Harnessing Metalloprotease Wss1 to Enhance Methanol Utilization

The research on synthetic methylotrophic bacteria for one-carbon (C1) feedstock assimilation has garnered substantial interest and is regarded as the forefront of biomanufacturing advancements. Nevertheless, the effective utilization of C1 feedstocks faces challenges due to inadequate tolerance toward C1 compounds. This study elucidates that the buildup of formaldehyde causes severe DNA-protein cross-linking (DPC), and thus hampers growth and methanol assimilation in <i>Escherichia coli</i>. To tackle this issue, we exploited a metalloproteinase, SpWss1, from <i>Schizosaccharomyces pombe</i>. By fine-overexpressing SpWss1 in the <i>E. coli</i> genome, we were able to alleviate DPC damage and enhance formaldehyde tolerance. Remarkably, the engineered strain displayed a 10-fold increase in the amount of methanol assimilated (142 mM) compared to that of the control strain lacking SpWss1 (14 mM). Moreover, through iterative substrate feeding of methanol and xylose in shake-flask experiments, the genetically modified strain exhibited improved consumption levels, reaching up to 309 mM (∼10 g/L), making it one of the highest methanol-consuming strains among all <i>E. coli</i> strains without adaptive evolution. Additionally, the modified strain significantly enhanced the sustainable production of valuable products, such as triacetic acid lactone and fatty acids, from methanol. Overall, our findings underscore the significant scientific and biotechnological importance of addressing DPC to optimize C1 assimilation, providing valuable insights for sustainable chemistry, engineering, and industrial biotechnology applications.