Diacetyl Reductase Research Articles

Production of highly pure R,R-2,3-butanediol for biological plant growth promoting agent using carbon feeding control of Paenibacillus polymyxa MDBDO

BackgroundChemical fertilizers have greatly contributed to the development of agriculture, but alternative fertilizers are needed for the sustainable development of agriculture. 2,3-butanediol (2,3-BDO) is a promising biological plant growth promoter.ResultsIn this study, we attempted to develop an effective strategy for the biological production of highly pure R,R-2,3-butanediol (R,R-2,3-BDO) by Paenibacillus polymyxa fermentation. First, gamma-ray mutagenesis was performed to obtain P. polymyxa MDBDO, a strain that grew faster than the parent strain and had high production of R,R-2,3-BDO. The activities of R,R-2,3-butanediol dehydrogenase and diacetyl reductase of the mutant strain were increased by 33% and decreased by 60%, respectively. In addition, it was confirmed that the carbon source depletion of the fermentation broth affects the purity of R,R-2,3-BDO through batch fermentation. Fed-batch fermentation using controlled carbon feeding led to production of 77.3 g/L of R,R-2,3-BDO with high optical purity (> 99% of C4 products) at 48 h. Additionally, fed-batch culture using corn steep liquor as an alternative nitrogen source led to production of 70.3 g/L of R,R-2,3-BDO at 60 h. The fed-batch fermentation broth of P. polymyxa MDBDO, which contained highly pure R,R-2,3-BDO, significantly stimulated the growth of soybean and strawberry seedlings.ConclusionsThis study suggests that P. polymyxa MDBDO has potential for use in biological plant growth promoting agent applications. In addition, our fermentation strategy demonstrated that high-purity R,R-2,3-BDO can be produced at high concentrations using P. polymyxa.

A chemical study of yoghurt produced under isostatic pressure during storage

Yoghurt fermented under sub-lethal high pressure (10, 20, 30 and 40 MPa at 43 °C), and afterward placed under refrigeration (4 °C for 23 days) was studied and compared with yoghurt fermented at atmospheric pressure (0.1 MPa). For a deeper analysis, metabolite fingerprinting by nuclear magnetic resonance (NMR), sugars and organic acids assessment by high performance liquid chromatography (HPLC), total fatty acids (TFA) determination and quantification by gas chromatography with a flame ionization detector (GC-FID) were performed. Metabolomic analyses revealed that only 2,3-butanediol, acetoin, diacetyl and formate vary with the increase of pressure and probable relation with pressure influenced diacetyl reductase, acetoin reductase and acetolactate decarboxylase. Yoghurts fermented at 40 MPa had the lowest content in lactose (39.7 % of total sugar reduction) and the less content in TFA (56.1 %). Further research is of interest to understand more about fermentation processes under sub-lethal high pressure.

Genomic and Transcriptional Characteristics of Strain Rum-meliibacillus sp. TYF-LIM-RU47 with an Aptitude of Directly Producing Acetoin from Lignocellulose

Rummeliibacillus sp. TYF-LIM-RU47, isolated from the fermentation substrate of grain vinegar, could produce acetoin using a variety of carbon sources, including pentose, hexose and lignocellulose. The draft genome of TYF-LIM-RU47 was constructed and the genomic information revealed that TYF-LIM-RU47 contains genes related to starch and sucrose metabolism, pyruvate metabolism, the oxidative phosphorylation metabolic pathway and lignocellulosic metabolism. The acetoin anabolic pathway of TYF-LIM-RU47 has been deduced from the sequencing results, and acetoin is produced from α-acetolactate via decarboxylation and diacetyl reductase catalytic steps. The results of quantitative real-time PCR tests showed that the synthesis and degradation of acetoin had a dynamic balance in acetoin metabolism, and the transcription of the α-acetolactate synthase gene might exist to the extent of feedback regulation. This study can help researchers to better understand the bioinformation of thermophilic-lignocellulosic bacteria and the mechanisms of the acetoin biosynthesis pathway.

