Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The first objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:CoA ligase (4CL) from Arabidopsis thaliana, and the chalcone synthase (CHS) from Hypericum androsaemum into Saccharomyces cerevisiae AH22 strain. Each gene was cloned and inserted into an expression vector under the control of separate individual GAL10 promoter. Besides PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase (TAL) activity, which enabled biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). The yeast AH22 strain co-expressing PAL, 4CL and CHS produced approximately 7 mg L-1 of naringenin and 0.8 mg L -1 of pinocembrin. Several byproducts, such as 2\u27,4\u27,6\u27-trihydroxydihydrochalcone and phloretin were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor L-tyrosine. The second objective was to synthesize flavones, apigenin and chrysin in yeast. Flavone synthase II (FNSII) from Gerbera hybrida, a plant cytochrome P450 monooxygenase, catalyzes naringenin to apigenin, liquiritigenin to 7,4\u27-dihydroxyflavone, and eriodictyol to luteolin. Our study is the first to show that FNSII is able to catalyze pinocembrin to chrysin, an anti-anxiety agent. Engineering the phenylpropanoid pathway from PAL, to FNSII enabled the production of apigenin from amino acid in yeast and the yield was 1.6 μM (0.3 μmol g CDW-1). Feeding studies showed that the production of apigenin was limited by aromatic precursors, L-phenylalanine and L-tyrosine, p-coumarate, and naringenin. Much significant higher yield of apigenin (27 μM or 9.2 pmol g CDW -1) was observed by feeding 400 μM of p-coumarate to the yeast co-expressing 4CL, CHS, and FNSII. The third objective was to synthesize novel flavonoids by feeding non-natural C4H substrate analogs to the yeast co-expressing C4H, 4CL, and CHS. Preliminary data have shown that 2-methylcinnamate, 2-fluorocinnamate, and 2-chlorocinnamate were converted to six novel flavonoids by the engineered pathway that have not been previously reported