Mattheos A.G. Koffas, Chemical and Biological Engineering, University at Buffalo, the State University of New York, North Campus, 904 Furnas Hall, Buffalo, NY 14260
With microbial production becoming the primary choice for natural product synthesis, increasing precursor and cofactor availability has become a chief concern for the generation of efficient production platforms. As such, we have employed a stoichiometric-based model to identify combinations of gene knockouts for improving NADPH availability in Escherichia coli. Specifically, two different model objectives were used to identify possible genotypes that exhibited either improved overall NADPH production or an improved flux through an artificial reaction coupling NADPH yield to biomass. The top single, double and triple gene deletion candidates were constructed and as a case study evaluated for their ability to produce two polyphenols, leucocyanidin and (+)-catechin from their common precursor dihydroquercetin using two recombinant NADPH-dependent enzymes: dihydroflavonol reductase and leucoanthocyanidins reductase. The best engineered strain carrying Δpgi, Δppc and ΔpldA deletions accumulated up to 817 mg/L of leucocyanidin but only 50 mg/L (+)-catechin in batch culture with 10 g/L glucose in modified M9 medium, a 5-fold increase compared to the wildtype control.
