Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase

Polyketides, one of the largest classes of natural products, are often clinically relevant. The ability to engineer polyketide biosynthesis to produce analogs is critically important. Acyltransferases (ATs) of modular polyketide synthases (PKSs) catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. ATs have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, limiting the scope of AT engineering. Here, molecular dynamics simulations are used to prioritize ~50 mutations within the active site of EryAT6 from erythromycin biosynthesis, leading to identification of two previously unexplored structural motifs. Exchanging both motifs with those from ATs with alternative extender specificities provides chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy are expected to advance our ability to engineer PKSs towards designer polyketides.

Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase

ABSTRACTAcyltransferases (ATs) of modular polyketide synthases catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. Subsequently, AT domains have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, critically limiting the efficiency and scope of AT engineering. Here, molecular dynamics simulations were used to prioritize ~50 mutations in the active site of EryAT6 from erythromycin biosynthesis. Following detailed in vitro studies, 13 mutations across 10 residues were identified to significantly impact extender unit selectivity, including nine residues that were previously unassociated with AT specificity. Unique insights gained from the MD studies and the novel EryAT6 mutations led to identification of two previously unexplored structural motifs within the AT active site. Remarkably, exchanging both motifs in EryAT6 with those from ATs with unusual extender specificities provided chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy is expected to advance our ability to engineer PKSs towards designer polyketides.

Phosphate regulator PhoP directly and indirectly controls transcription of the erythromycin biosynthesis genes in Saccharopolyspora erythraea

Background The choice of phosphate/nitrogen source and their concentrations have been shown to have great influences on antibiotic production. However, the underlying mechanisms responsible for this remain poorly understood. Results We show that nutrient-sensing regulator PhoP (phosphate regulator) binds to and upregulates most of genes (ery cluster genes) involved in erythromycin biosynthesis in Saccharopolyspora erythraea, resulting in increase of erythromycin yield. Furthermore, it was found that PhoP also directly interacted with the promoter region of bldD gene encoding an activator of erythromycin biosynthesis, and induced its transcription. Phosphate limitation and overexpression of phoP increased the transcript levels of ery genes to enhance the erythromycin production. The results are further supported by observation that an over-producing strain of S. erythraea expressed more PhoP than a wild-type strain. On the other hand, nitrogen signal exerts the regulatory effect on the erythromycin biosynthesis through GlnR negatively regulating the transcription of phoP gene. Conclusions These findings provide evidence that PhoP mediates the interplay between phosphate/nitrogen metabolism and secondary metabolism by integrating phosphate/nitrogen signals to modulate the erythromycin biosynthesis. Our study reveals a molecular mechanism underlying antibiotic production, and suggests new possibilities for designing metabolic engineering and fermentation optimization strategies for increasing antibiotics yield.