Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.     

Citrate metabolism by Enterococcus faecium and Enterococcus durans isolated from goat's and ewe's milk: influence of glucose and lactose

Citrate metabolism by Enterococcus faecium ET C9 and Enterococcus durans Ov 421 was studied as sole energy source and in presence of glucose or lactose. Both strains utilized citrate as the sole energy source. Enterococcus faecium ET C9 showed diauxic growth in the presence of a limiting concentration of glucose. Neither strain used citrate until glucose was fully metabolized. The strains showed co-metabolism of citrate and lactose. Lactate, acetate, formate, and flavour compounds (diacetyl, acetoin, and 2,3-butanediol) were detected in both strains. The highest production of flavour compounds was detected during growth of E. durans Ov 421 in media supplemented with citrate-glucose and citrate-lactose. Citrate lyase was inducible in both strains. Acetate kinase activities presented the highest values in LAPTc medium, with E. faecium ET C9 displaying a specific activity 2.4-fold higher than E. durans. The highest levels of alpha-acetolactate synthase specific activity were detected in E. durans grown in LAPTc+g, in accordance with the maximum production of flavour compounds detected in this medium. Diacetyl and acetoinreductases displayed lower specific activity values in the presence of citrate. Enterococcus faecium and E. durans displayed citrate lyase, acetate kinase, alpha-acetolactate synthase, and diacetyl and acetoin reductase activities. These enzymes are necessary for conversion of citrate to flavour compounds that are important in fermented dairy products.     

Characterization of a citrate-negative mutant ofLeuconostoc mesenteroides subsp.mesenteroides: metabolic and plasmidic properties

Summary Comparison of the parental strain of the Leuconostoc mesenteroides subsp. mesenteroides (19D) and its citrate-negative mutant, which has lost a 22-kb plasmid, has confirmed the energetic role of citrate. Fermentation balance analysis showed that citrate led to a change in heterolactic fermentation from glucose. High levels of enzyme activity in both mutant and parental strains were found for NADH oxidase, lactate dehydrogenase, acetate kinase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, although NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase were partly repressed by citrate. All these enzymes studied were not plasmid linked. In the parental strain, citrate lyase was induced by citrate. No citrate lyase activity was found in the citrate-negative mutant grown in presence of citrate, but this does not provide evidence that citrate lyase is linked to the 22-kb plasmid. Offprint requests to: C. Diviès     

Effect of varying citrate levels on C4 compound formation and on enzyme levels in Leuconostoc mesenteroides subsp. cremoris grown in continuous culture

Summary The effects of citrate on diacetyl, acetoin and 2,3-butylene glycol (2,3-BG) production by Leuconostoc mesenteroides subsp. cremoris grown in continuous culture at pH 5.2 were studied. In glucose alone end-product production agreed with the theoretical stoichiometry. In the presence of citrate, lactate and acetate production was higher than the theoretical stoichiometry from glucose. Lactate production was constant when the initial citrate concentration was increased whereas ethanol production strongly decreased. In the absence of citrate, citrate lyase (CL) exhibited weak activity. Diacetyl reductase (DR) and acetoin reductase (AR) exhibited basal activity. When varying citrate concentrations ranging from 10 to 75 mm were added to glucose broth, DR, AR, lactate dehydrogenase, NADH oxidase and alcohol dehydrogenase decreased as the initial citrate concentration increased suggesting that they were partly repressed by citrate. In contrast, CL increased and the specific citrate utilization rate also increased in the same way, indicating no saturation of the first step of citrate metabolism. Acetate kinase (AK) was slightly higher in the presence of citrate and increased when the initial citrate concentration increased. This result was correlated with an increase of acetate from the acetyl phosphate pathway. More ATP was produced in the presence of citrate, which could explain the increase in biomass formation. Citrate bioconversion into diacetyl, acetoin and 2,3-BG increased as the initial citrate increased. Correspondence to: C. Diviès     

Redirection of pyruvate catabolism in Lactococcus lactis by selection of mutants with additional growth requirements

Based on requirements for acetate or lipoic acid for aerobic (but not anaerobic) growth, Lactococcus lactis subsp. lactis mutants with impaired pyruvate catabolism were isolated following classical mutagenesis. Strains with defects in one or two of the enzymes, pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH) and the pyruvate dehydrogenase complex (PDHC) were obtained. Growth and product formation of these strains were characterized. A PFL-defective strain (requiring acetate for anaerobic growth) displayed a two-fold increase in specific lactate production compared with the corresponding wild-type strain when grown anaerobically. LDH defective strains directed 91-96% of the pyruvate towards alpha-acetolactate, acetoin and diacetyl production when grown aerobically in the presence of acetate and absence of lipoic acid (a similar characteristic was observed in an LDH and PDHC defective strain in the presence of both acetate and lipoic acid) and more than 65% towards formate, acetate and ethanol production under anaerobic conditions. Another strain with defective PFL and LDH was strictly aerobic. However, a variant with strongly enhanced diacetyl reductase activities (NADH/NAD+ dependent diacetyl reductase, acetoin reductase and butanediol dehydrogenase activities) was selected from this strain under anaerobic conditions by supplementing the medium with acetoin. This strain is strictly aerobic, unless supplied with acetoin.     

