A large family of cytotoxic cyclic peptides exemplified by the patellamides has been isolated from ascidians harboring the obligate cyanobacterial symbionts Prochloron spp.1,2,3,4,5. Genome sequence analysis of these symbionts has revealed that Prochloron spp. synthesize patellamides by a ribosomal pathway6. To understand how this pathway evolved to produce a suite of related metabolites, we analyzed 46 prochloron-containing ascidians from the tropical Pacific Ocean for the presence of patellamide biosynthetic genes and taxonomic markers. Here, we show that Prochloron spp. generate a diverse library of patellamides using small, hypervariable cassettes within a conserved genetic background. Each symbiont strain contains a single pathway, and mixtures of symbionts within ascidians lead to the accumulation of chemical libraries. We used this information to engineer the production of a new cyclic peptide in Escherichia coli, thereby demonstrating the power of comparative analysis of closely related symbiotic pathways to direct the genetic synthesis of new molecules.
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This work was supported by grants from the US National Science Foundation (EF-0412226) and the US National Institutes of Health (R01 GM071425-01A1), and by a Willard L. Eccles Fellowship to B.J.H. We thank D.J. Faulkner (University of California San Diego), C. Ireland (University of Utah), L. Matainaho (University of Papua New Guinea) and the governments of the Republic of Palau and Papua New Guinea for the opportunities to collect the samples used in these studies. We also thank K. Rai and D. Jones (University of Utah) and L. Jiang (TIGR) for their help in the quantitative PCR experiments. T. Bugni (University of Utah) helped with mass measurements, and J. Sims (University of Utah) aided with sample processing.
The authors declare no competing financial interests.
Complete list of patE variants (patE1–patE29) identified by sequencing clone libraries. (PDF 167 kb)
Amino acid sequences of the compounds coded by the variants in Supplementary Figure 1. (PDF 151 kb)
An example of the SNPs observed in the raw Sequencher file. (PDF 185 kb)
Cladogram of prochloron strains derived from maximum likelihood analysis. (PDF 107 kb)
Data from the best-characterized 11 samples from this study. (PDF 88 kb)
Data from other samples examined in this study. (PDF 83 kb)
Detailed methods, NMR and mass spectra, and tables of primers used in the study. (PDF 1370 kb)
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Donia, M., Hathaway, B., Sudek, S. et al. Natural combinatorial peptide libraries in cyanobacterial symbionts of marine ascidians. Nat Chem Biol 2, 729–735 (2006). https://doi.org/10.1038/nchembio829
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