Lunaemycins, New Cyclic Hexapeptide Antibiotics from the Cave Moonmilk-Dweller Streptomyces lunaelactis MM109T

Streptomyces lunaelactis strains have been isolated from moonmilk deposits, which are calcium carbonate speleothems used for centuries in traditional medicine for their antimicrobial properties. Genome mining revealed that these strains are a remarkable example of a Streptomyces species with huge heterogeneity regarding their content in biosynthetic gene clusters (BGCs) for specialized metabolite production. BGC 28a is one of the cryptic BGCs that is only carried by a subgroup of S. lunaelactis strains for which in silico analysis predicted the production of nonribosomal peptide antibiotics containing the non-proteogenic amino acid piperazic acid (Piz). Comparative metabolomics of culture extracts of S. lunaelactis strains either holding or not holding BGC 28a combined with MS/MS-guided peptidogenomics and H-1/C-13 NMR allowed us to identify the cyclic hexapeptide with the amino acid sequence (D-Phe)-(L-HO-Ile)-(D-Piz)-(L-Piz)-(D-Piz)-(L-Piz), called lunaemycin A, as the main compound synthesized by BGC 28a. Molecular networking further identified 18 additional lunaemycins, with 14 of them having their structure elucidated by HRMS/MS. Antimicrobial assays demonstrated a significant bactericidal activity of lunaemycins against Gram-positive bacteria, including multi-drug resistant clinical isolates. Our work demonstrates how an accurate in silico analysis of a cryptic BGC can highly facilitate the identification, the structural elucidation, and the bioactivity of its associated specialized metabolites.

Automated summary

Streptomyces lunaelactis strains isolated from moonmilk deposits exhibit significant heterogeneity in the content of biosynthetic gene clusters (BGCs) for specialized metabolite production. Specifically, the cryptic BGC 28a is carried only by a subgroup of S. lunaelactis strains and is predicted to produce nonribosomal peptide antibiotics containing piperazic acid (Piz). Through metabolomics and peptidogenomics analysis, the cyclic hexapeptide lunaemycin A, with a unique amino acid sequence, was identified as the main compound synthesized by BGC 28a. Lunaemycins demonstrated potent bactericidal activity against Gram-positive bacteria, including multi-drug resistant clinical isolates. This study highlights the importance of accurate in silico analysis in elucidating the structure and bioactivity of cryptic BGCs.