title: Exploring recombinant flavonoid biosynthesis in metabolically engineered Escherichia coli.
abstract: Flavonoids are important plant-specific secondary metabolites synthesized from 4-coumaroyl coenzyme A (CoA), derived from the general phenylpropanoid pathway, and three malonyl-CoAs. The synthesis involves a plant type III polyketide synthase, chalcone synthase. We report the cloning and coexpression in Escherichia coli of phenylalanine ammonia lyase, cinnamate-4-hydroxylase, 4-coumarate:CoA ligase, and chalcone synthase from the model plant Arabidopsis thaliana. Simultaneous expression of all four genes resulted in a blockage after the first enzymatic step caused by the presence of nonfunctional cinnamate-4-hydroxylase. To overcome this problem we fed exogenous 4-coumaric acid to induced cultures. We observed high-level production of the flavanone naringenin as a result. We were also able to produce phloretin by feeding cultures with 3-(4-hydroxyphenyl)propionic acid. Feeding with ferulic or caffeic acid did not yield the corresponding flavanones. We have also cloned and partially characterized a new tyrosine ammonia lyase from Rhodobacter sphaeroides. Tyrosine ammonia lyase was substituted for phenylalanine ammonia lyase and cinnamate-4-hydroxylase in our E. coli clones and three different growth media were tested. After 48 h induction, high-level production (20.8 mg L(-1)) of naringenin in metabolically engineered E. coli was observed for the first time.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108, USA.
authors.forename: Kevin T
authors.initials: KT
authors.lastname: Watts
authors.forename: Pyung Cheon
authors.initials: PC
authors.lastname: Lee
authors.forename: Claudia
authors.initials: C
authors.lastname: Schmidt-Dannert
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003373:Coumaric Acids / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D044950:Flavanones / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D005419:Flavonoids / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D005818:Genetic Engineering; D015394:Molecular Structure; D010666:Phenylpropionates / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011422:Propionates
chemical_list: D003373:Coumaric Acids; D017077:Culture Media, Conditioned; D044950:Flavanones; D005419:Flavonoids; D010666:Phenylpropionates; D011422:Propionates; C008869:phloretic acid; C005273:naringenin; C495469:p-coumaric acid