(2lv) Engineering and Adaptive Laboratory Evolution of Escherichia coli for Improving Methanol Assimilation Based on a Hybrid Xump Pathway

Research Interests developing methanol as an emerging next-generation feedstock for industrial biotechnology. While recent attempts to engineer E. coli as a synthetic methylotroph have achieved great success, most of these works are based on the engineering of the prokaryotic ribulose monophosphate (RuMP) pathway. In this study, we introduced a hybrid xylulose monophosphate (XuMP) pathway with a prokaryotic methanol dehydrogenase (MDH) and a eukaryotic dihydroxyacetone synthase (DAS) into E. coli and reprogrammed E. coli metabolism to improve methanol assimilation by combining rational design and adaptive laboratory evolution. By deletion and down-regulation of key genes in TCA cycle and glycolysis to increase the flux toward the cyclic XuMP pathway, methanol consumption and the assimilation of methanol to biomass were significantly improved. Further improvements in methanol utilization and cell growth were achieved via adaptive laboratory evolution and a final evolved strain can even grow on methanol in the presence of only 0.1 g/L yeast extract. ¹³C-methanol labeling assay demonstrated significantly higher labeling in intracellular metabolites in glycolysis, TCA cycle, pentose phosphate pathway, and amino acids. Transcriptomics analysis showed that the expression of fba, dhak, and part of pentose phosphate pathway genes were highly up-regulated, suggesting that the rational engineering strategies and adaptive evolution are effective for activating the cyclic XuMP pathway. This study demonstrated the feasibility and provided new strategies to construct synthetic methylotrophy of E. coli based on the hybrid XuMP pathway.