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.     

Genetic manipulation of the pathway for diacetyl metabolism in Lactococcus lactis.

Diacetyl is an important food flavor compound produced by certain strains of citrate-metabolizing lactic acid bacteria. Citrate is converted to pyruvate, from which diacetyl is produced via intermediate alpha-acetolactate. This paper reports the cloning and analysis of the gene (aldB) encoding alpha-acetolactate decarboxylase from Lactococcus lactis MG1363. Deletion of the MG1363 chromosomal aldB gene was achieved by double crossover homologous recombination. The mutant strain was found to produce diacetyl; alpha-acetolactate decarboxylase activity was eliminated. Overexpression of the cloned ilvBN genes (encoding an alpha-acetolactate synthase) in the aldB deletion strain produced even higher levels of alpha-acetolactate, acetoin, and diacetyl.     

Influence of citrate on the diacetyl and acetoin production by fully grown cells ofStreptococcus lactis subsp.diacetylactis

The effects of citrate on diacetyl and acetoin level by fully grown cells ofStreptococcus lactis subsp.diacetylactis CNRZ 124 were studied. In the absence of citrate, diacetyl synthase as well as acetolactate synthase and acetoin and diacetyl reductases exhibited a basal activity confirming their constitutive nature. However, when initial citrate concentration ranged from 8.8 to 59 mM, the enzyme levels increased in the same way, indicating no saturation rate of citrate metabolism. These results were reflected by a similar enhancement in acetoin and diacetyl production. When citrate was added in fed-batch conditions, its utilization by the fully grown cells led to a twofold increase in diacetyl yield over batch conditions.     

Effect of Initial Oxygen Concentration on Diacetyl and Acetoin Production by Lactococcus lactis subsp. lactis biovar diacetylactis

The production of aroma compounds (acetoin and diacetyl) in fresh unripened cheese by Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 was studied at 30°C at different initial oxygen concentrations (0, 21, 50, and 100% of the medium saturation by oxygen). Regardless of the initial O₂ concentration, maximal production of these compounds was reached only after all the citrate was consumed. Diacetyl and acetoin production was 0.01 and 2.4 mM, respectively, at 0% oxygen. Maximum acetoin concentration reached 5.4 mM at 100% oxygen. Diacetyl production was increased by factors of 2, 6, and 18 at initial oxygen concentrations of 21, 50, and 100%, respectively. The diacetyl/acetoin concentration ratio increased linearly with initial oxygen concentration: it was eight times higher at 100% (3.3%) than at 0% oxygen (0.4%). The effect of oxygen on diacetyl and acetoin production was also shown with other lactococci. At 0% oxygen, specific activity of α-acetolactate synthetase (0.15 U/mg) and NADH oxidase (0.04 U/mg) was 3.6 and 5.4 times lower, respectively, than at 100% oxygen. The increasing α-acetolactate synthetase activity in the presence of oxygen would explain the higher production of diacetyl and acetoin. The NADH oxidase activity would replace the role of the lactate dehydrogenase, diacetyl reductase, and acetoin reductase in the reoxidation of NADH, allowing accumulation of these two aroma compounds.     

Citrate permease gene expression in Lactococcus lactis subsp. lactis strains IL1403 and MG1363

Abstract Citrate permease gene expression in the plasmid-free Lactococcus lactis strains IL1403 and MG1363 was studied. The ability to transport citrate results in diacetyl and acetoin production in IL1403 but not in MG1363. Citrate lyase, α-acetolactate decarboxylase, diacetyl and acetoin reductase were detected in IL1403. These data show that L. lactis ssp. lactis strain IL1403 is a citrate permease mutant of the biovar. diacetylactis . Immunological analysis revealed the α-and β-subunits of citrate lyase not only in IL1403 but also in MG1363 where no citrate lyase activity was found.     

Citric acid metabolism in hetero- and homofermentative lactic acid bacteria.

The effect of citrate on production of diacetyl and acetoin by four strains each of heterofermentative and homofermentative lactic acid bacteria capable of utilizing citrate was studied. Acetoin was quantitatively the more important compound. The heterofermentative bacteria produced no acetoin or diacetyl in the absence of citrate, and two strains produced traces of acetoin in its presence. Citrate stimulated the growth rate of the heterofermentative lactobacilli. Acidification of all heterofermentative cultures with citric acid resulted in acetoin production. Destruction of accumulated acetoin appeared to coincide with the disappearance of citrate. All homofermentative bacteria produced more acetoin and diacetyl in the presence of citrate than in its absence. Citrate utilization was begun immediately by the streptococci but was delayed until at least the middle of the exponential phase in the case of the lactobacilli.     

