Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.
ACS Chem Biol. 2023 Jul 21;18(7):1500-1509. doi: 10.1021/acschembio.3c00074. Epub 2023 Jul 4.
Modular polyketide synthases (PKSs) are attractive targets for the directed, biosynthetic production of platform chemicals and pharmaceuticals by protein engineering. In this study, we analyze docking domains from the 6-deoxyerythronolide B synthase, SYNZIP domains, and the SpyCatcher:SpyTag complex as engineering tools to couple the polypeptides VemG and VemH to functional venemycin synthases. Our data show that the high-affinity interaction or covalent connection of modules, enabled by SYNZIP domains and the SpyCatcher:SpyTag complex, can be advantageous, , in synthesis at low protein concentrations, but their rigidity and steric demand decrease synthesis rates. However, we also show that efficiency can be recovered when inserting a hinge region distant from the rigid interface. This study demonstrates that engineering approaches should take the conformational properties of modular PKSs into account and establishes a three-polypeptide split venemycin synthase as an exquisite platform for the analysis and engineering of modular PKSs.
模块化聚酮合酶（PKSs）是通过蛋白质工程定向生物合成平台化学品和药物的有吸引力的目标。在这项研究中，我们分析了 6-脱氧赤藓醇 B 合酶、SYNZIP 结构域和 SpyCatcher:SpyTag 复合物的对接结构域，作为将多肽 VemG 和 VemH 与功能性 venemycin 合酶偶联的工程工具。我们的数据表明，SYNZIP 结构域和 SpyCatcher:SpyTag 复合物使模块的高亲和力相互作用或共价连接能够在低蛋白浓度下合成，但它们的刚性和空间需求会降低合成速率。然而，我们还表明，当在远离刚性界面的位置插入铰链区域时，效率可以恢复。这项研究表明，工程方法应该考虑模块化 PKSs 的构象特性，并建立了一个三多肽分裂 venemycin 合酶作为模块化 PKSs 分析和工程的精致平台。
