Acetoin Reductase Research Articles

Comparative transcriptomic analysis of the flavor production mechanism in yogurt by traditional starter strains

The synergistic fermentation of milk by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus is one of the key factors that determines the quality of yogurt. In this study, the mechanism whereby yogurt flavor compounds are produced by a mixture of S. thermophilus SIT-20.S and L. delbrueckii ssp. bulgaricus SIT-17.B were investigated by examining the flavor production, growth, and gene transcription of these strains. The results showed that yogurt produced by a 10:1 mixture of the aforementioned strains had the highest abundance of acetoin, whereas yogurt produced by a 1:1 mixture had the highest abundance of diacetyl and acetaldehyde. In addition, the growth of S. thermophilus SIT-20.S was enhanced in the 10:1 mixture. Transcriptomic analysis revealed differentially expressed genes in the flavor-compound-related pathways of S. thermophilus SIT-20.S and L. delbrueckii ssp. bulgaricus SIT-17.B in yogurts produced by 10:1 and 1:1 mixtures compared with those produced by either strain alone. Mixed fermentations regulated the expression of genes related to glycolysis, resulting in an increase of pyruvate, which is an important precursor for diacetyl and acetoin synthesis. The gene encoding the acetoin reductase (SIT-20S_orf01454) was decreased in S. thermophilus SIT-20.S, which ensured the accumulation of acetoin. In addition, the gene encoding the acetaldehyde dehydrogenase (SIT-20S_orf00949) was upregulated in S. thermophilus SIT-20.S, and the expression of alcohol dehydrogenase (SIT-20S_orf01479; SIT-17B_orf00943) was downregulated in both strains, maintaining the abundance of acetaldehyde. In addition, the gene encoding the NADH oxidase (SIT-17B_orf00860) in L. delbrueckii ssp. bulgaricus SIT-17.B were upregulated, which promoted the accumulation of diacetyl and acetoin. Overall, we characterized the mechanism by which S. thermophilus and L. delbrueckii ssp. bulgaricus synergistically generated yogurt flavor compounds during their production of yogurt and highlighted the importance of appropriate proportions of fermentation starters for improving the flavor of yogurts.

Development of the Cre-Cas 2.0 system for evaluating acetoin production in Bacillus subtilis

BackgroundAcetoin is a vital platform compound widely used in the food, pharmaceutical, and chemical industries. Since 2004, the United States Department of Energy has listed it as one of the 30 platform compounds for priority development. Bacillus subtilis, a generally recognized safe microorganism, is an ideal platform microorganism for the engineered production of valuable bio-based chemicals. MethodsThe Cre-Cas 2.0 system derived from the Cre-Cas system was applied to inactive, enhance, or recover native metabolic genes. This system was designed to evaluate which native metabolic genes affect acetoin production in B. subtilis. Significant findingsUtilizing the Cre-Cas 2.0 system, acetoin reductase (bdhA), l-lactate dehydrogenase (ldh), and acetoin dehydrogenase (acoA) were inactivated, while the native acetoin synthesis genes, alsSD operon and alsR, were enhanced. This approach resulted in the creation of sixteen recombinant strains for the assessment of acetoin production. Finally, the acetoin production of strain ACN11 was 55 % higher than the original strain B. subtilis DB428. Moreover, employing the Cre-Cas 2.0 concept for editing the chromosomal genes of B. subtilis to enhance acetoin and other biochemical production could offer flexibility and applicability.

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.

Structural and enzymatic characterization of Bacillus subtilis R,R-2,3-butanediol dehydrogenase

2,3-butanediol dehydrogenase (BDH, EC 1.1.1.76) also known as acetoin reductase (AR, EC 1.1.1.4) is the key enzyme converting acetoin (AC) into 2,3-butanediol (BD) and undertaking the irreversible conversion of diacetyl to acetoin in various microorganisms. The existence of three BDHs (R,R-, meso-, and S,S-BDH) product different BD isomers. Catalyzing mechanisms of meso- and S,S-BDH have been understood with the assistance of their X-ray crystal structures. However, the lack of structural data for R,R-BDH restricts the integral understanding of the catalytic mechanism of BDHs. In this study, we successfully crystallized and solved the X-ray crystal structure of Bacillus subtilis R,R-BDH. A zinc ion was found locating in the catalytic center and coordinated by Cys37, His70 and Glu152, helping to stabilize the chiral substrates observed in the predicted molecular docking model. The interaction patterns of different chiral substrates in the molecular docking model explained the react priority measured by the enzyme activity assay of R,R-BDH. Site-directed mutation experiments determined that the amino acids Cys37, Thr244, Ile268 and Lys340 are important in the catalytically active center. The structural information of R,R-BDH presented in this study accomplished the understanding of BDHs catalytic mechanism and more importantly provides useful guidance for the directional engineering of R,R-BDH to obtain high-purity monochiral BD and AC.