Citric acid metabolism in hetero- and homofermentative lactic acid bacteria.

The effect of citrate on production of diacetyl and acetoin by four strains each of heterofermentative and homofermentative lactic acid bacteria capable of utilizing citrate was studied. Acetoin was quantitatively the more important compound. The heterofermentative bacteria produced no acetoin or diacetyl in the absence of citrate, and two strains produced traces of acetoin in its presence. Citrate stimulated the growth rate of the heterofermentative lactobacilli. Acidification of all heterofermentative cultures with citric acid resulted in acetoin production. Destruction of accumulated acetoin appeared to coincide with the disappearance of citrate. All homofermentative bacteria produced more acetoin and diacetyl in the presence of citrate than in its absence. Citrate utilization was begun immediately by the streptococci but was delayed until at least the middle of the exponential phase in the case of the lactobacilli.     

Imbalance of leucine flux in Lactococcus lactis and its use for the isolation of diacetyl-overproducing strains.

Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen. L. lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene. We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium. Industrial dairy strains of L. lactis subsp. lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized. Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk. Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu

CitrLactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu. On the other hand, the Y(Glu) value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP(i). In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.     

Localization of Lactococcus lactis ssp lactis bv diacetylactis in alginate gel beads affects biomass density and synthesis of several enzymes involved in lactose and citrate metabolism

Summary Lactococcus lactis ssp lactis bv diacetylactis, immobilized in calcium alginate beads, was grown in synthetic medium in a continuous flow reactor. Cell distribution inside the gel, as well as the activity of various enzymes, was measured after 30 h of operation. The included biomass tended to concentrate at the periphery of the bead along a section of radius about 100 m long. ATPase activity was maximal in this zone. The activity of NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, which are repressed in the presence of citrate, were higher in the deeper zones than at the surface of the beads. This result shows that only the peripheral zone of the bead is responsible for the bioconversion of citrate into flavour compounds (diacetyl and acetoin).     

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation. Received: 6 December 1996 / Received revision: 14 February 1997 / Accepted: 14 February 1997     

Diacetyl, Acetoin, and Acetaldehyde Production by Mixed-Species Lactic Starter Cultures

Citrate utilization and acetoin, diacetyl, acetaldehyde, and lactic acid production in milk at 21 C by five different mixed-strain starters, containing Streptococcus diacetilactis (D type), Leuconostoc (B type), and S. diacetilactis and Leuconostoc (BD type), were measured. BD and D cultures utilized citrate more rapidly and produced more diacetyl, acetoin, and acetaldehyde than B types. All cultures produced much more acetoin than diacetyl, with the BD and D cultures producing four to five times larger amounts of acetoin than the B cultures. Reduction of diacetyl and acetoin toward the end of the normal incubation period was characteristic of BD and D cultures, whereas a similar reduction of acetaldehyde was characteristic of BD and especially of B cultures. Continued incubation of B cultures beyond 17 h also resulted in reduction of diacetyl and acetoin. Addition of citrate to the milk retarded diacetyl and acetoin reduction. Mn²⁺ had no effect on diacetyl production by a BD culture but increased citrate utilization and, as a consequence, caused greater diacetyl destruction in one of the B cultures.     

Citrate Fermentation by Lactococcus and Leuconostoc spp

Citrate and lactose fermentation are subject to the same metabolic regulation. In both processes, pyruvate is the key intermediate. Lactococcus lactis subsp. lactis biovar diacetylactis homofermentatively converted pyruvate to lactate at high dilution (growth) rates, low pH, and high lactose concentrations. Mixed-acid fermentation with formate, ethanol, and acetate as products was observed under conditions of lactose limitation in continuous culture at pH values above 6.0. An acetoin/butanediol fermentation with alpha-acetolactate as an intermediate was found upon mild aeration in continuous culture and under conditions of excess pyruvate production from citrate. Leuconostoc spp. showed a limited metabolic flexibility. A typical heterofermentative conversion of lactose was observed under all conditions in both continuous and batch cultures. The pyruvate produced from either lactose or citrate was converted to d-lactate. Citrate utilization was pH dependent in both L. lactis and Leuconostoc spp., with maximum rates observed between pH 5.5 and 6.0. The maximum specific growth rate was slightly stimulated by citrate, in L. lactis and greatly stimulated by citrate in Leuconostoc spp., and the conversion of citrate resulted in increased growth yields on lactose for both L. lactis and Leuconostoc spp. This indicates that energy is conserved during the metabolism of citrate.