Α-acetolactate Decarboxylase Research Articles

Thiamine-Starved Lactococcus lactis for Producing Food-Grade Pyruvate.

Lactococcus lactis is a safe lactic acid bacterium widely used in dairy fermentations. Normally, its main fermentation product is lactic acid; however, L. lactis can be persuaded into producing other compounds, e.g., through genetic engineering. Here, we have explored the possibility of rewiring the metabolism of L. lactis into producing pyruvate without using genetic tools. Depriving the thiamine-auxotrophic and lactate dehydrogenase-deficient L. lactis strain RD1M5 of thiamine efficiently shut down two enzymes at the pyruvate branch, the thiamine pyrophosphate (TPP) dependent pyruvate dehydrogenase (PDHc) and α-acetolactate synthase (ALS). After eliminating the remaining enzyme acting on pyruvate, the highly oxygen-sensitive pyruvate formate lyase (PFL), by simple aeration, the outcome was pyruvate production. Pyruvate could be generated by nongrowing cells and cells growing in a substrate low in thiamine, e.g., Florisil-treated milk. Pyruvate is a precursor for the butter aroma compound diacetyl. Using an α-acetolactate decarboxylase deficient L. lactis strain, pyruvate could be converted to α-acetolactate and diacetyl. Summing up, by starving L. lactis for thiamine, secretion of pyruvate could be attained. The food-grade pyruvate produced has many applications, e.g., as an antioxidant or be used to make butter aroma.

2-Hydroxyisovalerate production by Klebsiella pneumoniae

2-Hydroxyisovalerate is a valuable chemical that can be used in the production of biodegradable polyesters. In nature, it was only produced at a very low level by Lactococcus lactis. 2-Ketoisovalerate is an intermediate metabolite of the branched-chain amino acid biosynthesis pathway, and Klebsiella pneumoniae ΔbudAΔldhA (Kp ΔbudAΔldhA) was a 2-ketoisovalerate producing strain. In this research, 2-hydroxyisovalerate was identified as a metabolite of Kp ΔbudAΔldhA, and its synthesis pathway was revealed. It was found that 2-ketoisovalerate and 2-hydroxyisovalerate were produced by Kp ΔbudA and Kp ΔbudAΔldhA, but not by Kp ΔbudAΔldhAΔilvD in which the 2-ketoisovalerate synthesis was blocked. budA, ldhA, and ilvD encode α-acetolactate decarboxylase, lactate dehydrogenase, and dihydroxy acid dehydratase, respectively. Thus, it was deduced that 2-hydroxyisovalerate was synthesized from 2-ketoisovalerate. Isoenzymes of ketopantoate reductase PanE, PanE2, and IlvC were suspected of being responsible for this reaction. Kinetic parameters of these enzymes were detected, and they all hold the 2-ketoisovalerate reductase activities. PanE and PanE2 use both NADH and NADPH as co-factors. While IlvC only uses NADH as a co-factor. Over-expression of panE, panE2, or ilvC in Kp ΔbudAΔldhA all enhanced the production of 2-hydroxyisovalerate. Accordingly, 2-hydroxyisovalerate levels were reduced by knocking out panE or panE2. In fed-batch fermentation, 14.41 g/L of 2-hydroxyisovalerate was produced by Kp ΔbudAΔldhA-panE, with a substrate conversion ratio of 0.13 g/g glucose.

Efficient production of acetoin from lactate by engineered Escherichia coli whole-cell biocatalyst.

Acetoin, an important and high-value added bio-based platform chemical, has been widely applied in fields of foods, cosmetics, chemical synthesis, and agriculture. Lactate is a significant intermediate short-chain carboxylate in the anaerobic breakdown of carbohydrates that comprise ~ 18% and ~ 70% in municipal wastewaters and some food processing wastewaters, respectively. In this work, a series of engineered Escherichia coli strains were constructed for efficient production of acetoin from cheaper and abundant lactate through heterogenous co-expression of fusion protein (α-acetolactate synthetase and α-acetolactate decarboxylase), lactate dehydrogenase and NADH oxidase, and blocking acetate synthesis pathways. After optimization of whole-cell bioconversion conditions, the engineered strain BL-11 produced 251.97 mM (22.20 g/L) acetoin with a yield of 0.434 mol/mol in shake flasks. Moreover, a titer of 648.97mM (57.18 g/L) acetoin was obtained in 30 h with a yield of 0.484 mol/mol lactic acid in a 1-L bioreactor. To the best of our knowledge, this is the first report on the production of acetoin from renewable lactate through whole-cell bioconversion with both high titer and yield, which demonstrates the economy and efficiency of acetoin production from lactate. Key Points • The lactate dehydrogenases from different organisms were expressed, purified, and assayed. • It is the first time that acetoin was produced from lactate by whole-cell biocatalysis. • The highest titer of 57.18 g/L acetoin was obtained with high theoretical yield in a 1-L bioreactor.