Biotechnological Production of Optically Pure 2,3-Butanediol by Bacillus subtilis Based on Dissolved Oxygen Control Strategy

2,3-Butanediol (2,3-BD) is a promising platform chemical, produced from microbial cells. Oxygen availability is a crucial factor driving the formation and proportion of 2,3-BD and acetoin in 2,3-BD producing bacterial strains. In this study, the ability of B. subtills GD5 to produce 2,3-BD in optimized sucrose-based media was evaluated, by investigating the impact of carbon to nitrogen (C/N) ratio and the effectiveness of alternative low-cost nitrogen sources (corn steep liquor, soybean meal, and ammonium sulphate). Subsequently, different dissolved oxygen (DO) controlling regimes were assessed in batch bioreactor fermentations. The best fermentation outcomes were obtained with uncontrolled DO, achieving 5.88 g/L of optically pure (R,R)-2,3-BD (~100% purity), accompanied by a production yield of 0.43 g/g, and a productivity of 0.2 g/L/h. Additionally, the influence of the DO controlling regime on B. subtills key enzymes involved in the reverse activity of acetoin reductase was also monitored. A fed-batch process under the most suitable DO conditions was carried out to improve 2,3-BD production, achieving 42.31 g/L 2,3-BD with a production yield of 0.52 g/g. Thus, B. subtilis GD5 is a promising strain for the efficient production of pure chiral (R,R)-2,3-BD under uncontrolled DO conditions, using alternative low-cost nitrogen sources.

Regulation of carbon flux and NADH/NAD+ supply to enhance 2,3-butanediol production in Enterobacter aerogenes

As a valuable platform chemical, 2,3-Butanediol (2,3-BDO) has a variety of industrial applications, and its microbial production is particularly attractive as an alternative to petroleum-based production. In this study, the regulation of intracellular carbon flux and NADH/NAD+ was used to increase the 2,3-BDO production of Enterobacter aerogenes. The genes encoding lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) were disrupted using the λ-Red recombination method and CRISPR-Cas9 to reduce the production of several byproducts and the consumption of NADH. Knockout of ldh or pfl increased intracellular NADH/NAD+ by 111 % and 113 %, respectively. Moreover, two important genes in the 2,3-BDO biosynthesis pathway, acetolactate synthase (budB) and acetoin reductase (budC), were overexpressed in E. aerogenes to further amply the metabolic flux toward 2,3-BDO production. And the overexpression of budB or budC increased intracellular NADH/NAD+ by 46 % and 57 %, respectively. In shake-flask cultivation with sucrose as carbon source, the 2,3-BDO titer of the IAM1183-LPBC was 3.55 times that of the wild type. In the 5-L fermenter, the maximal 2,3-BDO production produced by the IAM1183-LPBC was 2.88 times that of the original strain. This work offers new ideas for promoting the biosynthesis of 2,3-BDO for industrial applications.

Detection of Acetoin and Diacetyl by a Tobacco Mosaic Virus-Assisted Field-Effect Biosensor

Acetoin and diacetyl have a major impact on the flavor of alcoholic beverages such as wine or beer. Therefore, their measurement is important during the fermentation process. Until now, gas chromatographic techniques have typically been applied; however, these require expensive laboratory equipment and trained staff, and do not allow for online monitoring. In this work, a capacitive electrolyte–insulator–semiconductor sensor modified with tobacco mosaic virus (TMV) particles as enzyme nanocarriers for the detection of acetoin and diacetyl is presented. The enzyme acetoin reductase from Alkalihalobacillus clausii DSM 8716T is immobilized via biotin–streptavidin affinity, binding to the surface of the TMV particles. The TMV-assisted biosensor is electrochemically characterized by means of leakage–current, capacitance–voltage, and constant capacitance measurements. In this paper, the novel biosensor is studied regarding its sensitivity and long-term stability in buffer solution. Moreover, the TMV-assisted capacitive field-effect sensor is applied for the detection of diacetyl for the first time. The measurement of acetoin and diacetyl with the same sensor setup is demonstrated. Finally, the successive detection of acetoin and diacetyl in buffer and in diluted beer is studied by tuning the sensitivity of the biosensor using the pH value of the measurement solution.

