生物合成甘油三酯嗜甲烷菌人工细胞构建及其碳代谢流调控机制解析

Bioconversion of low-cost methane (biogas) into value-added products is a promising alternative to reduce greenhouse gas emission and fossil fuel consumption. Most of the methane derived from biogas is burned to generate heat and electricity in a process referred to as CHP (combined heat and electricity process), which reduces its value significantly. This project aims at multi-scale control, rational design and construction of artificial engineered Methylomicrobium buryatense for producing the triglyceride, which is a precursor of aviation fuels. The concept of the combination of the bioelements-model strain-artificial regulation” is developed and applied in this project. The principle of strain construction and the biosynthetic mechanism of the triglyceride accumulation in M. buryatense are systemically analyzed by using strategies of the exploration of functional genes and bioelements, the assembly of functional modules, the optimization of functional modules, the adaption of functional modules and the model strain as well as the artificial metabolic manipulation. By introducing a heterotrophic partial-serine pathway and controlling the regeneration of cofactors, the regulation mechanism for high carbon flux from methane to key metabolites will be revealed and the interaction of different functional modules will be investigated in the meantime. The response mechanism of M. buryatense to restrictive culture conditions will be illuminated to provide a high-efficient triglyceride production system. These preliminary works will not only demonstrate that bioconversion of methane into triglyceride/jet fuel is feasible, but also form the basis for a wide application in the production of other value-added bio-based products derived from low-cost methane and biogas.

将低品位甲烷转化为液体燃料以实现其增值利用，是提高能源品质的一条重要途径。本项目以甲烷合成生物航油前体-甘油三酯(TAG)为研究对象，探究高碳通量异源生物合成TAG生化过程的优化、调控方法及策略。采用异源基因挖掘、功能模块设计、代谢路径构建、底盘细胞优化、辅因子再生路径改造、甲烷代谢通量调控等手段，解析产TAG嗜甲烷菌人工细胞的构建原理，阐释辅因子平衡与甲烷代谢流调节的协同作用及其动态调控机制。通过重构异源丝氨酸循环路径捕捉二氧化碳并将其导向酰基辅酶A合成路径，进一步对嗜甲烷菌代谢网络碳流进行人工调节，揭示该异源路径对人工细胞碳代谢网络及甲烷合成TAG代谢通量的定向分配、强化作用机制。基于TAG生物合成规律，探索嗜甲烷菌富集合成TAG的培养策略和过程控制方法。最终为实现嗜甲烷菌人工细胞高效利用甲烷合成TAG奠定科学基础。

航空生物燃料因具有低碳排放和可持续性得到高度关注，但其原料成本高、收集难、运输贵等问题制约了其产业化进程。将低品位甲烷转化为液体燃料实现其增值利用是提高能源品质的一条重要途径。好氧嗜甲烷菌Methylomicrobium buryatense代谢网络中具有天然完备的转化甲烷合成油脂的途径，由于积累的油脂主要是功能性脂质磷脂，积累有限，所以通过引入浑浊红球菌Rhodococcus opacus PD630的磷脂酸磷酸酶（RoPAP）和弯曲高温单胞菌Thermomonospora curvata DSM 43183的二酰基甘油转移酶（tDGAT）在底盘宿主M. buryatense 胞内构建甘油三酯（TAG）合成路径，通过敲除旁路dgkA基因以提高TAG合成前体化合物甘油二酯的代谢通量，敲除fadE基因以实现酰基辅酶A合成通量最大化，完成对底盘代谢网络的优化，获得优良TAG合成嗜甲烷菌人工细胞。在此基础上，通过在M. buryatense体内重构异源丝氨酸循环路径捕捉CO2，实现CO2有效回流，进一步加强嗜甲烷菌TAG合成碳通量。最后，通过连续高密度发酵工艺，实现甲烷高效转化并合成油脂，为规模放大生产提供数据支撑。