Download Citrate metabolism in lactic acid bacteria

FEMS Microbiology Reviews 12 (1993) 165-178
© 1993 Federation of European Microbiological Societies 0168-6445/93/$15.00
Published by Elsevier
165
FEMSRE 00327
Citrate metabolism in lactic acid bacteria
Jeroen Hugenholtz
Netherlands Institute for Dairy Research (NIZO), Ede, the Netherlands
Abstract: Citrate metabolism plays an important role in many food fermentations involving lactic acid bacteria. Since citrate is a
highly oxidized substrate, no reducing equivalents are produced during its degradation, resulting in the formation of metabolic end
products other than lactic acid. Some of these end products, such as diacetyl and acetaldehyde, have very distinct aroma properties
and contribute significantly to the quality of the fermented foods. In this review the metabolic pathways involved in product
formation from citrate are described, the bioenergetic consequences of this metabolism for the lactic acid bacteria are discussed
and detailed information on some key enzymes in the citrate metabolism is presented. The combined knowledge is used for
devising strategies to avoid, control or improve product formation from citrate.
Key words: Pyruvate metabolism; Citrate permease; Plasmid; Growth on citrate; a-Acetolactate; Diacetyl; Metabolic engineering
Introduction
Lactic acid bacteria that are involved in food
fermentations have one main property in common: a limited ability to metabolize food-borne
substrates. The main activity of the lactic acid
bacteria is to metabolize the sugars present as
fast as possible to the end product lactic acid.
Lactic acid bacteria are also able to ferment a
number of non-carbohydrates including citrate.
Citrate is present in many of the substrates which
are used for food fermentations such as fruit,
vegetables and milk and it is also used as additive
for the production of fermented sausages. It can
be fermented by a limited number of lactic acid
bacteria. Its degradation usually results in the
formation of unusual fermentation products such
Correspondence to: J. Hugenholtz, Netherlands Institute for
Dairy Research (NIZO), Erie, the Netherlands.
as diacetyl, acetoin, butanediol and acetaldehyde.
The formation of the aroma compound diacetyl
can have a distinct effect on the fermented food.
This effect can be positive as seen in dairy products such as butter(milk) and cottage cheese, but
it is detrimental in products such as beer, fermented sausage and wine. Therefore, there is a
strong need to control the production of diacetyi
in the food industry in general. This can only be
achieved by gaining complete insight in the lactic
acid bacteria metabolism leading to diacetyl production. In this review some of the progress in
this field will be discussed. Firstly, the metabolic
reactions involved in citrate degradation will be
described. Secondly, the benefit of citrate
metabolism to the microorganism will be discussed. Subsequently, some of the key enzymes in
citrate metabolism will be described in more detail, genetically and biochemically. Finally, all the
information available on the fermentation, biochemistry and genetics will be used to devise
strategies for the control a n d / o r improvement of
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citrate fermentation and subsequent aroma production by lactic acid bacteria.
Metabolic pathways
Most of the knowledge on the metabolic pathways involved in citrate metabolism has been
derived from dairy lactic acid bacteria. More than
a century ago the first aroma-producing bacteria
were recognized in the ripening of cream [1]. In
the following decades these bacteria were identified as either betacocci [2], later renamed Leuconostoc, or Streptococcus [3]. The latter microorganism was originally designated as a separate
species, Streptococcus diacetilactis but was later
reclassified as part of the Lactococcus lactis
species under the full name of L. lactis subsp.
lactis biovar, diacetylactis. The two groups of
'aromabacteria" were both discovered to have
specific citrate-utilizing abilities [4-6]. Collins and
co-workers [7] identified the enzyme reactions
that were specific for these citrate utilizers and
demonstrated the key role of the metabolic intermediate, pyruvate. Research in later years focussed on the location and regulation of the
genes responsible for citrate utilization and on
the exact mechanism of diacetyl production from
pyruvate. Some of these efforts will be described
here. A majority of this research has been done
on L. lactis subsp, lactis biovar, diacetylactis,
which will be referred to as citrate-utilizing L.
lactis in the text for simplicity.
Citrate permease
The instability of citrate utilization in Lactococcus was discovered in the early fifties by
Swartling [8] and later confirmed by Collins and
Harvey [9] who showed that lactococci could lose
the ability to transport citrate. The molecular
basis for this instability was provided by the work
of Kempler and McKay [10] who identified a
citrate plasmid in several citrate-utilizing L. lactis
strains that encoded the citrate permease. Subsequently. Gasson and co-workers [11] demonstrated that the citrate-fermenting ability was not
restored in a plasmid-free L. lactis strain by
introduction of the citrate plasmid, indicating that
also another (chromosome-encoded) enzyme,
presumably citrate lyase, is uniquely present in
citrate-utilizing L. lactis strains.
