大肠杆菌异源表达逆己二酸降解途径产庚二酸研究

庚二酸是一种七碳直链饱和二元羧酸，除用于生物素合成外，还是重要的有机化工原料和生化研究制剂。据报道，大肠杆菌等微生物可利用脂肪酸合成途径生产庚二酸或其衍生物，然而具体的机理还不清晰，且推定的反应步骤较长，不易进行改造。申请人前期利用逆己二酸降解途径获得了少量的庚二酸积累，然而其关键酶β-酮硫解酶底物谱较宽，可同时催化多种酰基辅酶A反应生成不同的二元羧酸。为了解决上述问题，申请人拟通过同工酶筛选和蛋白质工程改造，解析β-酮硫解酶突变位点与其催化专一性的匹配关系，获得庚二酸合成高亲和力专一性β-酮硫解酶；同时，基于组学技术分析，阐明菌体生长与庚二酸积累之间的互作机制；在此基础上，对关键节点和限速步骤进行代谢工程改造，最终实现大肠杆菌中庚二酸的高效合成。

Pimelic acid is a seven-carbon linear saturated dicarboxylic acid. In addition to biotin synthesis, pimelic acid is also an important chemical raw material and biochemical reagents. It is reported that microorganisms such as Escherichia coli can produce pimelic acid or its derivatives using the fatty acid synthesis pathway. However, the specific mechanism is still unclear, and the presumed reactions are complicated and difficult to modify. The applicant has obtained a small amount of pimelic acid by using the reversed adipic acid degradation pathway. However, its key enzyme β-ketothiolase has a broad substrate spectrum, which simultaneously catalyzes multiple acyl-CoAs to generate different kinds of dicarboxylic acids. The applicant intends to analyze the relationship between the mutation site of β-ketothiolase and its catalytic specificity through isozyme screening and protein engineering, thus obtaining the β-ketothiolase with high affinity for pimelic acid synthesis, meanwhile, the interaction between bacterial growth and pimelic acid accumulation will be elucidated using the omics technology. Based on these, metabolic engineering of E. coli, including key nodes and rate-limiting steps, will be applied to achieve the efficient synthesis of pimelic acid.