基于基因组规模代谢网络模型的产琥珀酸放线杆菌GXAS137代谢工程改造

Succinic acid is an important C4 platform chemical and can be widely used in chemical, food, and medicine fields. Currently, succinic acid is commercially produced from chemical synthesis, a process which consumes petrochemical resources and contributes to environmental problems. Actinobacillus succinogenes is one of the most promising succinic acid industrial producers. However, it has some disadvantages such as the low concentration of succinic acid and high concentration of by-product. It is difficult to breed the excellent succinic acid producing strains for traditional metabolic engineering due to lack of understanding the cellular metabolic. Our team has finished a series of work for the strain metabolic engineering such as one A. succinogens strains’ genome sequencing, vector construction, gene expression and knockout, etc. This project intends to adopt systems metabolic engineering approach to construct the strain. First, the genome-scale metabolic model will be constructed based on the genome sequence. On this basis, the mechanism of cell physiological metabolism and succinic acid production will be illuminated from the whole cell level. Furthermore, the target genes for enhancing succinic acid production will be simulated by the model. Then the target genes will be engineered by the method of metabolic engineering and coenzyme engineering approach to enhance intermediate metabolites transmission efficiency in metabolic pathways and reduce carbon flux loss by repressing byproducts production. These general conclusions will not only provide fundamental information for the rational engineering of A. succinogens, but also provide important and meaningful way for other microorganisms breeding.enhance intermediate metabolites transmission efficiency in metabolic pathways and reduce carbon flux loss by repressing byproducts production. These general conclusions will not only provide fundamental information for the rational engineering of A. succinogens, but also provide important and meaningful way for other microorganisms breeding.

丁二酸是一种重要C4平台化合物，可广泛应用于食品、化工、医药等领域。目前主要以化学法合成，不仅消耗石化资源而且污染环境。产琥珀酸放线杆菌是目前最具潜力实现工业化的菌种之一，但存在产量低、副产物高等问题。传统代谢工程改造由于缺乏对细胞全局代谢网络的理解，通常难以实现改造预期，给菌株选育带来困难。在前期已完成丁二酸高产菌株GXAS137基因组测序、功能注释等工作的基础上，本项目拟采用系统代谢工程的策略对菌株进行理性改造。首先构建基因组规模代谢网络模型（GSMM），利用模型解析菌体生理代谢和产物合成机理，预测出显著提高丁二酸合成或副产物降低的改造靶点；再采用代谢工程及辅酶工程手段对选取靶点进行改造，增强主代谢途径、降低副产物代谢通量、调控细胞氧化还原状态，从而实现提高丁二酸浓度、降低副产物的目的。本课题的研究结果为丁二酸高产菌株的理性改造奠定理论基础，同时为其它微生物高产菌株选育提供有意义指导。

丁二酸是一种重要平台化合物，传统化学合成法生产污染大，成本高，限制其发展，生物发酵法以可再生生物质资源为原料，具有绿色、低能耗、可持续等优点。产琥珀酸放线杆菌（Actinoabcillus succinogens）是目前最具潜力实现工业化的菌种之一，但存在产量低、副产物高等问题。传统代谢工程改造由于缺乏对细胞全局代谢网络的理解，通常难以实现改造预期，给菌株选育带来困难。本项目在前期已完成丁二酸高产菌株GXAS137基因组测序、功能注释等工作的基础上，首先构建基因组规模代谢网络模型（GSMM），包含724个代谢基因相关的1075个酶反应，涉及714个代谢产物；在该模型的基础上，解析了菌株代谢丁二酸及副产物的代谢网络，预测出显著提高丁二酸合成的关键位点磷酸烯醇式丙酮酸羧化酶基因（pepck）及副产物形成关键位点丙酮酸甲酸裂解酶基因（pflB）及乙酸激酶基因（ack）。利用代谢工程手段获得工程菌株GXAS137-pepck，为解决菌株还原力缺乏问题，串联来源于大肠杆菌编码葡萄糖-6-磷酸脱氢酶（G6PDH）的zwf基因，重组GXAS137-pepck-zwf的丁二酸产量增加了11.6%；利用同源重组技术获得敲除菌株GXAS137-∆pflB及GXAS137-∆ack，副产物乙酸均降低13%以上。在获得工程菌的基础上，通过PB试验、正交及响应面等试验设计方法对工程菌发酵产丁二酸条件（底物浓度、氮源、pH缓冲剂等）进行了优化，并对工程菌发酵木薯、糖蜜等广西的“非粮”生物质资源发酵产丁二酸进行了研究，丁二酸可达90 g/L。本课题实施过程中共发表论文8篇，其中SCI/EI 收录3篇，申请国家发明专利5项，其中3项获授权，培养硕士研究生2名。本课题完成各项研究任务及指标，综合利用系统代谢工程与发酵调控手段，建立了基于基因组规模代谢网络模型（GSMM）微生物菌株系统代谢工程改造流程。