The presence of a citrate permease is essential
for metabolism of citrate. In the absence of the
citrate plasmid no citrate metabolism was observed although all enzymes necessary for citrate
conversion were present inside the cell. The essential role of the pcrmease in citrate metabolism
is further evident from the pH dependency of the
process that it catalyses. The citrate permease of
L. lactis was found to have a narrow pH optimum
with only appreciable activity between pH 5.0 and
6.0 [12]. The permeasc of Leuconostoc was found
to be homologous to the L. lactis permease [13]
and is likely to show a similar pH dependency as
the L. lactis protein. Within this pH range (5.06.0) both L. lactis and Leuconostoc have their
highest citrate metabolizing activity [14,15]. Below pH 5.0 citrate utilization is low in these
microorganisms due to low activity of the citrate
permease and low metabolic activity in general.
Resting cells of L. lactis were shown to utilize
citrate at pH levels as low as 4.5 [16] and when
immobilized in a hollow-fibre reactor some activity was observed at pH values down to pH 4.0
[17]. For a few lactic acid bacteria citrate utilization has been reported at even lower pH. In
Lactobacillus plantarum optimal citrate utilization
was described at pH 4.0 [18] and even lower pH
values were applied for citrate utilization in Leuconostoc oenos (Ana Ramos, personal communication). At these low pH values it is arguable
whether a citrate permease is necessary for citrate uptake since a considerable fraction of the
citrate (pK~ = 3.14, 4.77 and 5.40) in the medium
is present in the uncharged, acidic form, which
can easily diffuse through bacterial membranes.
Initial citrate breakdown
In all citrate-utilizing lactic acid bacteria citrate is converted initially to oxaloacetate and
acetate by the enzyme citrate [yase, in earlier
years also named citritasc. This enzyme seems to
be unique for the citrate utilizers since it is not
found in non-citrate-utilizing lactic acid bacteria
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[7,9,10,11]. The acetate production is very typical
for citrate utilization and, if detected in citrate
containing cultures of homofermentative lactic
acid bacteria, is a good indication for the occurrence of citrate metabolism.
In Lactococcus and Leuconostoc spp. oxaloacetate is decarboxylated to pyruvate. In the production of some semi-hard cheeses this reaction
is essential because the carbon dioxide produced
results in eye formation. Oxaloacetate decarboxylase is also present in some non-citrate-utilizing
strains, although at much lower levels (Perdon
and Hugenholtz, unpublished results), The enzyme has been purified from L. lactis [19] and
has a similar structure as the enzyme found in
Salmonella typhimuriurn [20].
In the genus Lactobacillus at least two different mechanisms of citrate utilization have been
reported. Lactobacillus pentosus uses part of the
citric acid cycle to reduce oxaloacetate to succihate, using the enzymes malate dehydrogenase,
fumarase and fumarate reductase [21]. In Lactobacillus plantarum both citrate conversion to succinate and via pyruvate to acetate and formate
have been reported [18,22].
Pyrm,ate metabolism
Citrate metabolism in lactic acid bacteria has
long been known to be associated with the production of other metabolites than lactic acid.
Besides the formation of acetate and carbon dioxide in the initial breakdown of citrate, the compounds acetoin, diacetyl and butanediol are often
produced by citrate-degrading lactic acid bacteria. It was first reported more than 30 years ago
that these C4 compounds were formed from
pyruvate [6,7,9]. However, the exact sequence of
reactions leading to the production of these compounds was, until recently, a matter of (heated)
debate. Collins and co-workers have presented
some evidence that diacetyl can be synthesized
directly from two C2-intermediates, hydroxyethyl
thiaminpyrophosphate ('active acetaldehyde') and
acetyl-CoA, in a reaction catalysed by the enzyme
diacetyl synthase [23,24]. Acetoin and butanediol
could subsequently be formed by enzymatic re-
duction of diacetyl. This pathway was broadly
accepted, up to the 1980s [25,26,27], although the
biochemical evidence supporting its existence remained scarce and the 'diacetyl synthase' had
never been identified. In another school of
thought [28,29] it is assumed that the production
of C4 compounds by lactic acid bacteria proceeds
in a similar way as found in other organisms such
as Bacillus subtilis [30], Klebsiella aerogenes [31],
and Serratia marcescens [32]. In this pathway one
C5-intermediate, a-acetolactate, is synthesized
from two pyruvate molecules and subsequently
decarboxylated to acetoin. The enzyme catalysing
this reaction, a-acetolactate synthase, was identified in several lactic acid bacteria [33-35] and
purified from Lactococcus lactis [36]. Its genc
from L. lactis has also been cloned and sequenced [37,38]. c~-Acetolactate decarboxylase
was purified from Lactobacillus casei [39] and
from L. lactis (Starrenburg and Hugenholtz, unpublished results). The enzymes in both lactic
acid bacteria showed very similar properties as
evidenced by the identical molecular masses, 29
kDa, and similar instability. Acetoin is either
excreted as end product or is reduced to butanediol catalysed by the enzyme acetoin reductase.
