Functional Analysis Of Gene Cluster P450 Related To Biosynthesis Of Antibiotics In Micromonospora Carbonacea JXNU-1

Gene knockout is a commonly used method to verify gene function.It verify the function of the studied gene by compare the phenotype difference between gene deletion strains and wild-type strains.M.carbonacea JXNU-1 is a rare actinomycetes isolated from YaoHu.It can produced a broad-spectrum nucleoside antibiotic,JX.In previous,we preliminary determination cluster P450 is associated with antibiotic JX biosynthesis.In this experiment,we further exploration the correlations between cluster P450 and antibiotic JX biosynthesis.The results are as following:1.The temperature sensitive plasmid pKC1139 was used as the template.Build gene knockout vector pFD306 by imported the upstream and downstream segments of the genes of the microspore JXNU-1 gene were imported as the homologous exchange arm and the chloramphenicol resistance marker gene cat in plasmid pIJ790.The pFD306 was verify by gene sequencing,Imported receptor bacteria M.carbonacea JXNU-1 by electroporation,Constructing gene deletion strains M.carbonacea JXNU-?P450.PCR product verification and sequencing verification shows the gene knocked out successfully.2.The plasmid pKC1139 was used as the template to build gene knockout vector pFD307 by imported the upstream and downstream segments of the genes of the microspore JXNU-1 gene were imported as the homologous exchange arm and desired gene.The pFD307 was verify by gene sequencing,imported receptor bacteria M.carbonacea JXNU-?P450 by electroporation.Constructing reverse mutation strains JXNU-re.PCR product verification and sequencing verification shows the reverse mutation successfully.3.We through morphological observation,growth curve drawing,and the Oxford cup method for fermentation broth,the result shows the gene deletion strain JXNU-?P450 growth rate is slower than wildtype and the reverant,the inhibition zone of fermentation liquid is smaller.The result is fully prove the antibacterial effect in JXNU-?P450 fermentation broth is change.The correlation between gene cluster P450 and JXNU-1 antibiotic JX was verified.According to the above conclusions,the gene cluster P450 is involved in the regulation of antibiotic JX synthetic metabolic pathway,which lays a solid foundation for further elucidation of antibiotic JX synthesis mechanism.