代谢网络模型指导的非天然甲醇同化途径设计及构建

The efficient utilization of one-carbon compounds represented by methanol is of great significance to the development of China’s green economy. With natural assimilation pathways of methanol having defects as step redundancy, carbon losses and energy waste, efficient bioconversion of methanol is severely limited. In this project, we plan to design non-natural assimilation pathways of methanol that are superior to existing ones via computational systems biology. Iteration strategy of simulation and experiments will also be used to verify our pathways. Firstly, a high-quality chassis metabolic network model based on known reactions will be constructed. Selected non-natural reactions will be introduced into the chassis model, and the combinational and constraint-based algorithm will also be added to predict new pathways. Secondly, the thermal and kinetic feasibility of the pathways will be evaluated and suitable enzymes will be screened. Then, in vitro construction and optimization will be performed to verify pathway efficiency. Results obtained from experiments will be used as a feedback in the chassis model to guide next-round predictions. Eventually, the optimized non-natural assimilation pathways of methanol with less steps and less carbon losses or energy waste will be introduced into Escherichia coli to construct methanol utilization strains. The efficiency of methanol assimilation in vivo will be verified. Our preliminary results showed that the key idea of this proposal worked out just as expected. In conclusion, this project will lay a solid foundation for the efficient bio-utilization of methanol.

以甲醇为代表的碳一化合物的高效利用对于发展我国绿色经济意义重大。天然甲醇同化途径存在反应步骤多、碳损严重、能量消耗大等缺陷，是限制甲醇高效利用的重要因素。本项目拟通过计算系统生物学方法，结合模拟与实验循环迭代策略，设计并构建优于已有途径的非天然甲醇同化途径。首先，构建基于已知反应的高质量底盘代谢网络模型，将精选的非天然反应引入底盘模型，结合组合算法与约束算法进行途径预测；其次，评估途径的热力学和动力学可行性，挖掘途径相关酶；然后，进行体外组装及优化，验证途径效率，并将结果反馈模型，进而指导下一轮预测；最终，将优选的步骤短、碳损少、能耗低的非天然甲醇同化途径导入大肠杆菌细胞中，构建甲醇利用菌株，验证其实际甲醇同化效率。本项目已通过预研打通了关键技术障碍，预期成果将为甲醇高效生物利用奠定重要基础。

以甲醇为代表的碳一化合物的高效利用对于发展我国绿色经济意义重大。然而，天然碳一同化途径存在反应步骤多、碳损严重、能量消耗大等缺陷，是限制甲醇高效利用的重要因素。本项目过计算系统生物学方法，设计并构建优于已有途径的非天然甲醇同化途径。首先，本研究开发了非天然途径设计算法comb-FBA，并基于该算法对生化反应数据库MetaCyc和非天然反应数据库ATLAS进行碳一利用途径的系统计算挖掘，得到59条无碳损和能耗的目标途径。然后，通过动力学建模对途径进行分析，筛选得到3条有潜力的非天然人工途径，通过新酶挖掘、产物谱鉴定确定了碳一利用新途径GAA，通过酶和底物的分步添加，降低甲醛对多酶体系的毒害作用。通过上述多步优化，使人工途径GAA的实际效率提升至88%，超过已知的MCC途径80%的碳得率。进一步通过算法循环迭代，进行第二轮途径预测，得到9条新途径，并验证获得了GAPA途径，其碳转化效率进一步提升至90%。最后，在大肠杆菌中进行了碳一途径的体内构建。本项目通过comb-FBA算法开发、途径计算、实验验证，获得了多条无碳损、无能耗的新型碳一转化途径，并实验验证了四条途径的可行性，其中两条途径具有较高的转化效率，为非天然途径的系统挖掘和理性设计提供了可行方法和范例。

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