In this sequence of reactions diacetyl is only
produced as byproduct resulting from chemical
(aerobic) decarboxylation of the intermediate c~acetolactate. Recent studies backed by powerful
I~C nuclear magnetic resonance [40,41] and a
newly developed detection technique for c~acetolactate [14,36,42] have provided ample evidence that production of C4 compounds proceeds
via c~-acetolactate. No diacetyl production was
observed from either citrate- or pyruvate-utilizing
cells [14,40,41,43] or cell-free extracts [44] of L.
lactis. Diacetyl was only formed at low pH, under
aerobic conditions, even when pure a-acetolactate synthase was incubated with pyruvatc
[14,36,43]. This could only be a result of chemical
decarboxylation of a-acetolactate. This knowledge has been the basis for the development of
industrial processes leading to increased diacetyl
levels in butter [45] and margarine [46]. The persistent claims of direct diacetyl production by
lactic acid bacteria must be contributed to the
methodology that precludes determination of the
168
relative unstable metabolic intermediate aacetolactate [47-49].
The amount of C4 compounds that is produced from citrate is often (much) less than can
be expected from stoichiometric conversion of
pyruvate to a-acetolactate. This means that other
products such as acetate, formate and lactate are
formed from pyruvate, as confirmed in L. lactB
[14,40,41]. In batch cultivation, often in the presence of fermentable sugar, lactate and acetate
were the main fermentation products along with
the C4 compounds. In continuous culture under
lactose limitation citrate was converted primarily
into formate and acetate resulting from mixed
acid fermentation. At low externa[ pH and aerobic conditions the mixed acid fermentation was
A
B
lactate
f
acetate
~ . . . . . . ~ formate
PYRUVATE
,~./[~,,s~s,<:
PYRUVATE
ethanol
I
i
(X '
,i
j:~ :i,;
i~',
:!i x,: ~:!~,:
I
i
I ¸ :'~:
: ~ i',,,
I[]~
I
C
D
PYRUVATE
' I ~L~ li I,,11
[ac- rpp]
I
~-~ ~ ~:II ~ ~ ~j:,~4t~j~L~
~i~Jo~:~:]
(~-acetolactate
......
-:* diacetyl
i
1
~ f~J~a~ ~.~,/Ji ~:[
butanediol
Fig. l. Citrate and carbohydrate metabolism in L. lactis ssp. lactis biovar, diacetylactis involving four different pathways. (A)
Lactate i ~oduction via lactate dehydrogenase. (B) Formate and acetate or ethanol production via pyruvate formate lyase. (C)
Acetate and CO 2 production via pyruvate dehydrogenase. (D) a-Acetolactate (and, subsequently, acetoin and butanedioD
production via c~-acetolactate synthase.
169
replaced by C4-production a n d / o r (extra) acetate
and carbon dioxide production via pyruvate dehydrogenase activity. The variety of products that
can be formed is a result of four different pyruvate-converting reactions that are present in lactococci (Fig. 1):
(1) lactate dehydrogenase: pyruvate + N A D H -~
r,-lactate + NAD (Fig. 1A)
(2) pyruvate formate lyase: pyruvate --* acetyl-P +
formate (Fig. 1B)
(3) pyruvate dehydrogenase: pyruvate + NAD ---,
acetyl-CoA + CO 2 + N A D H (Fig. 1C)
(4) a-acetolactate synthase:
acetolactate + CO 2 (Fig. 1D)
2 pyruvate-~ a-
Shifts in product formation from lactose, glucose
and pyruvate upon aeration, changing of the pH
or cultivation under carbohydrate limitation are
generally observed in different homofermentative
lactic acid bacteria [50-53]. The complex mechanisms that are involved in regulation of pyruvate
metabolism L. lactis will be discussed below.
In Leuconostoc spp. isolated from dairy sources
pyruvate metabolism is (much) less complex than
in lactococci. The product formation from citrate
is similar under all cultivation conditions. The
pyruvate produced from citrate is primarily reduced to D-lactate. For this reaction to proceed it
is essential that co-fermentation with carbohydrates takes place providing the cells with the
necessary reducing power. The heterofermentative Leuconostoc is able to do this by producing
acetate instead of ethanol during sugar metabolism [14,54,55]. Only under extreme conditions, at
low pH and in the absence of sugar, some citrate
can be converted into the C4 compounds acetoin
and butanediol. In the acidophilic Leuconostoc
oenos product formation from citrate is also dependent on the external pH (Ana Ramos, personal communication).
Stability of citrate metabolism and effect on
growth
As described earlier, citrate metabolism is considered an unstable trait in lactic acid bacteria.
This instability is reportedly due to location of
the citrate permease gene on a plasmid [10-13].
