Co-metabolism of citrate and lactose by Leuconostoc mesenteroides subsp. cremoris

Citrate stimulated growth rate, increased the specific lactose consumption rate and enhanced the molar growth yield of Leuconostoc mesenteroides subsp. cremoris growing on lactose at pH 5.2 or 6.2 and at 22 or 30°C. As soon as citrate utilization began, diacetyl and acetoin were produced: 2,3-butylene glycol appeared later while acetoin decreased.     

Microbial Production of 2,3-Butylene Glycol from Cheese Whey

Six microorganisms that produced acetoin or diacetyl or both from glucose were tested for the production of 2,3-butylene glycol from lactose. Bacillus polymyxa and Streptococcus faecalis gave positive results and were tested in unmodified wheys. Cottage cheese whey was unsatisfactory, but B. polymyxa produced large amounts of the glycol in sweet whey, about 60 mmol of glycol per 100 mmol of lactose utilized. Aeration and an increased ratio of surface area to volume of whey enhanced the production of glycol. 2,3-Butylene was separated from the spent whey and from acetoin and diacetyl with a Sephadex G-10 column.     

Separation of diacetyl, acetoin, and 2,3-butylene glycol by ion-exchange chromatography

Mixtures of diacetyl, acetoin, and 2,3-butylene glycol were quantitatively separated by ion-exchange chromatography on Dowex 1-X8 resin in the bisulfite form. Initial elution with water removed 2,3-butylene glycol from the column. Further elution with 0.1 m NaCl separated acetoin from diacetyl. Sulfite in the eluates was deactivated with $\text{I}{}_2\text{KI}$ reagent. After oxidation by bromine, 2,3-butylene glycol was measured as acetoin. Excess bromine was neutralized by addition of 40% NaOH and saturated Na₂S₂O₅. After separation and conversion of the glycol to acetoin, the Westerfeld colorimetric method was used to determine the three components quantitatively.     

A descriptive model for citrate utilization by Lactococcus lactis ssp lactis bv diacetylactis

A model for the use of citrate by Lactococcus lactis ssp lactis bv diacetylactis CNRZ 125 is proposed. Citrate metabolism by this strain leads to the production of acetate, CO₂ and C4 compounds (diacetyl, acetoin, 2,3-butylene glycol). The model furnishes correct simulations, consistent with published results on the pathways used and on lactose-citrate co-metabolism. Citric acid is incorporated independently of growth. The production of flavoring compounds is a complex process, depending on the rate of citrate utilization, on the proportion of pyruvate arising from citrate and which condenses to form -acetolactate and CO₂, on the rate of transformation of -acetolactate to diacetyl and acetoin, as well as on the rate of reduction of these compounds to 2,3-butylene glycol.     

Incorporation of Radioactive Acetate into Diacetyl by Streptococcus diacetilactis

Streptococcus diacetilactis was grown in a partially defined, lipoic acid-free medium containing radioactive acetate with and without addition of 0.1% unlabeled sodium pyruvate. Labeled carbon was incorporated into diacetyl, but neither the amount of diacetyl produced nor its specific activity was influenced by addition of pyruvate. Acetoin had low specific activity, indicating that it was a mixture of radioactive and nonradioactive acetoin. The specific activity of acetoin was lower when pyruvate, a precursor of unlabeled acetoin, was added to the medium, which indicated that the radioactive acetoin was produced from radioactive diacetyl by diacetyl reductase. Results substantiate condensation of acetyl-coenzyme A with hydroxyethylthiamine pyrophosphate as the in vivo mechanism for synthesis of diacetyl.     

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     

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.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu2+

Citr⁺Lactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu²⁺. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu²⁺, 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 Cu²⁺. 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 Cu²⁺ 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 Cu²⁺ 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.     

Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation

Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.     

Characterization of a stereospecific acetoin(diacetyl) reductase from Rhodococcus erythropolis WZ010 and its application for the synthesis of (2S,3S)-2,3-butanediol

Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K _m values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.     

Use of Gas-Liquid Chromatography to Determine the End Products of Growth of Lactic Acid Bacteria

A simple gas-liquid chromatographic procedure for analyzing ethanol, acetic acid, acetoin, and racemic and meso-2,3-butylene glycol in broth media is described. Overnight broth cultures were filtered or centrifuged, and the filtrate or supernatant was treated with formic acid to aid separation of volatile fatty acids. Samples were then directly analyzed by gas-liquid chromatography on a 20% Tween 80-Chromosorb W-AW column and propionic acid as an internal standard. A complete analysis took ca. 8 min. The method can be used to distinguish homofermentative from heterofermentative lactic acid bacteria based on the level of ethanol produced and citrate-utilizing from non-citrate-utilizing lactic acid bacteria based on the levels of acetic acid produced. The method also has potential in distinguishing other bacterial fermentations. Of the 13 species of lactic acid bacteria tested, Streptococcus lactis subsp. diacetylactis was the major producer of 2,3-butylene glycol (total range, 0.3 to 3.5 mM), and, except for strain DRC1, both the racemic and meso isomers were produced in approximately equal amounts.     

