Genome-Guided Discovery of an Antimicrobial Peptide Scaffold from an Undescribed Antarctic Rhodococcus Lineage: A Proof-of-Concept Study.

Rhodococcus species display remarkable metabolic versatility and adaptation to extreme environments, yet their antimicrobial potential remains largely unexplored, particularly in strains representing undescribed genomic lineages. This study investigated whether the genome of Rhodococcus sp. GB-02, a strain isolated from Antarctic soil, encodes antimicrobial peptides with demonstrable activity against clinically relevant pathogens. Hybrid genome assembly revealed a 6.58 Mb high-quality draft genome belonging to a previously undescribed genomic lineage within the genus Rhodococcus, based on average nucleotide identity and digital DNA-DNA hybridization analyses. Genome mining identified 20 biosynthetic gene clusters, predominantly non-ribosomal peptide synthetases, several of which showed low or no similarity to known pathways. Eight putative antimicrobial peptides (AMPs) were computationally predicted; among these, three lacked detectable homology to sequences in current databases, and experimental validation of one predicted peptide, designated Peptide 5764, demonstrated antibacterial activity at high micromolar concentrations against Gram-positive and Gram-negative pathogens and concentration-dependent inhibition of biofilm formation, particularly in Pseudomonas aeruginosa. However, Peptide 5764 exhibited considerable hemolytic activity at antibacterial concentrations, indicating that optimization for membrane selectivity will be required before therapeutic application. These findings establish Rhodococcus sp. GB-02 as an underexplored Antarctic lineage with a diverse biosynthetic repertoire and provide proof-of-concept that genome-guided AMP prediction can identify experimentally active peptide scaffolds from this lineage.