谷氨酸棒杆菌的甲醇利用限制因素研究

China has world leading methanol production capacity and it is important to explore the potential of methanol bioconversion for developing methanol economy. However, it is difficult to optimize natural methanol-utilizing microorganisms for this purpose due to lack of efficient genetic manipulation tools. Engineering commonly used industrial strains such as Corynebacterium glutamicum by expanding their methanol utilization ability is promising, but yet unsuccessful after so many years’ study. Balance between regeneration of methanol assimilation precursor and synthesis of biomass, physiological inhibition caused by toxic intermediates, and many other factors are cross-linked and limit the overall methanol unitization capacity. Taking advantage of years’ experience in metabolic engineering of C. glutamicum, here we propose a complete solution to address this problem. First, a strain able to co-utilize ribose and xylose is developed and evolved to enhance the supply network of ribulose-5-phosphate, a precursor for methanol assimilation. Then, a second strain able to co-utilize methanol and xylose is designed and constructed, in which the methanol utilization capacity is positively correlated with cellular growth rate, allowing to enhance the methanol utilization through simple subcultivation-based adaptive evolution. Afterwards, genomics, transcriptomics and reverse metabolic engineering will be applied to understand why and how the genetic mutations associate with methanol utilization. At the end, the strain will be further improved by combining the molecular understanding to build a novel and versatile methanol transformation platform. Our preliminary results show that the key idea of this proposal works just as expected. Limiting factors for methanol utilization in C. glutamicum should be substantially understood after this project is completed, which will lay important groundwork for methanol biotransformation.

中国甲醇产能世界第一，探索甲醇生物转化对于发展甲醇经济意义重大。天然甲醇利用微生物遗传操作困难，以常用工业菌株如谷氨酸棒杆菌为平台，赋予其甲醇利用能力，是实现甲醇规模利用的重要路线之一，然而未有成功报道。甲醇同化前体再生与生物量合成的平衡、中间产物毒性等诸多因素交织在一起，破解甲醇利用的限制性因素困难重重。携谷氨酸棒杆菌多年分子改造经验，本项目拟首先构建核糖-木糖共利用菌，通过强化戊糖磷酸途径，提高甲醇同化前体的供给能力；进一步创造性地构建甲醇-木糖共利用菌，关联甲醇利用能力与菌体生长速度，通过适应性进化平衡甲醇利用诸多限制因素，率先实现甲醇快速利用；通过反向代谢工程研究突变体的基因组和转录组变化，揭示甲醇利用的限制性因素和作用机理；最终整合研究结果，进一步增强突变株利用甲醇作为唯一碳源的能力，获得甲醇生物转化新平台。本项目已通过预研解决了关键技术障碍，成果将为甲醇生物转化奠定重要基础。

甲醇能量密度高，可由煤炭、可燃冰等石化资源，生物质、二氧化碳等可再生资源规模化生产，是生物制造的理想原料。自然界存在天然甲醇利用菌，但由于缺乏清晰的遗传背景和高效的基因编辑工具，研究者对这些菌株的理解和应用还较初步。因此，改造已广泛应用的底盘菌，创建人工甲醇利用菌，是实现甲醇规模利用的重要路线之一。但是，目前报道的人工甲醇利用菌的甲醇生物转化效率较低，急需破解甲醇利用的限制性因素。本项目以谷氨酸棒杆菌为底盘菌，通过理性设计和代谢工程改造，敲除了核糖-5-磷酸异构酶Rpi、甲醛脱氢酶AdhE和Ald，表达了异源的木糖异构酶XylA、甲醇脱氢酶Mdh、3-己酮糖-6-磷酸合成酶Hps和6-磷酸-3-己酮糖异构酶Phi，构建了同时利用甲醇和木糖生长的甲醇依赖型菌株，将细菌生长速度与甲醇利用能力直接关联，并通过适应性进化平衡甲醇利用诸多限制因素，获得了高效利用甲醇的突变株。进一步，结合基因组、转录组等组学分析和反向代谢工程研究了突变菌的基因突变和基因表达调控变化对甲醇利用的影响，鉴定了位于cgl0653、cgl0833等基因的关键突变，解析了甲醇氧化、甲醇毒性、氧化还原平衡等人工菌株中甲醇利用的限制因素，并实现了生物转化甲醇合成谷氨酸产品。项目的研究成果为人工甲醇利用菌的优化提供了理论指导，为发展基于甲醇的生物制造奠定了基础。相关研究成果发表论文10篇，均标注该项目资助，其中5篇为第一标注，项目负责人为相关论文的第一（含共同一作）或通讯作者，申请中国发明专利3项，培养研究生2名，完成项目目标。