Whole-Cell Biocatalytic Production of Acetoin with an aldC-Overexpressing Lactococcus lactis Using Soybean as Substrate

Douchi is a traditional Chinese fermented soybean product, in which acetoin is a key flavor substance. Here, the α-acetolactate decarboxylase gene aldC was cloned from Lactiplantibacillus (L.) plantarum and overexpressed in Lactococcus (L.) lactis NZ9000 by nisin induction. The ALDC crude enzyme solution produced an enzyme activity of 35.16 mU. Next, whole cells of the recombinant strain NZ9000/pNZ8048-aldC were employed as the catalyst to produce acetoin in GM17 medium. An optimization experiment showed that an initial OD600 of 0.6, initial pH of 7.5, nisin concentration of 20 ng/mL, induction temperature of 37 °C and static induction for 8 h were the optimal induction conditions, generating the maximum acetoin production (106.93 mg/L). Finally, after incubation under the optimal induction conditions, NZ9000/pNZ8048-aldC was used for whole-cell biocatalytic acetoin production, using soybean as the substrate. The maximum acetoin yield was 79.43 mg/L. To our knowledge, this is the first study in which the aldC gene is overexpressed in L. lactis and whole cells of the recombinant L. lactis are used as a biocatalyst to produce acetoin in soybean. Thus, our study provides a theoretical basis for the preparation of fermented foods containing high levels of acetoin and the biosynthesis of acetoin in food materials.

Immobilization of Alpha Acetolactate Decarboxylase in Hybrid Gelatin/Alginate Support for Application to Reduce Diacetyl Off-Flavor in Beer

Beer production is the largest among alcoholic beverages. Its production process is complex and demands several steps. Lager beers commonly present an off-flavor of butter that is due to the presence of diacetyl, and to avoid such a problem, a long period of maturation (3–5 weeks) is required. Another way is the application of (α-acetolactate decarboxylase) ALDC to accelerate the process. The objectives of the present work were to develop a low-cost support using gelatin, a residue from capsules from the nutraceutical industry, to immobilize the ALDC enzyme. For this, the yield, efficiency and activity recovered, and the stability of free and immobilized enzymes at different temperatures and pH were evaluated. To evaluate the capacity of immobilized enzymes when applied directly to beer and their operational stability, three concentrations of glutaraldehyde (1%, 2.5% and 5%) were tested in distilled water as a cross-linking agent. The best results obtained were 95.6%, 27.0% and 23.6%, respectively, for yield, efficiency and activity recovery. Immobilization provided a high activity over a wide pH range. The immobilized enzyme showed greater stability at temperatures of 50 and 60 °C. The immobilized derivative showed adequate reuse capacity, and its dehydrated form had excellent activity after long periods of storage.

Dynamic correlation between tetramethylpyrazine and influencing factors in Bacillus subtilis-fermented dehulled adlay

Tetramethylpyrazine (TMP), a flavor compound with biological activity, naturally exists in a variety of roasted, fried or fermented foods. Biosynthesis has been green and effective practice to produce TMP, and high TMP level was obtained in Bacillus subtilis BJ3-2 fermented dehulled adlay (BDA) in previous study. To elucidate the correlation between TMP and influencing factors in this study, the dynamics of TMP and influencing factors in BDA were investigated, and the correlation between TMP and influencing factors was analyzed by Pearson's correlation coefficients. Results showed that a weakly acidic environment was found in BDA and B. subtilis CICC 20637 fermented dehulled adlay (CDA), but a weakly alkaline environment was observed in B. subtilis BJ3-2 fermented soybean (BSB). The higher levels of α-acetolactate decarboxylase, reducing sugar, pyruvate, diacetyl, leucine, acetoin and TMP were detected in BDA, while higher levels of α-acetolactate synthase, free amino acids and ammonium were obtained in BSB. At the end of fermentation, the TMP content in BDA was 6.13 mg/g dry weight, which was 204- and 12-fold higher than that in BSB and CDA, respectively. TMP synthesis in BDA and CDA was remarkable correlation with pyruvate, diacetyl, leucine, ammonium and acetoin (p < 0.05). In summary, a weakly acidic environment, moderate ammonium content, high leucine and acetoin levels synergistically promoted TMP efficient synthesis.

