A Cell-Free Tool for Rapid Screening of Antifungal Peptides

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 group
3,4. 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
activity
2,6
.
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 Syngenta
Project 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.