限铵条件下谷氨酸棒杆菌生产α-酮戊二酸机制的研究

α-ketoglutaric acid is widely used in chemical industry, food, medicine and daily chemicals. Corynebacterium glutamicum is considered as ideal chassis cell. In previous study, we found that glutamate producer Corynebacterium glutamicum GKG-047 produced considerable α-ketoglutaric acid under the condition of limited ammonium. Data of transcriptomics showed that level of genes and regulator thereof involved in carbon and nitrogen metablism varies in different degrees under the condtion. In this project, effect of limited ammonium on α-ketoglutaric acid sythesis as well as energy and coenzyme metablism of C. glutamicum GKG-047 will be studied; regulation of α-ketoglutaric acid sythesis by regulator of AmtR, SugR, ClgR and GntR1 will be investigated; ammonium-limit sensor of signaling molecules and its regulation on α-ketoglutaric acid sythesis and the former regulator will also be detected. And consequently, the regulatory mechanism of α-ketoglutaric acid synthesis undert the condition of limited ammonium is to be clarified. This project will help to understand regulatory mechanism and provide important reference and practice guidance for C. glutamicum metabolic engineering research.

α-酮戊二酸在食品、医药等领域具有广泛应用。谷氨酸棒杆菌是生物合成α-酮戊二酸的理想底盘生物。前期研究发现谷氨酸生产菌Corynebacterium glutamicum GKG-047在限NH4+条件下可大量积累α-酮戊二酸，经转录组分析发现在限铵条件下该菌株碳源代谢相关基因及其调控因子转录水平不同程度地发生改变。本项目在前期研究基础上首先考察限铵对C. glutamicum GKG-047α-酮戊二酸代谢、能量和酶代谢的影响；进而解析SugR等调控因子在限铵条件下GKG-047过量合成α-酮戊二酸的调控作用；明晰谷氨酸棒杆菌铵饥饿感应信号分子并考察其对调控因子及菌株生理和代谢的影响。最终解析限铵条件下谷氨酸棒杆菌过量合成α-酮戊二酸的调控机制。本项目对谷氨酸棒杆菌代谢调控机制的理解具有重要意义，同时可为谷氨酸棒杆菌的代谢工程应用研究提供重要参考和实践依据。

α-酮戊二酸参与生物体内氨基酸、维生素和有机酸的合成及能量代谢，具有重要的应用前景。前期研究发现，谷氨酸棒杆菌Corynebacterium glutamicum GKG-047在限铵条件下可大量积累α-酮戊二酸。为解析其调控机制，首先考察了谷氨酸脱氢酶等多个α-酮戊二酸代谢相关酶对其合成的影响，明确gdh、aceA、gogat、alaT、gltA均不同程度地影响α-酮戊二酸合成及菌体生长；发现限铵条件下，调控因子SugR、GntR1、ClgR和AmtR编码基因的转录水平受到不同程度的影响，从而影响其受控基因的转录，进而使得中心代谢途径的代谢流发生改变，促进α-酮戊二酸的合成；采用基因过表达或干扰的手段对SugR等调控因子编码基因进行反向代谢工程研究，发现均能不同程度地扰动菌株代谢网络，从而影响α-酮戊二酸合成；在此基础上， 确定胞内NH4+变化为引起上述差异的主要信号分子，并明确了NH4+对C. glutamicum GKG-047生理、代谢及其调控因子的调控作用。研究结果对于加深谷氨酸棒杆菌代谢调控和遗传机制的认识及理解具有重要意义，同时也将为谷氨酸棒杆菌代谢工程应用研究提供重要的技术支撑。