Production of R,R-2,3-butanediol of ultra-high optical purity from Paenibacillus polymyxa ZJ-9 using homologous recombination

The present study describes the use of metabolic engineering to achieve the production of R,R-2,3-butanediol (R,R-2,3-BD) of ultra-high optical purity (>99.99%). To this end, the diacetyl reductase (DAR) gene (dud A) of Paenibacillus polymyxa ZJ-9 was knocked out via homologous recombination between the genome and the previously constructed targeting vector pRN5101-L′C in a process based on homologous single-crossover. PCR verification confirmed the successful isolation of the dud A gene disruption mutant P. polymyxa ZJ-9-△dud A. Moreover, fermentation results indicated that the optical purity of R,R-2,3-BD increased from about 98% to over 99.99%, with a titer of 21.62 g/L in Erlenmeyer flasks. The latter was further increased to 25.88 g/L by fed-batch fermentation in a 5-L bioreactor.

Reduced production of diacetyl by overexpressing BDH2 gene and ILV5 gene in yeast of the lager brewers with one ILV2 allelic gene deleted.

Diacetyl causes an unwanted buttery off-flavor in lager beer. The production of diacetyl is reduced by modifying the metabolic pathway of yeast in the beer fermentation process. In this study, BDH2 and ILV5 genes, coding diacetyl reductase and acetohydroxy acid reductoisomerase, respectively, were expressed using a PGK1 promoter in Saccharomyces cerevisiae, which deleted one ILV2 allelic gene. Diacetyl contents and fermentation performances were examined and compared. Results showed that the diacetyl content in beer was remarkably reduced by 16.52% in QI2-KP (one ILV2 allelic gene deleted), 55.65% in QI2-B2Y (overexpressed BDH2 gene and one ILV2 allelic gene deleted), and 69.13% in QI2-I5Y (overexpressed ILV5 gene and one ILV2 allelic gene deleted) compared with the host strain S2. The fermentation ability of mutant strains was similar to that of S2. Results of the present study can lead to further advances in this technology and its broad application in scientific investigations and industrial beer production.

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 (kcat/Km≈0.4mM−1s−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 (Vmax≈6U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3g/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.

α,β-Dicarbonyl reduction is mediated by the Saccharomyces Old Yellow Enzyme.

The undesirable flavor compounds diacetyl and 2,3-pentanedione are vicinal diketones (VDKs) formed by extracellular oxidative decarboxylation of intermediate metabolites of the isoleucine, leucine and valine (ILV) biosynthetic pathway. These VDKs are taken up by Saccharomyces and enzymatically converted to acetoin and 3-hydroxy-2-pentanone, respectively. Purification of a highly enriched diacetyl reductase fraction from Saccharomyces cerevisiae in conjunction with mass spectrometry identified Old Yellow Enzyme (Oye) as an enzyme capable of catalyzing VDK reduction. Kinetic analysis of recombinant Oye1p, Oye2p and Oye3p isoforms confirmed that all three isoforms reduced diacetyl and 2,3-pentanedione in an NADPH-dependent reaction. Transcriptomic analysis of S. cerevisiae (ale) and S. pastorianus (lager) yeast during industrial fermentations showed that the transcripts for OYE1, OYE2, arabinose dehydrogenase (ARA1), α-acetolactate synthase (ILV2) and α-acetohydroxyacid reductoisomerase (ILV5) were differentially regulated in a manner that correlated with changes in extracellular levels of VDKs. These studies provide insights into the mechanism for reducing VDKs and decreasing maturation times of beer which are of commercial importance.

Diacetyl content reduction in industrial brewer’s yeast through ILV2 disruption and BDH1 expression

Diacetyl is crucial to beer maturation because of its characteristic buttery taste. New industrial brewer’s yeast strains, including single ILV2 allele disruption (QI2), single ILV2 allele disruption and BDH1 overexpression (QI2GB1), and BDH1 overexpression (B1Y), were constructed by disrupting the gene (ILV2) involved in a-acetolactate synthesis and introducing the diacetyl reductase gene (BDH1). The diacetyl content and fermentation performance of the three recombinant strains were examined and compared. Results showed that the diacetyl concentration in QI2, QI2GB1, and B1Y decreased by 17.83, 28.26, and 9.57 %, respectively, compared with that in the host strain S2. The fermentation ability in terms of fermentation degree and major flavors yield of the three recombinant strains was similar to that of the host. This study may serve as a reference for the brewing industry and for research on diacetyl reduction.

Production of diacetyl by metabolically engineered Enterobacter cloacae.

