Metabolic engineering of Escherichia coli for methanol-assisted resveratrol biosynthesis

Resveratrol is a high-value stilbenoid with diverse pharmaceutical and nutraceutical applications. However, its microbial production remains limited by insufficient precursor availability and inefficient carbon utilization. In this study, Escherichia coli was sequentially engineered to establish a methanol-assisted platform for resveratrol biosynthesis. The heterologous pathway was reconstructed by co-expressing Populus tomentosa 4-coumarate: CoA ligase (Pt4CL) and Arachis hypogaea stilbene synthase (AhSTS), followed by introduction of the matBC operon from Streptomyces coelicolor to enhance intracellular malonyl-CoA supply. A synthetic methylotrophic pathway comprising methanol dehydrogenase (mdh2) from Bacillus methanolicus together with 3-hexulose-6-phosphate synthase (hxlA) and 6-phospho-3-hexuloisomerase (hxlB) from Bacillus subtilis was subsequently introduced, and CRISPR interference (CRISPRi) targeting frmA was implemented to modulate formaldehyde metabolism and facilitate methanol utilization. Optimization of heterologous protein expression increased resveratrol production from 7.3 ± 0.2 to 14.1 ± 0.3 mg/L, while matBC-mediated precursor engineering further increased the titer to 20.2 ± 0.4 mg/L. Under glucose-methanol co-feeding conditions, the engineered strain produced approximately 23 ± 0.28 mg/L resveratrol in shake-flask cultivation. Scale-up in a 5-L bioreactor yielded 26.42 ± 0.31 mg/L (132.1 ± 2.4 mg/L total resveratrol) with a PCA conversion efficiency exceeding 95%, complete glucose consumption, and approximately 40.3% methanol utilization after 36 h of cultivation. To our knowledge, this is the first report of methanol-assisted resveratrol biosynthesis in engineered E. coli. These results demonstrate the potential of integrating synthetic methylotrophy, precursor engineering, and CRISPRi-mediated carbon redistribution to establish a methanol-assisted platform for microbial resveratrol production. This work provides a proof-of-concept framework for future development of microbial production systems utilizing renewable C₁ feedstocks.