恶臭假单胞菌中一条新的戊二酸代谢途径

In previous reports, glutaric acid was metabolized by glutaric acid dehydrogenation pathway. Based on the comparative genomic analysis and preliminary experimental results, we speculated that a new glutaric acid hydroxylation pathway exists in Pseudomonas putida KT2440. The new glutaric acid hydroxylation pathway involves three proteins; glutaric acid hydroxylase (GHS), L-2-hydroxyglutaric acid oxidase (LGO), and a regulatory protein of GntR family (GhsR). GHS catalyzes the hydroxylation of glutaric acid to produce L-2-hydroxyglutaric acid. LGO catalyzes the oxidation of L-2-hydroxyglutaric acid to produce 2- ketoglutarate. GhsR regulated the expression of LGO and GHS. In this project, the new glutaric acid hydroxylation pathway would be studied in depth. Based on the physiological and biochemical characteristics of each key enzyme in the new pathway, roles of LGO and GHS in glutaric acid metabolism and the regulating mechanism of GhsR would be clarified. This project could reveal the source of L-2-hydroxyglutaric acid, the reason for L-2-hydroxyglutaric acid accumulation, and the inhibition mechanism of L-2-hydroxyglutaric acid on glutaric acid metabolism in P. putida KT2440. The theoretical basis for revealing the organism glutaric acid metabolism and the related pathogenic processes would also be acquired through the accomplishment of the project.

已报道的生物体内戊二酸代谢均通过戊二酸脱氢途径，申请人基于比较基因组学分析与前期实验结果，推测Pseudomonas putida KT2440中存在一条新的戊二酸羟基化途径。该途径涉及戊二酸羟基化酶（GHS）、L-2-羟基戊二酸氧化酶（LGO）与GntR家族转录调控蛋白（GhsR），其中GHS催化戊二酸羟基化生成L-2-羟基戊二酸；LGO催化L-2-羟基戊二酸生成2-酮基戊二酸；GhsR调控GHS、LGO的表达。本项目拟以P. putida KT2440中该戊二酸代谢新途径为研究对象，基于对各关键酶的生理功能及生化特性分析，确定GHS、LGO在戊二酸代谢中的作用；阐明GhsR调控GHS、LGO表达的具体机制；解析P. putida KT2440中L-2-羟基戊二酸的来源、积累及其抑制戊二酸代谢的机理，为揭示生物体戊二酸代谢及相关致病过程奠定理论基础。

戊二酸是动物、植物和微生物中L-赖氨酸分解代谢的重要中间体，广泛分布于不同生境。在以往研究中，唯一报道的戊二酸分解代谢途径是戊二酰辅酶A脱氢途径。申请者基于比较基因组学分析与前期实验结果，推测恶臭假单胞菌KT2440中存在一条新的戊二酸羟基化代谢途径，其关键酶为CsiD和LhgO；外源表达并分离纯化CsiD和LhgO，确认CsiD为戊二酸羟化酶，催化戊二酸羟基化生成L-2-羟基戊二酸，LhgO为L-2-羟基戊二酸氧化酶，催化L-2-羟基戊二酸氧化生成2-酮基戊二酸；基于关键酶CsiD和LhgO的催化特性和生理功能分析，证明戊二酰辅酶A脱氢途径与戊二酸羟基化途径共同参与恶臭假单胞菌KT2440的戊二酸代谢，但戊二酸羟基化途径发挥更加关键的作用；确定CsiD和LhgO编码基因位于同一操纵子中，受转录调控蛋白CsiR调控，揭示了CsiR调控CsiD和LhgO编码基因表达的具体机制，并证明戊二酰辅酶A脱氢途径与戊二酸羟基化途径间不存在交叉调控；通过阻断戊二酰辅酶A脱氢途径与戊二酸羟基化途径，构建了戊二酸高产工程菌株，实现了基于L-赖氨酸分解代谢的戊二酸高效生产；上述结果一方面完善了研究者对生物体内戊二酸代谢途径和调控机制的认识，另一方面为重要生物基化学品戊二酸的高效生产奠定了理论基础。