Exploration Of Secondary Metabolism In Marine Streptomyces Sp. FJNU027

Streptomyces have the power to produce a wide variety of biologically active substances with novel structures,and is recognized as an important source of drug lead compounds.Genome sequencing data showed that Streptomyces haves a large number of secondary metabolite biosynthetic gene clusters for secondary metabolites.However,under laboratory conditions,the number of secondary metabolites isolated from Streptomyces is far less than the number of its biosynthetic gene clusters,suggesting that a lot of secondary metabolite biosynthetic gene clusters were remained silent or weakly expressed,as a result,activating these cryptic gene clusters is an important method to develop active natural products of Streptomyces.In this paper,we investigated the biosynthesis of tirandamycin in Streptomyces sp.FJNU027,and adopt the OSMAC strategy,including changing the culture conditions,adding chemical inducers or co-cultivating with other microorganisms to activate cryptic biosynthetic gene clusters.Moreover,their involved functions and metabolic pathways were analyzed with the aid of transcriptome sequencing data.In this paper,the genetic manipulation system of Streptomyces sp.FJNU027 was established.The results showed that the optimal condition for sporulation was the strain grew as a single colony on 0.2×YPD medium for 5～7 days;The optimal spore concentration was 10⁹/m L;Modified Gauze’s No.1 medium and ISP-4 medium are the optimal medium for conjugation and transformation.The orf6171 and orf6176 mutants of the NRPS/PKS hybrid gene（cluster 17）were constructed by the single-crossover homologous recombination.The tirandamycin in the mutants were not synthesized,which indicating that the synthesis of tirandamycin A and B was controlled by the orf6171 and orf6176,and cluster 17 is the biosynthetic gene cluster of tirandamycin.The structure of compound WLF08 is similar to that of tirandamycin,according to the analysis of the synthesis of tirandamycin compounds at different cultural times,compound WLF08 was speculated to be a degradation product of tirandamycin.Streptomyces sp.FJNU027 was fermented in modified Gauze’s No.1,oligotrophic,nutrient-rich and rice medium.The results showed that the strain produced differential secondary metabolites on oligotrophic medium.and then the differential product4,4’,5,5’-tetramethyl-[1,1’-biphenyl]-2,2’-diol was purified and identified.Transcriptome sequencing data showed that differentially expressed genes under oligotrophic conditions were mainly enriched in growth,metabolic process,biological regulation,signal transduction and response to external stimuli.Streptomyces sp.FJNU027 was fermented in modified Gauze’s No.1 supplemented with chemical inducers:100μM Ni Cl₂,25 m M N-acetylglucosamine,5.7μM Etoposide,50 m Mγ-butyrolactone,22.5μM Lincomycin and 28 m M Dimethyl sulfoxide.HPLC analysis of the secondary metabolites showed that Ni Cl₂increased the production of the tirandamycin A by 1.6 times,and this result was also verified by transcriptome sequencing data,Ni Cl₂could up-regulate the expression of genes orf6171 and orf6176which was the key genes of tirandamycin biosynthesis by 3 times and 5.5 times;In addition,the transcription levels of genes encoding fatty acid synthesis-related enzymes ACP-transacylase（Fab D）and enoyl-ACP reductase（Fab I）were decreased by 91.2%and94.6%under the Ni Cl₂condition.However,Glc NAc inhibited the production of the tirandamycin,and it could down-regulate the expression of genes orf6171 and orf6176 by80.1%and 83%,while the expression of orf6164 which was the negatively regulation gene of tirandamycin biosynthesis was up-regulated by 3.5 times.The research in this paper also showed that Streptomyces sp.FJNU027 had the ability to degrade the epigenetic modifier SAHA.Streptomyces sp.FJNU027 were cultivated in the modified Gauze’s No.1 medium supplemented with 75μM SAHA,which induced differential products N-phenylacetamide and 8-oxo-8-（phenylamino）octanoic acid appeared,both of which were degradation products of SAHA.In addition,the degradation process of lignin by Streptomyces FJNU027 was also explored.Using 2%lignin as the sole carbon source,it was found that the degradation of lignin by the strain reached 32.34%on the twelfth day,in particular,one of the components of lignin was degraded by more than 90%.In this paper,the research showed that pleiotropic methods such as changing the medium and adding chemical inducers could effectively increase the diversity of secondary metabolites of Streptomyces sp.FJNU027,and affect the synthesis and regulation of its secondary metabolites on the transcriptional level.The degradation of SAHA and lignin was also explored,which provided a new method for the biodegradation of aromatic compounds.