Study On Oligosaccharide Biosynthesis Of Micromonospora Enhanced By Recombinant Biological Components

Efficient biological components was assembled and integrated into the specific sites of biosynthetic gene cluster to finally enhance the synthesis of secondary metabolites.In this study,sisomicin-produced strain Micromonospora inyoensis was used as a model.By molecular genetic techniques,the powerful biological components from phage and the components of clear regulatory mechanisms from lac operon were integrated into the specific sites of sisomicin biosynthesis gene cluster in Micromonospora inyoensis.Study on the growth and development affected by these components in Micromonospora was carried out,especially the impact on the improvement magnitude of secondary metabolite.Integration of T7 promoter into the downstream specific site of sisomicin biosynthetic gene cluster(SBGC).Using sisomicin biosynthetic gene cluster(GenBank accession number:JF431003)as a reference template,a segment of DNA between genes CDS28 and CDS29 was designated as the site for integration of T7 promoter into M.inyoensis,the upstream and downstream sequences of the segment of DNA were amplified and were used as homologous exchange arms.Recombinant plasmid pHB202,based on temperature sensitive plasmid pKC1139,was constructed for integration T7 promoter into the M.inyoensisTS3388 Then,plasmid pHB202 was introduced into M.inyoensis TS388 by conjugation.Two single-crossover strains DTS202-1,DTS202-2 were obtained.T7 promoter was successfully integrated into the site between genes CDS28 and CDS29.Double crossover mutant TS202 was obtained from mixtures of DTS202 by replica plating.The results showed that its growth morphology had no difference from the original strain TS388.Moreover,no significant difference existed between fermentation titer,and they were only 228u/ml.It was verified that significantly affect would not be produced on biosynthesis by integration of T7 promoter into M.inyoensis.Integration of recombinant biological components into the upstream specific site of sisomicin biosynthetic gene cluster(SBGC).T7promoter and T7RNApolymerase gene from phage and the components(lacI-lacP-lacO)from lac operon was recombined to form recombinant biological components(lacI-lacP-lacO-T7RNApol-T7promoter).Using the same method,recombinant plasmid pHB213 containing recombinant biological components was constructed for recombinant components into M inyoensisTS202.Then,plasmid pHB213 was introduced into M.inyoensis TS202 by conjugation.One engineered strains DTS213 was obtained.Recombinant biological components were successfully integrated into the site between genes 16S rRNA and CDS2.Study on biological characteristic of M.inyoensis DTS213.The characteristics of strain DTS213 was investigated using the mutant TS202 and TS388 as a control group,the results showed as following:M.inyoensis DTS213 grew fast and began to turn color in short time.Its mycelium turned to slender,and the fermentation broth was dark red.The results of fermentation product analysis of DTS213 revealed as following:without IPTG,its fermentation titer was 114u/ml.It was indicated that it might be affected by insertion of large exogenous DNA fragment.The average fermentation units of DTS213 still decreased to 58u/ml when fermentation medium was added with lactose to induce.It was roughly suggested that M.inyoensis may be lack of lactose permease or ?-galactosidase,and pH will declined by lactose metabolism,which leaded to that product was degradated.However,the fermentation titer of M.inyoensis DTS213 increased up to 556u/ml,nearly about 2.5 times of the original strain,while IPTG was added at a final concentration of 0.01mmol/mL.It was indicated that repressor lacl was lift by IPTG,and protein T7RNApolymerase was expressed.Thus,the genes for sisomicin biosynthesis were efficiently transcribed and over-expressed,finally improving the secondary metabolite of the DTS213.Integration of recombinated components(lacP-lacO-T7RNApol-T7promoter)into the upstream specific site of sisomicin biosynthetic gene cluster(SBGC).A segment of DNA between 16SrRNA and CDS2 was also designated as the site for integration of(lacP-lacO-T7RNApol-T7promoter)into the upstream site of sisomicin biosynthetic gene cluster.Recombinant plasmid pHB224 was constructed for integration of recombinated components into M.inyoensisTS202.Plasmid pHB224 was then introduced into M.inyoensis TS202 by conjugation.Howover,the single-crossover strains were not obtained after several times of conjugation.