The Research Of Functional Gene Combination Of Micromonospora-Streptomyces

Streptomyces tenebrarius Tt-49 mainly produces apramycin,tobramycin,carbamoyltobramycin and other compounds.The single compound carbamoyltobramycin or apramycin producing strain was obtained by using genetic engineering techniques.On this basis,to explore the possibility of combinating the gene coding C3',4'-dehydroxylation enzyme or methylase gene with apramycin and tobramycin biosynthetic gene.The details are as follows:Firstly,the construction of engineering strain producing carbamoyltobramycin.The pKB5 was constructed based on the shuttle vector pKC1139 and was introduced into Streptomyces tenebrarius Tt-49 by conjugation to obtain the single crossover mutant ST315.The aprK gene deletion strain ST316 was selected by replica plating and PCR analysis after ST315 was continuously cultured without any antibiotic.The metabolites were analyzcd by using TLC and MS.Compared with the original strain Tt-49,ST316 mainly produced carbamoyltobramycin,apramycin was no longer produced.ST316 can be used as gene combination mode strain.Secondly,the construction of engineering strain producing apramycin.Taking the same method and parent strain Tt-49 as the aprK research,the tobM2 in-frame deletion engineering strain TW402 was obtained.The metabolites were analyzed by using TLC and MS,the result indicated that TW402 mainly produced apramycin and carbamoyltobramycin was no longer produced.Thirdly,the combination of 3',4'-dehydroxylation genes from Micromonospora with apramycin biosynthesis genes of Streptomyces tenebrarius:taking the gene aprD4 as gene combination locus of C3',4'-dehydroxylation,sisl from Micromonospora inyoensis and genP from micromonospora purpurea respectively replaced the aprD4 of Streptomyces tenebrarius ST316 to obtained the TW414 which gene aprD4 was replaced by gene sisl and TW416 which gene aprD4 was replaced by gene genP The metabolites of TW414 and TW416 were analyzed by using MS,the result indicated that TW414 and TW416 mainly produced carbamoylkanamycin B and kanamycin B,and carbamoyltobramycin was no longer produced.But,dibekacin and other compounds were not obtained as expected.Fourth,the combination of methylase gene from Micromonospora with tobramycin biosynthesis genes of Streptomyces tenebrarius:The homologous recombination plasmid pKM5 which gene forK replaced the tobM2 was constructed and introduced into Streptomyces tenebrarius Tt-49 by conjugation.The engineering strain TW420 was obtained which biosynthetic gene of tobramycin combined with gene forK The metabolites were analyzed by using MS,the results showed that TW420 had the similar results with the strain TW402,mainly produced apramycin and carbamoyltobramycin was no longer produced,.Fifthly,the discovery of the genB4 function research.During the preparation process of designing the method to combinate the genB4 of Micromonospora purpurea with biosynthesis genes of Streptomyces tenebrarius,in order to elucidate gene function of genB4 by the disruption of genB4,a Micromonospora purpurea GbKB4 which mainly produed sisomicin was obtained.Therefore,turning attention to the research of the engineering strain productivity features from the gene combination.Using the GbKB4 as parent strain,GbKB4-2 which the level of fermentation was three times of the original strain was obtained by natural separation and UV mutagenesis.Studied the genetic stability and fermentation characteristics of GbKB4-2,drawn a fermentation metabolism graph of sisomicinin,and the final fermentation was up to 970u/mlL.It had excellent prospects for industrial application.The gene combination would be as a follow-up study.Dibekacin or other new components was not synthesized as expected as the combination of biosynthesis genes of Micromonospora with biosynthesis genes of Streptomyces tenebrarius.It suggested that 3',4'-dehydroxylation gene or methyltransferase gene did not work alone,it required its own environmental impact.The biosynthetic enzymes systems of Micromonospora and Streptomyces tenebrarius were quite different,so the gene of micromonospora failed to express normally.