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

Abstract

Doc number: 16

Abstract

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 Δbud 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.