPP synthase isoform
works in the carotenoid pathway?
At3g29430
Migliori correlatori tra tutti i
geni di Arabisopsis
(R value in linear plots)
At3g29430
At3g29410
At4g33720
At5g15180
At1g53940
At2g24400
At5g59680
At5g24410
At1g73780
At3g47210
At3g59370
At1g33900
At5g03570
At3g32040
At1g21210
At5g37450
At1g11540
At3g49860
At2g31085
At1g49030
At1g66020
At3g05950
At5g15725
At3g01190
At4g31875
At2g38600
At3g46400
1.0000
0.8313
0.7440
0.7352
0.7341
0.7081
0.7022
0.6942
0.6873
0.6867
0.6865
0.6857
0.6855
0.6847
0.6831
0.6827
0.6810
0.6770
0.6739
0.6734
0.6725
0.6709
0.6668
0.6644
0.6620
0.6569
0.6532
geranylgeranyl pyrophosphate synthase, putative
terpene synthase/cyclase family protein
pathogenesis-related protein, putative
peroxidase, putative
GDSL-motif lipase/hydrolase family protein
auxin-responsive protein, putative / small auxin up RNA (SAUR_D)
leucine-rich repeat protein kinase, putative
glucosamine/galactosamine-6-phosphate isomerase-related
protease inhibitor/seed storage/lipid transfer protein
expressed protein
expressed protein
avirulence-responsive protein, putative
iron-responsive transporter-related
geranylgeranyl pyrophosphate synthase, putative
wall-associated kinase 4
leucine-rich repeat transmembrane protein kinase, putative
expressed protein
ADP-ribosylation factor, putative
Clavata3 / ESR-Related-6 (CLE6)
expressed protein
terpene synthase/cyclase family protein
germin-like protein, putative
expressed protein
peroxidase 27 (PER27) (P27) (PRXR7)
expressed protein
acid phosphatase class B family protein
leucine-rich repeat protein kinase, putative
At3g29430 is possibly involved in terpene synthesis
Calvin cycle
At4g26520
At4g26530
At4g38970
At2g21330
At5g56630
At5g47810
At4g32840
At2g22480
At4g26390
At3g55440
At2g29560
At1g07110
At1g13440
At1g42970
At3g26650
At3g04120
At3g12780
At1g58150
At1g56190
At1g22170
At1g78040
At3g08590
At5g04120
At3g22960
At5g52920
At2g21170
At5g61410
At1g71100
At3g04790
At2g45290
At3g60750
At1g32060
At1g43670
At3g54050
At3g55800
At5g35790
At1g09420
At5g24420
At5g24410
At3g49360
At1g13700
At5g44520
At2g01290
At5g39320
At5g64290
At5g35630
At4g37930
At1g23310
At3g19710
At1g32450
3.5 in log scale  >3000
fructose-bisphosphate aldolase, cytoplasmic
fructose-bisphosphate aldolase, putative
fructose-bisphosphate aldolase, putative
fructose-bisphosphate aldolase, putative
phosphofructokinase family protein
phosphofructokinase family protein
phosphofructokinase family protein
phosphofructokinase family protein
pyruvate kinase, putative
triosephosphate isomerase, cytosolic, putative
enolase, putative
fructose-6-phosphate 2-kinase / fructose-2,6-bisphosphatase (F2KP)
glyceraldehyde 3-phosphate dehydrogenase, cytosolic, putative
glyceraldehyde-3-phosphate dehydrogenase B, chloroplast (GAPB)
glyceraldehyde 3-phosphate dehydrogenase A, chloroplast (GAPA)
glyceraldehyde-3-phosphate dehydrogenase, cytosolic (GAPC)
