Engineering Acetoin and meso-2,3-Butanediol Biosynthesis in E.coli

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 The functional reconstruction of acetoin and meso-2,3-butanediol biosynthetic pathways in E. coli have been systematically explored. Pathway construction involved the in vivo screening of prospective pathway isozymes of yeast and bacterial origin. After substantial engineering of the host background to increase pyruvate availability, E. coli YYC202(DE3) ldhA ilvC expressing ilvBN from E. coli and aldB from L. lactis (encoding acetolactate synthase and acetolactate decarboxylase activities, respectively) was able to produce up to 870 mg/L acetoin, with no co-production of diacetyl observed. These strains were also found to produce small quantities of meso-2,3-butanediol, suggesting the existence of endogenous 2,3-butanediol dehydrogenase activity that has not before been characterized. Finally, the co-expression of bdh1 from S. cerevisiae, encoding 2,3-butanediol dehydrogenase, in this strain resulted in the production of up to 1120 mg/L meso-2,3-butanediol, with glucose a yield of 0.29 g/g. While disruption of the native lactate biosynthesis pathway increased pyruvate precursor availability to this strain, increased availability of NADH for acetoin reduction to meso-2,3-butanediol was found to be the most important consequence of ldhA deletion.