Discovery of the Azaserine Biosynthetic Pathway Uncovers a Biological Route for a-Diazoester Production

Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling alpha-diazoester functional group. Here, we report the discovery of the azaserine (aza) biosynthetic gene cluster from Glycomyces harbinensis. Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine production. Notably, this gene cluster encodes homologs of hydrazonoacetic acid (HYAA)-producing enzymes, implicating HYAA in alpha-diazoester formation. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2-electron oxidation of a hydrazonoacetyl intermediate is required for alpha-diazoester formation, constituting a distinct logic for diazo biosynthesis. Uncovering this biological route for alpha-diazoester production now enables the production of a highly versatile carbene precursor, enhancing approaches for engineering complete carbene-mediated biosynthetic transformations in vivo.

重氮丝氨酸（Azaserine）是一类细菌来源的代谢产物，其含有生物学上罕见且具备合成应用价值的α-重氮酯（alpha-diazoester）官能团。本研究报道了从哈尔滨糖霉菌（Glycomyces harbinensis）中发掘的重氮丝氨酸（简称aza）生物合成基因簇。对相关基因簇的发掘揭示了一批此前未被认知的重氮丝氨酸产生菌，而对aza基因簇的异源表达验证了其在重氮丝氨酸生物合成中的功能。值得注意的是，该基因簇编码了腙基乙酸（hydrazonoacetic acid，简称HYAA）合成酶的同源蛋白，提示HYAA参与了α-重氮酯的形成过程。同位素示踪饲喂与生化实验验证了这一假说。上述研究结果表明，α-重氮酯的生物合成需要对腙乙酰基中间体进行两电子氧化，这为重氮类化合物的生物合成提供了一种独特的机制逻辑。如今，该α-重氮酯生物合成途径的阐明，可用于制备应用范围极广的卡宾（carbene）前体，从而优化体内完整卡宾介导生物转化反应的工程化策略。