ME: Metabolic Engineering of Anthocyanin Production in Saccharomyces cerevisiae and Escherichia coli

Suny At Buffalo, Amherst NY

Investigators

Abstract

This exploratory project will focus on two targets: 1. Elucidate the enzymatic properties of proteins involved in anthocyanin biosynthesis, and 2. Construct and achieve the heterologous expression of the anthocyanin biosynthesis pathway that leads from flavanones and phenylalanine to the first stable colored anthocyanin, anthocyanidin 3-glucoside (flavylium ion) in yeast. The primary goal in the first target is the enzyme dihydroflavonol 4-reductase (DFR) that converts dihydroflavonols to leucoanthocyanidins. The reasons why this enzyme is perhaps the most important and intriguing in the whole anthocyanin pathway are: (i) it does not functionally express in Escherichia coli (but expresses in yeast), (ii) it can utilize various other substrates, such as flavanones and even anthocyanidins and (iii) it is one of the few flavonoid biosynthesis enzymes that appears in the genome of prokaryotic species. The Principal Investigator (PI) now has experimental evidence showing that plant and prokaryotic dfr genes successfully translate into protein that appears in the cytoplasm of Escherichia coli. That eliminates the possibility of codon usage problems and inclusion body formation suggested by other researchers in the very recent past. The PI is currently working on the enzymatic characterization of the recombinant proteins using E. coli and yeast crude extracts and permeabilized cells, and is also working on elucidating possible post-translational modifications this enzyme might be undergoing in yeast (where it appears to be functional), such as glycosylation and phosphorylation. The goal on the second target is the construction of a functional anthocyanin biosynthesis pathway in yeast. The PI now has available in his lab a complete set of four genes from various plants that convert flavanones (naringenin) to anthocyanin, and will be putting these four genes together in yeast and investigate the conversion of naringenin (provided in the fermentation medium) to anthocyanidin 3-glucoside. In addition the PI will be acquiring the complete set of five genes that convert phenylalanine to naringenin in Arabidopsis thaliana (provided by the RIKEN and Kazusa Research Institutes in Japan). The pathway construction will be achieved by homologous recombination using plasmid and chromosome integration.