α-Acetolactate decarboxylase immobilized in chitosan: A highly stable biocatalyst to prevent off-flavor in beer.

In this work, we studied the covalent immobilization of α-acetolactate decarboxylase (ALDC) on glutaraldehyde activated chitosan beads for application in beers to prevent off-flavor. The immobilized preparation was characterized regarding yield, efficiency, activity recovery, thermal and pH stabilities, and operational stability in beer. The concentration of activating agent was evaluated and at the best condition an immobilization yield of 84% and activity recovery of 9.1% were verified. Optimal pH was 6.0 for free and immobilized enzymes, however, the immobilized enzyme showed higher relative activity at the tested pH, with emphasis on the pH range from 5.0 to 6.5. Besides, immobilized enzyme showed stabilization factor of almost sevenfold at 60°C compared to free form. The immobilized biocatalyst was tested in repeated batches in beer for α-acetolactate conversion to acetoin, and after 12 cycles the enzyme presented 80% of its initial activity. To date, this is the most stable immobilized preparation of ALDC reported.

Blocking the 2,3-butanediol synthesis pathway of Klebsiella pneumoniae resulted in L-valine production.

Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of L-valine production strain was studied in this paper. The first step of 2,3-butanediol synthesis and branched-chain amino acid synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. However, the two pathways are existing in parallel and do not interfere with each other in the wild-type strain. A knockout of budA blocked the 2,3-butanediol synthesis pathway and resulted in the L-valine production. The budA coded an α-acetolactate decarboxylase and catalyzed the acetoin formation from α-acetolactate. Furthermore, blocking the lactic acid synthesis by knocking out of ldhA, which is encoding a lactate dehydrogenase, improved the L-valine synthesis. 2-Ketoisovalerate is the precursor of L-valine, it is also an intermediate of the isobutanol synthesis pathway, while indole-3-pyruvate decarboxylase (ipdC) is responsible for isobutyraldehyde formation from 2-ketoisovalerate. Production of L-valine has been improved by knocking out of ipdC. On the other side, the ilvE, encoding a transaminase B, reversibly transfers one amino group from glutamate to α-ketoisovalerate. Overexpression of ilvE exhibited a distinct improvement of L-valine production. The brnQ encodes a branched-chain amino acid transporter, and L-valine production was further improved by disrupting brnQ. It is also revealed that weak acidic and aerobic conditions favor L-valine production. Based on these findings, L-valine production by metabolically engineered K. pneumonia was examined. In fed-batch fermentation, 22.4g/L of L-valine was produced by the engineered K. pneumoniae ΔbudA-ΔldhA-ΔipdC-ΔbrnQ-ilvE after 55h of cultivation, with a substrate conversion ratio of 0.27mol/mol glucose.

High-yield production of (R)-acetoin in Saccharomyces cerevisiae by deleting genes for NAD(P)H-dependent ketone reductases producing meso-2,3-butanediol and 2,3-dimethylglycerate

Acetoin is widely used in food and cosmetics industries as a taste and fragrance enhancer. To produce (R)-acetoin in Saccharomyces cerevisiae, acetoin biosynthetic genes encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) from Bacillus subtilis and water-forming NADH oxidase (NoxE) from Lactococcus lactis were integrated into delta-sequences in JHY605 strain, where the production of ethanol, glycerol, and (R,R)-2,3-butanediol (BDO) was largely eliminated. We further improved acetoin production by increasing acetoin tolerance by adaptive laboratory evolution, and eliminating other byproducts including meso-2,3-BDO and 2,3-dimethylglycerate, a newly identified byproduct. Ara1, Ypr1, and Ymr226c (named Ora1) were identified as (S)-alcohol-forming reductases, which can reduce (R)-acetoin to meso-2,3-BDO in vitro. However, only Ara1 and Ypr1 contributed to meso-2,3-BDO production in vivo. We elucidate that Ora1, having a substrate preference for (S)-acetoin, reduces (S)-α-acetolactate to 2,3-dimethylglycerate, thus competing with AlsD-mediated (R)-acetoin production. By deleting ARA1, YPR1, and ORA1, 101.3 g/L of (R)-acetoin was produced with a high yield (96% of the maximum theoretical yield) and high stereospecificity (98.2%).

