(210g) Developing Metabolic Workhorses through in Silico Evolutionary Optimization: Case Studies in Flavonoid Biosynthesis

Breakthroughs in systems models of cellular metabolism have greatly enhanced the understanding of biological network interactions on a genome-scale, specifically using stoichiometric based flux analysis. As such, we have previously described a Cipher of Evolutionary Design (CiED) to investigate the impact of gene deletions and other network modifications on the metabolite profile of microorganisms. Here we report the use of CiED to investigate the metabolic potential of Escherichia coli to channel carbon towards malonyl-CoA and NADPH in an effort to generate recombinant strains with elevated flavonoid production capacity. Flavonoids, along with several of their substituted unnatural analogues, have potential therapeutic value in the treatment of various chronic diseases such as cancer, obesity, diabetes and heart disease. Our engineered strains were modified using the predicted gene deletion targets identified by CiED as well as selected enzyme overexpressions. As a result, the specific flavanone production from an optimally engineered E. coli strain for malonyl-CoA was increased by over 660% for naringenin and by over 420% for eriodictyol. In a similarly engineered E. coli strain for NADPH, a 4-fold and 2-fold increase in leucocyanidin and catechin production was seen, respectively. These efforts demonstrate the utility of an evolutionary model based solely on stoichiometry in predicting improved microbial phenotypes for natural product production.