Diacetyl, a high value product that can be extensively used as a food ingredient, could be produced from the non-enzymatic oxidative decarboxylation of α-acetolactate during 2,3-butanediol fermentation. In this study, the 2,3-butanediol biosynthetic pathway in Enterobacter cloacae subsp. dissolvens strain SDM, a good candidate for microbial 2,3-butanediol production, was reconstructed for diacetyl production. To enhance the accumulation of the precursor of diacetyl, the α-acetolactate decarboxylase encoding gene (budA) was knocked out in strain SDM. Subsequently, the two diacetyl reductases DR-I (gdh) and DR-II (budC) encoding genes were inactivated in strain SDM individually or in combination to decrease the reduction of diacetyl. Although the engineered strain E. cloacae SDM (ΔbudAΔbudC) was found to have a good ability for diacetyl production, more α-acetolactate than diacetyl was produced simultaneously. In order to enhance the nonenzymatic oxidative decarboxylation of α-acetolactate to diacetyl, 20 mM Fe3+ was added to the fermentation broth at the optimal time. In the end, by using the metabolically engineered strain E. cloacae SDM (ΔbudAΔbudC), diacetyl at a concentration of 1.45 g/L was obtained with a high productivity (0.13 g/(L·h)). The method developed here may be a promising process for biotechnological production of diacetyl.

Identification and characterization of a mycobacterial NAD⁺-dependent alcohol dehydrogenase with superior reduction of diacetyl to (S)-acetoin.

An enzyme capable of reducing acetoin in the presence of NADH was purified from Mycobacterium sp. B-009, a non-clinical bacterial strain of soil origin. The enzyme is a homotetramer and can be classified as a medium-chain alcohol dehydrogenase/reductase based on the molecular weight of the monomer. Identification of the structural gene revealed a limited distribution of homologous genes only among actinomycetes. In addition to its activity as a reductase specific for (S)-acetoin (EC 1.1.1.76), the enzyme showed both diacetyl reductase (EC 1.1.1.304) and NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1) activities. (S)-Acetoin and diacetyl reductases belong to a group of short-chain alcohol dehydrogenase/reductases but do not have superior abilities to dehydrogenate monoalcohols. Thus, the purified enzyme can be readily distinguished from other enzymes. We used the dual functionality of the enzyme to effectively reduce diacetyl to (S)-acetoin, coupled with the oxidation of 1-butanol.

Production of S -acetoin from diacetyl by Escherichia coli transformant cells that express the diacetyl reductase gene of Paenibacillus polymyxa ZJ-9

S-acetoin (S-AC) is an important four-carbon chiral compound that has unique industrial applications in the asymmetric synthesis of valuable chiral specialty chemicals. However, previous studies showed that the usually low yield and optical purity of S-AC as well as the very high substrate cost have hindered the application of this compound. In the current work, a gene encoding diacetyl reductase (DAR) from a Paenibacillus polymyxa strain ZJ-9 was cloned and expressed in Escherichia coli. Whole cells of the recombinant E.coli were used to produce S-AC from diacetyl (DA). Under optimal conditions, S-AC with high optical purity (purity >99·9%) was obtained with a yield of 13·5±0·24 and 39·4±0·38gl(-1) under batch and fed-batch culture conditions, respectively. This process featured the biotransformation of DA into S-AC using whole cells of engineered E.coli. The result is a considerable increase in the yield and optical purity of S-AC, which in turn facilitated the practical application of the compound. This study demonstrated a highly efficient new method to produce S-acetoin with higher than 99·9% optical purity from diacetyl using whole cells of engineered Escherichia coli. It will therefore decrease the production cost of S-acetoin and highlight its application in asymmetric synthesis of highly valuable chiral compounds.

Overexpressed acetohydroxyacid reductoisomerase (ILV5) gene in Saccharomyces cerevisiae reduces diacetyl contents in Korean Campbell Early and Muscat Bailey a grape wines

An ILV5 (acetohydroxyacid reductoisomerase) gene expression vector (designated pILV5) was constructed by cloning the yeast ILV5 gene into a yeast expression vector (pLG669Z) containing yeast CYC1 promoter. S. cerevisiae cells harboring the pILV5 plasmid showed ∼3.7-fold higher level of the diacetyl reductase activity compared with the control yeast cells. When they were applied to wine fermentation using Campbell Early and Muscat Baily A grapes, the diacetyl content level was reduced to ∼35.0–39.0% of those obtained with the control yeasts.

