Study On The Gene Recombination Characteristics Of The Amino-oligosaccharide Biosynthesis

Amino-oligosaccharide is a kind of clinically important aminoglycoside antibiotic.This study starts with genetic engineering,introduction of the biology methods,to study on the biosynthesis of the amino-oligosaccharide and its modification.On this basis,to explore the possibility of Micromonospora purpurea methylase gene of single gene heterologous expression in Streptomyces tenebrarius,the possibility of the combination of oligo gene,and the possibility of protoplast fusion between M.purpurea and S.tenebrarius.The main research contents and result are as follows:Firstly,study on the heterologous combination of single gene genK:Using the temperature-sensitive plasmid pKC1139 as vector,construction of the recombinant plasmid pKM103 which gene genK combined with tobM2,and introduced into S.tenebrarius Tt49 by taking conjugation.The single crossover strain TKM201 was received by using erythromycin as a selective marker,the desired double crossover mutant TKM202 was selected by copying the tablet and PCR analysis after TKM201 was keeping cultivating in the absence of erythromycin.The metabolites were analyzed by using MS,the results showed that TKM202 had the similar results with the strain TW402(?tobM2),mainly produced apramycin and other methylated antibiotic were not obtained as we except.Secondly,study on the combination of double gene:Using the same process and parent as the engineering strain TKM202,the engineering strain TDM202 was obtained which gene genDl that modified with gentamicin C-methylated combined with tobM2.The metabolites were analyzed by using MS,the results showed that TKM202 mainly produced apramycin.Taking the engineering strain TDM202 as parent strain,the engineering strain TLX 102 was obtained which gene genX combined with tobL.The metabolites were analyzed by using MS,the results showed that TLX 102 had the similar results with the strain TDM202,mainly produced apramycin,and other new compounds were not obtained.Gene combinations were successful,but no functional expression was achieved.Thirdly,study on the construction and fermentation characteristics of Kanamycin B producing strain:To construct a Kanamycin B industrialization overproducing strain.The tobZ deletion mutant ST314 was constructed in S.tenebrarius312 which mainly produces carbamoykanamycin B,the mutant strain only produced Kanamycin B.Then the biosynthetic potential of ST314 was studied,the amplification experiment was carried out on a 200L fermentor,and the parameters were measured finally the fermentation metabolic curve was drawn.The results showed that the characteristic would be steadily inherited,and the production capacity of the strain was nearly 2 times higher than the parent strain.According to the fermentation metaboliccurve the fermentation level of kanamycin B reached 4500 u/mL,it could be transferred to the industrial production.Fourth,exploration of multiple genes combination between Micromonosporaceae and Streptomycetaceae:Exploration on the feasibility of the combination of specific gentamicin biosynthetic gene clusters into the genome of S.tenebrarius by protoplast fusion,construction of the' engineering strain GEB202 that having erythromycin selective marker which gene ermE combined with genB2by using genetic engineering techniques.The metabolites were analyzed by using MS,the results showed that GEB202 mainly generating gentamicin C1a,and a little gentamicin C2a and X2.Research on the condition of the protoplasts formation and regeneration in M.purpurea GEB202 and S.tenebrarius Tt49:It was found that mycelium preparation it was better to use spore suspension into the seed culture medium,and then transferred into the mycelium culture medium.In the mycelium culture medium supplemented with glycine to enhance mycelium sensitivity to lysozyme,easily to dissolve the cell wall by lysozyme.Dissolve the cell wall of the M.purpurea GEB202,the preparation of the protoplasts by the concentration of lysozyme 8 mg/mL,35? temperature to maintain 6 h is appropriate.Dissolved S.tenebrarius Tt49 cell wall,the preparation of protoplasts by the concentration of lysozyme 2 mg/mL,32 ?temperature to maintain 3 h is appropriate.Research on the cell fusion by using double resistance markers,after repeated exploration,the fusion of directional gene has not yet been obtained,which needs to be further explored.