Metabolic engineering design to enhance (R,R)-2,3-butanediol production from glycerol in Bacillus subtilis based on flux balance analysis.

BACKGROUND: Glycerol is a desirable alternative substrate for 2,3-butanediol (2,3-BD) production for sustainable development in biotechnological industries and non-food competitive feedstock. B. subtilis, a "generally recognized as safe" organism that is highly tolerant to fermentation products, is an ideal platform microorganism to engineer the pathways for the production of valuable bio-based chemicals, but it has never been engineered to improve 2,3-BD production from glycerol. In this study, we aimed to enhance 2,3-BD production from glycerol in B. subtilis through in silico analysis. Genome-scale metabolic model (GSM) simulations was used to design and develop the metabolic pathways of B. subtilis. Flux balance analysis (FBA) simulation was used to evaluate the effects of step-by-step gene knockouts to improve 2,3-BD production from glycerol in B. subtilis. RESULTS: B. subtilis was bioengineered to enhance 2,3-BD production from glycerol using FBA in a published GSM model of B. subtilis, iYO844. Four genes, ackA, pta, lctE, and mmgA, were knocked out step by step, and the effects thereof on 2,3-BD production were evaluated. While knockout of ackA and pta had no effect on 2,3-BD production, lctE knockout led to a substantial increase in 2,3-BD production. Moreover, 2,3-BD production was improved by mmgA knockout, which had never been investigated. In addition, comparisons between in silico simulations and fermentation profiles of all B. subtilis strains are presented in this study. CONCLUSIONS: The strategy developed in this study, using in silico FBA combined with experimental validation, can be used to optimize metabolic pathways for enhanced 2,3-BD production from glycerol. It is expected to provide a novel platform for the bioengineering of strains to enhance the bioconversion of glycerol into other highly valuable chemical products.

The anti-cancer effect of flaxseed lignan derivatives on different acute myeloid leukemia cancer cells.

Flaxseeds have been known for their anti-cancerous effects due to the high abundance of lignans released upon ingestion. The most abundant lignan, secoisolariciresinol diglucoside (SDG), is ingested during the dietary intake of flax, and is then metabolized in the gut into two mammalian lignan derivatives, Enterodiol (END) and Enterolactone (ENL). These lignans were previously reported to possess anti-tumor effects against breast, colon, and lung cancer. This study aims to investigate the potential anti-cancerous effect of the flaxseed lignans SDG, END and ENL on acute myeloid leukemia cells (AML) in vitro and to decipher the underlying molecular mechanism. AML cell lines, (KG-1 and Monomac-1) and a normal lymphoblastic cell line were cultured and treated with the purified lignans. ENL was found to be the most promising lignan, as it exhibits a significant selective dose- and time-dependent cytotoxic effect in both AML cell lines, contrary to normal cells. The cytotoxic effects observed were attributed to apoptosis induction, as revealed by an increase in Annexin V staining of AML cells with increasing ENL concentrations. The increase in the percentage of cells in the pre-G phase, in addition to cell death ELISA analysis, validated cellular and DNA fragmentation respectively. Analysis of protein expression using western blots confirmed the activation of the intrinsic apoptotic pathway upon ENL treatment. This was also accompanied by an increase in ROS production intracellularly. In conclusion, this study demonstrates that ENL has promising anti-cancer effects in AML cell lines in vitro, by promoting DNA fragmentation and the intrinsic apoptotic pathway, highlighting the protective health benefits of flax seeds in leukemia.

Optimal design of a simulated-moving-bed chromatographic process for high-purity separation of acetoin from 2,3-butanediol in a continuous mode.

For the high-purity production of acetoin or 2,3-butanediol (BD) from related fermentation processes, it is essential to accomplish a detailed separation between acetoin and BD in an economical mode. To address this issue, we aimed to develop a highly-efficient simulated-moving-bed (SMB) process for the continuous-mode separation of acetoin from BD with high purity and small loss. As a first step for this task, the adsorption and mass-transfer parameters of acetoin and BD on a proven adsorbent were estimated while assuming that BD isomers (meso-BD and DL-BD) would be identical in adsorption and mass-transfer behaviors. The resultant parameters from such estimation were applied to the optimal design of the acetoin-BD separation SMB. The designed SMB was then experimentally investigated, which revealed that some sign of BD isomerism occurred in the SMB column-profile data and thus had an adverse effect on the SMB separation performance. To resolve this problem, the individual parameters of BD isomers were determined on the basis of the SMB column-profile data and an inverse-method principle. The resulting parameters of BD isomers were used in the re-design of the target SMB, which was then experimentally checked for its separation performance. It was confirmed that such SMB re-designed in consideration of BD isomerism was quite effective in the continuous-mode separation of acetoin from BD with high purity (> 99.2%) and small loss (< 1.52%).