However, quantitative data on the loss of citrate
plasmids by Lactococcus and Leuconostoc have
not been published. Furthermore, some of the
observations on the instability of citrate
metabolism in starter cultures have been made in
mixed populations and loss of citrate utilization
could merely be due to overgrowth by non-citrate
utilizers [56]. Recently, Smith and co-workers [15]
looked at the stability of citrate utilization in a
pure culture of L. lactis subsp, lact& var. diacetylactis C17 upon continuous cultivation for extended time periods. They found that the ability
to utilize citrate was completely retained in the
cultures without selective pressure while another
plasmid-encoded function, lactose metabolism,
was lost when cells were grown without lactose
over the same period of time. Even the rate of
citrate uptake was unaltered after growth in the
absence of citrate. Apparently, citrate metabolism
is much more stable under these conditions than
the lactose metabolism. The question can now be
asked why citrate metabolism is retained in lactic
acid bacteria, while it does not seem to be beneficial to cells themselves. Numerous authors have
reported that citrate metabolism does not support growth in the lactic acid bacteria [7,25,27,35].
This claim was based on the inability of the lactic
acid bacteria to grow in batch culture with citrate
added, in excess, as only energy source. However,
several observations have appeared in the literature recently that growth of some citrate utilizers
on carbohydrate-containing media is stimulated
in the presence of citrate [14,35,54-56]. In Lactobacillus plantarurn [18] and in Lactococcus lactis
[14], growth on citrate even seemed to occur in
the absence of a fermentable carbohydrate. The
growth stimulation by citrate as seen in Leuconostoc was first reported by Cogan [54] and
later confirmed by other research groups [14,55].
It is explained by a change in product formation
from lactose or glucose. As mentioned above,
citrate is almost stoichiometrically converted to
D-lactate by these microorganism. The reducing
power that normally would be used for production of ethanol is used instead for reduction of
pyruvate (produced from citrate) to D-lactate. The
170
acetyI-P that is produced from the carbohydrate
by heterofermentative degradation is now converted into acetate and ATP via the acetate kinase reaction. So, by the operation of citrate (or
pyruvate) as electron acceptor, more energy
(ATP) is generated during carbohydrate fermentation (see reactions 5-7):
(5)
glucose -~ 1 D-lactate + 1 ethanol + 2 C O ,
+ 1 ATP
glucose -~ 1 D-lactate + 1 acetate + 1 CO~
+ 2 ATP + 4 [H]
citrate + 2 [H] ~ 1 D-lactate + l acetate
+1 C O ,
(6)
glucose + 2 citrate --, 3 D-lactate + 3 acetate
+ 3 CO~ + 2 ATP
or
(7)
2 glucose + 2 citrate ~ 4 D-lactatc
+ 3 acetate + 1 ethanol + 4 CO~ + 3 A T P
The same p h e n o m e n o n has been reported in
Leuconostoc spp. using pyruvate and oxygen as
externally added electron acceptors [57].
In Lactococcus lactis the situation is quite
different. Carbohydrates and citrate are both metabolized with pyruvate as the central intermediate. Although L. lactis is characterized as a homofermentative lactic acid bacteria, several different products in addition to lactate can be
formed by pyruvate metabolism. Four different
pyruvate-utilizing pathways have been described
above: two leading directly to ATP production,
namely the conversion via p y r u v a t e / f o r m a t e lyase
with formate and acetate or ethanol as products;
and the conversion via the pyruvate dehydrogenase complex with acetate and carbon dioxide as
end products. In both cases A T P is formed by the
acetate kinase reaction. The pyruvate which is
formed from citrate breakdown will be converted
by one or more of the pyruvate-utilizing enzymes
depending on the cultivation conditions. Lactate
will not be produced by mere citrate metabolism
since no reducing equiwtlents arc formed during
citrate metabolism. Conversion of citrate into form a t e / a c e t a t e or a c e t a t e / c a r b o n dioxide has been
reported and indicates that metabolic energy
(ATP) can be produced during citrate metabolism
in L. lactis. This could explain the observed
growth of L. lactis on citrate in the absence of
fermentable carbohydrates. However, recent
studies [19] indicate that more energy is conserved by citrate metabolism than can be explained from substrate level phosphorylation by
the acetate kinase reaction. Even under conditions that pyruvate was not further metabolized
by L. lactis or under conditions that pyruvate was
converted, quantitatively, into aeetoin, growth of
L. lactis on citrate could still be observed. Apparently, additional energy is produced during initial
breakdown of citrate into pyruvate (and a c e t a t e /
CO2). The immediate generation of a proton
motive force upon citrate utilization, the relatively low production of ATP and the rapid uptake of a-aminoisobutyrate but not of glutamate
indicate that energy is generated by direct ion
movements over the m e m b r a n e as a result of
citrate metabolism [19]. A role of a membrane-associated Na t-pumping oxaloacetate decarboxylase, such as found in Klebsiella aerogenes [58],
was investigated and shown to be highly unlikely.
The L. lactis enzyme appeared to be a cytoplasmic and no dependency for Na ~ could be found.
The proposed mechanism of ion movements is by
the involvement of a s u b s t r a t e / p r o d u c t antiport
system which exchanges the di- or trivalent citrate
ion for a monovalent product in analogy to the
energy generation by malolactic fermentation in
L. lactis as described by Poolman in this volume
(59).