Enhanced Production of 2,3-Butanediol in Recombinant Escherichia coli Using Response Regulator DR1558 Derived from Deinococcus radiodurans

2,3-Butanediol (2,3-BDO) is a promising bio-based chemical for its wide range of applications in industrial areas as synthetic rubber precursor, food additives, and cosmetics. In this study, Escherichia coli DH5α was metabolically engineered for enhanced production of 2,3-BDO by expressing Bacillus subtilis alsS, alsD, and ydjL genes encoding α-acetolactate synthase, α-acetolactate decarboxylase, and acetoin reductase/2,3 -butanediol dehydro¬genase, respectively, along with Deinococcus radiodurans dr1558 gene encoding a response regulator. When recombinant E. coli DH5α strain expressing only B. subtilis alsS, alsD, ydjL genes was cultured in LB medium containing 20 g/L glucose, 3.14 g/L of 2,3-BDO was produced. Additional expression of D. radiodurans dr1558 gene in E. coli DH5α expressing alsS, alsD, and ydjL genes resulted in the production of 7.81 g/L of 2,3-BDO under the same culture conditions, which is 2.5 fold higher than that produced by the strain without DR1558. Transcriptional analysis of E. coli DH5α expressing DR1558 suggested that the expression levels of the genes related to 2,3-BDO pathways were enhanced, while those of genes related to by-product pathways were suppressed, compared with control strain expressing only 2,3-BDO synthesis genes. These results strongly suggest that introduction of the stress tolerant response regulator DR1558 can modulate metabolic pathways to favor production of the target product.

N-Acyl-Homoserine Lactone Quorum Sensing Switch from Acidogenesis to Solventogenesis during the Fermentation Process in Serratia marcescens MG1.

N-acyl-homoserine lactone quorum sensing (AHL-QS) has been shown to regulate many physiological behaviors in Serratia marcescens MG1. In the current study, the effects of AHL-QS on the biosynthesis of acid and neutral products by S. marcescens MG1 and its isogenic ∆swrI with or without supplementing exogenous N-hexanoyl-L-homoserine lactone (C6-HSL) were systematically investigated. The results showed that swrI disruption resulted in rapid pH drops from 7.0 to 4.8, which could be restored to wild type by supplementing C6-HSL. Furthermore, fermentation product analysis indicated that ∆swrI could lead to obvious accumulation for acidogenesis products such as lactic acid and succinic acid, especially excess acetic acid (2.27 g/l) produced at the early stage of fermentation, whereas solventogenesis products by ∆swrI appeared to noticeably decrease by an approximate 30% for acetoin during 32-48 h and by an approximate 20% for 2,3-butanediol during 24-40 h, when compared to those by wild type. Interestingly, the excess acetic acid produced could be removed in an AHL-QS-independent manner. Subsequently, quantitative real-time PCR was used to determine the mRNA expression levels of genes responsible for acidogenesis and solventogenesis and showed consistent results with those of product synthesis. Finally, by close examination of promoter regions of the analyzed genes, four putative luxI box-like motifs were found upstream of genes encoding acetyl-CoA synthase, lactate dehydrogenase, α-acetolactate decarboxylase, and Lys-like regulator. The information from this study provides a novel insight into the roles played by AHL-QS in switching from acidogenesis to solventogenesis in S. marcescens MG1.

Effect of Respiratory Growth on the Metabolite Production and Stress Robustness of Lactobacillus casei N87 Cultivated in Cheese Whey Permeate Medium.

