吡啶霉素是具有抗结核分枝杆菌活性的环酯肽类化合物，包括3-羟基吡啶羧酸（3-HPA）和吡啶丙氨酸（PAA）两个独特的含吡啶结构单元。吡啶霉素通过与分枝菌酸合成关键酶InhA的辅因子及底物结合区域结合，从而抑制结核分枝杆菌细胞壁合成，两个吡啶结构单元对其活性发挥了关键作用。二者的生物合成明显不同于以往的报道，它们来源于天冬氨酸，而不是常见的赖氨酸。本项目拟对由两个吡啶结构单元的生物合成机制展开研究：①通过体内遗传学研究确定3-HPA和PAA的生物合成相关基因，②体外生化研究阐明生物合成途径中关键酶的催化机制，③体外重构高效的3-HPA和PAA片段合成体系。本研究对两种特殊吡啶结构单元生物机制和关键酶催化反应机理的阐释，以及对构建体外合成体系的探索，不仅能够丰富现有天然产物中吡啶结构单元的生物合成机制和相关酶学功能，还可为合成生物学研究提供构建此类结构单元的理论依据和催化元件。

Pyridomycin is an antimycobacterial cyclodepsipeptide natural product harboring 3-hydroxypicolinyl acid (3-HPA) and pyridyl-L-alanine (PAA), two structurally unique pyridine containing building blocks. Pyridomycin specifically targets InhA, the NADH-dependent enoyl acyl carrier protein reductase, which is a critical enzyme in the tuberculosis bacteria. It functions as a competitive inhibitor of both the NADH cofactor binding site and the lipid substrate-binding pocket of InhA further inhibit the cell wall formation of tuberculosis bacteria. The two unique pyridine containing building blocks contribute to its bioactivity, yet the biosynthetic pathways of the two aspartic acid derived pyridyl building blocks remain unclear. Here we will confirm the related biosynthetic genes by in vivo experiments, characterize and elucidate the mechanisms of key enzymes from the pathways by in vitro work, and explore the efficient reconstitution systems of 3-HPA and PAA. This work will not only represent new models for the pyridine containing building blocks, but also expand the function of enzymes from these pathways, and might be exploited to develop catalytic elements of these structure moieties in synthetic biology.