Regulation of key enzymes in citrate metabolism
The initial breakdown of citrate and the conversion of the intermediate pyruvate into specific
fermentation products can be regulated on different levels, depending on the microorganism. The
first step in citrate metabolism, the uptake of
citrate, is regulated by the pH of the growth
medium. The protein is constitutively expressed
in both Lactococcus and Leuconostoc but has a
171
narrow pH optimum as mentioned above. In this
section other key enzymes in citrate metabolism,
such as citrate lyase, all pyruvate-utilizing enzymes and acetoin reductase, will be discussed in
relation to possible regulation mechanisms.
Citrate lyase
In citrate-utilizing bacteria that have an intact
citric acid cycle, citrate cleaving and citrate synthesizing enzymes are present at the same time.
A very strict regulation of citrate lyase can be
expected in these organisms. In Enterobacter
aerogenes [60] citrate lyase is regulated by activity
of an electron transport chain and in Rhodopseudomonas gelatinosa the enzyme is activated by
acetylation induced by the presence of citrate in
the growth medium [61]. Although lactic acid
bacteria do not have a functional citric acid cycle,
regulation of citrate lyase has been observed in
some lactic acid bacteria. Mellerick and Cogan
[62] observed total induction of citrate lyase in
several Leuconostoc spp. and in Lactobacillus
t,iridescens upon addition of citrate to growing
cultures. Hugenholtz and Starrenburg reported a
20-fold increase in specific activity of citrate lyase
when Leuconostoc was grown in citrate-containing growth media [43]. In Lactococcus lactis no
regulation of citrate lyase was found in all strains
tested [35,43]. As discussed above, citrate lyase is
only present in the citrate-utilizing strains of L.
lactis.
Lactate dehydrogenase (Reaction 1)
Although lactate dehydrogenase (LDH) is not
directly involved in citrate metabolism, its regulation plays a crucial role in product formation
from citrate. L-Lactate dehydrogenase is regulated by two positive activators, fructose-l,6-diphosphate (FDP) and N A D H [63,64,65]. The first
is present at relatively high levels during active
glycolysis under carbohydrate excess and ensures
high L D H activity and rapid production of lactic
acid. The second is produced during glycolysis
and in addition to being an activator, it is also a
direct substrate for reduction of pyruvate to lactate. Neither of the activators of LDH is pro-
duced during metabolism of citrate resulting in
low activity of this enzyme when citrate is present
as only growth substrate. Subsequently, products
other than lactate are formed from citrate. Similar product profiles were observed in lactic acid
bacteria growing under lactose limitation in continuous cultures. Under these conditions internal
FDP levels are low resulting in reduced activity of
LDH [14,50,51].
D-Lactate dehydrogenase, which is present in
most heterofermentative lactic acid bacteria including Leuconostoc spp. [66] is not subject to
regulation. In citrate utilizers containing I~-LDH,
citrate is converted to lactate under all conditions
[14,54,55].
P);rut'ate formate lyase (Reaction 2)
Pyruvate formate lyase (PFL) is in many
(facultative) anaerobic bacteria responsible for
the production of formate, acetate and ethanol
from pyruvate; the so-called mixed acid fermentation [67,68]. The enzyme is present in most homofermentative lactic acid bacteria, but has not
been found in the heterofermentative Leuconostoc [14,54,55]. The enzyme is inactivated at low
oxygen levels and for that reason extensive biochemical information on this enzyme is lacking
from most microorganisms. In Escherichia co#
[69], an elaborate regulation has been described
involving anaerobic induction of transcription and
post-translational interconversion between an active and inactive form. The effect of oxygen on
PFL activity does present a powerful regulation
mechanism of pyruvate metabolism in lactic acid
bacteria. A switch from anaerobic to aerobic conditions has led to dramatic changes in product
profiles in several lactic acid bacteria with complete disappearance of ethanol and formate
[14,43,52,53]. This regulation by oxygen is also
observed in product formation from citrate in L.
lactis (Fig. 2). Another factor which regulates the
activity of PFL is the (internal) pH. Mixed acid
fermentations are usually observed when lactic
acid bacteria are grown at pH values near neutrality. When the pH drops to values below 6.0 no
more formate is produced from citrate or lactose.
This is in agreement with the narrow pH opti-
172
B
[ ..~.:_] Acetain
AL
6
E
5
quite different from other PDCs from other
sources in its unusual low activity of enzyme 3.
Because of this low activity the sensitivity for
N A D H was relatively high and in vivo P D C activity was only observed under strong aeration
[14,43].
~4
"O
O
Acetolactate synthase (Reaction 4)
It)
a-Acetolactate synthase (ALS) is present in
many different lactic acid bacteria [33,34,36,43].