Cheese whey permeate (WP) is a low-cost feedstock used for the production of biomass and metabolites from several lactic acid bacteria (LAB) strains. In this study, Lactobacillus casei N87 was cultivated in an optimized WP medium (WPM) to evaluate the effect of anaerobic and respiratory conditions on the growth performances (kinetics, biomass yield), consumption of sugars (lactose, galactose, glucose) and citrate, metabolite production [organic acids, volatile organic compounds (VOCs)] and stress survival (oxidative, heat, freezing, freeze-drying). The transcription of genes involved in the main pathways for pyruvate conversion was quantified through Real Time-PCR to elucidate the metabolic shifts due to respiratory state. Cultivation in WPM induced a diauxic growth in both anaerobic and respiratory conditions, and L. casei N87 effectively consumed the lactose and galactose present in WPM. Genomic information suggested that membrane PTS system and tagatose-6-P pathway mediated the metabolism of lactose and galactose in L. casei N87. Respiration did not affect specific growth rate and biomass production, but significantly altered the pyruvate conversion pathways, reducing lactate accumulation and promoting the formation of acetate, acetoin and diacetyl to ensure the redox balance. Ethanol was not produced under either cultivation. Pyruvate oxidase (pox), acetate kinase (ack), α-acetolactate decarboxylase (ald), acetolactate synthase (als) and oxaloacetate decarboxylase (oad) genes were up-regulated under respiration, while L-lactate dehydrogenase (ldh), pyruvate formate lyase (pfl), pyruvate carboxylase (pyc), and phosphate acetyltransferase (pta) were down regulated by oxygen. Transcription analysis was consistent with metabolite production, confirming that POX-ACK and ALS-ALD were the alternative pathways activated under aerobic cultivation. Respiratory growth affected the production of volatile compounds useful for the development of aroma profile in several fermented foods, and promoted the survival of L. casei N87 to oxidative stresses and long-term storage. This study confirmed that the respiration-based technology coupled with cultivation on low-cost medium may be effectively exploited to produce competitive and functional starter and/or adjunct cultures. Our results, additionally, provided further information on the activation and regulation of metabolic pathways in homofermentative LAB grown under respiratory promoting conditions.

Structure and catalytic mechanistic insight into Enterobacter aerogenes acetolactate decarboxylase

α-Acetolactate decarboxylase (ALDC) catalyses α-acetolactate into acetoin (3-hydroxy-2-butanone, AC) and is considered to be the rate-limiting enzyme in the synthesis of 2,3-butanediol. In this work, the enzymatic activity of ALDC from Enterobacter aerogenes ALDC (E.a.-ALDC) was fully characterized with enzyme kinetics, indicating a Km of 14.83 ± 0.87 mM and a kcat of 0.81 ± 0.09 s−1. However, compared with the activities of ALDCs reported from other bacteria, the activity of E.a.-ALDC was determined to present a relatively lower value of 849.08 ± 35.21 U/mg. The enzyme showed maximum activity at pH 5.5. In addition, the activity of E.a.-ALDC was promoted by Mg2+. The crystal structure of E.a.-ALDC firstly solved by X-ray crystallography at resolution of 2.4 Å revealed a chelated zinc ion with conserved His199, His201, His212, Glu70 and Glu259. In the active center, the conservative Arg150 was particularly proven to deviate from the zinc ion of the active centre, by adopting a flexible conformational change, resulting in a weak interaction network of the enzyme and the substrate. Further in silico docking of E.a.-ALDC with two enantiomers, (R)-acetolactate and (S)-acetolactate, unaltered the interaction network of E.a.-ALDC from the apo structure, which confirmed the weakened role of Arg150 in the catalytic properties of E.a.-ALDC. Our results reveal a unique structure-function relationship of acetolactate decarboxylase and provide a fundamental basis for the enzymatic synthesis of acetoin.

Safety evaluation of the food enzyme acetolactate decarboxylase from a genetically modified Bacilluslicheniformis (strain NZYM-JB).

The food enzyme acetolactate decarboxylase (α‐acetolactate decarboxylase; EC 4.1.1.5) is produced with a genetically modified Bacillus licheniformis strain NZYM‐JB by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and recombinant DNA. This acetolactate decarboxylase is intended to be used in distilled alcohol production and brewing processes. Residual amounts of total organic solids (TOS) are removed by distillation; consequently, dietary exposure was not calculated for distilled alcohol products. For other brewery products, based on the maximum use level recommended for the brewing processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme–TOS was estimated to be up to 0.003 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests with the food enzyme did not indicate a genotoxic concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level at the highest dose of 1,018 mg TOS/kg bw per day. When compared with the dietary exposure, this results in a sufficiently high margin of exposure (at least 300,000). The amino acid sequence of the food enzyme did not match to those of known allergens. The Panel considered that, under the intended condition of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is considered low. Based on the data provided, the Panel concluded that this food enzyme does not raise safety concerns under the intended conditions of use.