Continuous-mode separation of fucose and 2,3-butanediol using a three-zone simulated moving bed process and its performance improvement by using partial extract-collection, partial extract-recycle, and partial desorbent-port closing.

If a multi-component monosugar mixture including fucose was used as the substrates for the Klebsiella oxytoca fermentation, it could offer the following two benefits simultaneously; (i) the removal of all monosugars other than fucose, and (ii) the acquisition of 2,3-butanediol (BD). To utilize such two benefits in favor of the economical efficiency of the fucose production process, it is essential to accomplish a high-purity separation between fucose and BD on the basis of a highly-economical mode. To address this issue, we aimed to develop a simulated moving bed (SMB) process for continuous-mode separation of fucose and BD with high purities. It was first found that an Amberchrom-CG71C resin could become a suitable adsorbent for the separation of interest. The intrinsic parameters of fucose and BD on such proven adsorbent were determined, and then applied to the optimal design of the fucose-BD separation SMB. The capability of the designed SMB in ensuring high purities and high yields was experimentally verified. Finally, we devised two potential strategies to make a further improvement in product concentrations and/or desorbent usage while keeping the purities and yields of fucose and BD almost unchanged. The first strategy was based on partial extract-collection and partial extract-discard, which was found to result in 33% higher BD product concentration. The second strategy was based on partial extract-collection, partial extract-recycle, and partial desorbent-port closing, which could lead to 25% lower desorbent usage, 33% higher BD product concentration, and 7% higher fucose product concentration.

Anionic Extraction for Efficient Recovery of Biobased 2,3-Butanediol-A Platform for Bulk and Fine Chemicals.

2,3-Butanediol (BDO) presents a promising platform molecule for the synthesis of basic and fine chemicals. Biotechnological production of BDO from renewable resources with living microbes enables high concentrations in the fermentation broth. The recovery of high-boiling BDO from an aqueous fermentation broth presents a subsequent challenge. A method is proposed for BDO isolation based on reversible complexation with phenylboronate in an anionic complex. BDO can be recovered by back-extraction into an acidic solution. The composition of the extracted species was determined by NMR spectroscopy, MS, and GC-MS methods. The conditions of extraction and back-extraction were optimized by using commercial BDO and finally applied to different fermentation broths. Up to 72-93 % BDO can be extracted and up to 80-90 % can be back-extracted under the optimized conditions. Purified bio-BDO was used in the presence of sulfuric acid for the synthesis of methyl ethyl ketone, an established organic solvent and discussed tailor-made biofuel.

Alashinols F and G, two lignans from stem bark of Syringa pinnatifolia.

Two new lignans, alashinols F and G (1 and 2), together with two known analogues (-)-secoisolariciresinol (3) and meso-secoisolariciresinol (4) were isolated from the stem bark of Syringa pinnatifolia, a Mongolian folk medicine with anti-myocardial ischaemic effects. Their structures were elucidated on basis of spectroscopic data analyses, including MS and 1D and 2D NMR, and their absolute configurations were elucidated on the basis of experimental and calculated electronic circular dichroisms. The in vitro anti-inflammation and anti-hypoxia evaluations were also discussed.

Rational engineering of diol dehydratase enables 1,4-butanediol biosynthesis from xylose.

