Biochemical characterization of unusual meso-2,3-butanediol dehydrogenase from a strain of Bacillus subtilis

Hao, Wenbo; Ji, Fangling; Wang, Jingyun; Zhang, Yue; Wang, Tianqi; Bao, Yongming

doi:10.1016/j.molcatb.2014.08.015

Cited by 7 publications

(4 citation statements)

References 29 publications

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“…Noticeably, d -(−)-2,3-BD dehydrogenase coded by bdhA was thought to be the major AR in B. subtilis, and its deletion abolished 2,3-BD production in early stationary phase [ 28 , 51 ]; however, the other 2,3-BD dehydrogenases remained unknown. In a recent study [ 52 ], meso-2,3-BD dehydrogenase was identified from a strain of B. subtilis , and it was suggested that a nonconservative amino acid substitution from Asp to Gly of this dehydrogenase caused the loss of its catalytic function and enzymatic activity. This result might further illustrate the fact that wild-type B. subtilis 168 generated only d -(−)-2,3-BD (purity >99 %).…”

Section: Resultsmentioning

confidence: 99%

Metabolic engineering of Bacillus subtilis for chiral pure meso-2,3-butanediol production

Huo

et al. 2016

Biotechnol Biofuels

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Background2,3-Butanediol (2,3-BD) with low toxicity to microbes, could be a promising alternative for biofuel production. However, most of the 2,3-BD producers are opportunistic pathogens that are not suitable for industrial-scale fermentation. In our previous study, wild-type Bacillus subtilis 168, as a class I microorganism, was first found to generate only d-(−)-2,3-BD (purity >99 %) under low oxygen conditions.ResultsIn this work, B. subtilis was engineered to produce chiral pure meso-2,3-BD. First, d-(−)-2,3-BD production was abolished by deleting d-(−)-2,3-BD dehydrogenase coding gene bdhA, and acoA gene was knocked out to prevent the degradation of acetoin (AC), the immediate precursor of 2,3-BD. Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate. We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiellapneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (ΔacoAΔbdhAΔptaΔldh) to achieve the efficient production of chiral meso-2,3-BD. Finally, the pool of NADH availability was further increased to facilitate the conversion of meso-2,3-BD from AC by overexpressing udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-BD with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct AC was no more than 1.1 g/L.ConclusionThis work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis. To our knowledge, this is the first report indicating that metabolic engineered B. subtilis could produce chiral meso-2,3-BD with high purity under limited oxygen conditions. These results further demonstrated that B. subtilis as a class I microorganism is a competitive industrial-level meso-2,3-BD producer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0502-5) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Metabolic engineering of Bacillus subtilis for chiral pure meso-2,3-butanediol production

Huo

et al. 2016

Biotechnol Biofuels

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“…It revealed that meso ‐2,3‐BDH possess a hydrophobic tunnel connecting the protein exterior to the catalytic center. Previous studies from our lab have shown that the Asp194Gly mutation resulted in a dramatic decrease in the meso ‐2,3‐BDH enzymatic activity with no apparent effect to substrate binding . Through our MD analysis, it is revealed that Asp194 was not the key residues that make up the channel, but Asp194Gly mutation disrupted hydrogen bond with Trp190 (Table S2).…”

Section: Resultsmentioning

confidence: 73%

“…The interconversion between (3R)‐AC and meso ‐2,3‐BD or (3S)‐AC and (2S,3S)‐2,3‐BD was catalyzed by meso ‐2,3‐butanediol dehydrogenase ( meso ‐2,3‐BDH). Previous studies from our lab have shown that the Asp194Gly mutation away from the active site resulted in a dramatic decrease in the activity . In this work, we start from the structure of meso ‐2,3‐BDH from K. pneumoniae to increase the activity of enzyme.…”

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confidence: 99%

Rational design of Meso‐2,3‐butanediol dehydrogenase by molecular dynamics simulation and experimental evaluations

Zhang

Wang

et al. 2017

FEBS Letters

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Meso-2,3-butanediol dehydrogenase (meso-2,3-BDH) catalyzes NAD -dependent conversion of meso-2,3-butanediol to acetoin, a crucial external energy storage molecule in fermentive bacteria. In this study, the active tunnel of meso-2,3-BDH was identified. The two short α helixes positioned away from the α4-helix possibly expose the hydrophobic ligand-binding cavity, gating the exit of product and cofactor from the activity pocket. Further MM/GBSA-binding free energy analysis shows that Phe212 and Asn146 function as the key product-release sites. Site-directed mutagenesis experiments targeted to the sites show that the k of Phe212Tyr is enhanced up to (4-8)-fold. The original activity of Asn146Gln is retained, but the activity of Asn146Ala mutation is lost. These results could provide helpful guidance on rational design of short-chain dehydrogenases/reductases.

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“…(Wayne 2008; Hao et al 2014). The reaction was initiated by adding 20 μl crude extract to 1 ml reaction buffer which contained 67 mM phosphate buffer (pH 7.4), 5 mM of AC, and 0.2 mM of NADH.…”

Section: Methodsmentioning

confidence: 99%

Metabolic engineering of Serratia marcescens MG1 for enhanced production of (3R)-acetoin

Dai

Bai

et al. 2016

Bioresour. Bioprocess.

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BackgroundOptically pure acetoin (AC) is an important platform chemical which has been widely used to synthesize novel optically active α-hydroxyketone derivatives and liquid crystal composites.ResultsIn this study, slaC and gldA encoding meso-2,3-butanediol dehydrogenase (meso-2,3-BDH) and glycerol dehydrogenase (GDH), respectively, in S. marcescens MG1 were knocked out to block the conversion from AC to 2,3-butanediol (2,3-BD). The resulting strain MG14 was found to produce a large amount of optically pure (3R)-AC with a little 2,3-BD, indicating that another enzyme responsible for 2,3-BD formation except meso-2,3-BDH and GDH existed in the strain MG1. Furthermore, SlaR protein, a transcriptional activator of AC cluster, was overexpressed using P C promoter in the strain MG14, leading to enhancement of the (3R)-AC yield by 29.91%. The recombinant strain with overexpression of SlaR, designated as S. marcescens MG15, was used to perform medium optimization for improving (3R)-AC production.ConclusionUnder the optimized conditions, 39.91 ± 1.35 g/l (3R)-AC was produced by strain MG15 with the productivity of 1.11 g/l h and the conversion rate of 80.13%.

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Biochemical characterization of unusual meso-2,3-butanediol dehydrogenase from a strain of Bacillus subtilis

Cited by 7 publications

References 29 publications

Metabolic engineering of Bacillus subtilis for chiral pure meso-2,3-butanediol production

Metabolic engineering of Bacillus subtilis for chiral pure meso-2,3-butanediol production

Rational design of Meso‐2,3‐butanediol dehydrogenase by molecular dynamics simulation and experimental evaluations

Metabolic engineering of Serratia marcescens MG1 for enhanced production of (3R)-acetoin

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