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黑暗链霉菌H6(Streptomyces tenebrarius H6)——一种氨基糖苷类抗生素产生菌中dTDP-葡萄糖-4,6-脱水酶基因及其连锁基因簇的鉴定与功能分析

Identification and functional analysis of dTDP-glucose-4,6-dehydratase gene and its linked gene cluster in an aminoglycoside antibiotics producer of Streptomyces tenebrarius H6.

作者信息

Du Yu, Li Tianbo, Wang Yiguang G, Xia Huanzhang

机构信息

Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking Union Medical College, Tiantan Xili No1, Beijing 100050, China.

出版信息

Curr Microbiol. 2004 Aug;49(2):99-107. doi: 10.1007/s00284-004-4212-z.

Abstract

Streptomyces tenebrarius H6 produces a variety of aminoglycoside antibiotics, such as apramycin, tobramycin, and kanamycin B. Primers were designed according to the highly conserved sequences of the dTDP-glucose-4,6-dehydratase genes, and a 0.6-kb PCR product was obtained from S. tenebrarius H6 genomic DNA. With the 0.6-kb PCR product as a probe, a BamHI 7.0-kb fragment was isolated. DNA sequence analysis of the 7.0-kb fragment revealed four ORFs and an incomplete ORF. In search of databases, the deduced product of one ORF (orfE) showed 62% identity to the dTDP-glucose-4,6-dehydratase, StrE of S. griseus. Three other ORFs (orfG1, orfG2, and orfGM) showed 55%, 62%, and 42% similarities, respectively, to glycosyltransferase from Clostridium acetobutylicum and mannosyltransferase from Xanthomonas axonopodis pv. citri str. 306 and glycosyltransferase from Pseudomonas putida KT2440. Upstream of the orfE was an incomplete ORF, and the deduced product showed 56% similarity to dTDP-4-dehydrorhamnose, StrL from S. griseus. The function of the orfE gene was studied by targeted gene disruption. The resulting mutant failed to produce tobramycin and kanamycin B, but still produced apramycin, suggesting that the orfE gene and linked gene cluster are essential for the biosynthesis of tobramycin and kanamycin B in S. tenebrarius H6.

摘要

黑暗链霉菌H6能产生多种氨基糖苷类抗生素,如阿泊拉霉素、妥布霉素和卡那霉素B。根据dTDP - 葡萄糖 - 4,6 - 脱水酶基因的高度保守序列设计引物,从黑暗链霉菌H6基因组DNA中获得了一个0.6 kb的PCR产物。以该0.6 kb的PCR产物为探针,分离出一个7.0 kb的BamHI片段。对该7.0 kb片段进行DNA序列分析,发现有四个开放阅读框(ORF)和一个不完整的ORF。在数据库搜索中,一个ORF(orfE)的推导产物与灰色链霉菌的dTDP - 葡萄糖 - 4,6 - 脱水酶StrE有62%的同一性。另外三个ORF(orfG1、orfG2和orfGM)分别与丙酮丁醇梭菌的糖基转移酶、柑桔溃疡病菌306菌株的甘露糖基转移酶以及恶臭假单胞菌KT2440的糖基转移酶有55%、62%和42%的相似性。orfE上游是一个不完整的ORF,其推导产物与灰色链霉菌的dTDP - 4 - 脱氢鼠李糖StrL有56%的相似性。通过靶向基因破坏研究了orfE基因的功能。所得突变体不能产生妥布霉素和卡那霉素B,但仍能产生阿泊拉霉素,这表明orfE基因及其相连的基因簇对黑暗链霉菌H6中妥布霉素和卡那霉素B的生物合成至关重要。

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