Production of Rosmarinic Acid in Escherichia coli by a Novel Biosynthetic Route

Hydroxycinnamic acid esters (HCEs) are key metabolites in plants and an important class of biologically-active compounds found in foods.  Among HCEs, rosmarinic acid (RA) has exhibited significant anti-inflammatory and anti-oxidant activity as well as potential for use as a psychoactive therapeutic.  Currently, RA is produced by extraction from plant tissue.  Engineering a microbial biocatalyst could provide a low-cost and tunable means of producing RA and other HCEs.  In plants, the RA biosynthetic pathway requires ten enzymatic steps, including catalysis by cytochrome P450s, which can be problematic in industrial microbes.  By incorporating microbial enzymes, I have designed a novel RA biosynthetic pathway in Eshcherichia coli which requires only six enzymes, four from bacteria, some of which exhibit previously uncharacterized activities.  The final enzyme, RA synthase (RAS), has been shown to accept a range of acyl donor and acceptor substrates to synthesize a number of HCEs in vitro.  In this work, assembly of subsets of the novel pathway in E. coli led to the de novo biosynthesis of RAS acyl-acceptor substrates 4-hydroxyphenyllactate and 3,4-dihydroxyphenyllactate (HPL and DHPL), as well as caffeic acid, the Co-A ester of which is an acyl donor substrate of RAS.  Coexpression of these pathway subsets with RAS and a 4-coumaroyl-CoA ligase led to the de novo biosynthesis of RA, the ester of caffeic acid and DHPL.  Optimization of this pathway for high production titers and incorporation of various acyl donor and acceptor substrate pathways could lead to a commercially viable microbial production platform for RA and other HCEs.