It catalyses the TPP-dependent condensation reaction of two pyruvate molecules to the C5 component a-acetolactate with the release of carbon
dioxide. Actually, two different reactions with
both pyruvate as substrate are catalysed by this
enzyme. One pyruvate molecule is decarboxylated
with TPP acting as the coenzyme, resulting in the
formation of hydroxyethyl-TPP, also called 'active
acetaldehyde'. This enzyme-bound intermediate
reacts with the other pyruvate molecule to synthesize a-acetolactate. ALS from L. lactis was
purified and characterized [36]. The native enzyme measures 172 kDa and consists of three
identical 62-kDa monomers. Although the decarboxylation of pyruvate catalysed by ALS is an
identical reaction to the enzym~ 1 reaction of
PDC, no common components and no interactions were observed between P D C and ALS. The
ALS showed a very low affinity (Km = 50 raM)
and a strong positive cooperativity for pyruvate
interaction. This is an important regulating factor
in the production of acetoin and diacetyl, aAcetolactate will only be produced by the lactic
acid bacteria when pyruvate is accumulated inside the cells. Pyruvate accumulation can occur
when other, more effective, pyruvate-utilizing enzymes are inhibited or when the rate of pyruvate
production or influx exceeds its conversion rate.
Both situations occur in lactic acid bacteria,
sometimes simultaneously. As described in this
section L D H , PFL, and P D C are all regulated
and under certain conditions one or more of
these three enzymes can be inhibited leading to
pyruvate accumulation inside the cells. When L.
lactis cultures were aerated, inhibiting the PFLreaction, almost stoichiometric conversion of citrate (and even lactose) into a-acetolactate a n d / o r
¢..) 2
,q.
o
1
o ~
~
Nitrogen
Low Air
High Air
Gas Atmosphere
Fig. 2. Effect of aeration on citrate (and lactose) fermentation
in L. lactis Ru4 in lactose-limited continuous culture on
Ml7-medium with 10 mM citrate.
mum of 7.5 as reported for P F L in the lactic acid
bacterium Streptococcus mutans [68].
Pyrut,ate dehydrogenase (Reaction 3)
The pyruvate dehydrogenase complex (PDC) is
found in most aerobic and facultative anaerobic
microorganisms [70-72]. It consist of three different enzymes with enzyme 1 responsible for decarboxylation of pyruvate, enzyme 2 for regeneration
of cofactors and enzyme 3 for the oxidation reaction [73]. The activity of PDC is strongly dependent on the presence of the cofactors coenzyme
A and thiamin pyrophosphate (TPP). Reducing
power in the form of N A D H is produced as a
result of the reaction. This N A D H has a strong
inhibitory effect on enzyme activity, in particular
on enzyme 3 [74]. Under anaerobic conditions
when N A D H levels are relatively high in the
cytoplasm, usually no P D C activity is observed.
However, in Enterococcus faecalis clear evidence
has been presented that the enzyme complex is
indeed operative under anaerobic conditions [75].
Characterization of the E. faecalis enzyme showed
a lower sensitivity for N A D H inhibition compared to PDCs from other sources [76]. In several
other lactic acid bacteria the enzyme has also
been identified [77]. The L. lactis P D C has been
purified and characterized [36]. It appeared to be
173
acetoin was observed [43,78]. High rates of pyruvate production or influx can also occur in lactic
acid bacteria. When citrate is added under appropriate conditions to active cultures of L. lactis,
rapid uptake and conversion takes place resulting
in internal accumulation of pyruvate to concentrations of 50 mM and higher [78]. These conditions favour the production of a-acetolactate and
subsequent formation of acetoin, diacetyl or butanediol. This explains the observations that considerable amounts of pyruvate (upto 10 mM) can
be excreted into the growth media under conditions that the C4 compounds are formed
[14,19,43]. A similar situation can occur in the
lactic acid bacteria that can actively take up pyruvate such as E. faecalis [75] and Lactobacillus
plantarum [79].
Recently, Godon et al. [80] identified the gene
for another ALS in L. lactis. This enzyme is part
of the branched-chain amino acid biosynthesis
operon and is very similar to ALS present in
bacteria [81], plants [82] and animal tissue [83].
This biosynthetic ALS is consists of two subunits
and is regulated by the amino acids valine and
isoleucine by feedback inhibition. This enzyme is,
presumably, rarely present in L. lactis, since most
strains have an absolute growth requirement for
valine, leucine and isoleucine.
In different Leuconostoc spp. a 2-10-fold induction of ALS was observed during growth on
citrate [43,61]. Even under induced conditions the
ALS activity was much ( > 40 fold) lower than in
citrate-utilizing L. lactis strains [43]. This agrees
with the observed low production of C4 compounds from citrate by Leuconostoc [14,54-56].
