甲醇高值化转化是当前生物化工的重要研究热点，但因甲醇生物催化过程一碳中间物（甲醛或甲酸）低效同化和甲醇高效氧化不匹配，仍然面临大量二氧化碳外排及还原力损耗的巨大挑战。提高甲醇转化率要解决如何扩大同化代谢流，拉动一碳代谢平衡向下游途径转移的核心问题。本项目以甲基杆菌为底盘宿主，以其同化甲醇合成丁醇为对象，提出构建异源一碳协作同化通道的策略，尝试通过理性改造与适应性进化将焦磷酸依赖核酮糖单磷酸途径和甲醛聚合途径组合为相互协作的同化模块，扩大同化通量和还原力供给，严密耦合氧化过程，降低二氧化碳足迹，并利用Mu噬菌体转座技术将重构通道有效可控插入基因组，提高同化稳定性；然后借助生物传感器感应丁醇，调控同化下游代谢流的分配，提高丁醇产量。本项目构建的同化通道协作分工、双管齐下提高碳效率与还原力供给，为甲醇高值转化提供新的思路，为甲醇/甲烷为碳同化中心的绿色生物催化开拓新的策略。

Currently, the bioconversion of methanol to high value-added products is an important hot topic in the field of biochemical engineering. However, the current low efficiency of C1 assimilation and poor matching of methanol oxidation result in the release of large amount of carbon dioxide from bacterial cells and an obvious loss of reducing power. The approach to increase the flux of C1 assimilation and then to pull one carbon metabolism balance to downstream pathway is important to solve the essential problem. To this end, we propose to engineer a previously studied methylotrophic bacteria of Methylobacterium extorquens to biocatalyze methanol conversion to 1-butanol. The methodologies of rational engineering with adaptive evolution will be carried out to construct an efficiently heterologous and synergic assimilation channel of one carbon. The synergic channel combining a PPi dependent ribulose monophosphate pathway and methanol formolase pathway will enhance the flux of C1 assimilation and closely couple to methanol oxidative pathway, then decrease the release of greenhouse gas, CO2. Moreover, Mu transposable phage will be carried out to controllably integrate the constructed channel into genome of Methylobacterium extorquens, which will further increase the stability of one carbon assimilation. Next, the biosensor will be engineered to sense the 1-butanol and to regulate the distribution of assimilated flux for increasing the titer of 1-butanol. This proposal provides a synergic assimilation channel to highly enhance carbon conversion rate and reducing power efficiency. The proposed new strategies will allow the chassis cells to utilize methanol more efficiently. Ultimately, the technologies provide the new and green biocatalytic direction of high value-added products based on the C1 methanol or methane.