链霉菌Streptomyces tenebrarius H6中与抗生素有关的糖生物合成基因的克隆

链霉菌S.tenebrarius H6产生多种氨基糖甙类抗生素,主要有阿普霉素、妥普霉素及卡那霉素B,其中阿普霉素因含有8碳糖的一种特殊结构令人注目,它的抗菌谱广,特别是对革兰氏阴性菌有较强的抗菌活性,不容易产生耐药性,对已有的耐药菌产生的氨基糖苷转移酶等失活酶仍有抵抗力.主要用于牛、猪、鸡等的大肠杆菌、沙门氏菌和支原体所引起的白痢、腹泻和肺炎等疾病.迄今有关八碳糖生物合成基因簇的研究在国内外尚无报道,在该菌株开展有关糖合成代谢基因的研究有着一定的意义.

Cloning of the Sugar Related Biosynthesis Gene Cluster from Streptomyces tenebrarius H6

Streptomyces tenebrarius H6 produces a complex of aminoglycoside antibiotics, such as apramycin, tobramycin and kanamycin B etc. To study the apramycin biosynthetic genes the genomic library from the Streptomyces tenebrarius H6 was established using E. coli/Streptomyces shuttle vector pKC505 by in vitro packing. The probability of finding a specific gene from the library composed of 3,000 colonies was over 99.9%. According to the highly conserved sequence of the genes involved in 6-deoxyhexose biosynthesis, primers were designed and 0.6 kb fragment homologous to strE gene was obtained by PCR. 30 positive clones were found from the genomic library of S. tenebrarius H6 with the 0.6 kb fragment as a probe. Overlapped regions were localized by Southern hybridization and putative sugar related biosynthetic gene cluster was mapped by restriction enzyme digestions. An ORF of dTDP-glucose-4,6-dehydratase gene consisted of 1,132 bp, designated as aprE, was obtained and submitted to GenBank under the accession number of AF306787. A DNA sequence highly homologous to strL coding dTDP-4-dehydrorhamnose reductase was found linked with aprE gene.