Construction and optimization of p-coumaric acid-producing Saccharomyces cerevisiae

Abstract

p-Coumaric acid is an important precursor of various natural compounds, such as flavonoids and stilbenes. It has been widely used in biomedicine, food, nutrition and health care industries. Compared with traditional plant extracts and chemical synthesis, microbial synthesis of natural compounds such as p-coumaric acid has attracted wide attention due to its short production cycle and high conversion efficiency. Here a p-coumaric acid-producing Saccharomyces cerevisiae platform strain was developed. First, the tyrosine synthesis competition pathway genes ARO10 and PDC5 were knocked out, and ARO4(K229L) and ARO7(G141S) were mutated to release negative feedback inhibition from tyrosine. The tyrosine ammonia-lyase coding gene TAL from Flavobacterium johnsoniaeu was then integrated into genome and obtained C001 with yield of p-coumaric acid 296.73 mg/L. To further increase the accumulation of p-coumaric acid precursors, 8 genes encoding amino acids and carbohydrate transporters were knocked out and the gluconeogenesis pathway was enhanced. The results showed that GAL2 knockout and overexpression of EcppsA increased the yield of p-coumaric acid to 475.11 mg/L. Finally, the effect of FjTAL anchoring to yeast vacuoles on product accumulation was analyzed, and the highest titer of p-coumaric acid of 593.04 mg/L was obtained after intracellular vacuolar localization of FjTAL. It provided an efficient p-coumaric acid-producing platform strain for the subsequent synthesis of flavonoids and stilbene compounds by enhancing the supply of precursors, blocking the competitive bypass pathway, and using the strategy of subcellular localization.