Enantiopure meso-2,3-butanediol production by metabolically engineered Saccharomyces cerevisiae expressing 2,3-butanediol dehydrogenase from Klebsiella oxytoca

2,3-Butanediol (2,3-BDO) is a functional C4 compound with various industrial applications. It exists as three isomers, and racemic mixtures can be produced through chemical synthesis and fermentation using natural producers. In this study, Saccharomyces cerevisiae was engineered to produce enantiopure meso-2,3-BDO by eliminating BDH1 encoding (2 R,3 R)-butanediol dehydrogenase and introducing budC coding for acetoin reductase from Klebsiella oxytoca. The resulting strain produced 69.2 g/L of enantiopure meso-2,3-BDO production with a productivity of 1.5 g meso-2,3-BDO/L•h using cassava hydrolysates. Furthermore, improved titer and productivity of meso-2,3-BDO were achieved by resolving C2-auxotrophy. To decrease the acetoin accumulation, the budC gene was stably and strongly expressed throughout the chromosomal integration. The resulting strain produced 171 g/L of meso-2,3-BDO with 0.49 g meso-2,3-BDO /g glucose, which is 99.8 % of theoretical yield and a productivity of 1.8 g meso-2,3-BDO/L•h. These results will help facilitate the commercial production of enantiopure meso-2,3-BDO using the GRAS strain.

(IMCS Second Place Best Paper Award) Acetoin Biosensor Based on Acetoin Reductase-Modified Field-Effect Sensor Applied for Acetoin Detection in Beer Samples

Introduction In food industry, the use and dosing of flavoring substances is an important aspect during fabrication processes. Acetoin and diacetyl are flavoring agents which can be found in many food products, like yogurt, cheese, milk, wine and beer [1]. In beer, acetoin/diacetyl is a critical parameter for the maturation degree of the beverage during fabrication. They are formed during the first phase of beer fermentation and getting reduced during the later phase of fermentation and the maturation. Both can have a negative effect on the beer flavor, when they are above a certain concentration. Typical acetoin concentrations in beer are in the range of 10-50 µM. Measuring the acetoin/diacetyl concentration during the production can serve as a quality control and unnecessary maturation time could be avoided [2]. Until now, the quantitative determination of the acetoin/diacetyl concentration is mostly done “off-line” by gas chromatography. Therefore, a sample has to be sent to an external laboratory, which takes time till results are achieved, or the brewery needs an own laboratory, whereby enormous costs arise and trained staff is needed. Because of that, we recently developed a biosensor for the detection of acetoin based on an enzyme-modified electrolyte-insulator-semiconductor (EIS) sensor, which was characterized in buffer solutions [3]. In this work, the novel biosensor was applied for acetoin measurements in beer samples for the first time. Materials and Methods As sensor structures, capacitive Al-p-Si-SiO2-Ta2O5 field-effect sensors have been used, schematically shown in Fig. 1(a). The Ta2O5 transducer surface was modified with a novel acetoin reductase by means of cross-linking. The acetoin reductase (from B. clausii DSM 8716T) catalyzes the reduction of acetoin to 2,3-butanediol, while NADH serves as a cofactor [4]. The fabricated acetoin biosensors have been electrochemically characterized by capacitance-voltage- and constant-capacitance (ConCap) measurements in buffer solution (0.2 mM Tris-HCl) with varying acetoin concentration and beer samples, spiked with different amounts of acetoin. Results and Conclusions The acetoin sensitivity was investigated in beer solutions and the biosensors showed a linear behavior in the concentration range between 10 µM and 90 μM with a sensitivity of ca. 30 mV/dec, see Fig. 1(b). A detailed description of experiments and the obtained results will be presented and discussed. Acknowledgments The authors thank the ministry of innovation, science and research of the state of North-Rhine-Westphalia for financial support (funding program FH-Struktur 2016).

