Biosynthesis Of The Polyene Antifungal Antibiotic Jinanmycin In Streptomyces NetropsisSD-07 And The Preliminary Study Of Gene Knockout

Streptomyces netropsis SD-07 was isolated in 2007 from Jinan,Shandong Porvince.The production of polyene macrolide antibiotics is a mixture of pentaene macrolides and heptaene macrolides,the highest content of pentaene macrolides,and pentaene macrolides have been analyzed for chemical structure,named as Jinanmycin.Compared to Amphotericin B and Nystatin,Jinanmycin have stronger antifungal activity,It has the potential to develop new antifungal antibiotics.Therefore,it is necessary to explore the biosynthetic pathway of Jinanmycin in SD-07 and the gene knockout of its synthesis-related PKS gene cluster.The results of the study are as follows:1.Scanning the complete genome sequence of Streptomyces netropsis SD-07,and analyzing the biosynthesis of Jinanmycin in SD-07.The genome of Streptomyces netropsis SD-07 has about 7593656bp,obtained 46 gene clusters through analysed by antismash,among them,cluster 32 is most likely to be a coding gene cluster of S.netropsis SD-07 polyene macrolide antibiotics,located at 4266507-4419580 bp,153074 bp in total,PKS-related genes located in 4286507-4388174 bp,101667bp in total,we speculate that it is the gene cluster for the Jinanmycin.Then the biosynthetic pathways of Jinanmycin in SD-07 were analyzed.The synthesis of macrolide rings beginning with JinA,after polymerization of JinB,JinC,JinD,JinE and JinF,Finally,the long chain of polyketone was hydrolyzed from PKS and cyclized into a macrolide ring by JinF-TE.Followed by post-modification,JinI oxidizes C16 methyl branch to carboxyl group,The fructose-6-phosphate was converted to GDP-actinomycin glucoside by the GDP-mannose dehydrating enzyme JinGIII,aminotransferase JinGII and some primary metabolizing enzymes,and then by JinGI GDP-actinomycin glycoside and amphotericin aglycone core combination.Finally,JinMI and JinMII ABC-type transporter responsible for the transport of Jinanmycin to the active strain of S.netropsis SD-07.This study laid the foundation for the gene knockout of SD-07.2.Two SD-07 gene knockout vectors were successfully constructed and attempted for gene knockout.The knockout vector pJTU1278-?5883 and pJTU1278-?2166 were successfully constructed.We try to make a series of experiments on the gene knockout of S.netropsis SD-07 by using the conjugation,electroporation and PEG-mediated transformation of protoplasts.And the experimental conditions were optimized,including the proportion of the recipient,the heat shock conditions and germination time of the spores,antibiotic action time and concentration,voltage and resistance,knockout the concentration of the carrier,the recovery time after the electric turn,medium selection,electrotransfer buffer,lysozyme concentration etc.But we did not get the correct clones eventually.For the above results,we believe that the replication plasmid is not suitable for S.netropsis SD-07.3.The cloning of PKS gene cluster in SD-07 was explored by Red/ET recombineering.The PKS gene cloning of polyene macrolide antibiotics was attempted by Red/ET reorganization.The PKS gene cluster about 110 kb was cloned into two parts by enzyme digestion,the smaller DNA fragment is about 40kb,recombined correctly and successfully screened.However,when attempting to recombine larger DNA fragment about 65kb,although it grows a lot of clones,but when we screen them one by one,we do not get the right clones;after analysis we think may be 65kb DNA is too larger to clone.So the 65 kb DNA was digested into about 20 kb and about 40 kb to clone,the 40 kb DNA also did not get the right clones.The reason is likely to be that the expression of certain genes of S.netropsis SD-07 is toxic to E.coli,so lead to the correct clones died.This situation happen sometime by querying the literature.Although gene knockout of SD-07 did not complete,but this study provides a series of guidance for future research.We will continue to overcome this problem that gene knockout of SD-07.