title: Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases.
abstract: 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. 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. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4'-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3beta-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.
authors.affiliation: Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA.
authors.forename: Lyle
authors.initials: L
authors.lastname: Ralston
authors.forename: Senthil
authors.initials: S
authors.lastname: Subramanian
authors.forename: Michiyo
authors.initials: M
authors.lastname: Matsuno
authors.forename: Oliver
authors.initials: O
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D004789:Enzyme Activation; D020224:Expressed Sequence Tags; D005419:Flavonoids / Q000096:biosynthesis*; D020869:Gene Expression Profiling; D017343:Genes, Plant; D005818:Genetic Engineering; D019753:Intramolecular Lyases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D007529:Isoflavones / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics
chemical_list: D018744:DNA, Plant; D005419:Flavonoids; D007529:Isoflavones; D011994:Recombinant Proteins; D019753:Intramolecular Lyases; C019865:chalcone isomerase