Enhanced methanol metabolism via reinforced cellular energy and reducing power supply for sustainable carbon conversion.

Methanol, derived from abundant natural gas, biogas methane, coal gasification, biomass conversion, and CO2 reduction processes, presents a promising substrate for bioproduction of chemicals, making its assimilation a key focus of this study. In this study, to enhance the methanol assimilation efficiency of Escherichia coli (E. coli), we increased the intracellular supply of ATP and NADPH, significantly improving cellular metabolism and thereby enhancing methanol utilization. The endogenous transhydrogenase gene sthA and ATP synthase gene atpAGD were initially overexpressed individually in the genome, leading to a 15-fold increase in methanol assimilation efficiency for sthA and a 21-fold increase in methanol utilization for atpAGD. Subsequently, by screening various RBS sequence combinations for the co-expression of sthA and atpAGD, the methanol utilization capacity of the engineered strain was further enhanced by 28-fold. Furthermore, intracellular levels of ATP, NADH, and NADPH were analyzed, revealing a marked increase in ATP levels, while NADH levels decreased and continued to decline throughout the cultivation process. In contrast, NADPH levels gradually increased. These findings indicate that methanol metabolism requires an elevated supply of ATP and NADPH to mitigate cellular metabolic stress. Finally, the engineered strain with co-expression was employed for the biosynthesis of 3-hydroxypropionic acid (3-HP) and triacetic acid lactone (TAL). Notably, under conditions without any additional metabolic modifications, the engineered strain achieved a 3-HP titer of 2014 mg/L, reflecting a relatively high production level when methanol and xylose were used as mixed carbon sources, demonstrating its significant industrial application potential.