The effect of regulatory system on the engineered biosynthetic pathway in chassis cells remains incompletely understood in microorganisms. Acyl-CoAs function as key precursors for the biosynthesis of various natural products and the dominant donors for protein acylation. The polyphenol pinosylvin, with high antimicrobial and antifungal activities, is biosynthesized with malonyl-CoA as its direct precursors. But correlation between lysine malonylation and pinosylvin biosynthesis remains unknown. Herein, we found that the malonyl-CoA-driven lysine malonylation plays an important role in interaction between the engineered pathway of pinosylvin synthesis and E. coli chassis cell. Oversupply of malonyl-CoA leads to an increase in malonylation level of global proteome as well as the enzymes in the artificial pathway, thereby decreasing yield of pinosylvin. The results revealed that the intricate balance of cellular acyl-CoA concentrations is critical for the yields of acyl-CoA-derived natural products. We next modified the enzymes in the biosynthetic pathway to adjust their acylation level and successfully improved the yield of pinosylvin. Our study uncovers the effect of protein acylation on the biosynthetic pathway, helps optimization of synthetic constructs, and provides new strategies in metabolic engineering and synthetic biology at the protein post-translational level.