phosphoglycerate kinase, putative
hypothetical protein
phosphoglycerate kinase, putative
phosphoglycerate/bisphosphoglycerate mutase family protein
pollen Ole e 1 allergen and extensin family protein
2,3-biphosphoglycerate-independent phosphoglycerate mutase
phosphoglycerate/bisphosphoglycerate mutase family protein
pyruvate kinase, putative
pyruvate kinase, putative
triosephosphate isomerase, chloroplast, putative
ribulose-phosphate 3-epimerase, chloroplast, putative /
ribose 5-phosphate isomerase-related
ribose 5-phosphate isomerase-related
transketolase, putative
transketolase, putative
phosphoribulokinase (PRK) / phosphopentokinase
fructose-1,6-bisphosphatase, putative
fructose-1,6-bisphosphatase, putative
sedoheptulose-1,7-bisphosphatase, chloroplast
glucose-6-phosphate 1-dehydrogenase / G6PD (APG1)
glucose-6-phosphate 1-dehydrogenase, putative / G6PD, putative
glucosamine/galactosamine-6-phosphate isomerase-related
glucosamine/galactosamine-6-phosphate isomerase-related
glucosamine/galactosamine-6-phosphate isomerase family protein
glucosamine/galactosamine-6-phosphate isomerase family protein
ribose 5-phosphate isomerase-related
expressed protein
UDP-glucose 6-dehydrogenase, putative
oxoglutarate/malate translocator, putative
glutamine synthetase (GS2)
glycine hydroxymethyltransferase
glutamate:glyoxylate aminotransferase 1 (GGT1)
branched-chain amino acid aminotransferase, putative
proton-dependent oligopeptide transport (POT) family protein
Reducing glucosinolates in Arabidopsis
Glucosinolates are sulphur rich
compounds from brassicas
Some beneficial, other toxic (quantity!)
Upon wounding are converted into toxic
products
Two branches
Mutants isolated
Short chain
Aliphatic GSL
Long chain
Indolic GSL
Beekwilder et al., (2008) PLoS 3:e2068.
Glucosinolate pathway
Phase 2 - core structure synthesis
Step 1: Oxidation
Amino Acid
Step 2: Oxidation
Aldoxime
CYP79s
CYP83s
Aci-Nitro
compound
Step 3: Conjugation
GSTs
S-Alkyl
Thioidroximate
Cytoplasm
C-S Lyase
Step 6: Sulfatation
Glucosinolate
ST5s
Step 5: Glucosylation
Desulfoglucosinolate
Step 4: C-S Clevage
Thioidroximate
UGTs
Glucosinolates: sulfur-rich
secondary metabolites
Amino acid
Oxo-acid
Chloroplast
Transamination
Amino acid
(n+1)C
Several rounds of chain
elongation are possible
Condensation
2-alkyl-malic acid
Isomerization
Export
Oxo-acid
3-alkyl-malic acid
Oxydative
decarboxylation
Phase 1 - side chain elongation
Kroymann et al., Plant Physiology (2001) 127:1077–1088,
Phase 3 - Side Chain Modification
Various oxidations on the side chain
Cytoplasm
compartimentation -transport
TRYPTOPHAN BIOSYNTHESIS
GLUCOSINOLATE FROM
TRYPTOPHAN AND PHENYLALANINE
SHARED GENES
(PAPS BIOSYNTHESIS,C-S
LYASE AND GLUCOSYL
TRANSFERASE)
CYP79A2 At5g05260
CYP79B2 At4g39950
CYP79B3 At2g22330
CYP83B1 At4g31500
At1g74100
ST5a
ATGSTF10 At2g30870