Acetoin / diacetyl reductase
The lactic acid bacteria that produce acetoin
and diacetyl are also able to reduce these compounds to butanediol. Originally it was thought
that, in dairy lactic acid bacteria, two different
enzymes were involved in the two-step reduction
of diacetyl [62,84]. However, recent studies with
L. lactis have demonstrated clearly that one enzyme, acetoin reductase or butanediol dehydrogenase, catalyses both the irreversible reduction of
diacetyl (to acetoin) and the reversible reduction
of acetoin (to butanediol) [85,86]. In lactobacilli
and lactococci the enzyme uses N A D H as reducing cofactor [62,85,86] while in most Leuconostoc
spp. N A D P H is used for reduction [43,62]. The
enzyme was reported to be (slightly) repressed in
Lactococcus lactis and Leuconostoc in the presence of citrate [35,43,62]. When both acetoin and
diacetyl are present in the reaction mixture, both
substrates compete for reduction with acetoin
being the preferred substrate. The Knl for acetoin (0.2 mM) is much lower than for diacetyl (9
mM). At concentrations above 1 mM, acetoin has
a inhibitory effect on enzyme activity. The higher
affinity of acetoin reductase for acetoin than for
diacetyl together with the non-competitive inhibition of enzyme activity by acetoin is probably the
reason for the observed low rates of diacetyl
reduction in dairy products such as butter, buttermilk and cheese. These products, usually, contain
much higher amounts of acetoin than of diacetyl.
However, in some products diacetyl reduction
presents a problem. The rate of diacetyl reduction in these products could, possibly, be reduced
by increasing the acetoin levels.
Strain variation
In lactic acid bacteria a large variation is found
in product formation during fermentation. A
well-known example is the differences in lactose
conversion between homofermentative and heterofermentative lactic acid bacteria. As described
above, these basic differences also result in different product profiles during citrate metabolism.
Also, within the homofermentative lactic acid
bacteria complete different strategies are observed for citrate conversion. In Lactobacillus
pentosus and Lactobacillus plantarum citrate conversion to succinate (and acetate) was reported
[21,22], while in Lactococcus lactis acetoin, diacetyl, formate and acetate are the main products
from citrate metabolism [14,17,40]. Even within
one species, large variations between strains are
observed. In a strain of Lactobacillus plantarum
isolated from orange juice, acetate but no succinate was produced [18] from citrate. The best
documented strain differences are found within
174
the Lactococcus lactis species. The mesophilic
starter cultures that are used for production of
cheese, quark, sour cream, buttermilk and butter,
are all largely composed of L. lactis strains [29,87].
During the fermentation of these dairy products
the lactic acid bacteria utilize both citrate and
lactose simultaneously. However, for diacetyl production, in butter and buttermilk, specific starter
cultures are used which result in relatively high
diacetyl production [45,88]. In these starter cultures, apparently, some strains are present with
the ability to convert citrate effectively into diacetyl. From one high diacetyl-producing starter
culture, N I Z O 4 / 2 5 [45] different research groups
[14,16,25,37,40,43,44,89] have isolated a L. &otis
strain that accumulated large amounts of ~acetolactate upon citrate metabolism (Fig. 2).
Biochemical characterization of this strain (L.
lactis strain Ru4 = SD806 = 425A) showed that it
differed from other, non-diacetyl-producing,
strains only in one respect; it lacked the enzyme
~-acetolactate decarboxylase [43]. In this strain
citrate conversion to acetoin and butanediol is
blocked and the metabolic intermediate ~facetolactate is accumulated. Since ~-acetolactatc
is relatively unstable and is chemically decarboxylatcd to diacetyl ( a n d / o r acetoin), high levels of
diacetyl are found in dairy products fermented
with this strain.
A classic example of mutations leading to altered product profiles was reported by McKay
and Baldwin [90]. They described a naturally occurring L. lactis strain (KB) which lacked the
enzyme lactate dehydrogenase. In this bacterium
lactose was converted, in batch culture, into formatc, acetate, ethanol and large amounts of acetoin instead of lactate. The product profile of this
strain showed that even non-citrate-utilizing L.
lactis strains can effectively produce (74 compounds.
Other interesting variations within the L. lactis
species are the large strain differences in acetoin/diacetyl reductase activity. In citrate-utilizing strains high activity of this enzyme is always
observed [35,43,84]. In some non-citrate utilizers
(L. lactis E8 and S K l l ) no activity could be
detected, while in others such as L. lactis KB and
the related strain L. lactis" C2 activc acetoin
Table I
Activity in lactic acid b a c t e r i a of key e n z y m e s
metabolism
l,actic acid b a c t e r i u m
l.actococcus biovar.
diaccO'lactis (717 b
Lactococcus biovar.
diacety'lacti.~ Ru4 i,
l.actococcus laclis KB
Lactococcus lactic E8
E n z y m e activity
(p.molmin ling
in c i t r a t e
i)
CP '
LDH
ALS
ALD
DR
+
16
2.1)
0.08
0.0
+
18
0
15
2.4
1.5
0.4
0
+
+
0.8
0.1
I)
16
IO '
2.4
11.115
+
+
0
0.4 d
23
0,3
t-
0
(~
I). I
+
0.3
Lactococctt.~ lacti.s
SKI 1
]X'llCOIIOSIoc sp 60 i,
La('[O('OCCIIS plant a r u m N I Z O 4-1
n.d. ~'
[,llCtOCOCCIL~casit'
NIZO R
n.d.
