Single-module nonribosomal peptide synthetases (NRPSs) have nonclassical domain compositions and play versatile roles in the biosynthesis of natural products. AstC, a prototypical single-module NRPS-like d-alanyltransferase with the architecture of adenylation-thiolation-thioesterase (A-T-TE) tridomain, was identified as the catalyst in the d-alanylation process during the post-polyketide synthase (PKS) modifications of ansatrienin biosynthesis. In this study, the function of the TE domain of AstC was elucidated in intermolecular esterification, and its substrate promiscuity was revealed for both acyl donors and polyketide acceptors. Through genome mining, a newly characterized AstC homolog was identified, SmAstC, and it was shown that SmAstC exhibited the highest catalytic activity for d-alanylation of N-desmethyl-4,5-desepoxymaytansinol (DDM), a precursor of the antitumor agent ansamitocins, obtained from Actinosynnema pretiosum HGF052. Harnessing the broad substrate selectivity of the TE domains and the spontaneous hydrolysis propensity of the d-alanylated products, the post-PKS modification of ansamitocin biosynthesis was reprogrammed through introducing the gene encoding SmAstC into A. pretiosum HGF052. The ansamitocin biosynthetic pathway in this engineered strain was switched toward the production of maytansinol that was a critical intermediate of maytansine-derived antibody-drug conjugates. The SmAstC-catalyzed d-alanylation of DDM interrupted the stringent post-PKS modification steps within the original biosynthesis of ansamitocins, which produced 3-O-d-alanyl maytansinol that readily underwent spontaneous hydrolysis to afford maytansinol. The innovative application of the single-module NRPS-like d-alanyltransferases combined with rational strain engineering has surmounted the longtime hurdle in converting DDM to maytansinol in vivo, enabling the biomanufacturing of maytansinol without precedent.

中文翻译：

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单模块 NRPS 样 d-丙氨酰转移酶在美登醇生物合成中的功能应用

单模块非核糖体肽合成酶（NRPS）具有非经典结构域组成，在天然产物的生物合成中发挥多种作用。 AstC 是一种原型单模块 NRPS 样d-丙氨酰转移酶，具有腺苷酸化-硫醇化-硫酯酶 (AT-TE) 三域结构，被确定为聚酮合酶 (PKS) 后修饰过程中d-丙氨酰化过程中的催化剂安莎三宁的生物合成。在本研究中，阐明了 AstC TE 结构域在分子间酯化中的功能，并揭示了其对于酰基供体和聚酮化合物受体的底物混杂性。通过基因组挖掘，鉴定出新鉴定的AstC同源物SmAstC，并且表明SmAstC对抗肿瘤剂安丝菌素的前体N-去甲基-4,5-去环氧美登醇（DDM）的d-丙氨酰化表现出最高的催化活性，从Actinosynnema pretiosum HGF052获得。利用 TE 结构域的广泛底物选择性和d-丙氨酰化产物的自发水解倾向，通过将编码 SmAstC 的基因引入A. pretiosum HGF052中，对安丝菌素生物合成的 PKS 后修饰进行了重新编程。该工程菌株中的安丝菌素生物合成途径转向生产美登醇，这是美登素衍生的抗体-药物缀合物的关键中间体。 SmAstC 催化的DDM d-丙氨酰化中断了安丝菌素原始生物合成中严格的 PKS 后修饰步骤，产生了 3- O - d-丙氨酰美登醇，很容易自发水解得到美登醇。单模块NRPS类d-丙氨酰转移酶的创新应用与合理的菌株工程相结合，克服了DDM在体内转化为美登醇的长期障碍，实现了美登醇的生物制造，史无前例。