A Cell-Free Tool for Rapid Screening of Antifungal Peptides

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To date, only one antifungal RiPP has been identified (e.g., pinensin), which is a significant area of opportunity for the discovery of novel RiPPs. As an enabling technology, we will use cell-free transcription-translation (TX-TL) systems, developed by the Moore group3,4. TX-TL systems require DNA, a cell extract, and an energy solution to make recombinant peptides/proteins within minutes to hours, and up to a milligram scale. This method of production also overcomes key challenges and time scale problems with engineering RiPPs in cell-based systems, including known peptide solubility issues. Additionally, TX-TL reactions are microscale and compatible with automation5. TX-TL has been used to create new RiPPs with improved biological activity2,6.Overall, our project will address the following question: can we use TX-TL to discover novel antifungal RiPPs that inhibit plant pathogens? The specific aims and objectives (1-4) of this proposal are:Objective 1 - Produce a model antibacterial RiPP using TX-TL Objective 2 - Develop an automated RiPP screening platform at Syngenta Objective 3 - Characterise new antifungal RiPPs Objective 4 - Automate screening of antifungal RiPP Biosynthetic Gene Clusters at SyngentaProject impact: This project will (i.) provide a tool to make different RiPP variants for bioactivity screening; (ii.) characterise novel enzymes and secondary metabolites from RiPP biosynthesis; (iii.) develop an automated platform for discovery of antifungal RiPP natural products as a biological control measure for emerging crop pathogens.