Poster: Secondary metabolism

Abs # 267: In vivo reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using type I and type II chalcone isomerases from soybean

Presenter:	Matsuno, Michiyo , mmatsuno@danforthcenter.org
Authors	Matsuno, Michiyo  (A)   Ralston, Lyle  (A) (B)  Subramanian, Senthil  (A)   Walker, Laura  (A)   Yu, Oliver  (A)
Affiliations:	(A): Donald Danforth Plant Science Center, St. Louis, MO (B): current address: Sigma-Aldrich Biotechnology, St. Louis, MO
Web Site:	http://www.danforthcenter.org/yu/

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes, serving as cell wall components, UV-protectants, pigments, and phytoalexins. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women’s health. Chalcone isomerase (CHI) is essential in the biosynthesis of these compounds and acts together with isoflavone synthase (IFS), flavanone-3â-hydroxylase (F3H) and flavone synthase II (FNSII) to produce isoflavones, dihyroflavonols and flavones respectively. In this study, five CHI cDNAs were isolated from soybean and the kinetic properties of the encoded enzymes were analyzed. Gene expression and kinetics analyses indicated that type I CHI, which uses naringenin-chalcone as substrate, is coordinately regulated with F3H. In addition, type II CHIs, which use a variety of chalcone substrates, were coordinately regulated with IFS and specifically activated by nodulation signals. We have attempted to engineer yeast (Saccharomyces cerevisiae) to produce flavonoids and isoflavonoids by co-expressing type I or type II CHIs along with IFS, F3H or FNSII. Co-expression of type II CHI and IFS in yeast resulted in the production of different isoflavones (including novel compounds) from various chalcone substrates. We also co-expressed type I or type II CHIs with IFS and FNSII. The ratio of genistein and apigenin, produced from naringenin chalcone, suggested that both type I and type II CHIs preferentially interact with IFS. Furthermore, the subcellular localization of CHIs appears to be crucial for the distribution of common substrates and the direction of metabolic flux. Thus, the in vivo yeast system not only produced interesting novel compounds, but also provided a unique platform to study enzyme interactions and metabolic channeling.