Engineering Bacillus licheniformis for the production of meso-2,3-butanediol.

Yimin Qiu,Christopher T Nomura,Shuilin Wu,Shouwen Chen,Lu Li,Zhiyou Wen,Jinyan Zhang

doi:10.1186/s13068-016-0522-1

Abstract

Background2,3-Butanediol (2,3-BD) can be used as a liquid fuel additive to replace petroleum oil, and as an important platform chemical in the pharmaceutical and plastic industries. Microbial production of 2,3-BD by Bacillus licheniformis presents potential advantages due to its GRAS status, but previous attempts to use this microorganism as a chassis strain resulted in the production of a mix of D-2,3-BD and meso-2,3-BD isomers.ResultsThe aim of this work was to develop an engineered strain of B. licheniformis suited to produce the high titers of the pure meso-2,3-BD isomer. Glycerol dehydrogenase (Gdh) was identified as the catalyst for D-2,3-BD biosynthesis from its precursor acetoin in B. licheniformis. The gdh gene was, therefore, deleted from the wild-type strain WX-02 to inhibit the flux of acetoin to D-2,3-BD biosynthesis. The acoR gene involved in acetoin degradation through AoDH ES was also deleted to provide adequate flux from acetoin towards meso-2,3-BD. By re-directing the carbon flux distribution, the double-deletion mutant WX-02ΔgdhΔacoR produced 28.2 g/L of meso-2,3-BD isomer with >99 % purity. The titer was 50 % higher than that of the wide type. A bench-scale fermentation by the double-deletion mutant was developed to further improve meso-2,3-BD production. In a fed-batch fermentation, meso-2,3-BD titer reached 98.0 g/L with a purity of >99.0 % and a productivity of 0.94 g/L–h.ConclusionsThis work demonstrates the potential of producing meso-2,3-BD with high titer and purity through metabolic engineering of B. licheniformis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0522-1) contains supplementary material, which is available to authorized users.

Highlights

A significant research has been conducted for microbial production of 2,3-butanediol (2,3-BD), as a potential liquid fuel additive for petroleum replacement
Identification of the enzyme catalyzing D‐AC to D‐2,3‐BD in B. licheniformis To produce meso-2,3-BD with high optical purity in B. licheniformis, we proposed to block the synthesis of D-2,3-BD by knocking out the gene encoding the enzyme catalyzing the conversion of D-AC to D-2,3-BD, so that the carbon flux from D-AC would be diverted into meso2,3-BD (Fig. 1)
It has been confirmed that different 2,3-BD isomers arise from the same precursor D-AC, and the enzyme catalyzing the reduction of D-AC to meso-2,3-BD has been identified in B. licheniformis WX-02 [13]

Summary

Introduction

A significant research has been conducted for microbial production of 2,3-butanediol (2,3-BD), as a potential liquid fuel additive for petroleum replacement. 2,3-BD exists in three isomeric forms, such as D-2,3-BD, L-2,3-BD, and meso-2,3-BD, with each isomer having its own unique applications. Optically active D-2,3-BD and L-2,3-BD can be used as building blocks in the synthesis of chiral compounds [3], Genetic engineering has been used to produce specific 2,3-BD isomers with high purity. D-2,3-BD was produced with a purity >97.5 % through an engineered Enterobacter cloacae strain to titers of 152.0 g/L [10]. Engineered Escherichia coli has been used to produce L-2,3-BD from diacetyl with a purity >99 % [3]. A high purity (~98 %) of meso-2,3-BD was reported through a recombinant E. coli strain [11].

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