Structure-guided engineering of 4-coumarate: CoA ligase for efficient production of rosmarinic acid in Saccharomyces cerevisiae

The utilization of genetically modified microbial cells for rosmarinic acid (RA) production is gaining increased attention as a cost-effective and sustainable approach. However, the substrate promiscuity of 4-coumarate: CoA ligase and RA synthase has been considered as a critical factor for low RA yields. In this study, we rationally engineered the substrate preference of 4-coumarate: CoA ligase (OPc4CL2) from Petroselinum crispum , resulting in a significant enhancement in RA production. Particularly, the introduction of the Y240C mutation led to a remarkable 176 % increase in RA yield. Subsequent enzymatic analysis of OPc4CL2 variants revealed diminished activity towards p -coumaric acid, resulting in insufficient time for the transformation of p -coumaric acid to 4-coumaroyl CoA to generate byproduct. Furthermore, to minimize the formation of undesired byproducts, the overexpression of 4-hydroxyphenylacetate 3-monooxygenase (OHpaB) and NADPH-flavin oxidoreductase (HpaC) was carried out to facilitate the conversion of p -coumaric acid to caffeic acid and 4-hydroxyphenyllactate to salvianic acid A, thus achieving a significant increase in RA yield of up to 329.9 mg/L (16.5 mg/g yield on glucose) in shake-flask cultivation. Finally, the engineered strain YRA113–24BHM achieved a notable RA production of 3.6 g/L (about 20.2 mg/g yield on glucose) by fed-batch fermentation. This study serves as a foundation for the sustainable biosynthesis of RA and other caffeic acid derivatives.