Engineering Corynebacterium glutamicum for the production of 2,3-butanediol

Abstract

Background2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving α-acetolactate synthase, α-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions.ResultsA gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum ΔldhA, C. glutamicum ΔaceEΔpqoΔldhA and C. glutamicum ΔaceEΔpqoΔldhAΔmdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,PtufbutA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol−1 yield on glucose, an overall productivity of 0.2 g L−1 h−1 and a titer of 6.3 g L−1.ConclusionsWe have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0362-x) contains supplementary material, which is available to authorized users.

Fermentative production of L‐pipecolic acid from glucose and alternative carbon sources

“…The pTuf promoter, which often is employed in metabolic engineering of C. glutamicum , is considered as strong constitutive promoter . The IPTG inducible promoter pTac, a hybrid of the E. coli trp and lac promoters , is present in the vector pVWEx1 and allows for inducible and very high gene expression in C. glutamicum .…”

Recent advances in metabolic engineering of Corynebacterium glutamicum as a potential platform microorganism for biorefinery

“…It can be also used as feedstock for biochemical production owing to its advantageous properties, including (i) high production, i.e., approximately 259 000 t in 2012, and (ii) convenient production of the important intermediate pyruvate via a one‐step enzymatic reaction mediated by lactate dehydrogenase . For the production of pyruvate‐derived platform chemicals, such as ethanol, isobutanol, and 2,3‐butanediol, utilization of lactic acid as feedstock is an effective strategy . C. glutamicum naturally utilizes lactate and harbors three genes involved in lactate metabolism, NCgl2816 (encoding lactate permease), lldD ( NCgl2817 ; encoding l ‐lactate dehydrogenases), and dld ( NCgl0865 ; encoding d ‐lactate dehydrogenase) (Fig.…”

Shake flask methodology for assessing the influence of the maximum oxygen transfer capacity on 2,3-butanediol production

“…Consequently, non-pathogenic 2,3-butanediol producers are promising candidates for industrial production processes [15, 16]. Genetic engineering enables 2,3-butanediol production with well-characterized strains like Escherichia coli [17–20], Saccharomyces cerevisiae [21, 22], Zymomonas mobilis [23] and Corynebacterium glutamicum [24, 25]. Furthermore, some Bacillus and Paenibacillus strains naturally produce 2,3-butanediol [15, 26].…”