Establishing novel synthetic routes for microbial production of chemicals often requires overcoming pathway bottlenecks by tailoring enzymes to enhance bio-catalysis or even achieve non-native catalysis. Diol dehydratases have been extensively studied for their interactions with C2 and C3 diols. However, attempts on utilizing these insights to enable catalysis on non-native substrates with more than two hydroxyl groups have been plagued with low efficiencies. Here, we rationally engineered the Klebsiella oxytoca diol dehydratase to enable and enhance catalytic activity toward a non-native C4 triol, 1,2,4-butanetriol. We analyzed dehydratase's interaction with 1,2-propanediol and glycerol, which led us to develop rationally conceived hypotheses. An in silico approach was then developed to identify and screen candidate mutants with desired activity. This led to an engineered diol dehydratase with nearly 5 fold higher catalytic activity toward 1,2,4-butanetriol than the wild type as determined by in vitro assays. Based on this result, we then expanded the 1,2,4-butanetriol pathway to establish a novel 1,4-butanediol production platform. We engineered Escherichia coli's xylose catabolism to enhance the biosynthesis of 1,2,4-butanetriol from 224mg/L to 1506mg/L. By introducing the complete pathway in the engineered strain we achieve de novo biosynthesis of 1,4-butanediol at 209mg/L from xylose. This work expands the repertoire of substrates catalyzed by diol dehydratases and serves as an elucidation to establish novel biosynthetic pathways involving dehydratase based biocatalysis.

Synthesis of (3R)-acetoin and 2,3-butanediol isomers by metabolically engineered Lactococcus lactis.

The potential that lies in harnessing the chemical synthesis capabilities inherent in living organisms is immense. Here we demonstrate how the biosynthetic machinery of Lactococcus lactis, can be diverted to make (3R)-acetoin and the derived 2,3-butanediol isomers meso-(2,3)-butanediol (m-BDO) and (2R,3R)-butanediol (R-BDO). Efficient production of (3R)-acetoin was accomplished using a strain where the competing lactate, acetate and ethanol forming pathways had been blocked. By introducing different alcohol dehydrogenases into this strain, either EcBDH from Enterobacter cloacae or SadB from Achromobacter xylosooxidans, it was possible to achieve high-yield production of m-BDO or R-BDO respectively. To achieve biosustainable production of these chemicals from dairy waste, we transformed the above strains with the lactose plasmid pLP712. This enabled efficient production of (3R)-acetoin, m-BDO and R-BDO from processed whey waste, with titers of 27, 51, and 32 g/L respectively. The corresponding yields obtained were 0.42, 0.47 and 0.40 g/g lactose, which is 82%, 89%, and 76% of maximum theoretical yield respectively. These results clearly demonstrate that L. lactis is an excellent choice as a cell factory for transforming lactose containing dairy waste into value added chemicals.

Microwave-assisted on-spot derivatization for gas chromatography-mass spectrometry based determination of polar low molecular weight compounds in dried blood spots.

Dried blood spot (DBS) sampling and analysis is increasingly being applied in bioanalysis. Although the use of DBS has many advantages, it is also associated with some challenges. E.g. given the limited amount of available material, highly sensitive detection techniques are often required to attain sufficient sensitivity. In gas chromatography coupled to mass spectrometry (GC-MS), derivatization can be helpful to achieve adequate sensitivity. Because this additional sample preparation step is considered as time-consuming, we introduce a new derivatization procedure, i.e. "microwave-assisted on-spot derivatization", to minimize sample preparation of DBS. In this approach the derivatization reagents are directly applied onto the DBS and derivatization takes place in a microwave instead of via conventional heating. In this manuscript we evaluated the applicability of this new concept of derivatization for the determination of two polar low molecular weight molecules, gamma-hydroxybutyric acid (GHB) and gabapentin, in DBS using a standard GC-MS configuration. The method was successfully validated for both compounds, with imprecision and bias values within acceptance criteria (<20% at LLOQ, <15% at 3 other QC levels). Calibration lines were linear over the 10-100µg/mL and 1-30µg/mL range for GHB and gabapentin, respectively. Stability studies revealed no significant decrease of gabapentin and GHB in DBS upon storage at room temperature for at least 84 days. Furthermore, DBS-specific parameters, including hematocrit and volume spotted, were evaluated. As demonstrated by the analysis of GHB and gabapentin positive samples, "microwave-assisted on-spot derivatization" proved to be reliable, fast and applicable in routine toxicology. Moreover, other polar low molecular weight compounds of interest in clinical and/or forensic toxicology, including vigabatrin, beta-hydroxybutyric acid, propylene glycol, diethylene glycol, 1,4-butanediol and 1,2-butanediol, can also be detected using this method.

Combining metabolic engineering and biocompatible chemistry for high-yield production of homo-diacetyl and homo-(S,S)-2,3-butanediol.

