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Open Access Publications from the University of California

Biosynthesis and Engineering of Cyclomarin and Cyclomarazine: Prenylated, Non-Ribosomal Cyclic Peptides of Marine Actinobacterial Origin


Cyclomarins are potent anti-inflammatory cyclic peptides originally encountered in the estuarine streptomycete, strain CNB-982, and subsequently isolated from the marine obligate actinobacterium Salinispora arenicola CNS-205 along with the truncated antibacterial diketopiperazine cyclomarazines. The cyclomarins are composed of seven amino acid residues, two of which are of unique biosynthetic interest, 2-amino-3,5-dimethylhex-4-enoic acid (ADH), which has never been reported as a constituent of any peptide, and N-(1,1-dimethyl-1-allyl)-tryptophan, which is a rare example of the prenylation of a bacterial nonribosomalpeptide. The N-prenylated tryptophan residue, along with δ-hydroxyleucine, is present in both the cyclomarins and cyclomarazines, suggesting a common biosynthetic origin.

Because of the unique structural features of ADH and N-(1,1-dimethyl-1-allyl)-tryptophan, along with the extensive tailoring of the remaining residues of cyclomarin, we initiated the interrogation of the biosynthetic machinery behind cyclomarin and cyclomarazine synthesis. We were able to determine the basic steps of cyclomarin and cyclomarazine production, along with insights into how the two unique amino acids are synthesized. Furthermore, we were able to identify the oxidative enzymes involved in tailoring both peptides. Analysis of the genome sequence of S. arenicola CNS-205 revealed the cym biosynthetic gene cluster, which is dominated by cymA, encoding for a seven module nonribosomal peptide synthetase. Downstream of cymA are the genes for N-(1,1- dimethyl-1-allyl)- tryptophan synthesis via the prenyltransferase CymD, and ADH via cymE-H. CymEF is an aldolase/dehydrogenase pair utilized for the key C-C bond forming step of ADH synthesis, along with a methyltransferase CymG, and a dehydrogenase CymH. Upstream of cymA is the Fe2+/α-ketoglutarate dependent leucine δ-oxygenase CymW, and three cytochrome P450s oxidases, CymO, CymS, and CymV. Bioinformatic analysis coupled with in vivo mutagenesis confirmed the role of Cym W along with CymS as a N-(1,1-dimethyl-1-allyl)- tryptophan β-hydroxylase, and CymV as a N-(1,1-dimethyl-2,3- epoxypropyl)-tryptophan epoxidase, and suggested the function of CymO as a phenylalanine β-hydroxylase . In addition to establishing the function of the xxiv aforementioned enzymes, we were able to generate the novel N-(1-methyl)-tryptophan and N-(1-propargyl)-tryptophan analogs of both sized peptides in the cymD deficient mutant, confirming the preference for N-alkylated tryptophan residues and the amenability of cyclomarin and cyclomarazine to derivatization via mutasynthesis.

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