Genome sequencing, annotation and exploration of the SO2-tolerant non-conventional yeast Saccharomycodes ludwigii

BackgroundSaccharomycodes ludwigii belongs to the poorly characterized Saccharomycodeacea family and is known by its ability to spoil wines, a trait mostly attributable to its high tolerance to sulfur dioxide (SO2). To improve knowledge about Saccharomycodeacea our group determined whole-genome sequences of Hanseniaspora guilliermondii (UTAD222) and S. ludwigii (UTAD17), two members of this family. While in the case of H. guilliermondii the genomic information elucidated crucial aspects concerning the physiology of this species in the context of wine fermentation, the draft sequence obtained for S. ludwigii was distributed by more than 1000 contigs complicating extraction of biologically relevant information. In this work we describe the results obtained upon resequencing of S. ludwigii UTAD17 genome using PacBio as well as the insights gathered from the exploration of the annotation performed over the assembled genome.ResultsResequencing of S. ludwigii UTAD17 genome with PacBio resulted in 20 contigs totaling 13 Mb of assembled DNA and corresponding to 95% of the DNA harbored by this strain. Annotation of the assembled UTAD17 genome predicts 4644 protein-encoding genes. Comparative analysis of the predicted S. ludwigii ORFeome with those encoded by other Saccharomycodeacea led to the identification of 213 proteins only found in this species. Among these were six enzymes required for catabolism of N-acetylglucosamine, four cell wall β-mannosyltransferases, several flocculins and three acetoin reductases. Different from its sister Hanseniaspora species, neoglucogenesis, glyoxylate cycle and thiamine biosynthetic pathways are functional in S. ludwigii. Four efflux pumps similar to the Ssu1 sulfite exporter, as well as robust orthologues for 65% of the S. cerevisiae SO2-tolerance genes, were identified in S. ludwigii genome.ConclusionsThis work provides the first genome-wide picture of a S. ludwigii strain representing a step forward for a better understanding of the physiology and genetics of this species and of the Saccharomycodeacea family. The release of this genomic sequence and of the information extracted from it can contribute to guide the design of better wine preservation strategies to counteract spoilage prompted by S. ludwigii. It will also accelerate the exploration of this species as a cell factory, specially in production of fermented beverages where the use of Non-Saccharomyces species (including spoilage species) is booming.

Capacitive Field‐Effect Biosensor Applied for the Detection of Acetoin in Alcoholic Beverages and Fermentation Broths

An acetoin biosensor based on a capacitive electrolyte–insulator–semiconductor (EIS) structure modified with the enzyme acetoin reductase, also known as butane‐2,3‐diol dehydrogenase (Bacillus clausii DSM 8716T), is applied for acetoin detection in beer, red wine, and fermentation broth samples for the first time. The EIS sensor consists of an Al/p‐Si/SiO2/Ta2O5 layer structure with immobilized acetoin reductase on top of the Ta2O5 transducer layer by means of crosslinking via glutaraldehyde. The unmodified and enzyme‐modified sensors are electrochemically characterized by means of leakage current, capacitance–voltage, and constant capacitance methods, respectively.

Proteomic comparison of biofilm vs. planktonic Staphylococcus epidermidis cells suggests key metabolic differences between these conditions

Previous studies have shown that biofilm-forming bacteria are deficient in tricarboxylic acid (TCA) cycle metabolites, suggesting a relationship between these cellular processes. In this work, we compared the proteomes of planktonic vs biofilm cells from a clinical strain of Staphylococcus epidermidis using LC-MS/MS. A total of 168 proteins were identified from both growth conditions. The biofilm cells showed enrichment of proteins participating in glycolysis for the formation of pyruvate; however, the absence of TCA cycle proteins and the presence of lactate dehydrogenase, formate acetyltransferase, and acetoin reductase suggested that pyruvate was catabolized to their respective products: lactate, formate and acetoin. On the other hand, planktonic cells showed proteins participating in glycolysis and the TCA cycle, the pentose phosphate pathway, gluconeogenesis, ATP generation and the oxidative stress response. Functional networks with higher interconnection were predicted for planktonic proteins. We propose that in S. epidermidis, the relative absence of TCA cycle proteins is associated with the formation of biofilms and that lactate, formate and acetoin are the end products of partial glucose metabolism.

