Periodic Reporting for period 1 - PrediKSion (PrediKSion: An evolutionary guided and experimentally validated computational pipeline to unravel new polyketide synthase functionality)
Periodo di rendicontazione: 2022-01-01 al 2023-12-31
Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. These natural products have found widespread use as, e.g. antibiotics, anticancer therapeutics and antifungals. To combat rising antimicrobial resistance and produce these complex chemicals in more sustainable ways, engineering of the PKS architecture to diversify polyketide structures has been a longstanding goal. However, notwithstanding successes made with textbook, cis-acyltransferase (cis-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans-AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this project, we analyzed amino acid coevolution to identify a common module site that yields functional engineered trans-AT PKSs. We have used this site to insert and delete diverse modules and create 22 engineered trans-AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides. As such, this project has achieved an important step towards sustainable production of complex designer polyketides with diverse potential applications in human health and agriculture.
Work towards discovering the engineering guidelines of trans-AT PKSs consisted of 3 WPs. WP consisted of developing a structure-independent bioinformatic analysis pipeline to analyze amino acid coevolution in trans-AT PKSs. This WP yielded a software tool that performs statistical coupling analysis on PKS sequences. Using this tool, we identified an amino acid motif downstream of the ketosynthase domain that appears to be a conserved boundary between groups of coevolving amino acids. In WP2, we employed this amino acid motif as a fusion site to engineer trans-AT PKSs. Using the oocydin biosynthetic gene cluster (BGC) in Serratia plymuthica 4Rx13 as a model system, we introduced 15 foreign PKS modules from different organisms into a truncated oocydin BGC. These engineering efforts resulted in the production of 4 new designer polyketides. Finally, in WP3, we show the generality of this engineering approach by employing the amino acid motif as engineering site to produce more than 11 engineered lacunalides by truncating the lacunalide BGC in Gynuella sunshinyii. As such, this project has discovered a widely applicable engineering strategy to produce designer polyketides. We are currently in the process of disseminating these results and anticipate to share our findings with a wide scientific audience. By achieving the long-standing goal of engineering PKSs, this project has enabled a new line of research that aims to unravel the wider scope of this engineering strategy. We expect many new exciting findings along these lines in the near future.
Engineering PKSs for the production of designer polyketides has been a long-standing goal since the discovery of the modular architecture of PKSs in the early 90s. Many attempts at engineering these enzymes were structure-based, but suffered from the large size and little available structural data of these enzymes. In this project, we show that a sequence-based approach discovers an evolutionary conserved amino acid motif that serves as successful site for enzyme engineering. This structure-independent approach goes beyond the current state of the art in PKS engineering. Moreover, this engineering strategy enabled the biosynthesis of highly complex, potentially bioactive polyketides, providing a considerable contrast to the small model compounds produced by engineered cis-AT PKSs. As such, our engineering strategy provides an experimentally shown viable method to diversify polyketide natural products with pharmaceutically relevant structures. This ability can potentially greatly expedite the investigation of structure-activity relationships of novel antibiotic or anticancer compounds. By facilitating the more efficient discovery and diversification of such pharmaceuticals, the results presented in this project can provide society with better, cheaper and more diverse and sustainable pharmaceuticals.