Construction Of Micromonospora Engineering Strain And Study On Its Functional Gene

Gentamicin producted by micromonospora purpurea, had been widely used in clinical. However the study on gentamicin biosynthesis and gene function has just begun. In this study, we explored the relationship of gentamicin biosynthesis and function of genes at the molecular level. And the micromonospora engineering strains (GKB3226, GKN27, GKP116, GO91) were constructed by gene knockout method. There are three aspects involved in this study:Firstly, bioinformatics analysis. The functions of the key genes from gentamicin biological gene cluster were predicted by bioinformatics analysis. This study was focus on four functional genes on the base of industrial application and gentamicin biosynthetic pathway. The functions of genes (genN, genO, genP, genB3) were speculated by homologous comparison, domain analysis and homology modeling research. The gene genN belongs to the methyltransferase superfamily; The gene genB3 was associated with gentamicin C6’aminotransferase, The gene genO enconding tRNA queosine ribosyl transferase, is related with the reaction of methylation in gentamicin biosynthetic pathway; The gene genP may be charge of 3’-phosphotransferase.Secondly, the research of functional genes (genN, genO, genP). According to the principle of homologous recombination, the experimental method of gene (genN, genO, genP) frame knockout was designed. The micromonospora engineering strain GKN27 (deletion of genN gene) was successfully constructed by genetic engineering technology. Instead of gentamicin C complex, the engineering strain M. Purpurea GKN27 generates four intermediates of gentamicin biosynthesis pathway. And it indicated that genN was not associated with methyl transferase. The study parper has been recieved by the journal of industial microbiology. The engineering strain M. Purpurea GKP116, the gene genP of which was knocked out from M. purpurea GK1101, was constructed by the same method. The analysis of metabolites from GKP116 suggested that genP is closely related to gentamicin biosynthesis. The engineering strain In. Purpurea G091, the genO gene of which was knocked out from M. purpurea G1008. The results showed that genO gene is association with the reaction of 6’C-N methylation.Thirdly, construction of gentamicin X2 micromonospora engineering strain. Gentamicin X2 is an important precursor of antiprotozoal, antiviral and antitumor drugs. The homologous fragments were amplified with the chromosome DNA of M. purpurea GK1101. The recombinant plasmid pFU803 was constructed by molecular cloning technology. Then the recombinant plasmid pFU803 was introduced into the M. purpurea GK1101 through the conjugal transfer method. After repeated screening, we successfully obtained double exchange engineering strain M. purpurea GKB3226. According to the results of TLC and EIS-MS analysis, we substantiated that the engineering strain GKB3226 accumulates gentamicin X2 by inactivation of genB3. The micromonospora engineering strains M. purpurea GKB3226, special biosynthesis gentamicin X2, fills the gaps in the domestic and foreign research.