- 3:35 PM

Engineering of Synthetic Cytochrome P450s for Plant Estrogen Isoflavone Biosynthesis from Escherichia Coli

Effendi Leonard and Mattheos Koffas. Chemical and Biological Engineering, University at Buffalo, the State University of New York, 914 Furnas Hall, Buffalo, NY 14260

An expansive collection of scientific publications has affirmed the potential clinical importance of plant estrogen isoflavones to promote health and prevent or delay the onset of certain chronic diseases. Currently, isoflavones are popular dietary supplements, and are engaged in chemoprevention clinical trials sponsored by the National Cancer Institute. We aim at developing efficient recombinant microbial platforms for isoflavone production, since traditional isolation from plant materials is inefficient, especially when taking into account the significant losses during processing. Isoflavone biosynthesis in plants is mediated by isoflavone synthase (IFS), the membrane bound cytochrome-P450 enzyme that performs a novel aryl-ring migration of the flavanone substrates. The bacterium Escherichia coli is a robust whole-cell biocatalyst for the production of secondary metabolites. However, the metabolic engineering endeavor of isoflavone biosynthesis through implantation of the plant metabolic pathway is hindered due to the absence of suitable membranes and P450-electron transport protein to support the functionality and catalytic activities of IFS. Here, 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 yeast, Saccharomyces cerevisiae. Moreover, the chimera also exhibited oxidative activities towards several unnatural substrates. Introducing the chimera into an E. coli strain pre-engineered for flavanone biosynthesis resulted in the production of isoflavones from the phenylpropanoic acid precursor. A similar pathway was also constructed in S. cerevisiae for isoflavone synthesis from the inexpensive precursor.