Biocompatible chemistry is gaining increasing attention because of its potential within biotechnology for expanding the repertoire of biological transformations carried out by enzymes. Here we demonstrate how biocompatible chemistry can be used for synthesizing valuable compounds as well as for linking metabolic pathways to achieve redox balance and rescued growth. By comprehensive rerouting of metabolism, activation of respiration, and finally metal ion catalysis, we successfully managed to convert the homolactic bacterium Lactococcus lactis into a homo-diacetyl producer with high titer (95mM or 8.2g/L) and high yield (87% of the theoretical maximum). Subsequently, the pathway was extended to (S,S)-2,3-butanediol (S-BDO) through efficiently linking two metabolic pathways via chemical catalysis. This resulted in efficient homo-S-BDO production with a titer of 74mM (6.7g/L) S-BDO and a yield of 82%. The diacetyl and S-BDO production rates and yields obtained are the highest ever reported, demonstrating the promising combination of metabolic engineering and biocompatible chemistry as well as the great potential of L. lactis as a new production platform.

2,3 Butanediol production in an obligate photoautotrophic cyanobacterium in dark conditions via diverse sugar consumption.

Cyanobacteria are under investigation as a means to utilize light energy to directly recycle CO2 into chemical compounds currently derived from petroleum. Any large-scale photosynthetic production scheme must rely on natural sunlight for energy, thereby limiting production time to only lighted hours during the day. Here, an obligate photoautotrophic cyanobacterium was engineered for enhanced production of 2,3-butanediol (23BD) in continuous light, 12h:12h light-dark diurnal, and continuous dark conditions via supplementation with glucose or xylose. This study achieved 23BD production under diurnal conditions comparable to production under continuous light conditions. The maximum 23BD titer was 3.0gL(-1) in 10d. Also achieving chemical production under dark conditions, this work enhances the feasibility of using cyanobacteria as industrial chemical-producing microbes.

Ionic Liquid-Based Ultrasonic-Assisted Extraction of Secoisolariciresinol Diglucoside from Flaxseed (Linum usitatissimum L.) with Further Purification by an Aqueous Two-Phase System.

In this work, a two-step extraction methodology of ionic liquid-based ultrasonic-assisted extraction (IL-UAE) and ionic liquid-based aqueous two-phase system (IL-ATPS) was developed for the extraction and purification of secoisolariciresinol diglucoside (SDG) from flaxseed. In the IL-UAE step, several kinds of ILs were investigated as the extractants, to identify the IL that affords the optimum extraction yield. The extraction conditions such as IL concentration, ultrasonic irradiation time, and liquid-solid ratio were optimized using response surface methodology (RSM). In the IL-ATPS step, ATPS formed by adding kosmotropic salts to the IL extract was used for further separation and purification of SDG. The most influential parameters (type and concentration of salt, temperature, and pH) were investigated to obtain the optimum extraction efficiency. The maximum extraction efficiency was 93.35% under the optimal conditions of 45.86% (w/w) IL and 8.27% (w/w) Na2SO4 at 22 °C and pH 11.0. Thus, the combination of IL-UAE and IL-ATPS makes up a simple and effective methodology for the extraction and purification of SDG. This process is also expected to be highly useful for the extraction and purification of bioactive compounds from other important medicinal plants.

Genome sequence of type strain Paenibacillus polymyxa DSM 365, a highly efficient producer of optically active (R,R)-2,3-butanediol.

Paenibacillus polymyxa DSM 365, an efficient producer of (R,R)-2,3-butanediol, is known to show the highest production titer and productivity reported to date. Here, the first draft genome sequence of this promising strain may provide the genetic basis for further insights into the molecular mechanisms underlying the production of (R,R)-2,3-butanediol with high optical purity and at a high titer. It will also facilitate the design of rational strategies for further strain improvements, as well as construction of artificial biosynthetic pathways through synthetic biology for asymmetric synthesis of chiral 2,3-butanediol or acetoin in common microbial hosts.

Enhanced production of 2,3-butanediol by a genetically engineered Bacillus sp. BRC1 using a hydrolysate of empty palm fruit bunches.