ATGSTU13 At1g27130
ATGSTF9 At2g30860
F17I23 At4g30530
ASA1
At5g05730
TSA1
At5g17990
TRP 1 At3g54640
IGPS
At2g04400
DHS1
At4g39980
SAT52 At5g56760
OASC At3g59760
PEN2
PEN3
SUR1
UGT74B1
AKN2
AKN1
BCAT3
BCAT4
MAM1
F17J16
T9E8
MFL8
CYP83A1
ATGSTF11
ATGSTU20
ST5b
ST5c
B5 #1
F12P19
T3P18
F16J13
MYB28
F28J8
AOP2
AOP3
At2g04400
At1g59870
At2g20610
At1g24100
At4g39940
At2g14750
At3g49680
At3g19710
At5g23010
At3g58990
At4g13430
At2g43100
At4g13770
At3g03190
At1g78370
At1g74090
At1g18590
At2g46650
At1g65860
At1g62560
At4g12030
At5g61420
At1g21440
At4g03060
At4g03050
HOMOMETHIONINE BIOSYNTHESIS
GLUCOSINOLATE FROM HOMOMETIONINE
Aromatic
branch
CYP79A2
CYP79B2
CYP79B3
CYP83B1 Phase II –
ST5a
- Sulfotransferase
GLS
from Trp and Phe
Glutathione S-Transferase
Glutathione S-Transferase
Glutathione S-Transferase
Anthranilate synthase
ASA1 -Anthranilate synthase α subunit
TSA1 - Trp synthase, alpha subunit
TRP Biosynthesis
TRP1- P-ribosyl-anthranilate synthase
IGPS Indole-3-glycerol p synthase
DHS1 – DAHP synthetase 1
SAT 52 – Serine O-acetyltrasferase
Cysteine Synthase
Glycosil hydrolase family 1 protein
ABC Transporter
SUR1 - C-S Lyase
UGT74B1
S-Glucosil Trasferase
(PAPS–Biosynthesis,
C-S
AKN2 – Adenylylsulfate kinase 2
Lyase,
Glucosyl
Transferase)
AKN1 – Adenylylsulafte kinase 1
BCAT3
Branched-chain amino
BCAT4
acid aminotransferase
MAMPhase
1 – 2 isopropylmalate
synthase 3
I - Homomet
Aconitase C-terminal domain
Biosynthesis
Aconitase family
protein
Aconitase C-terminal domain
CYP83A1
Glutathione-S Transferase
Phase II
– GLS from
Glutathione-S
Transferase
ST5b – Sulfotransferase
Homomet
ST5c – Sulfotransferase
Cytochrome b5
Flavin-contaning monooxygenase
Phase III, monooxygenase
transport and
Flavin-contaning
Bile
acid Sodium symporter
regulation
– GLS from
MYB 28
HOMOMET
Mutase family
protein
AOP2 - Dioxygenase
AOP3 -Dioxygenase
Phase II Shared genes
Aliphatic
branch
GLUCOSINOLATE
BIOSYNTHESIS
METHIONINE SIDE-CHAIN
ELONGATION
At4g13770
At3g03190
At1g78370
At2g20610
At1g18590
At1g74090
At3g19710
At5g23010
At3g58990
At2g43100
At4g13430
At4g12030
At5g61420
At2g46650
At1g62560
At1g21440
CYP83A1
Monooxygenase “GLUCOSINOLATE FROM FENIL.-OMOMET.”
ATGSTF11 Glutathione S-transferase
SUR1
Phase II - GLS biosynthesis
(Met derived)
C-S Lyase “GLUCOSIN. FROM PHENILAL-TRYPT-HOMOMET.”
ST5c
Sulfotransferase “GLUCOSINOLATE FROM HOMOMET.”
ST5b
Sulfotransferase “GLUCOSINOLATE FROM HOMOMET.”
ATGSTU20 Glutathione S-transferase
BCAT4
Aminotransferase“HOMOMET.–LEUCINE BIOSYNTHESIS”
MAM1
Phase I -BIOSYNTHESIS”
GLS biosynthesis
2-isopropylmalate Synthase “HOMOMET
F17J16
derived) SIDE-CHAIN
Aconitase C-terminal domain(Met
“LEUC.-HOMOMET.BIOSYNTHESIS”
MFL8
ELONGATION
Aconitase C-terminal domain“HOMOMET.
BIOSYNTHESIS”
T9E8
Aconitate hydratase
F16J13
Sodium symporter family protein
MYB28
Transcription factor
T3P18
Candidate genes for transport,
regulation...
