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Background   D -2,3-butanediol has many industrial applications such as chiral reagents, solvents, anti-freeze agents, and low freezing point fuels. Traditional  D -2,3-butanediol producing microorganisms, such as  Klebsiella pneumonia  and  K. xoytoca , are pathogenic and not capable of producing  D- 2,3-butanediol at high optical purity.  Bacillus licheniformis  is a potential 2,3-butanediol producer but the wild type strain (WX-02) produces a mix of  D - and  meso -type isomers.  BudC  in  B. licheniformis  is annotated as 2,3-butanediol dehydrogenase or acetoin reductase, but no pervious experiment was performed to verify this hypothesis.    Results  We developed a genetically modified strain of  B. licheniformis  (WX-02 Δ bud C) as a  D -2,3-butanediol producer with high optimal purity. A marker-less gene deletion protocol based on a temperature sensitive knock-out plasmid T2-Ori was used to knock out the  bud C gene in  B. licheniformis  WX-02. The  budC  knock-out strain successfully abolished  meso -2,3-butanediol production with enhanced  D -2,3-butanediol production. No  meso -BDH activity was detectable in cells of this strain. On the other hand, the complementary strain restored the characteristics of wild strain, and produced  meso -2,3-butanediol and possessed  meso -BDH activity. All of these data suggested that  bud C encoded the major  meso -BDH catalyzing the reversible reaction from acetoin to  meso -2,3-butanediol in  B. licheniformis . The  bud C knock-out strain produced  D- 2,3-butanediol isomer only with a high yield of 30.76 g/L and a productivity of 1.28 g/L-h.    Conclusions  We confirmed the hypothesis that  bud C gene is responsible to reversibly transfer acetoin to  meso -2,3-butanediol in  B. licheniformis . A mutant strain of  B. licheniformis  with depleted  bud C gene was successfully developed and produced high level of the  D -2,3-butanediol with high optimal purity.

Deletion of meso-2,3-butanediol dehydrogenase gene bud C for enhanced D-2,3-butanediol production in Bacillus licheniformis