Expression of dar Gene in Lactic Acid Bacteria and Effect on Metabolite Formation in Feta-Type Cheese Quality

The dar gene from Lactococcus lactis subsp. cremoris MG1363, encoding the enzyme diacetyl reductase (DAR) was cloned into the expression vector pMG36e under the control of the P32 promoter, and Lc. lactis MG1363 was transformed with this recombinant plasmid by electroporation. Metabolic end products (e.g., pyruvate, lactate, formate, acetoin) of this strain grown in glucose medium were analyzed by HPLC. The overexpression of the plasmid-encoded dar gene in Lc. lactis MG1363 led to the shift from homolactic to mixed acid fermentation and the production of a high level of acetoin, confirming the overexpression of the diacetyl reductase. In addition, the strains overexpressing dar gene and wild type were used as starter cultures in cheese. Different levels of metabolic end products were detected in cheese during the two weeks of ripening compared to the wild type strain.

New acetoin reductases from Bacillus stearothermophilus: meso- and 2R,3R-butanediol as fermentation products

The fermentation of sucrose with Bacillus stearothermophilus ATCC 2027 afforded 2R,3R- and meso-2,3-butanediol together with R-acetoin following the bacterial catabolic pathway. This metabolic route was confirmed by the conversion of pyruvate to R-acetoin with the cell free extract of B. stearothermophilus. On the other hand the reduction of 3R-acetoin to 2R,3R- and meso-butanediol with the same cell free extract allowed at least the presence of two NADH-dependent reductases. Together with the S-stereospecific diacetyl reductase (BSDR), purified in a previous work, a new S-stereospecific acetoin reductase (S-ACR) was partially purified and a fraction containing R-stereospecific acetoin reductase (R-ACR) was obtained. On the other hand in B. stearothermophilus fermentation of sucrose the “butanediol cycle” is negligible because the acetylacetoin synthase, responsible of the first step of this cycle, is an inducible enzyme that needs low concentration of sugar and high concentration of acetoin to be expressed.

Redirection of Pyruvate Pathway of Lactic Acid Bacteria to Improve Cheese Quality

Metabolic engineering in Lactic acid bacteria (LAB) has focused on changing of pyruvate metabolism to increase production of desired flavor compounds. A constructed mutant strain should contain no foreign DNA and antibiotic resistance genes. Therefore, food grade lactate dehydrogenase (ldh d) and diacetyl reductase (dar d) mutant strains were created using two plasmid system in this study. Metabolic end products (pyruvate, lactate, formate and acetoin) of these strains in glucose medium and in cheese were determined using HPLC. Created mutant and wild type strains were used as a starter culture in cheese. Compared to the wild type strain, different levels of metabolites were observed in cheese during three weeks of ripening. The ldh d strains produced less lactate but high acetoin as a result of gene deletion. Deletion of dar gene decreased the production of acetoin. The dar deficient strains have low diacetyl reductase activity and are able to reduce significant amounts of acetoin but not terminate it completely. Genetic modification made the shift from homolactic to mixed acid fermentation, but the desired compound production hardly improved. The basis of these results and techniques are promising for the further studies.

Inhalation Dosimetry of Diacetyl and Butyric Acid, Two Components of Butter Flavoring Vapors

Occupational exposure to butter flavoring vapors (BFV) is associated with significant pulmonary injury. The goal of the current study was to characterize inhalation dosimetric patterns of diacetyl and butyric acid, two components of BFV, and to develop a hybrid computational fluid dynamic-physiologically based pharmacokinetic model (CFD-PBPK) to describe these patterns. Uptake of diacetyl and butyric acid vapors, alone and in combination, was measured in the upper respiratory tract of anesthetized male Sprague-Dawley rats under constant velocity flow conditions and the uptake data were used to validate the CFD-PBPK model. Diacetyl vapor (100 or 300 ppm) was scrubbed from the airstream with 76-36% efficiency at flows of 100-400 ml/min. Butryic acid (30 ppm) was scrubbed with >90% efficiency. Concurrent exposure to butyric acid resulted in a small but significant reduction of diacetyl uptake (36 vs. 31%, p < 0.05). Diacetyl was metabolized in nasal tissues in vitro, likely by diacetyl reductase, an enzyme known to be inhibited by butyric acid. The CFD-PBPK model closely described diacetyl uptake; the reduction in diacetyl uptake by butyric acid could be explained by inhibition of diacetyl reductase. Extrapolation to the human via the model suggested that inspired diacetyl may penetrate to the intrapulmonary airways to a greater degree in the human than in the rat. Thus, based on dosimetric relationships, extrapulmonary airway injury in the rat may be predictive of intrapulmonary airway injury in humans. Butyric acid may modulate diacetyl toxicity by inhibiting its metabolism and/or altering its inhalation dosimetric patterns.

