A Cell-Free Tool for Rapid Screening of Antifungal Peptides

到目前为止，只发现了一种抗真菌RIPP(如Pinensin)，这是发现新型RIPP的一个重要机会。作为一项使能技术，我们将使用由Moore Group 3，4开发的无细胞转录-翻译(TX-TL)系统。TX-TL系统需要DNA、细胞提取物和能量溶液来在几分钟到几小时内制造重组多肽/蛋白质，最高可达毫克规模。这种生产方法还克服了细胞系统中工程RIPP的关键挑战和时间尺度问题，包括已知的多肽溶解问题。此外，TX-TL反应是微规模的，并与自动化兼容5。TX-TL已经被用来创造具有更高生物活性的新的RIPP 2，6.总的来说，我们的项目将解决以下问题：我们是否可以使用TX-TL来发现新的抑制植物病原体的抗真菌RIPP？本提案的具体目的和目标(1-4)是：目标1-使用TX-TL生产抗菌RIPP模型目标2-在先正达开发RIPP自动筛选平台目标3-表征新的抗真菌RIPPS目标4-在先正达项目影响：本项目将(I)自动筛选抗真菌RIPP生物合成基因簇提供用于生物活性筛选的不同RIPP变体的工具；(Ii)鉴定RIPP生物合成的新型酶和次生代谢物；(Iii)开发一个发现抗真菌RIPP天然产物的自动化平台，作为对新出现的作物病原体的生物防治措施。

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.