Effect of ILV6 Deletion and Expression of aldB from Lactobacillus plantarum in Saccharomyces uvarum on Diacetyl Production and Wine Flavor.

Diacetyl generates an aromatic off-flavor in wine at a high level. The present study expressed α-acetolactate decarboxylase (ALDB) from Lactobacillus plantarum and/or inactivated acetohydroxyacid synthase (Ilv6) in Saccharomyces uvarum, and the effects on diacetyl production and wine flavor in mutants were investigated through sequential fermentation and cofermentation in mixed cultures of S. uvarum and L. plantarum. The diacetyl content of WYDΔ6 (disrupted one ILV6 allele), WYSΔ6 ( ILV6 complete deletion), WYADΔ6 (disrupted one ILV6 allele with aldB expression), and WYASΔ6 ( ILV6 complete deletion with aldB expression) decreased by 25.71%, 41.30%, 47.77%, and 50.00%, respectively, after sequential fermentation and decreased by 15.15%, 26.72%, 35.26%, and 43.80%, respectively, after cofermentation, compared with that of the parental strain. In addition, Ilv6 inactivation not only decreased the acetic acid content but also balanced the flavor profile in wine effectively. This work provided a valuable insight into the metabolic pathway of diacetyl and wine flavor in S. uvarum.

Studies on structure-function relationships of acetolactate decarboxylase from Enterobacter cloacae.

Acetoin is an important bio-based platform chemical with wide applications. Among all bacterial strains, Enterobacter cloacae is a well-known acetoin producer via α-acetolactate decarboxylase (ALDC), which converts α-acetolactate into acetoin and is identified as the key enzyme in the biosynthetic pathway of acetoin. In this work, the enzyme properties of Enterobacter cloacae ALDC (E.c.-ALDC) were characterized, revealing a Km value of 12.19 mM and a kcat value of 0.96 s−1. Meanwhile, the optimum pH of E.c.-ALDC was 6.5, and the activity of E.c.-ALDC was activated by Mn2+, Ba2+, Mg2+, Zn2+ and Ca2+, while Cu2+ and Fe2+ significantly inhibited ALDC activity. More importantly, we solved and reported the first crystal structure of E.c.-ALDC at 2.4 Å resolution. The active centre consists of a zinc ion coordinated by highly conserved histidines (199, 201 and 212) and glutamates (70 and 259). However, the conserved Arg150 in E.c.-ALDC orients away from the zinc ion in the active centre of the molecule, losing contact with the zinc ion. Molecular docking of the two enantiomers of α-acetolactate, (R)-acetolactate and (S)-acetolactate allows us to further investigate the interaction networks of E.c.-ALDC with the unique conformation of Arg150. In the models, no direct contacts are observed between Arg150 and the substrates, which is unlikely to maintain the stabilization function of Arg150 in the catalytic reaction. The structure of E.c.-ALDC provides valuable information about its function, allowing a deeper understanding of the catalytic mechanism of ALDCs.

Hydroxy-pentanones production by Bacillus sp. H15-1 and its complete genome sequence

Acyloins are useful organic compounds with reactive adjacent hydroxyl group and carbonyl group. Current research is usually constrained to acetoin (i.e. 3-hydroxy-2-butanone) and the biological production of other acyloins was scarcely reported. In this study, two hydroxy-pentanone metabolites (3-hydroxy-2-pentanone and 2-hydroxy-3-pentanone) of Bacillus sp. H15-1 were identified by gas chromatography–mass spectrometry and authentic standards. Then the complete genome of this strain was sequenced and de novo assembled to a single circular chromosome of 4,162,101bp with a guanine-cytosine content of 46.3%, but no special genes were found for the biosynthesis of the hydroxy-pentanones. Since hydroxy-pentanones are the homologues of acetoin, the two genes alsD and alsS (encoding α-acetolactate decarboxylase and α-acetolactate synthase, respectively) responsible for acetoin formation in this strain were respectively expressed in Escherichia coli. The purified enzymes were found to be capable of transforming pyruvate and 2-oxobutanoate to the two hydroxy-pentanones. This study extends the knowledge on the biosynthesis of acyloins and provides helpful information for further utilizing Bacillus sp. H15-1 as a source of valuable acyloins.

