Study On The Secondary Metabolites From Two Strains Of Streptomvces And The Biosynthesis Of Fervenulin

In this paper,secondary metabolites of jys28 and ATCC53615 were isolated and identified.The biosynthesis of fervenulin in ATCC53615 was described through molecular genetic manipulation such as genome sequencing,gene library construction and so on.Firstly,two strains of Streptomyces jys28 and ATCC53615 were selected from the library of strains in our laboratory though fermentation by HPLC analysis and antibacterial activity screening.After fermentation,extraction,separation and identification,8 compounds were obtained from jys28,in which 6 were new compounds including 2 carbazole structure compounds,1 rubromycin structure compound and 3 peptide structure compounds.Four known compounds were isolated and identified from ATCC53615,including fervenulin,an analogue of toxoflavin.Fervenulin,toxoflavin and reumycin are the pathogenic factors of rice yellow wilt.The biosynthesis of toxoflavin has been reported in B.ghumae BGR1,but the biosynthesis in Streptomyces has not been reported.The formation of N-N bond is still unclear,and the methyltransferase of fervenulin has not been reported.Subsequently,the biosynthesis of fervenulin in Streptomyces was studied using ATCC53615 as the original strain.Due to the difficulty in gene knockout of the original strain ATCC53615,the method of heterogenous expression was selected to study the biosynthesis of fervenulin.Through gene sequencing and homology comparison,possible gene cluster for biosynthesis of fervenulin was found in ATCC53615 genome.After the construction and screening of the gene library,plasmids containing possible gene cluster were found and heterogeneously expressed in S.lividans M1152.Toxoflavin was isolated from S.lividans M1152/p5F11 and plasmid p5F 11 was chosed for gene knockout.Fervenulin was detected in the fermentation broth of the mutant strain in which gene Shi4237 was knocked out.Therefore,p5F11 was confirmed to contain gene cluster of fervenulin and toxoflavin.Secondly,the macroporous resin phase of the fermentation broth of the mutant strains were analyzed by HPLC.D-Ribitol.l-deoxy-l-[1,3,4,7-tetrahydro-6-(1H-indol-3-yl)-2,4,7-trioxo-8(2H)-pteridinyl](compound 22)was isolated from M1152/p5F11 and compound 23 and 24 were isolated from M1152/p5F11?Shi4234.Compound 23 was derivative of unoxidized amino metabolites of compound 22.Compound 24 was derivative of unopened ribose and unoxidized amino metabolites of compound 22.Shi4234 is the isozyme of RibD,which is responsible for the deamination and redox bifunctional enzymes.The function of Shi4234 was verified in terms of metabolites first time.Finally,by the analysis of the genes belong and inside of the gene cluster,four methyltransferases Shi4222,Shi4227,Shi4232 and Shi4237 were found.The function of those methyltransferases was verfied in vitro.Shi4232 and Shi4237 respectively methylated the N6 and N1 of Pyrimido[5,4-e]-as-Triazine-5,7(6H,8H)-dione(compound 15)to generate toxoflavin without order.Shi4222 or Shi4227 can catalyze compound 15 to generate 8-methylpyrimido[5,4-e]-as-triazin-5,7(6H,8H)-dione(compound 20)and fervenulin.Shi4232 catalyzed compound 15 to generate reumycin,and it can also catalyze the formation of fervenulin from compound 20.This is the first report for methylation network of fervenulin.In conclusion,this paper has completed the separation and identification of secondary metabolites of jys28 and ATCC53615.The biosynthesis of fervenulin in ATCC53615 strain was studied by heterologous expression method.By heterogenous expression and gene knockout,the biosynthesis of fervenulin and its analogues was basically consistent in ATCC53615 as same as in B.glumae BGR1.The function of Shi4234 was firstly verified by intermediate metabolites.The methylation network of fervenulin biosynthesis was reported for the first time through protein expression,enzyme assay and gene knockout.This paper also proved that the methylation process of the toxoflavin biosynthetic pathway in ATCC53615 is different from that in B.glumae BGR1,which provided a research example for the synthesis of the same compound by different pathways in the biosynthetic process of natural products.