(MET derived GLS)
Flavin conteining monooxygenase
family protein
F28J8
Mutase family protein
B5 #1
Cytochrome b5
Phase I and II enzymes are co-regulating
H
C
3
O
H O
S
O

-C
h
e
to
g
lu
ta
r
a
to
S
H
C
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O
A
na
m
in
oa
c
id
C
o
A
A
c
e
ty
lC
o
A
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H
O
S
O
H
H
C
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N
H
2
A
m
in
o
tr
a
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sfe
r
a
se
M
e
th
io
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in
e
A
t3
g
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9
7
1
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O
H
O
2o
x
o4m
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th
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lth
iob
u
ta
n
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ica
c
id
M
e
th
y
lth
io
a
lk
y
lm
a
la
tesy
n
th
a
se
O
H
A
t5
g
2
3
1
0
0
2
-(2
'm
e
th
y
tio
)e
th
y
lm
a
lica
c
id
BCAT4
A
c
o
n
ita
se
MAM1
A
t3
g
5
8
8
9
0
BCAT3
A
t2
g
4
3
1
0
0
O
O

-C
h
e
to
g
lu
ta
r
a
to
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H
A
na
m
in
oa
c
id
H
C
3
S
H
C
3
H
o
m
o
m
e
th
io
n
in
e
N
H
2
N
A
D
H
O
+
N
A
D
S
H
C
3
O
H
O
H
O
S
H
O
O
2o
x
o5m
e
th
y
lth
iop
e
n
ta
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ica
c
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-(2
'm
e
th
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th
y
lm
a
lica
c
id
Myb28 (At5g61420)
LBa1
SALK_136312
LB51
BRC_H161Lb
ATG
PROM
1
ATG
EX3
134 214 344 484
1623
TGA
MAM-L
400
350
300
250
RGE
Effect of knocking
out Myb28?
200
150
100
50
0
RT-PCR on 2 controls and 2 KOs
-50
C
2
6
9
C
Leaf
2
6
9
6
9
6
9
Root
MYB 28
CYP83A1
16
14
1.8
1.6
10
1.4
8
1.2
6
1.0
RGE
RGE
12
4
0.8
0.6
2
0.4
0
C
2
6
9
C
Leaf
2
6
0.2
9
0.0
Root
-0.2
C
2
6
9
C
Leaf
2
Root
MYB 29
Aconitase
2.0
3.0
1.5
2.5
2.0
RGE
RGE
1.0
0.5
0.0
C
2
6
9
C
2
6
9
1.5
1.0
0.5
0.0
-0.5
Leaf
Root
C
2
6
9
C
2
-0.5
Leaf
Root
5.31
279
565.0460
-2
5.00
M09107
2.74
144
422.0223
%
98
37.26
1962
487.1212
39.66
2088
223.0983
Wt and Myb28-KO metabolome
10.00
4.42
232
436.0179
5.24
275
565.0455
27.46
1445
505.1335
10.88
572
323.1347
15.00
14.40
757
478.0881
20.00
18.96
998
739.1794
25.00
20.06
1055
492.0634
30.00
26.45
1392
339.0452
35.00
45.00
50.00
55.00
1: TOF MS ESBPI
54.25;2856;791.4733
3.43e4
32.86
1729
477.0614
27.44
1444
505.1348
10.88
572
323.1338
40.00
46.99
2474
333.1882
42.60
2242
478.0856
37.26
1962
487.1232
51.52
2712
476.1041
46.97
2473
333.1925
-2
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
1: TOF MS ESBPI
54.22;2854;791.4753 3.42e4
M09106
20.17;1062;447.0292
4.38
230
436.0205
%
98
5.29
278
565.0436
16.79
883
385.1132
26.41
1390
339.0430
32.80
1727
477.0533
27.44
1444
389.1235
10.91
574
323.1335
37.26
1962
487.1258
39.61
2085
223.0984
51.50
46.97
2711
2473 1046.5104
333.1924
ko
-2
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
M09105
98
%
2.78
145
422.0237
4.40
231
436.0169
18.98
999
739.1732
14.40
GSL unknown
757
20.12
26.40
1059
1389
492.0636 339.0451
32.86
1729
477.0608
478.0864
5.27
277
565.0471
55.00
1: TOF MS ESBPI
54.34;2861;791.4747
3.43e4
Methylsulfinyloctyl
42.62
Methylsulfinylheptyl
2243
27.46
1445
389.1248
10.91
574
323.1330
37.26
1962
487.1224
478.0875
46.99
2474
333.1934
50.00
51.60
2717
492.0991
51.14
2692
552.2348
-2
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
wt
Time
55.00
myb28, myb29 and myb28myb29
Mutating Myb28 and Myb29
Regulators
Beekwilder et al., (2008) PLoS 3:e2068.
