Supplementary file 2_A novel Streptomyces species producing thiolutin with anti-MRSA activity and insights into its biosynthetic gene cluster.xlsx

Exploring microbial diversity from extreme environments, combined with targeted strategies, is a promising approach to uncover novel antimicrobial agents. In order to discover compounds with antimicrobial activity, the sulfur-containing antibiotic thiolutin was isolated and identified from strain HMX112T using a targeted antibacterial screening strategy. Strain HMX112T was isolated from a soil sample collected from the Turpan-Hami Basin in China and was characterized as an actinobacterium. 16S rRNA gene sequence analysis revealed that strain HMX112T showed the highest similarity to Streptomyces lateritius CGMCC 4.1427T, Streptomyces narbonensis CGMCC 4.1737T, and Streptomyces purpureus DSM 43362T, with sequence similarities of 98.83, 98.83, and 98.76%, respectively. Whole-genome analysis indicated that the average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain HMX112T and its closest related type strains were well below the thresholds for novel species delineation (95 and 70%, respectively). Based on polyphasic taxonomic analysis, strain HMX112T was confirmed to represent a novel species of the genus Streptomyces, for which the name Streptomyces flavimicrosus sp. nov. was proposed. Based on its observed antibacterial activity against Gram-positive bacteria, the bioactive components of strain HMX112T were investigated, resulting in the isolation and identification of the secondary metabolite thiolutin. Antimicrobial assays demonstrated that thiolutin possesses broad-spectrum inhibitory activity, showing MIC values of 16 μg/mL against two MRSA strains and inhibition rates exceeding 70% against nine plant pathogenic fungi at a concentration of 50 μg/mL. Furthermore, the biosynthetic pathway of thiolutin was elucidated through genomic analysis.