Metabolic engineering of Escherichia coli to utilize methanol as a co-substrate for the production of (R)-1,3-butanediol

Due to its abundance, cost-effectiveness, and high reducibility, methanol has gained considerable attention in the biomanufacturing industry as a nonfood feedstock for the production of value-added chemicals. The range of chemicals that can be derived from methanol, however, remains constrained and is currently in the concept validation phase. This study aimed to develop and evaluate a hybrid methanol assimilation pathway in Escherichia coli to improve the production of (R)-1,3-butanediol ((R)-1,3-BDO) by utilizing methanol and sugars as co-substrates. By combining the methanol dehydrogenase (MDH) from the prokaryotes with the dihydroxyacetone synthase (DAS) from the eukaryotes, the hybrid pathway facilitates methanol conversion into the central metabolism while generating NADH at the same time. Through pathway optimization and targeted gene deletions, we have successfully developed an E. coli strain capable of producing 5.79 g/L (R)-1,3-BDO in shake flask experiments and 13.71 g/L (R)-1,3-BDO with a yield of 0.35 C-mol/C-mol in batch fermentation using methanol and glucose as co-substrates. Our study also showed the incorporation of ¹³C-methanol into cellular intermediates and an increase in NAD(P)H concentration, confirming the role of methanol as a co-substrate and supplier of NADH. In addition, our study also demonstrated the co-utilization of methanol with xylose for the production of (R)-1,3-BDO, expanding the substrate spectrum for sustainable 1,3-BDO production.