Reducing glucosinolate content...
...stimulates pest growth and damage!
Beekwilder et al., (2008) PLoS 3:e2068
Insect feeding
Effect of the double KO
Too late!
2828 genes
What is the distribution of all the R values in the matrix?
La spalla di valori alti e positivi di R all’interno dei geni
metabolici è la testimonianza che esiste molta coregolazione
Open issues
Explore enzyme subsets
Pathway identification
Clustering of enzymes
Shared cis-elements / regulators
Suggest substrate for enzymes / trasporters
Limitations
Other levels of regulation
Co-regulation does not mean necessarily…
One vs. all analysis for At5g57800 CER1 protein, putative (WAX2) (Log)
At5g57800
At5g20270
At2g26250
At3g43720
At1g17840
At1g68530
At4g39330
At2g26910
At5g13400
At4g25960
At5g14410
At1g02205
At1g51500
At2g04570
At5g57800
1
0.8287
0.8044
0.7894
0.7892
0.7864
0.7792
0.7755
0.7735
0.7679
0.766
0.7563
0.7379
0.7234
CER1 protein, putative (WAX2)
expressed protein
beta-ketoacyl-CoA synthase family (FIDDLEHEAD) (FDH)
protease inhibitor/seed storage/lipid transfer protein (LTP) family protein
ABC transporter family protein
very-long-chain fatty acid condensing enzyme (CUT1)
mannitol dehydrogenase, putative
ABC transporter family protein
proton-dependent oligopeptide transport (POT) family protein
multidrug resistance P-glycoprotein, putative
expressed protein
CER1 protein (another?)
ABC transporter family protein
GDSL-motif lipase/hydrolase family protein
CUT1 (very-long-chain fatty acid condensing enzyme, At1g68530) shows
good correlation with At1g51500 (R=0.815), an ABC transporter protein
Transporters
WT
cer5
cer5
Wax analyses of Arabidopsis
stem surface (cuticle) or
epidermal peel extracts (total
epidermis).
Cer5 (At1g51500)
Pighin et al., Science (2004) 306:622-625
Programma
Ripasso di cinetica enzimatica e approccio classico al controllo dei flussi [1,6].
Fondamenti di Analisi del Controllo Metabolico (MCA): proprietà locali e
sistemiche, elasticità e coefficienti di controllo del flusso e delle concentrazione
[1,6,7]. Trattazione dei sistemi Supply-Demand in generale [8] e dell’ATP in
particolare [9]. Rate limiting steps e ingegneria metabolica [10, 11 e 12].
Tipi di ingegneria metabolica: a- Inattivazione di enzimi e allergeni (via del
gossipolo [13], ODAP e glucosidi cianogenici) e review generale [14]); bCreazione di vie metaboliche ex novo o potenziamento di vie endogene già
presenti (Glucosidi cianogenici [15,16], Vitamina E [17, 18], Folato [19], laurato
[20, 21]); c- Aumento del demand (aumento del contenuto in aa, aumento del
contenuto in zucchero) [22-24]; e- Amido in patata: strategie diverse [25]; fUtilizzo dei fattori di trascrizione (Terpenoid Indole Alkaloyd, Flavonoidi, cuticola,
glucosinolati...) [10,11,26].
Bibliografia (ref 2-4 sono testi generali sul metabolismo delle piante e la sua manipolazione)
Generali (MCA e metabolismo):
[1] Fell, Understanding the control of Metabolism Portland Press (1997) (in Biblioteca biologica)
[2] Dennis/Turpin Plant Metabolism (1998) Longman; nuova edizione.
[3] Lea/Leegood Plant Biochemistry and Molecular Biology (1993) Wiley & sons.
[4] Foyer e Quick (Eds) A molecular approach to primary metabolism in higher plants; Taylor and Francis
(1997)
Articoli originali
[6] Kacser, Burns, & Fell, The control of flux (1995) Biochem. Soc. Trans. 23, 341-366 (art. del 1973).
[7] Kacser e Acerenza, Eur. J. Biochem. (1993) 216:361-367
[8] Hofmeyr & Cornish-Bowden (2000) Regulating the cellular economy of supply and demand. FEBS Lett.