Optimization and mechanism of diacetyl accumulation by Enterobacter aerogenes mutant UV-3

A mutant designated as UV-3 was obtained from wild-type Enterobacter aerogenes 10293 through u.v. radiation. The activities of a-acetolactate decarboxylase (Ald), lactate dehydrogenase (Ldh) and diacetyl reductase (Dr) in UV-3 were strongly attenuated, with the lowest activities at pH 7.0-7.5, and temperature between 36 and 39C. Compared to the wild-type, the yield of diacetyl by UV-3 was increased 18.7-fold, up to 1.05 ± 0.01 g l -1 . Acetoin and ethanol productions were decreased by 48.4 and 71.4%, respectively, but acetate yield was increased by 34.6%. Optimum medium for diacetyl production by UV-3 contained 10% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.01% (NH4)2SO4, 0.1% citric acid, 0.2% MnSO4 and 0.2% MgSO4, and this was determined by one-factor-at- a-time approach. Data from the five level central composite designs demonstrated that initial pH of 7.0, temperature of 37C and rotational speed of 180 rev/min were optimum processing parameters for diacetyl production. The maxi- mum yield of diacetyl could reach 1.35 g l -1 in a 5-l bioreactor. These results showed an enhancement of the non-enzymatic oxidative decarboxylation of a-acetolactate and a decrease in the activities of Ald, Ldh and Dr as a consequence of diacetyl accumulation in UV-3.

Insertion sequence elements as mediators of strain diversity in Lactobacillus helveticus

Insertion sequence (IS) elements were found to be associated with the truncation of predicted cellobiose transport, acetaldehyde dehydrogenase and diacetyl reductase genes in the genome of Lactobacillus helveticus DPC 4571. The conservation of the IS elements in these different genomic locations among L. helveticus cheese isolates was determined by amplification with gene-specific and IS element-specific primers. The presence of two of the IS elements was found to follow a genotypic profile of the strains generated by randomly amplified polymorphic DNA (RAPD)–PCR and strains that clustered by RAPD–PCR tended to have the IS element in the same position. However, the IS element that interrupted the cellobiose transport gene was found to be common to all strains tested. This conserved genotype suggests the insertion event occurred early in the evolution of L. helveticus as a separate species.

Expression of citrate permease gene of plasmid pCM1 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7 in Lactobacillus casei L-49-4

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man-Rogosa-Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.

Aerobic glycerol catabolism by Pediococcus pentosaceus isolated from wine

Glycerol catabolism was studied in Pediococcus pentosaceus N 5p isolated from wine, growing on different glycerol concentrations in aerobic conditions. Enzymatic activities of the glycerol kinase and glycerol dehydrogenase pathways were detected but the levels demonstrated allow to establish that glycerol was mainly degraded by glycerol kinase pathway. The highest levels of the activities of this pathway were expressed at the lowest concentration of glycerol, decreasing with the increase in glycerol concentration in the growth media. Glycerol was transformed in d-lactate, acetate, diacetyl and 2,3-butanediol. The enzymatic activities involved in the formation of these products were also modulated by glycerol. NAD-dependent lactate dehydrogenases were not affected by glycerol. Diacetyl reductase and acetoin reductase were inhibited by glycerol, whereas NAD-independent lactate dehydrogenases, NADH oxidase as CoA-independent acetaldehyde dehydrogenase, and acetate kinase were stimulated. The behavior of the enzymatic activities was in conformity with the fermentation balances; we propose a probable pathway for glycerol catabolism in P. pentosaceus N 5p.