The Research Of Cloning And Expression Of Genes Involved In Gentamicin Biosynthesis

Firstly,Micromonospora purpurea Gbk10(?genK)was used as the research object,by using bioinformatics tools,four genes orf2060?orf2390?orf2810 and orf5507 which probably modify the gentamicin with C6'-N methylation were selected from the whole genome sequence of Micromonospora to analyze the flux in the biosynthesis of gentamicin by means of in-frame knock-out of target genes.The details are as follows:Using pKC1139 as the shuttle vector,the upstream and downstream homologous exchange arms of orf2060,orf2390,orf2810 and orf5507 were amplified,and the four homologous recombinant plasmids with deleted target sequences were constructed.By means of conjugation and transfer,the four homologous recombinant plasmids were introduced into Micromonospora sp.Gbk10.The four mutant strains with the target gene-deleted GbkN102(?orf20(50),GbkN202(?orf2590),GbkN302(?orf2810)and GbkN402(?orf5507)thereafter were obtained through plating and resistance screening,Next,the secondary metabolites of the starting strain and the four mutant strains were extracted and analyzed through TLC and MS.The results showed that the main metabolites produced by mutant strains GbkN102,GbkN202,GbkN302 and GbkN402 were gentamicin Cia,gentamicin C2b and gentamicin X2,which means that no significant changes were found by comparing with the starting strain Gbk10,indicating that the deletion of the gene orf2060,orf2390,orf2810 and orf5507 did not block the synthesis of gentamicin C2b,and also did not affect the other biosynthesis pathways of gentamicin.Therefore,the genes orf2060,orf2390,orf2810 and orf5507 neither be involved in gentamicin biosynthesis genes,nor be the key genes for modifying C6'-N methylation of purpurosamine.Secondly,Using Streptmyces tenebrarius as a model bacterium and combined gentamicin-specific genes to its target sites to explore the feasibility of gentamicin-specific genes in the modification of key groups of the natural products of Streptomyces tenebrarius.The details are as follows:Using pKC 1139 as a shuttle plasmid,we constructed the homologous recombinant plasmids with genB3(EcoRV)-genX replacing aprD4 and genW-genB3(EcoRV)replacing aprD3-aprQ,and then the plasmids were transferred into Streptomyces tenebrarius Tt49 to form the combined engineering strain TGM104 with gentamicin biosynthesis gene subset genW-genB4-genP-genB3-genK-genB2-genX,which replaced the gene subset aprD3-aprQ-aprD4 in Streptomyces tenebrarius.The metabolites of the mutant strain TGM104 extracted and analyzed by MS showed the products were carbamoylkanamycin B and kanamycin B.Compared with the metabolites of the starting strain Tt49,the carbamoyltobramycin,tobramycin and apramycin components are no longer synthesized,meanwhile no new expected compounds such as 3',4'-dideoxycarbamoylkanamycin B or dibekacin were detected,suggesting that the products of gentamicin genes do not modify the corresponding group in carbamoylkanamycin B or kanamycin B.Therefore,despite that we successfully obtained the combined engineering strain with gentamicin-kanamycin B biosynthetic genes,the expression of gentamicin-specific genes with heterologous functions remains to be further explored and developed.