Bacterial natural products (secondary metabolites) act as chemical communication signals in nature, serve as lead compounds for pharmaceuticals and agrochemicals, and inspire organic syntheses. Reactions leading to these molecules are catalyzed by specialized enzymes encoded by bacterial genomes. Characterization of these biosynthetic pathways both illuminates the chemical capabilities of bacteria and enables implementation of these capabilities to expand chemical diversity. The 2,5-diketopiperazine (DKP) family of natural products, formed by linkage of two amino acids via two amide bonds, is structurally diverse and commonly bioactive. Cyclo(Trp-Trp) DKP scaffolds are tailored with an array of functionalities to offer myriad biological activities, rendering the cyclo(Trp-Trp) scaffold important for biosynthetic understanding and manipulation. Applying the nocardioazines as model cyclo(Trp-Trp) DKP derivatives, we aimed to establish the biosynthetic pathway yielding the cyclo(Trp-Trp) core and to develop a biosynthetic platform for expanding the chemical diversity of DKPs. Bioinformatics analyses of the nocardioazine-producing Nocardiopsis sp. bacterial genome revealed two cyclodipeptide synthase (CDPS) homologues as candidate catalysts of cyclo(Trp-Trp) assembly. Metabolite profiling of E. coli expressing these genes and in vitro assays with purified recombinant enzymes revealed that both CDPSs catalyze cyclo(Trp-Trp) biogenesis from tryptophanyl-tRNA. To evaluate these two CDPSs as tools for expanding DKP biosynthetic diversity, E. coli expressing these genes were fed with Trp analogs that included selected constitutional isomers of fluoro-, chloro-, and methyl-functionalized Trp. Evaluation of resulting metabolite profiles by LC/MS2 revealed incorporation of each of these precursors into cyclo(Trp-Trp) analogs. To date, our novel in vivo DKP biosynthetic platform has yielded over 30 different cyclo(Trp-Trp) analogs. These data establish CDPSs as tools for expanding DKP biosynthetic diversity beyond the standard 20 amino acids, and open doors for the biosynthetic generation of analogs of medicinally promising DKPs such as the nocardioazines.

Unleashing bacterial biosynthetic pathways to expand diketopiperazine chemical diversity

255th National Meeting (2018)

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