Developing a highly efficient whole-cell biotransformation of 3,4-dihydroxyacetophenone into Apocynin by engineered Escherichia coli expressing caffeic acid O-methyltransferase
摘要
Apocynin (Ap), a bioactive compound from the roots of Picrorhiza kurroa, faces challenges in production. This study developed a whole-cell biotransformation approach using engineered Escherichia coli expressing caffeic acid O-methyltransferase to convert 3,4-dihydroxyacetophenone into Ap. Caffeic acid O-methyltransferase from Medicago sativa (MsCOMT) showed the highest activity, yielding 90.75 mg·L⁻¹ and 97.04 mg·L⁻¹ Ap after 24 h and 48 h, while the mutant I319A enhanced titers to 198.32 mg·L⁻¹ and 228.37 mg·L⁻¹ by optimizing H269-D270 catalytic mechanisms. Besides, this study explored the impact of biotransformation conditions on the activity of MsCOMT and the yield of Ap. TB medium was found to be the most effective, with yields of 158.52 mg·L⁻¹ and 174.75 mg·L⁻¹ after 24 h and 48 h. The in vivo SAM regeneration system, less effective than in vitro SAM supplementation, still improved Ap yield when the genes mtn, luxS, and MsCOMT were arranged in a pseudo-operon configuration. Orthogonal experiments showed the importance order of transformation factors as: transformation temperature > induction temperature > substrate concentration > IPTG concentration, with optimal conditions being 35 °C transformation temperature, 15 °C induction temperature, 8 mmol·L⁻¹ substrate concentration, and 0.1 mmol·L⁻¹ IPTG concentration. Using mutant MsCOMTI319A under these optimal conditions, Ap yield increased steadily with transformation time, reaching a maximum of 544 mg·L⁻¹ after 72 h. This research successfully achieved the whole-cell biotransformation of 3,4-dihydroxyacetophenone into Ap for the first time, providing a foundation for further optimization of Ap biosynthesis.