476:47-51.
[9] Koebmann et al. (2002) The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J.
Bacteriol. 184:3909-16
[10] Morandini & Salamini (2003) Plant biotechnology and Breeding, allied for years to come Trends Pl. Sci.
8:70-5.
[11] Morandini, Salamini & Gantet, (2005) Engineering of Plant Metabolism for Drug and Food. Curr. Med.
Chem. – Immun., Endoc. & Metab. Agents 5:103-112
[12] Morandini (2009) Rethinking metabolic control. Plant Science 176:441-451
[13] Sunilkumar et al., (2005) Engineering cottonseed for use in human nutrition by tissue-specific reduction
of toxic gossypol. P.N.A.S. 103:18054–18059.
[14] Morandini (2010) Inactivation of allergens and toxins. N Biotechnol. 27:482-93.
[15] Tattersall DB et al., (2001) Resistance to an herbivore through engineered cyanogenic glucoside
synthesis. Science 293:1826-8.
[16] Nielsen et al., (2008) Metabolon formation in dhurrin biosynthesis. Phytochemistry 69:88-98.
[17] DellaPenna D. (2005) Progress in the dissection and manipulation of vitamin E synthesis. Trends Plant
Sci 10:574-9.
[18] Valentin (2006) The Arabidopsis vitamin E pathway gene5-1 mutant reveals a critical role for phytol
kinase in seed tocopherol biosynthesis. Plant Cell. 18:212-24.
[19] Hossain et al. (2004) Enhancement of folates in plants through metabolic engineering. Proc Natl Acad
Sci USA 101:5158–5163.
[20] Knutzon et al., (1999) LPAAT from coconut endosperm mediates the insertionof laurate at the sn-2
position of triacylglycerols in Lauric rapeseed oil and can increase total laurate levels. Plant Physiology
120:739746.
[21] Thelen JJ, Ohlrogge JB. (2002) Metabolic engineering of fatty acid biosynthesis in plants. Metab Eng.
4:12-21.
[22] Chong et al. (2007) Growth and metabolism in sugarcane are altered by the creation of a new hexosephosphate sink. Plant Biotechnol J. 5:240-53.
[23] Wu (2007) Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. Pl.
Biotech. J. 5:109-17.
[24] Basnayake S. (2012) Field performance of transgenic sugarcane expressing isomaltulose synthase.
Plant Biotechnology Journal 10:217-225
[25] Geigenberger et al., (2004) Metabolic control analysis and regulation of the conversion of sucrose to
starch in growing potato tubers. Plant, Cell and Environment 27:655–673.
[26] Broun P. (2004) Transcription factors as tools for metabolic engineering in plants. Curr Opin Plant Biol.
7:202-9.
In rosso sono evidenziati quelli da leggere con attenzione ai fini dell’esame.
Ulteriori riferimenti bibliografici si trovano nei singoli file di powerpoint delle lezioni.
Chiunque desiderasse gli articoli originali basta me li chieda.
Cosa è naturale?
L’uomo fa parte della natura?
Da cosa viene la specialità dell’uomo?
Su cosa si fonda?
Gli esseri umani e la tecnologia sono una cosa sola?
Un compito...
Il docente universitario ha il compito non solo di indagare la verità e di
suscitarne perenne stupore, ma anche di promuoverne la conoscenza in
ogni sfaccettatura e di difenderla da interpretazioni riduttive e distorte.
Porre al centro il tema della verità non è un atto meramente speculativo,
ristretto a una piccola cerchia di pensatori; al contrario, è una questione
vitale per dare profonda identità alla vita personale e suscitare la
responsabilità nelle relazioni sociali.
Di fatto, se si lascia cadere la domanda sulla verità e la concreta
possibilità per ogni persona di poterla raggiungere, la vita finisce per
essere ridotta ad un ventaglio di ipotesi, prive di riferimenti certi.
BENEDETTO XVI
Pontificia Università Lateranense, Sabato, 21 ottobre 2006
http://www.vatican.va/holy_father/benedict_xvi/speeches/2006/october/documents/hf_ben-xvi_spe_20061021_lateranense_it.html