Isobutanol and 2-ketoisovalerate production by Klebsiella pneumoniae via a native pathway

Isobutanol is a valuable chemical and is considered a new generation biofuel. Construction of isobutanol synthesis pathways in bacteria is a hot topic in isobutanol production. Here, we show that an isobutanol synthesis pathway exists naturally in Klebsiella pneumoniae; however, this pathway is dormant in the wild-type bacterium. K. pneumoniae is a 2,3-butanediol producer, and the synthesis pathways of 2,3-butanediol, valine and isobutanol all start from condensation of two pyruvate molecules to yield α-acetolactate. Inactivation of α-acetolactate decarboxylase (encoded by budA) resulted in α-acetolactate flowing into the valine pathway, which led to synthesis of isobutanol and 2-ketoisovalerate (a precursor of isobutanol). ldhA (lactate dehydrogenase) deletion further increased the isobutanol and 2-ketoisovalerate production. In the first step of this pathway, BudB (α-acetolactate synthase) was identified as responsible for most of the α-acetolactate synthesis. Complementation of ilvBN or ilvIH (isoenzymes of budB) both resulted in a remarkable increase in 2-ketoisovalerate production. Thus, α-acetolactate formation is the rate-limiting step of 2-ketoisovalerate production. ilvC (acetohydroxy acid isomeroreductase) and ilvD (dihydroxy acid dehydratase) were identified responsible for 2-ketoisovalerate synthesis from α-acetolactate. ipdC, which encodes an indole-3-pyruvate decarboxylase, was identified responsible for most of the isobutyraldehyde formation from 2-ketoisovalerate, and isobutanol production was increased 15.7 fold in the ipdC complementation strain, with a final titer of 2.45g/L. Isobutanol dehydrogenase activity is distributed across multiple alcohol dehydrogenase enzymes expressed by K. pneumoniae. BudC, DhaT, DhaD and YqhD all had isobutanol dehydrogenase activity in vitro. YqhD uses NADPH as the coenzyme, while the other dehydrogenases use NADH. However, inactivating one or two of these dehydrogenases had no effect on isobutanol production in vivo with isobutyraldehyde as the substrate. These results reveal a novel method for biological production of isobutanol and 2-ketoisovalerate.

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate

Acetoin (3-hydroxy-2-butanone) is an important bio-based platform chemical with wide applications. In vitro enzyme catalysed synthesis exhibits great feasibility in the production of chemicals with high purity. In the present work, a synthetic pathway involving a two-step continuous reaction was constructed in vitro for acetoin production from pyruvate at improved temperature. Thermostable candidates, acetolactate synthase (coAHASL1 and coAHASL2 from Caldicellulosiruptor owensensis OL) and α-acetolactate decarboxylase (bsALDC from Bacillus subtilis IPE5-4) were cloned, heterologously expressed, and characterized. All the enzymes showed maximum activities at 65–70 °C and pH of 6.5. Enzyme kinetics analysis showed that coAHASL1 had a higher activity but lower affinity against pyruvate than that of coAHASL2. In addition, the activities of coAHASL1 and bsALDC were promoted by Mn2+ and NADPH. The cascade enzymatic reaction was optimized by using coAHASL1 and bsALDC based on their kinetic properties. Under optimal conditions, a maximum concentration of 3.36 ± 0.26 mM acetoin was produced from 10 mM pyruvate after reaction for 24 h at 65 °C. The productivity of acetoin was 0.14 mM h−1, and the yield was 67.80% compared with the theoretical value. The results confirmed the feasibility of synthesis of acetoin from pyruvate with a cell-free enzyme catalysed system at improved temperature.

Efficient production of 2,3-butanediol by recombinant Saccharomyces cerevisiae through modulation of gene expression by cocktail δ-integration

In this study, the expression of 4 genes encoding α-acetolactate synthase, α-acetolactate decarboxylase, 2,3-butanediol dehydrogenase, and NADH oxidase was modulated using a previously developed cocktail δ-integration strategy. The resultant strain, YPH499/dPdAdG/BD6-10, was used in a fed-batch cultivation for the production of 2,3-butanediol. The concentration, production rate, and yield obtained were 80.0g/L, 4.00g/L/h, and 41.7%, respectively. The production rate and yield of the compound obtained are higher for this strain compared to reports published for Saccharomyces cerevisiae so far. The cocktail δ-integration strategy allows for modulation of multiple gene expression, without the exact knowledge of rate-limiting steps, and therefore, could be used as a promising strategy for the production of bio-based chemicals in recombinant S. cerevisiae.