Green acetyl modification of puerarin to form puerarin 6′′-O-acetate using engineered Escherichia coli with favorable pathways and elevated acetyl-CoA supply

Abstract

Puerarin 6′′-O-acetate (P6′′A), a puerarin derivative with a high stability and an improved permeability, exhibits a promising potential in drug development. The large-scale production of P6′′A through green synthesis procedures has never been achieved owing to the low availability of acetyl-CoA. Herein, the enzymatic synthesis of P6′′A in Escherichia coli cell factories overproducing acetyl-CoA has been achieved. Firstly, the effect of three acetyl-CoA-producing pathways on P6′′A conversion in E.coli was compared comprehensively. The pathways containing acetyl-CoA synthetase (ACS) or the pyruvate dehydrogenase (PDH) complex were thereby identified as favorable fluxes yielding more acetyl-CoA. Hence, cells co-expressing ACS and the PDH complex were selected as cell factories overproducing acetyl-CoA owing to the positive cooperativity of ACS and PDH. Subsequently, the maa gene encoding maltose acetyltransferase (MAT) was introduced into these acetyl-CoA-producing cell factories to form engineered strains Q1–Q14. After condition optimization, the engineered strain Q5 got a P6′′A yield of 0.98 g L⁻¹ in M9P-Glc medium supplemented with 2.5 mM puerarin and 1.5% glucose. To further improve P6′′A production, the maa gene in Q5 was replaced by a mutant maaE125N gene to generate another engineered strain (Q15). Microbial production of P6′′A in Q15 was achieved through the centrifuge tube culture, the shake-flask culture and the 6-L reactor culture and the highest P6′′A yield of up to 2.02 g L⁻¹ was obtained in the reactor fermentation. Enzymatically synthesized P6′′A displayed a high stability in human and mouse plasma, a dose-dependent cytotoxicity in HepG2 cells and an improved liposolubility. This research provides a feasible procedure for the green synthesis of puerarin monoesters and lays a foundation for druggability research on P6′′A.