(263a) Biosynthesis of Novel Flavonoids through an Engineered Phenylpropanoid Pathway in Saccharomyces Cerevisiae

The phenylpropanoid pathway is a key pathway leading to many important compounds such as lignins, flavonoids, and coumarins. Our group has cloned the first several enzymes of this pathway in Saccharomyces cerevisiae. The engineered pathway we constructed includes cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL) from Arabidopsis thaliana, chalcone synthase (CHS) from Hypericum androsaemum, and flavone synthase II (FNSII) from Gerbera hybrida. This engineered pathway leads to the synthesis of some important compounds such as naringenin, phloretin, apigenin, and pinocembrin. Besides the natural substrates, we found this engineered yeast could metabolize several substrate analogs and thus produce novel flavonoids. The substrate selectivity of the enzymes in this pathway was investigated separately. More than 10 and 12 analogs were shown to be metabolized by C4H and 4CL respectively. Based on the analysis of a C4H homology model, some key amino acid residues were identified in the active site. With the intention of expanding the substrate range of C4H, modifications were created through site directed mutagenesis of these residues. The activity of the mutants towards reactive analogs was tested in vivo. No new activity was identified and most mutants showed reduced activity towards both the substrates and analogues. To further investigate the substrate selectivity of the engineered pathway, substrate analogs were fed to two engineered yeasts containing 4CL-CHS and C4H-4CL-CHS respectively. Several of the substrate analogs were metabolized by one or both of the engineered yeasts. The resulting products from these multi-step biotransformations will be tested as substrates of FNSII to produce novel flavones.