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.
Highlights
Concern over exhaustion of fossil fuel resources, emission of CO2 linked with petroleum-derived products, and accumulation of non-degradable synthetic polymers urged the development of environmentally friendly processes for production of chemicals
The three genes were cloned into pEKEx2 expression vector under control of the isopropyl β-D-1-thiogalactopyranoside (IPTG)inducible Ptac promoter (Additional file 1: Fig. S1), and transformed into the three selected background strains C. glutamicum ΔldhA, ΔaceEΔpqoΔldhA and ΔaceEΔpqoΔldhAΔmdh (Table 1)
Induced expression of als and acetolactate decarboxylase (aldB) genes in C. glutamicum ΔldhA(pEKEx2-als,aldB,butanediol dehydrogenase (butA)) led to 61and 15-fold increase in ALS/ALDC activity when compared to control C. glutamicum Δldh(pEKEx2) grown on glucose and acetate, respectively; this corresponded to an increase from 0.024 ± 0.003 to 1.47 ± 0.42 U−1 in glucose grown cells, and from 0.037 ± 0.002 to 0.54 ± 0.09 U−1 in acetate grown cells
Summary
Concern over exhaustion of fossil fuel resources, emission of CO2 linked with petroleum-derived products, and accumulation of non-degradable synthetic polymers urged the development of environmentally friendly processes for production of chemicals. A solution offered by the rising field of white biotechnology is to produce. 2,3-Butanediol (2,3-BD) is an important chemical used in the production of plasticizers and fumigants, as an antifreeze agent, a fuel and octane booster, among other applications [2]. The range is still expanding and the market size is expected to reach 74 kilo tons by 2018 [3]. The 2,3-BD derivative, 1,3-butanediene, can be used in synthetic rubber production, while 2-butanone (methyl ethyl ketone) is a fuel additive and solvent for resins and lacquers. Ester-derivatives are used in the pharmaceutical and cosmetics industries [2].
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