Functional expression of an Arabidopsis p450 enzyme, p-coumarate-3-hydroxylase, in the cyanobacterium Synechocystis PCC 6803 for the biosynthesis of caffeic acid

Caffeic acid, which exhibits strong anticancer activities, is a natural phenolic compound found in small amounts in plants. Production of caffeic acid by bacterial systems is technically challenging due to difficulties in functionally expressing p-coumarate-3-hydroxylase (C3H), a cytochrome P450 enzyme that converts p-coumaric acid into caffeic acid. Here, we report for the first time that the cyanobacterium Synechocystis PCC 6803 is able to produce caffeic acid from p-coumaric acid upon heterologous expression of C3H. The Arabidopsis thaliana ref8 gene, which encodes a C3H, was synthesized and codon-optimized for enhanced expression in Synechocystis. Expression of the synthetic ref8 gene was driven by a native psbA2 promoter and confirmed at the transcriptional and translational levels. This heterologous pathway enabled Synechocystis to produce caffeic acid at a concentration of similar to 7.2 mg L-1 from p-coumaric acid under oxygenic photosynthetic growth conditions. This study demonstrates that cyanobacteria are well suited for the bioproduction of plant secondary metabolites that are difficult to produce in other bacterial systems.