~' CP, c i t r a t e p c r m c a s e : D R , diacety[ r e d u c t a s c .
b F r o m ref. [43]
D-lactate d e h y d r o g e n a s e .
d NADPH-dependcnt.
c Not d e t e r m i n e d .
reductasc was present ([84]; Starrenburg and
Hugenholtz, unpublished results). More subtle
differences are observed in activity of aacetolactatc synthase. Difference in specific ALS
activity will not be observed under ordinary conditions, but will be important tinder conditions
leading to high internal accumulation of the toxic
pyruvate.
These differences between species and strains
are summarized in Table 1. They demonstrate
how different mutations and variations can effect
metabolism in lactic acid bacteria and serve as
examples for metabolic engineering.
Metabolic engineering
The extensive microbiological, biochemical and
genetic information that is now available on citrate metabolism in lactic acid bacteria can be
used to control or improve diacetyl production
lot dairy application or to avoid diacetyl production in products such as beer. The development
of a fermentation procedure with high production
175
lactate
,:'~:, : ~ ),:°l~
(citrate)
~L
• ~
!2
PYRUVATE
acetate
~ formate
ethanol
C02
(acetaldehyde)
(x a c e t o l a c t a t e
3
) DIACETYL
acetoin "
4
butanediol
Fig. 3. Strategies fl~r metabolic engineering of diacetyl production in homofermentative lactic acid bacteria. Strategy 1,
LDH inactivation by lactose-limited growth, by low NADH or
by mutation/genetic engineering; strategy 2, PFL inactivation
by aeration and/or by low pH: strategy 3, ALD inactivation
by mutation/genetic engineering; strategy 4, DR inactivation
by mutation/genetic engineering or by acetoin inhibition:
strategy 5, ALS overproduction by genetic engineering
of diacetyl (up to 15 mM) from citrate on industrial scale, as reported by Wagendorp and
Hugenholtz [16], was based on the available
knowledge. The naturally occurring ALD-negative mutant L. lactis Ru4 was chosen as production strain and fermentation conditions were designed to achieve optimal citrate conversion into
a-acetolactate and subsequently into diacetyl. Interestingly, also some acetaldehyde (upto 0.5 mM)
was produced during the process (see Fig. 3).
Marugg and co-workers [38] employed genetic
engineering techniques to improve production of
a-acetolactate. They combined the strategies 3
and 5 in Fig. 3 by overproduction of a-acetolactate synthase in the ALD-negative L. lactis SD806
( - Ru4) leading to increased rates of aacetolactate production from citrate and pyruvate
[91]. Another approach to increase diacetyl production would be to combine the ALD-defective
phenotype with inactivation of lactate dehydrogenase and, possibly, other pyruvate-metabolizing
enzymes. This would lead to efficient diacetyl
production from lactose (Fig. 3). The inactivation
of L D H can, partially, be achieved by cultivation
under lactose limitation, but total inactivation is
only possible using genetic techniques. With the
known sequence of the L. lactis L D H [92] it
should be possible to combine the A L D and
L D H mutations. This engineered L. lactis is expected to produce considerable amounts of aacetolactate from lactose. However, as observed
in the L D H mutant L. lactis KB, high production
of formate and ethanol can also be expected. In
strain KB this production was greatly reduced by
aeration of the cultures (Hugenholtz, unpublished results) resulting in higher production of
acetoin. So, by combining the strategies (1), (2)
and (3) of Fig. 3 high production of ~-acctolactate from lactose should be possible by metabolic
engineering.
The available knowledge on a-acetolactate
production can also be used to reduce diacetyl
production in food products. In beer the
metabolic activity of yeasts is responsible for production of a-acetolactate. Since these organisms
lack a-acetolactate decarboxylase, ~-acetolactate
is excreted in the medium (beer) where it is then
converted into diacetyl. Addition of a-acetolactate decarboxylase isolated from different sources
to the beer fermentations led to a decrease of
diacetyl accumulation in beer [39]. The enzymes
from Bacillus brecis and Enterobacter aerogenes
have been cloned and sequenced [93-95]. This
progress has made it possible to apply metabolic
engineering in beer production. The A L D gene
of E. aerogenes was cloned and expressed in
Saccharornyces carlsbergensis, the brewer's yeast
[94]. This engineered yeast was successfully applied in wort fermentation for reduction of diacetyl formation.
It can be concluded that a combination of
metabolic regulation and genetic engineering is a
powerful procedure for directing metabolic fluxes
in industrial organisms. This metabolic engineering has been applied successfully in controlling
and improving diacetyl production of food products.
Acknowledgements
I would like to thank Marjo Starrenburg for
her assistance in our research on citrate metabo-
176
lism, and Willem M. de Vos, Ton Weerkamp and
Tjakko Abee for stimulating discussions throughout the work and Mark Smith for critically reading the manuscript. Finally, I am most grateful to
Walter Verhue, Joey Marugg and Aria Ramos for
providing me with information prior to publication.
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