The Research Of Cloning And Expression About Amino Sugars Methylation Gene

Currently,the research of functional expression about combination between methylation gene and Streptomyces tenebrarius gene has not been reported.If the methylation gene will be put into appropriate sites of Streptomyces tenebrarius and the expression is expected to produce a new compound.The advantages of quick growth,good ability of producing antibiotic,many spores and relatively clear genetic background can be found in Streptomyces tenebrarius Tt49.Thus it can be used as a model bacteria to study the possibility of heterologous gene expression.Tt49 can produces tobramycin,carbamoyltobramycin,apramycin and other compounds,Components of secondary metabolites are more,but knockouted tobM2 mainly produces apramycin and neper amine,component becomes relatively single,thus it is a rational choice to replace tobM2 with exogenous gene.Constructing the vector including homologous sequences double sides of tobM2 and methylation gene,by transconjunction technology to transform recombinant vector into Tt49,the methylation gene is integrated into a specific site,in order to study the possibility of methylation gene heterologous expression.This research selects methylation gene eryB?,eryC? and eryG from Saccharopolyspora erythraea,ctcK from Streptomyces aureofaciens as the research object.The details are as follows:Firstly,to explore the possibility of gene heterologous expression.genP and forP all have C3',C4'-didehydroxylation function.Microspora as a starting strain,according to genetic recombination principle to construct TP322 that forP replace genP.Metabolites of TP322 analyzes by TLC and MS shows engineering bacteria produces gentamicin Cl,which illustrates the forP realized the heterologous expression.Secondly,the constrution of the engineering strain which eryB? replaced tobM2.The homologous recombination plasmid pBM6 which eryB? replaced tobM2 was constructed and introduced into Streptomyces tenebrarius Tt49 by conjugation.The engineering strain TB322 was obtained.TB322 metabolites were analyzed by TLC and MS,whose result showed that it produced apramycin and neper amine and no longer produced tobramycin,carbamoyltobramycin.But the desired methylated product was not got.Thirdly,expression of EryC? protein and Construction of engineering strain TC322.The expression vector successfully expressed protein EryCVI by induced.To get engineering strain TC322 that tobM2 was replaced by eryC?,as the same way of constructing TB322.The fermented product was detected by TLC and MS and found to be similar to the strain TB322.The replacement of the gene was only equivalent to knocking out tobM2 and the function of methylation gene did not reflect.Fourth,expression of EryG protein and construction of engineering strain TG322.The expression vector successfully expressed protein EryG by induced.Application of homologous recombination principle to construct engineering bacteria that eryG replace tobM2.Its secondary metabolites analyze by MS,which showed the same result with engineering bacteria TC322.TLC analysis of single exchange engineering bacteria showed that the methylation gene did not work.Fifthly,construction of engineering bacteria TK322 and TKQ322.Application of genetic engineering technology to consrtuct engineering bacterium ctcK(aureomycin C-6 methylation gene)replace tobM2,the fermentation products analyze by TLC and MS,the result did not express about methylation gene.Considering that may be the reason for the methylation gene little expression,then the erythromycin promoter was inserted before the methylation gene and got another engineering bacterium TKQ322.But it didn't find secondary metabolites is decorated by methylation by MS.