A Bacillus species that produces 2,3-butanediol (2,3-BD), termed BRC1, was newly isolated, and a 2,3-BD dehydrogenase (Bdh) from this species was identified and characterized at the molecular and biochemical level. Sequence analysis revealed that Bdh is homologous to D-2,3-BD dehydrogenases. An analysis of the enzymatic properties of Bdh overexpressed in Escherichia coli confirmed the molecular results, showing preferred activity toward D-2,3-BD. Optimum pH, temperature, and kinetics determined for reductive and oxidative reactions support the preferential production of 2,3-BD during cell growth. Overexpression of bdh under the control of a xylose-inducible promoter resulted in increased enzyme activity and enhanced 2,3-BD production in Bacillus sp. BRC1. Additionally, a hydrolysate of cellulosic material, (empty palm fruit bunches), was successfully used for the enhanced production of 2,3-BD in the recombinant Bacillus strain.

Neural cell activation by phenolic compounds from the Siberian larch (Larix sibirica).

Small organic phenolic compounds from natural sources have attracted increasing attention due to their potential to ameliorate the serious consequences of acute and chronic traumata of the mammalian nervous system. In this contribution, it is reported that phenols from the knot zones of Siberian larch (Larix sibirica) wood, namely, the antioxidant flavonoid (+)-dihydroquercetin (1) and the lignans (-)-secoisolariciresinol (2) and (+)-isolariciresinol (3), affect migration and outgrowth of neurites/processes from cultured neurons and glial cells of embryonic and early postnatal mice. Compounds 1-3, which were available in preparative amounts, enhanced neurite outgrowth from cerebellar granule neurons, dorsal root ganglion neurons, and motoneurons, as well as process formation of Schwann cells in a dose-dependent manner in the low nanomolar range. Migration of cultured astrocytes was inhibited by 1-3, and migration of neurons out of cerebellar explants was enhanced by 1. These observations provide evidence for the neuroactive features of these phenolic compounds in enhancing the beneficial properties of neurons and reducing the inhibitory properties of activated astrocytes in an in vitro setting and encourage the further investigation of these effects in vivo, in animal models of acute and chronic neurological diseases.

Isolation, characterization and antihyperlipidemic activity of secoisolariciresinol diglucoside in poloxamer-407-induced experimental hyperlipidemia.

CONTEXT: Linum usitatissimum L. (Linaceae), commonly known as flaxseed, is a good source of dietary fiber and lignans. Earlier we reported cardioprotective, antihyperlipidemic, and in vitro antioxidant activity of flax lignan concentrate (FLC) obtained from flaxseed. OBJECTIVES: To isolate secoisolariciresinol diglucoside (SDG) from FLC and to evaluate the antihyperlipidemic activity of SDG in poloxamer-407 (P-407)-induced hyperlipidaemic mice. MATERIAL AND METHODS: FLC was subjected to column chromatography and further subjected to preparative HPTLC to isolate SDG. The chemical structure of the isolated compound was elucidated by UV, IR, (1)H NMR, (13)C NMR, DEPT, COSY, HSQC, HMBC, ROESY, MS, and specific optical rotation was recorded. Further, we have investigated the antihyperlipidaemic effect of SDG (20 mg/kg) in P-407-induced hyperlipidaemic rats. Hyperlipidaemia was induced by intraperitoneal administration of P-407 (30% w/v). Serum lipid parameters such as total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) levels were measured. RESULTS AND DISCUSSION: The structure and stereochemistry of the isolated compound were confirmed on the basis of 1D and 2D spectral data and characterized as SDG. Finally, isolated pure SDG was screened using a P-407-induced mice model for its antihyperlipidemic action using serum lipid parameters. The isolated SDG (20 mg/kg) significantly reduced serum cholesterol, triglyceride (p < 0.001), very low-density lipoprotein (p < 0.05), and non-significantly increased HDL-C. CONCLUSION: Finally, it was concluded unequivocally that SDG showed antihyperlipidaemic effects in P-407-induced hyperlipidaemic mice. Isolated pure SDG confirms that SDG is beneficial in the prevention of experimental hyperlipidemia in laboratory animals.

Permeability and conjugative metabolism of flaxseed lignans by Caco-2 human intestinal cells.

