Discovery Of Cyclic Lipopeptide Marihysin A And Study On Its Biosynthetic Gene Cluster In Streptomyces Thermolilacinus SPC6

Abuse of antibiotics leads to the emergence of drug resistance to the bacterial,which threaten human health seriously,therefore,it is extremely urgent to find safe and effective antibiotics.The complex and varied natural products are always an important source of drug development,but it is difficult to find new natural products from microorganisms separated from the traditional environment,and the extremophiles have special physiological and biochemical properties because they adapt to special environment,and finding novel and well-active natural products from their secondary metabolites becomes an effective way for drug development.In this paper,a salt-tolerant Streptomyces thermolilacinus SPC6 isolated from the Badain Jaran Desert was selected,and suitable spore medium and fermentation medium were screened at the beginning,and after activity of the fermentation product was tested preliminarily,a large amount of fermentation was carried out,and the active compounds were obtained by separation and purification,and analyzed by HPLC,LC-MS and NMR,and a compound was identified as cyclic lipopeptide Marihysin A.Bioactivity test has shown good antifungal activity and so far there have been no reports of biosynthetic gene clusters associated with this compound.Most of the cyclic lipopeptide compounds belong to non-ribosomal polypeptides which are mainly catalyzed by non-ribosomal polypeptide synthetase(NRPS).Bioinformatics analysis S.thermolilacinus SPC6 genome shows that it can produce a wealth of secondary metabolites,including ribosomal peptides,non-ribosomal peptides,polyketones,lanthionine and other natural products with great medicinal potential.Based on the characteristics of cyclic lipopeptide compounds and bioinformatics analysis results,in this study,three gene clusters(two NRPS and one heterozygous Cf_fatty_acid-T1pks-Ladderane-Nrps)were selected which may be responsible for the synthesis of Marihysin A and the gene disrupted were carried out respectively.The recombinant plasmids C9-pOJ260,C14-pOJ260 and C28-pKC1139 were successfully constructed,and three gene disrupted mutant strains of C9-pOJ260-SPC6,C14-pOJ260-SPC6 and C28-pKC1139-SPC6 were finally obtained.Fermentation mutant strains and wild type showed that the mutants still produced Marihysin A by HPLC analysis,which showed that none of these candidate gene clusters were involved in the biosynthesis of Marihysin A,and the biosynthetic gene cluster of the compound needs further study.In this paper,the fermentation products of Streptomyces SPC6 were isolated and purified,and a cyclic lipopeptide compound marihysin A was successfully found and identified,and the activity test showed that it had good antifungal activity,and its biosynthetic gene cluster was studied by genomic sequence analysis combined with gene disruption.None of the three candidate NRPS gene clusters are responsible for biosynthesis of Marihysin A,the discovery of Marihysin A and the study of its biosynthetic gene clusters for the further combination of Marihysin A with more stable biological structures,providing precursors for drug screening.