The Research Of Modifier Genes On Gentamicin Oligosaccharide

The study of the biosynthesis metabolic flux of gentamicin, a kind of clinically important amino-oligosaccharide antibiotic, has achieved some progress. However, almost all modifier genes refer to the gentamicin biosynthesis have not been confirmed by experiment. Based on the analysis of the bioinformatics and report, the genQ, genB2 and genD2, which were speculated to be modifier genes in gene cluster, were regarded as taget genes. With the gentamicin production strain micromonospora purpurea as research object, this dissertation focused on gene disruption research by molecular genetics technology. The catalytic function of each gene was confirmed by the variation analysis of gentamicins biosynthesis when the relevant gene was disrupt, and the corresponding engineering strains were obtained.Research of the function of genQ:Using the temperature-sensitive plasmid pKC1139 as vector, the recombinant plasmid pQB303 was constructed and introduced into the micromonospora purpurea G1008 by conjugation. Then the single crossover mutant GQ1 was obtained. The disruption mutant GQ175 was screened out by replica plating and PCR analysis after GQ1 was continuously cultured in seed medium without apramicin. The result of fermentation of GQ175 in shake flask shown that its level of antibiotic production was 728 μ/ml as well as the parent strain’s. The result of TLC and MS analysis indicated that the metabolic flow of gentamicin biosynthesis was black out in GQ175, which only accumulated intermediate species G418. This demonstrated that the genQ participates in the amination of C-6’. The genQ can be responsible for C-6’dehydrogenation based on homology analysis. Simultaneously, a engineering strain GQ175 for producing G418 single component was obtained and applied for national invention patent.Study on the function of genB2:Taking the same method and parent strain as the genQ research, the plasmid pZB303 was constructed for the genB2 disruption. The engineering strain GB102 was obtained. The antimicrobial activity assay of GB102 fermentation broth showed that its antibiotic production was about 856 μ/ml and the pigment content was litter. The result of TLC, HPLC and MS analysis indicated the gentamicin metabolic flux of GB102 was different from parent strain G1008. GB102 mainly produced gentamicin C2a and a extremely small amount of gentamicinCla and C1. This result demonstrated that the genB2 is responsible for C-6’ epimerization.Study of the function of genD2:Using the same method and parent strain as the genB2 disruption, the plasmid pDB303 was successfully constructed for the genD2 disruption and the genD2 disruption mutant GD238 was successively obtained. The antibiotic production of GD238 was only 100 μ/ml. The MS analysis showed that GD238 only accumulated gentamicin A2 and A2e, instead of gentamicin Cs. This demonstrated the genD2 is responsible for C-3" dehydrogenation and it was speculated that gentamicin A2 was converted to A2e through further modifition by methylation enzyme GenK.Research of gentamicin A2e biosynthesis gene:Take the same method with the genQ research, but the parent strain is GD238. Using the plasmid pFD308 for genK disruption, the engineering strain GDK3 was constructed by further disrupted. The MS analysis of Metabolites indicated that GDK3 only accumulated gentamicin A2 without A2e. This result demonstrated that GenK can not only catalyze the methylation of gentamicin X2 to G418, but also A2 to A2e. Although the catalysis site and function is identical, the selectivity to substrates is irrigorous.