甲基杆菌异源一碳协作同化通道的构建及其强化甲醇转化

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.

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。

甲醇高值化转化是当前生物化工的重要研究热点。甲基杆菌是以甲醇作为唯一碳源和能源生长的甲基细胞工厂，但因其利用丝氨酸循环同化甲醇过程中存在代谢步骤多、高耗能、高耗还原力和大量碳外排等的限制，不利于高还原度化学品的有效合成。为了扩大甲醇同化代谢流，拉动碳一代谢平衡向下游途径转移，本项目以甲基杆菌模式菌---扭脱甲基杆菌作为细胞底盘，以其同化甲醇转化成还原度产品3-羟基丙酸及已丁醇为对象，首先基于基因组尺度模拟计算，整合缺陷型突变菌快速筛选技术、途径设计与基因元件优化、转录物及代谢组分析等代谢工程策略，构建了核酮糖单磷酸途径（RuMP pathway）和丝氨酸循环途径（Serine Cycle）组合的高效碳一协同同化途径，获得重塑甲醇同化代谢底盘的甲基细胞工厂，生长速率提高16%，达到0.134 h-1，构建了3-羟基丙酸生产菌在摇瓶下产量提高到90 mg/L，优化的两阶段发酵条件下3-羟基丙酸产量达3.7 g/L；为了进一步解决还原力不足的问题，在动态梯度连续降低甲醇浓度的同时连续升高甲酸浓度的适应性进化过程中筛选获得一株兼顾甲酸耐受和高效利用的进化菌，在高浓度甲酸盐作为碳源培养条件下可以生长，且在甲醇上生长速率达到0.175 h-1，在甲酸和甲醇作为混合碳源下摇瓶下3-羟基丙酸产量提高到175 mg/L；基于此，我们利用基因元件优化、代谢组分析、竞争通路阻断和限速途径强化等策略，在扭脱甲基杆菌中建立了催化甲醇转化成异丁醇的甲基工程菌；此外为了从mRNA和蛋白质两个层面动态调控基因表达水平，提高甲基细胞工厂合成目标化学品的能力，我们首次建立了CRISPRi和small RNA技术，并成功应用于类胡萝卜素合成途径调控和高通量快速挖掘类胡萝卜素合成的未知功能基因。本项目实施期间的研究成果在代谢工程主流刊物（Metabolic Engineering、Applied and Environmental Microbiology、Applied Microbiology and Biotechnology和Biotechnology Journal）发表论文4篇，一般SCI期刊论文3 篇；申请国家发明专利 2件；培养硕士研究生 9 名、青年教师4名。本项目实施为甲醇高值绿色转化提供新的思路，为甲醇/甲烷为碳同化中心的绿色生物催化开拓新的策略。