Exploring Recombinant Flavonoid Biosynthesis in Metabolically Engineered Escherichia coli

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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.