Selection of an endogenous 2,3-butanediol pathway in Escherichia coli by fermentative redox balance

Fermentative redox balance has long been utilized as a metabolic evolution platform to improve efficiency of NADH-dependent pathways. However, such system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. In this study, endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. In this case, feeding of the 2,3-BD precursor acetoin effectively restored anaerobic growth of the mixed-acid fermentation mutant that remained otherwise inhibited even in the presence of a functional 2,3-BD pathway. Using established 2,3-BD dehydrogenases as model enzyme, we verified that the redox-based selection system is responsive to NADPH-dependent reactions but with lower sensitivity. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol/1,2-propanediol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity (k_cat/K_m≈0.4 mM⁻¹ s⁻¹) observed in E. coli. Significant shift of 2,3-BD configuration upon withdrawal of the heterologous acetolactate decarboxylase revealed that the endogenous synthesis of acetoin occurs via diacetyl. Among the predicted diacetyl reductase in E. coli, Ec-UcpA displayed the most significant activity towards diacetyl reduction into acetoin (V_max≈6 U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3 g/L without introduction of foreign genes. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme bioprospecting and metabolic evolution.     

Effect of lactic acid on diacetyl and acetoin production byLactobacillus casei subsp.rhamnosus ATCC 7469

Lactic acid or its acidity apparently play an important role in the regulation of the biosynthesis of flavor compounds inLactobacillus casei subsp.rhamnosus ATCC 7469. In pyruvate-containing media,L. casei produces lactic acid, acetoin, and diacetyl. A specific pH-dependent system is necessary for both the use of pyruvate and the induction of acetoin and diacetyl production. In cell extracts ofL. casei, lactic acid inhibits the enzymatic activity of acetolactate decarboxylase (ALD) and acetolactate synthetase (ALS); this effect does not occur in whole cells under standard physiological conditions. Lactic acid prevents the use of pyruvate, and the induction of acetoin and diacetyl production. When pyruvate-containing media are used, the pH must be kept close to 6.0 in order to obtain the best production of acetoin and diacetyl.     

Cloning,expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K _m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation–reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe²⁺ and for (3S/3R)-acetoin reduction in the presence of Mn²⁺, while several cations inhibited its activity, particularly Fe²⁺ and Fe³⁺ for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .     

Metabolic engineering of Bacillus sp. for diacetyl production

Diacetyl, a highly valuable product that is extensively used as an ingredient of food, tobacco, and daily chemicals such as perfumes, can be produced from the nonenzymatic oxidative decarboxylation of α-acetolactate during bacterial fermentation and converted to acetoin and 2,3-butanediol by 2,3-butanediol dehydrogenase. In the present study, Bacillus sp. DL01, which gives high acetoin production, was metabolically engineered to improve diacetyl production. After the deletion of α-acetolactate decarboxylase (ALDC)-encoding gene (alsD) by homologous recombination, the engineered strain, named Bacillus sp. DL01-ΔalsD, lost ALDC activity and produced 1.53 g/L diacetyl without acetoin and 2,3-butanediol accumulation. The channeling of carbon flux into diacetyl biosynthetic pathway was amplified by an overexpressed α-acetolactate synthase (ALS)-encoding gene (alsS) in Bacillus sp. DL01-ΔalsD-alsS, which produced 4.02 g/L α-acetolactate and 1.94 g/L diacetyl, and the conversion from α-acetolactate to diacetyl was increased by 1-fold after 20 mM Fe³⁺ was added to the fermentation medium. A titer of 8.69 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation in optimal conditions using the metabolically engineered strain of Bacillus sp. DL01-ΔalsD-alsS. These results are of great importance as a new method for the efficient production of diacetyl by food-safe bacteria.     

