A Phosphopantetheinyl Transferase from <i>Dictyobacter vulcani</i> sp. W12 Expands the Combinatorial Biosynthetic Toolkit

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 <i>holo</i> 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 <i>Dictyobacter vulcani</i> 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 <i>apo</i> to <i>holo</i> 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.