Hsu Kenneth K, Ferguson Charlie M, McBride Christina M, Mostaghim Nicholas B, Mabry Kelsey N, Fairman Robert, Cho Yae In, Charkoudian Louise K
Department of Chemistry, Haverford College, Haverford, Pennsylvania 19041, United States.
Department of Biology, Haverford College, Haverford, Pennsylvania 19041, United States.
ACS Omega. 2025 Aug 12;10(33):37276-37283. doi: 10.1021/acsomega.5c02708. eCollection 2025 Aug 26.
The value of microbial natural product pathways extends beyond the chemicals they produce, as the enzymes they encode can be harnessed as biocatalysts. Microbial type II polyketide synthases (PKSs) are particularly noteworthy, as these enzyme assemblies produce complex polyaromatic pharmacophores. Combinatorial biosynthesis with type II PKSs has been described as a promising route for accessing never-before-seen bioactive molecules, but this potential is stymied in part by the lack of functionally compatible noncognate proteins across type II PKS systems. Acyl carrier proteins (ACPs) are central to this challenge, as they shuttle reactive intermediates and malonyl building blocks between the other type II PKS domains during biosynthesis. To perform this essential role within PKSs, ACPs must first be activated to their state via the phosphopantetheinyl transferase (PPTase)-catalyzed installation of a coenzyme A (CoA)-derived phosphopantetheine (Ppant) arm. The installation of the Ppant arm is critical to effectively study and strategically engineer type II PKSs, yet not all ACPs can be activated using conventional PPTases. Here, we report the discovery of a previously unexplored nonactinobacterial PPTase from sp. W12 (vulcPPT). We explored its compatibility with both native and non-native ACPs, observing that vulcPPT activated all ACPs tested in this study, including a noncognate, nonactinobacterial ACP that was not readily activated by the prototypical broad substrate PPTases AcpS and Sfp. Strategic optimization of phosphopantetheinylation reaction conditions increased the to conversion catalyzed by vulcPPT. In addition to identifying a promising new PPTase that is easy to prepare and use, this work establishes a roadmap for further investigation of PPTase compatibility and increases access to functional synthase components for use in combinatorial biosynthesis.
微生物天然产物途径的价值不仅在于它们所产生的化学物质，还在于它们所编码的酶可作为生物催化剂加以利用。微生物II型聚酮合酶（PKSs）尤其值得关注，因为这些酶组装体可产生复杂的多芳基药效基团。II型PKSs的组合生物合成已被描述为获取前所未见的生物活性分子的一条有前景的途径，但这一潜力在一定程度上受到II型PKS系统中缺乏功能兼容的非同源蛋白的阻碍。酰基载体蛋白（ACPs）是这一挑战的核心，因为它们在生物合成过程中在其他II型PKS结构域之间穿梭反应性中间体和丙二酰构建模块。为了在PKSs中发挥这一关键作用，ACPs必须首先通过磷酸泛酰巯基乙胺基转移酶（PPTase）催化安装辅酶A（CoA）衍生的磷酸泛酰巯基乙胺（Ppant）臂而被激活至其 状态。Ppant臂的安装对于有效研究和策略性改造II型PKSs至关重要，但并非所有ACPs都能用传统的PPTases激活。在此，我们报告了从 sp. W12中发现的一种以前未被探索的非放线菌PPTase（vulcPPT）。我们研究了它与天然和非天然ACPs的兼容性，观察到vulcPPT激活了本研究中测试的所有ACPs，包括一种非同源的非放线菌ACPs，它不容易被典型的宽底物PPTases AcpS和Sfp激活。磷酸泛酰巯基乙胺化反应条件的策略性优化提高了vulcPPT催化的 至 转化率。除了鉴定出一种易于制备和使用的有前景的新PPTase外，这项工作还为进一步研究PPTase兼容性建立了路线图，并增加了用于组合生物合成的功能性合酶组件的可及性。