Acetoin Biosensor Based on Acetoin Reductase-Modified Field-Effect Sensor Applied for Acetoin Detection in Beer Samples

Introduction In food industry, the use and dosing of flavoring substances is an important aspect during fabrication processes. Acetoin and diacetyl are flavoring agents which can be found in many food products, like yogurt, cheese, milk, wine and beer [1]. In beer, acetoin/diacetyl is a critical parameter for the maturation degree of the beverage during fabrication. They are formed during the first phase of beer fermentation and getting reduced during the later phase of fermentation and the maturation. Both can have a negative effect on the beer flavor, when they are above a certain concentration. Typical acetoin concentrations in beer are in the range of 10-50 µM. Measuring the acetoin/diacetyl concentration during the production can serve as a quality control and unnecessary maturation time could be avoided [2]. Until now, the quantitative determination of the acetoin/diacetyl concentration is mostly done “off-line” by gas chromatography. Therefore, a sample has to be sent to an external laboratory, which takes time till results are achieved, or the brewery needs an own laboratory, whereby enormous costs arise and trained staff is needed. Because of that, we recently developed a biosensor for the detection of acetoin based on an enzyme-modified electrolyte-insulator-semiconductor (EIS) sensor, which was characterized in buffer solutions [3]. In this work, the novel biosensor was applied for acetoin measurements in beer samples for the first time. Materials and Methods As sensor structures, capacitive Al-p-Si-SiO2-Ta2O5 field-effect sensors have been used, schematically shown in Fig. 1(a). The Ta2O5 transducer surface was modified with a novel acetoin reductase by means of cross-linking. The acetoin reductase (from B. clausii DSM 8716T) catalyzes the reduction of acetoin to 2,3-butanediol, while NADH serves as a cofactor [4]. The fabricated acetoin biosensors have been electrochemically characterized by capacitance-voltage- and constant-capacitance (ConCap) measurements in buffer solution (0.2 mM Tris-HCl) with varying acetoin concentration and beer samples, spiked with different amounts of acetoin. Results and Conclusions The acetoin sensitivity was investigated in beer solutions and the biosensors showed a linear behavior in the concentration range between 10 µM and 90 μM with a sensitivity of ca. 30 mV/dec, see Fig. 1(b). A detailed description of experiments and the obtained results will be presented and discussed.Figure 1: Schematic measurement set-up with the layer structure of the EIS sensor modified with acetoin reductase (a). Exemplary ConCap-measurement curve recorded in buffer-beer mixture (ratio 20:1) spiked with different acetoin concentrations, respectively (b). Acknowledgments The authors thank the ministry of innovation, science and research of the state of North-Rhine-Westphalia for financial support (funding program FH-Struktur 2016).

A simple biosynthetic pathway for 2,3-butanediol production in Thermococcus onnurineus NA1.

The biosynthetic pathway of 2,3-butanediol (2,3-BDO) production from pyruvate under anaerobic conditions includes three enzymes: acetolactate synthase (ALS), acetolactate decarboxylase (ALDC), and acetoin reductase (AR). Recently, in anaerobic hyperthermophilic Pyrococcus furiosus, it has been reported that acetoin, a precursor of 2,3-BDO, is produced from pyruvate by ALS through a temperature-dependent metabolic switch. In this study, we first attempted to produce 2,3-BDO from Thermococcus onnurineus NA1 using a simple biosynthetic pathway by two enzymes (ALS and AR) at a high temperature. Two heterologous genes, acetolactate synthase (alsS) from Pyrococcus sp. NA2 and alcohol dehydrogenase (adh) from T. guaymacensis, were introduced and expressed in T. onnurineus NA1. The mutant strain produced approximately 3.3 mM 2,3-BDO at 80 °C. An acetyl-CoA synthetase IIIα (TON_1001) was further deleted to enhance 2,3-BDO production, and the mutant strain showed a 25% increase in the specific production of 2,3-BDO. Furthermore, when carbon monoxide (CO) gas was added as a reductant, specific production of 2,3-BDO increased by 45%. These results suggest a new biosynthetic pathway for 2,3-BDO and demonstrate the possibility of T. onnurineus NA1 as a platform strain for 2,3-BDO production at high temperatures.

