Metabolic engineering of Escherichia coli for enhanced production of p-coumaric acid via L-phenylalanine biosynthesis pathway.

Abstract

p-Coumaric acid (p-CA), an invaluable phytochemical, has novel bioactivities, including antiproliferative, anxiolytic, and neuroprotective effects, and is the main precursor of various flavonoids, such as caffeic acid, naringenin, and resveratrol. Herein, we report the engineering of Escherichia coli for de novo production of p-CA via the PAL-C4H pathway. As the base strain, we used the E. coli H-02 strain, which was previously engineered for sufficient supplementation of L-phenylalanine, the main precursor of p-CA. For the bioconversion of L-Phe to p-CA, we constructed and optimized an expression system for phenylalanine ammonia lyase (SmPAL), codon-optimized cinnamate 4-hydroxylase (AtC4H), and its redox partner, cytochrome P450 reductase (AtCPR1). We confirmed that the engineered cell showed higher production of p-CA at 30 °C and the addition of 0.5 mM 5-aminolevulinic acid could increase the production titer further. Subsequently, the main pathways of acetic acid (poxB and pta-ackA) were eliminated to reduce its accumulation and restore cell growth. Next, to increase the available pool of cofactor (NADPH), the co-expression system of the zwf gene in the pentose phosphate pathway (PPP) was integrated into genome and the expression level was optimized with synthetic promoters. Finally, by optimizing fed-batch culture in a 5 L-scale bioreactor, the engineered strain achieved 1.5 g/L p-CA with a productivity of 31.8 mg/L/h.