Metabolic engineering of acetoin and meso-2, 3-butanediol biosynthesis in E. coli

The functional reconstruction of acetoin and meso-2,3-butanediol (meso-2,3-BD) biosynthetic pathways in Escherichia coli have been explored systematically. Pathway construction involved the in vsivo screening of prospective pathway isozymes of yeast and bacterial origin. After substantial engineering of the host background to increase pyruvate availability, E. coli YYC202(DE3) ldhA⁽ ilvC( expressing ilvBN from E. coli and aldB from L. lactis (encoding acetolactate synthase and acetolactate decarboxylase activities, respectively) was able to produce up to 870 mg/L acetoin, with no coproduction of diacetyl observed. These strains were also found to produce small quantities of meso-2,3-BD, suggesting the existence of endogenous 2,3-BD dehydrogenase activity. Finally, the coexpression of bdh1 from S. cerevisiae, encoding 2,3-BD dehydrogenase, in this strain resulted in the production of up to 1120 mg/L meso-2,3-BD, with glucose a yield of 0.29 g/g. While disruption of the native lactate biosynthesis pathway increased pyruvate precursor availability to this strain, increased availability of NADH for acetoin reduction to meso-2,3-BD was found to be the most important consequence of ldhA deletion.     

Conversion of Pyruvate to Acetoin Helps To Maintain pH Homeostasis in Lactobacillus plantarum

Pyruvate is the substrate for diacetyl and acetoin synthesis by lactobacilli. Exogenous pyruvate stimulates acetoin production when glucose is present as an energy source. In Lactobacillus plantarum ATCC 8014, the energy derived from glucose via glycolysis generated a constant proton motive force of about -120 mV. At a low external pH, energized cells rapidly transported and accumulated pyruvate but did not do so when they were deenergized by nigericin. When large amounts of pyruvate were transported and subsequently accumulated internally, the cotransported protons rapidly lowered the internal pH. The conversion of pyruvate to acetoin instead of acidic end products contributed to the maintenance of pH homeostasis. This is the first report showing that the conversion of pyruvate to acetoin serves as a mechanism of pH homeostasis.     

High-Efficiency Conversion of Pyruvate to Acetoin by Lactobacillus plantarum during pH-Controlled and Fed-Batch Fermentations

The influence of pH on the type and concentration of metabolites produced from pyruvate by Lactobacillus plantarum ATCC 8014 was examined in pH-controlled fermentors at pH values of 4.5 to 6.5. Specific growth rates, cell dry weights, and diacetyl concentrations were highest at pH 5.5, with values of 0.78 h⁻¹, 190 mg/liter, and 1.2 mM, respectively. While the conversion efficiency (millimoles of acetoin formed per millimoles of pyruvate utilized) was highest (94.6%) at pH 4.5, acetoin levels were similar (20 mM) between pH 4.5 and 5.5. Feeding stationary-phase cells exogenous pyruvate increased acetoin levels to 78 mM.     

Reducing diacetyl production of wine by overexpressing <Emphasis Type="Italic">BDH1</Emphasis> and <Emphasis Type="Italic">BDH2</Emphasis> in <Emphasis Type="Italic">Saccharomyces uvarum</Emphasis>

As a byproduct of yeast valine metabolism during fermentation, diacetyl can produce a buttery aroma in wine. However, high diacetyl concentrations generate an aromatic off-flavor and poor quality in wine. 2,3-Butanediol dehydrogenase encoded by BDH1 can catalyze the two reactions of acetoin from diacetyl and 2,3-butanediol from acetoin. BDH2 is a gene adjacent to BDH1, and these genes are regulated reciprocally. In this study, BDH1 and BDH2 were overexpressed in Saccharomyces uvarum to reduce the diacetyl production of wine either individually or in combination. Compared with those in the host strain WY1, the diacetyl concentrations in the recombinant strains WY1-1 with overexpressed BDH1, WY1-2 with overexpressed BDH2 alone, and WY1-12 with co-overexpressed BDH1 and BDH2 were decreased by 39.87, 33.42, and 46.71%, respectively. BDH2 was only responsible for converting diacetyl into acetoin, but not for the metabolic pathway of acetoin to 2,3-butanediol in S. uvarum. This study provided valuable insights into diacetyl reduction in wine.     

Radioimmunoassay of rat liver metallothionein

A simple, inexpensive and convenient radioimmunoassay for rat liver metallothionein has been developed. The double-antibody assay involves the labeling of homogeneous, rat liver zinc thionein with trace amounts of ¹⁰⁹Cd(II) to a specific activity of 1–2 × 10⁶ cpm/μg protein; the binding of this antigen by rabbit anti-rat liver metallothionein antiserum; the displacement of this antigen by unlabeled zinc thionein or cadmium, zinc-thionein; the precipitation of the rabbit antibody-rat antigen complex by goat anti-rabbit IgG immunoglobulins; and the binding of this precipitate to cellulose acetate filters. The radioimmunoassay is useful in the range of concentration of metallothionein of 10–500 ng protein. The assay is accurate as compared with a previous technique of quantitating metallothionein in extracts of rat liver. A radial immunodiffusion assay for metallothionein is also described.