Construction Of A Gentamicin C1a-overproducing Strain And Heterologous Expression Of AprD3,aprD4 Gene

Most of the genes in aminoglycoside antibiotic biosynthetic pathway have been characterized,and there are many successful examples in improving strain through gene engineering.So we attempted to improve strains of kanamycin-producing and gentamicin-producing using gene engineering techniques in this paper.We obtained a disruption strain-SPU503 through gacD-inactivation in Micromonospora purpurea by gene engineering method.The fermentation products of SPU503 strain are Cla and C2b.It is the first time to prove that the function of gacD is responsible for C-6' methyltransferase of X2 to produce G418,which would be contribute to elucidate the gentamicin biosynthetic pathway;Moreover,we obtained a genetically stable recombination strain.The gentamicin C1a component content in the fermentation product of the recombination strain increased more than 10-fold compared to that of the wild type strain.The recombination strain produced only 2 components,C1a and C2b,making the purification of gentamicin C1a easier.Gentamicin C1a has the highest antibacterial activity in the gentamicin C complex and could also be used as the precursor of the semi-synthetic antibiotic etimicin.The obtained recombination strain should be useful for the industrial production of gentamicin C1a.The prerequisite for genetic engineering is to establish transformation system,there is no any report about the transformation of Streptomyces kanamyceticus so far.At first,we investigated the sensitivity of Streptomyces kanamyceticus to generally used antibiotics,and drew the conclusions that the appropriate antibiotics and dose are 20?parameters g/ml apramycin or 100?g/ml erythromycin,and it is not sensitive to thiostreptone.Then we investigated of relative to conjugation efficiency including the heat treatment temperature of spores,the MgCl2 concentration,the ratio of donor and recipient,and different plasmid including pKC1139,pSET152,pKC1218,pSOK804.When the temperature of heat treatment is 40? and 10mmol/1 MgC12 added into the MS medium,there is the highest conjugation efficiency.The ratio of donor and recipient has little effect when the donor is excessive.The pKC1139,pSET152,pKC1218 could be transformed into the Streptomyces kanamyceticus.These data establish the basis for gene engineering of Streptomyces kanamyceticus.3'-OH is one of the attacking sites of aminoglycoside antibiotics-resistant bacteria.Blast analysis of biosynthetic gene cluster of containing 2-DOS aminoglycoside antibiotics shows that there are several unique genes in 3'-deoxygen aminoglycoside antibiotics,which are aprD3,aprD4,livY,livW,gntY.Gene blocking analysis of aprD3 and aprD4 showed that the two genes were related to 3' deoxygenation reaction of the apramycin or tobramycin biosynthesis.The secondary metabolite products of Streptomyces tenebrarius and Streptomyces kanamyceticus are similar,so it is speculated that there is a high similarity in internal environment of Streptomyces tenebrarius and Streptomyces kanamyceticus.So the aprD3 and aprD4 were expressed in Streptomyces kanamyceticus to verfity their functions further.