(425b) Engineering Microbial Factories for the Production of Plant-Specific Flavonoids

Flavonoids are plant-derived secondary metabolites with various health promoting properties, and are being evaluated in preliminary trials for treatment of various diseases from cancer to diabetes. In light of the pharmacological importance, strategies for efficient production of flavonoids are important to pursue. Since microbial fermentation is an important cost-saving alternative to chemical synthesis or plant extraction, we aim to develop recombinant microbes for efficient plant flavonoid production. We present the metabolic engineering of bacterium Escherichia coli and yeast Saccharomyces cerevisiae of flavonoid biosynthesis from the inexpensive phenylpropanoic precursors and glucose. Implantation of artificial flavonoid biosynthetic pathways through simultaneous episomal expression of plant-derived genes resulted in the biosynthesis of a library of flavonoid molecules, which includes flavanones, flavones, flavonols, flavanols, and anthocyanins. The biosynthesis of the plant estrogen isoflavones requires the oxidation and aryl-ring migration of the flavanone substrates by the membrane bound cytochrome-P450 isoflavone synthase (IFS). However, metabolic engineering E. coli of isoflavone biosynthesis is hindered due to the lack of suitable membranes and P450-redox partner for the in vivo functionality and catalysis of IFS. We present the design for a series of soybean IFS chimeras to search for progenies that simultaneously exhibit functionality and high turnover catalysis in E. coli. Isoflavone productions from E. coli harboring the most effective IFS variant were superior when compared to that from the natural plant resources and the recombinant S. cerevisiae. The minute amount of malonyl-CoAs in E. coli, which are required as flavonoid building blocks, limits the high-level production of flavonoids. In the present work, we also present the frontier of cellular engineering the flavonoid producing E. coli strains by manipulations of the intracellular fatty acid metabolism. The metabolic manipulation to increase mlonyl-CoA contents through the over expression of acetyl-CoA carboxylase and the biotinylation protein, biotin ligase, increased the flavonoid production up to 3-folds. In parallel, introduction of a novel malonate synthase and carrier protein from a plant nitrogen-fixing bacterium to allow the direct conversion of an inexpensive malonate carbon source into malonyl-CoA elevated flavonoid production up to 4-folds. In conjunction with cessation of the recycling of malonyl-CoA into acetyl-CoA by inhibition of β-ketoacyl acyl carrier proteins, flavonoids were produced at high levels, up to hundreds of milligram per liter quantity.