The Study On The Regulation Of AurJ3M And AurT Gene On The Biosynthesis Of Aureofuscin

Aureofuscin is a secondary metabolite produced by Streptomyces aureofuscus,which belongs to polyene macrolide antibiotics and has antifungal activity,and looking forward to application in medical,health care and a wider range of areas such as food processing.The regulation mechanism of biosynthesis has not been studied clearly and its further application is restricted,so increasing the aureofuscin production has become more important.In the previous experiment,the aurJ3M gene was overexpressed in S.aureofuscus,and the fermentation product was detected by high performance liquid chromatography?HPLC?.The fermentation product of aureofuscin was greatly increased by 5-6 times.This study conducts in-depth research on the function and regulation of aurJ3M and aurT.?1?Bioinformatics analysis showed that AurJ3M?EU697915.1?,a regulator of the PAS-LuxR family,the knockout plasmid pC1203-11599 of aurJ3M gene was constructed.After the observation of the morphology of the fermentation product,the mutant strain??aurJ3M?could not synthesize the aureofuscin and it was confirmed that the aurJ3M gene positively regulates the biosynthesis of the aureofuscin;Further analysis by reverse transcription PCR showed that compared with the wild-type strain?SYAU0709?,the gene S0,S1 and B,C,G,F,D genes were not transcribed,the transcription levels of I,J,and A genes were significantly decreased,and the expression of R,E,and H genes were not affected.The absence of S0 and S1 genes expression inhibits the production of aureofuscin.The results showed that aurJ3M is a pathway-specific regulator of aureofuscin biosynthesis,which affects the synthesis of aureofuscin by regulating the transcriptional expression of structural genes in gene clusters.?2?The new gene aurT?Genbank accession number:MH247109.1?was obtained by molecular cloning technology,the bioinformatics analysis of AurT protein structure indicated that AurT belongs to the RhtB family regulatory factor,is an amino acid transporter of S.aureofuscus,and after the aurT gene is over-expressed,the yield of aureofuscin was increased by 1.3 times in the strain??aurT?,simultaneously,the aureofuscin production of the strain??aurT?lost 65%,but the production of aureofuscin was still not completely inhibited.This is different from the regulation of the pathway-specific regulator AurJ3M.?3?Using the wild-type strain S.aureofuscus SYAU0709 as a control,four different PI factor structural analogues?1,2-propanediol,glycerol,Triethanolamine,Ethylene glycol?were screened,the results showed that glycerol had the best synthesis effect on the regulation of aureofuscin biosynthesis,and the concentration of glycerol was 0.4 mL.When the time reached 96h,the yield of aureofuscin was the largest.In addition,after adding glycerol to?aurT strain,the fermentation product was detected and the production of aureofuscin was restored.The yield of wild type strain S.aureofuscus SYAU0709 was 693?g·mL^-1,the yield of?aurT strain was 243?g·mL^-1,and the yield of?aurT strain increased to 784?g·mL^-1 after exogenous addition of Glycerine,which was close to that of wild-type strain S.aureofuscus SYAU0709.It indicated that the PI-factor structural analog with a concentration of 0.4mL binds to the specific receptor in the gene cluster,initiates the expression of related genes,and participates in the biosynthesis regulation of aureofuscin.