P82: A Metabolic Engineering Approach to Improve NADPH Bioavailability in E. coli

Enzymatic synthesis of some industrially important compounds depends heavily on cofactor NADPH as the reducing force. This is especially true in the synthesis of chiral compounds often used as pharmaceutical intermediates to generate the correct stereochemistry in the bioactive product. In this study we use three examples: in the synthesis of lycopene, 16 moles of NADPH are consumed, in the monooxygenase reaction chiral lactone products can be formed, and in the reduction of the double bond in the substituted acrylate, a chrial acid is formed.  However, the high cost and technical difficulty of cofactor regeneration often pose a challenge for biocatalytic reactions.  To increase NADPH bioavailability, we replaced the native NAD-dependent GAPDH GapA in E. coli with a NADP-dependent GAPDH. We also enhanced biosynthesis of NADP by co-expression with an E. coli NAD kinase. The recombinant strains were tested in three reporter systems, namely cyclohexanone monooxygenase (CHMO), biosynthesis of lycopene, and an anaerobic system utilizing the 2-haloacrylate reductase. In all the reporting systems, recombinant strains showed greatly increased synthesis of NADPH-dependent compounds.  The increase was more pronounced when NAD kinase was also overexpressed.  These results validate this novel approach to improve NADPH bioavailability in E. coli and indicate its use in E. coli or other bacterium-based production of compounds requiring NADPH.