Reports in the literature associate the dietary intake of flaxseed lignans with a number of health benefits. The major lignan found in flaxseed, secoisolariciresinol diglucoside (1), undergoes metabolism principally to secoisolariciresinol (2), enterodiol (3), and enterolactone (4) in the human gastrointestinal tract. Systemically, lignans are present largely as phase II enzyme conjugates. To improve understanding of the oral absorption characteristics, a systematic evaluation of the intestinal permeation was conducted and the conjugative metabolism potential of these lignans using the polarized Caco-2 cell system was analyzed. For permeation studies, lignans (100 µM) were added to acceptor or donor compartments and samples were taken at 2 h. For metabolism studies, lignans (100 µM) were incubated in Caco-2 for a maximum of 48 h. Cell lysates and media were treated with ß-glucuronidase/sulfatase, and lignan concentrations were determined using HPLC. Apical-to-basal permeability coefficients for 2-4 were 8.0 ± 0.4, 7.7 ± 0.2, and 13.7 ± 0.2 (×10(-6)) cm/s, respectively, whereas efflux ratios were 0.8-1.2, consistent with passive diffusion. The permeation of compound 1 was not detected. The extent of conjugation after 48 h was <3%, ∼95%, ∼90%, and >99% for 1-4, respectively. These data suggest 2-4, but not 1 undergo passive permeation and conjugative metabolism by Caco-2 cells.

2,3-Butanediol recovery from fermentation broth by alcohol precipitation and vacuum distillation.

This study presents a new and effective downstream process to recover 2,3-butanediol (2,3-BD) from fermentation broth which is produced by a recombinant Klebsiella pneumoniae strain. The ldhA-deficient K. pneumoniae strain yielded about 90 g/L of 2,3-BD, along with a number of by-products, such as organic acids and alcohols, in a 65 h fed-batch fermentation. The pH-adjusted cell-free fermentation broth was firstly concentrated until 2,3-BD reached around 500 g/L by vacuum evaporation at 50°C and 50 mbar vacuum pressure. The concentrated solution was further treated using light alcohols, including methanol, ethanol, and isopropanol, for the precipitation of organic acids and inorganic salts. Isopropanol showed the highest removal efficiency, in which 92.5% and 99.8% of organic acids and inorganic salts were precipitated, respectively. At a final step, a vacuum distillation process enabled the recovery of 76.2% of the treated 2,3-BD, with 96.1% purity, indicating that fermentatively produced 2,3-BD is effectively recovered by a simple alcohol precipitation and vacuum distillation.

Biodegradation-inspired bioproduction of methylacetoin and 2-methyl-2,3-butanediol.

Methylacetoin (3-hydroxy-3-methylbutan-2-one) and 2-methyl-2,3-butanediol are currently obtained exclusively via chemical synthesis. Here, we report, to the best of our knowledge, the first alternative route, using engineered Escherichia coli. The biological synthesis of methylacetoin was first accomplished by reversing its biodegradation, which involved modifying the enzyme complex involved, switching the reaction substrate, and coupling the process to an exothermic reaction. 2-Methyl-2,3-butanediol was then obtained by reducing methylacetoin by exploiting the substrate promiscuity of acetoin reductase. A complete biosynthetic pathway from renewable glucose and acetone was then established and optimized via in vivo enzyme screening and host metabolic engineering, which led to titers of 3.4 and 3.2 g l(-1) for methylacetoin and 2-methyl-2,3-butanediol, respectively. This work presents a biodegradation-inspired approach to creating new biosynthetic pathways for small molecules with no available natural biosynthetic pathway.

Selective production of 2,3-butanediol and acetoin by a newly isolated bacterium Klebsiella oxytoca M1.

A newly isolated bacterium, designated as Klebsiella oxytoca M1, produced 2,3-butanediol (2,3-BDO) or acetoin selectively as a major product depending on temperature in a defined medium. K. oxytoca M1 produced 2,3-BDO mainly (0.32~0.34 g/g glucose) at 30 °C while acetoin was a major product (0.32~0.38 g/g glucose) at 37 °C. To investigate factors affecting product profiles according to temperature, the expression level of acetoin reductase (AR) that catalyzes the conversion of acetoin to 2,3-BDO was analyzed using crude protein extracted from K. oxytoca M1 grown at 30 and 37 °C. The AR expression at 37 °C was 12.8-fold lower than that at 30 °C at the stationary phase and reverse transcription PCR (RT-PCR) analysis of the budC (encoding AR) was also in agreement with the AR expression results. When AR was overexpressed using K. oxytoca M1 harboring pUC18CM-budC, 2,3-BDO became a major product at 37 °C, indicating that the AR expression level was a key factor determining the major product of K. oxytoca M1 at 37 °C. The results in this study demonstrate the feasibility of using K. oxytoca M1 for the production of not only 2,3-BDO but also acetoin as a major product.