Discovery, Biosynthesis and Engineering of Side-Chain-Macrocyclic Plant Peptides

项目总结

PROJECT SUMMARY Plants have been an important source of medicinal natural products, as ~25% of FDA-approved drugs are inspired by plant chemicals. However, discovery and development of new pharmaceuticals based on plant natural products is challenging due to three main bottlenecks: (1) rediscovery of known compounds from complex plant extracts during bioactivity-guided discovery approaches, (2) difficult scaled production of chemically complex lead structures by organic synthesis or source plant extraction, and (3) limited diversification of lead structures due to molecular complexity. Recent advances in plant omics-technologies and plant synthetic biology offer solutions to these general bottlenecks of natural product drug discovery by a gene-guided discovery approach. Herein, prioritization of new plant metabolites can be enabled by mass spectrometry (MS)-based metabolomics of plant extracts and prediction of biosynthetic genes from plant genomes. Heterologous expression of biosynthetic genes allows source-independent production of target plant natural products in microbial and eukaryotic host organisms. Genetic modification of biosynthetic pathways enables the formation of target natural product analogs. Cyclic plant peptides are a class of plant natural products with potential applications in antiviral and anticancer therapy due to oral bioavailability, stability and diversifiability. Our lab recently characterized a new plant-specific peptide cyclase called the BURP domain, which is an autocatalytic copper-dependent enzyme family involved in the biosynthesis of ribsomally encoded and posttranslationally modified peptides (RiPPs) with side-chain-macrocyclizations via tyrosines and tryptophans. Our objective is to overcome the aforementioned bottlenecks of plant-based drug discovery by developing a workflow for the systematic discovery of cyclic peptides from plants, characterizing BURP domain peptide cyclases for scaled in vivo or in vitro production of cyclic plant peptides and establishing a biocatalytic platform for the diversification of cyclic plant peptides via BURP domain cyclases for therapeutic evaluation. Herein, systematic identification of new side-chain-macrocyclic RiPP classes will be accomplished by connecting tandem MS spectra of candidate peptide analytes to BURP domain genes in plant genomes and transcriptomes. We will investigate the structure and function of known autocatalytic classes of BURP domain peptide cyclases, characterize BURP domain peptide cyclases, which act on separate precursor peptide substrates and identify enzymes involved in N- and C-terminal protection of BURP-domain-derived RiPPs. Finally, libraries of antiviral cyclopeptide alkaloids from Chinese date tree (Ziziphus jujuba) and anticancer celogentin peptides from Celosia argentea will be generated by design and optimization of biocatalytic routes and evaluated in in vivo cell-based assays. As plant genomic resources are rapidly growing, the proposed research will set the stage for using plant genetic information for the discovery and development of medicinal plant natural products.