(485z) Production of Enantiopure Hydroxyacids in Recombinant Escherichia Coli

Hydroxyacids have the potential to serve as useful chiral building block molecules for a diverse range of products, including biopolymers and optically-active fine chemicals, such as pharmaceuticals, vitamins, antibiotics, and flavor compounds. Through metabolic engineering efforts, we have constructed unique pathways for the biosynthesis of both enantiomers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) in E. coli, where the chirality was controlled by utilizing two enzymes of opposite stereoselectivity. In addition, several enzyme homologs were profiled to promote production. Given that dissimilar E. coli strains possess intrinsic differences in metabolic capacity, we further explored two representative strains, BL21Star(DE3) (B strain) and MG1655(DE3) (K-12 strain), for their abilities of hydroxyacid production. Moreover, since the two enantio-selective enzymes possess different pyridine nucleotide cofactor (NADPH/NADH) requirements, we measured intracellular levels of reduced and oxidized forms of each cofactor to gain additional insights into how the physiological ratios of NADH/NAD+ and NADPH/NADP+ could influence yields of (R)-enantiomer and (S)-enantiomer. Overall, we have achieved production of enantiopure (R)-3HB and (S)-3HB with titers of both in excess of 2.5 g/L in shake flask cultures within two days, and are currently investigating this microbial system for chiral 3HV production. To our knowledge, this work reports the highest titers of optically pure (S)-3HB achieved in shake flask E. coli cultures to date.