Discovery and production improvement of new pentamycin-producing bacteria

Abstract:

[Background] Pentamycin is a polyene macrolide with significant inhibitory activities against Candida albicans, Trichomonas vaginalis, and several other vaginal pathogens and has been registered in Switzerland for treating vaginal candidiasis, trichomoniasis, and mixed infections. [Objective] To explore new biosynthetic gene clusters and production strains and improve the yield of pentamycin by genetic engineering, providing new ideas and a theoretical basis for further increasing the yield and promoting the industrial production and application of pentamycin. [Methods] BLASTp was used to search for the homologous proteins of known enzymes involved in the biosynthesis of pentamycin from bacterial genomes, and new gene clusters for synthesizing pentamycin and its analogues were identified. Then, multi-locus sequence analysis (MLSA) was performed to compare the genomic similarity among the strains possessing the biosynthetic gene clusters. The constitutive promoter kasOp* was used to overexpress the positive transcriptional regulatory gene ptnF for increasing pentamycin production. [Results] A total of 27 new strains containing the biosynthetic gene clusters of pentamycin/filipin were discovered from the bacterial genome. The results from medium screening, fermentation, and metabolite analysis indicated that Streptomyces misionensis can synthesize multiple compounds such as pentamycin and filipin III. The genetic engineering increased the yield of pentamycin by 4.34 folds (reaching 101.7 mg/L) compared with that of the original strain. The total production of pentamycin and filipin analogues by the engineered strain reached 146.1 mg/L, which was 3.28 times higher than that of the original strain. [Conclusion] This study systematically analyzed the biosynthetic gene clusters of pentamycin in bacterial genomes and the diversity of the strains carrying these gene clusters. The experimental data confirmed that S.misionensis can produce natural products including pentamycin and filipin. In addition, the production of pentamycin was improved by genetic engineering. This study provides new elements, pathways, strains, and theoretical foundations for constructing elite strains for the industrial production of pentamycin.