Development and characterization of a field-effect biosensor for the detection of acetoin

A capacitive electrolyte-insulator-semiconductor (EIS) field-effect biosensor for acetoin detection has been presented for the first time. The EIS sensor consists of a layer structure of Al/p-Si/SiO2/Ta2O5/enzyme acetoin reductase. The enzyme, also referred to as butane-2,3-diol dehydrogenase from B. clausii DSM 8716T, has been recently characterized. The enzyme catalyzes the (R)-specific reduction of racemic acetoin to (R,R)- and meso-butane-2,3-diol, respectively. Two different enzyme immobilization strategies (cross-linking by using glutaraldehyde and adsorption) have been studied. Typical biosensor parameters such as optimal pH working range, sensitivity, hysteresis, linear concentration range and long-term stability have been examined by means of constant-capacitance (ConCap) mode measurements. Furthermore, preliminary experiments have been successfully carried out for the detection of acetoin in diluted white wine samples.

Systematic Characterization of the Metabolism of Acetoin and Its Derivative Ligustrazine in Bacillus subtilis under Micro-Oxygen Conditions.

Bacillus subtilis is an important microorganism for brewing of Chinese Baijiu, which contributes to the formation of flavor chemicals including acetoin and its derivative ligustrazine. The first stage of Baijiu brewing process is under micro-oxygen conditions; however, there are few studies about B. subtilis metabolism under these conditions. Effects of various factors on acetoin and ligustrazine metabolism were investigated under these conditions, including key genes and fermentation conditions. Mutation of bdhA (encoding acetoin reductase) or overexpression of glcU (encoding glucose uptake protein) increased acetoin concentration. Addition of Vigna angularis powder to the culture medium also promoted acetoin production. Optimal culture conditions for ligustrazine synthesis were pH 6.0 and 42 °C. Ammonium phosphate was shown to promote ligustrazine synthesis in situ. This is the first report of acetoin and ligustrazine metabolism in B. subtilis under micro-oxygen conditions, which will ultimately promote the application of B. subtilis for maintaining Baijiu quality.

A shortened, two-enzyme pathway for 2,3-butanediol production in Escherichia coli.

The platform chemical 2,3-butanediol (2,3-BDO) is produced by a number of microorganisms via a three-enzyme pathway starting from pyruvate. Here, we report production of 2,3-BDO via a shortened, two-enzyme pathway in Escherichia coli. A synthetic operon consisting of the acetolactate synthase (ALS) and acetoin reductase (AR) genes from Enterobacter under control of the T7 promoter was cloned in an episomal plasmid. E. coli transformed with this plasmid produced 2,3-BDO and the pathway intermediate acetoin, demonstrating that the shortened pathway was functional. To assemble a synthetic operon for inducer- and plasmid-free production of 2,3-BDO, ALS and AR genes were integrated in the E. coli genome under control of the constitutive ackA promoter. Shake flask-level cultivation led to accumulation of ~1g/L acetoin and ~0.66g/L 2,3-BDO in the medium. The novel biosynthetic route for 2,3-BDO biosynthesis described herein provides a simple and cost-effective approach for production of this important chemical.

Optimized whole cell biocatalyst from acetoin to 2,3‐butanediol through coexpression of acetoin reductase with NADH regeneration systems in engineered Bacillus subtilis

AbstractBACKGROUND2,3‐Butanediol (2,3‐BD) has a wide range of applications in chiral molecular synthesis, biofuel additives, and in food flavor additive manufacturing. Fermentation is a favorable method for 2,3‐BD production. However, it requires much time and produces several NADH related byproducts which compete with 2,3‐BD production. Bacillus subtilis has an excellent ability for 2,3‐BD production by biocatalysis. However, its production is limited by low intracellular NADH and the reversible property of acetoin reductase (AR/2,3‐BDH). The whole cell biocatalyst process with two different NADH regeneration systems was designed for efficient production of 2,3‐BD in B. subtilis 168.RESULTSFormate dehydrogenase and glucose dehydrogenase for NADH regeneration were successfully co‐expressed with acetoin reductase in B. subtilis 168. After optimization of biocatalyst bioconversion conditions, B. subtilis 168/pMA5‐bdhA‐HpaII‐fdh yielded 74.5 g L−1 of 2, 3‐BD with 9.3 g L−1 h−1 productivity by fed batch and 115.4 g of 2,3‐BD was achieved using same batch bacterium by three repeated batch bioconversions. On the other hand, 63.7 g L−1 of 2, 3‐BD was produced with 7.92 g L−1 h−1 productivity by B. subtilis 168/pMA5‐bdhA‐HpaII‐gdh. To our knowledge, the volume productivity obtained here is the highest ever reported for biocatalysis.CONCLUSIONA higher productivity of 2,3‐BD from acetoin was achieved by whole cell biocatalysis with NADH regeneration systems in B. subtilis 168. This approach can be applied for NADH related bio‐based chemicals production to improve titer, yield and productivity. © 2017 Society of Chemical Industry

Enhanced 2,3-Butanediol Production by Optimizing Fermentation Conditions and Engineering Klebsiella oxytoca M1 through Overexpression of Acetoin Reductase.

Microbial production of 2,3-butanediol (2,3-BDO) has been attracting increasing interest because of its high value and various industrial applications. In this study, high production of 2,3-BDO using a previously isolated bacterium Klebsiella oxytoca M1 was carried out by optimizing fermentation conditions and overexpressing acetoin reductase (AR). Supplying complex nitrogen sources and using NaOH as a neutralizing agent were found to enhance specific production and yield of 2,3-BDO. In fed-batch fermentations, 2,3-BDO production increased with the agitation speed (109.6 g/L at 300 rpm vs. 118.5 g/L at 400 rpm) along with significantly reduced formation of by-product, but the yield at 400 rpm was lower than that at 300 rpm (0.40 g/g vs. 0.34 g/g) due to acetoin accumulation at 400 rpm. Because AR catalyzing both acetoin reduction and 2,3-BDO oxidation in K. oxytoca M1 revealed more than 8-fold higher reduction activity than oxidation activity, the engineered K. oxytoca M1 overexpressing the budC encoding AR was used in fed-batch fermentation. Finally, acetoin accumulation was significantly reduced by 43% and enhancement of 2,3-BDO concentration (142.5 g/L), yield (0.42 g/g) and productivity (1.47 g/L/h) was achieved compared to performance with the parent strain. This is by far the highest titer of 2,3-BDO achieved by K. oxytoca strains. This notable result could be obtained by finding favorable fermentation conditions for 2,3-BDO production as well as by utilizing the distinct characteristic of AR in K. oxytoca M1 revealing the nature of reductase.

Metabolic engineering of Bacillus subtilis for redistributing the carbon flux to 2,3-butanediol by manipulating NADH levels.

BackgroundAcetoin reductase (Acr) catalyzes the conversion of acetoin to 2,3-butanediol (2,3-BD) with concomitant oxidation of NADH to NAD+. Therefore, intracellular 2,3-BD production is likely governed by the quantities of rate-limiting factor(s) Acr and/or NADH. Previously, we showed that a high level of Acr was beneficial for 2,3-BD accumulation.Results Metabolic engineering strategies were proposed to redistribute carbon flux to 2,3-BD by manipulating NADH levels. The disruption of NADH oxidase (YodC, encoded by yodC) by insertion of a formate dehydrogenase gene in Bacillussubtilis was more efficient for enhancing 2,3-BD production and decreasing acetoin formation than the disruption of YodC by the insertion of a Cat expression cassette. This was because the former resulted in the recombinant strain AFY in which an extra NADH regeneration system was introduced and NADH oxidase was disrupted simultaneously. On fermentation by strain AFY, the highest 2,3-BD concentration increased by 19.9 % while the acetoin titer decreased by 71.9 %, relative to the parental strain. However, the concentration of lactate, the main byproduct, increased by 47.2 %. To further improve carbon flux and NADH to 2,3-BD, the pathway to lactate was blocked using the insertional mutation technique to disrupt the lactate dehydrogenase gene ldhA. The resultant engineered strain B. subtilis AFYL could efficiently convert glucose into 2,3-BD with little acetoin and lactate accumulation.ConclusionsThrough increasing the availability of NADH and decreasing the concentration of unwanted byproducts, this work demonstrates an important strategy in the metabolic engineering of 2,3-BD production by integrative recombinant hosts.