Open AccessAn efficient approach for cloning the dNDP‐glucose synthase gene from actinomycetes and its application in Streptomyces spectabilis , a spectinomycin producer
Author(s) -
Hyun ChangGu,
Kim Sang Suk,
Sohng Jae Kyung,
Hahn JaeJin,
Kim JongWoo,
Suh JooWon
Publication year - 2000
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2000.tb08955.x
Subject(s) - spectinomycin , biology , gene , dehydratase , atp synthase , escherichia coli , biochemistry , streptomyces , enzyme , cloning (programming) , dna , microbiology and biotechnology , dna sequencing , homology (biology) , sequence analysis , molecular cloning , genetics , peptide sequence , bacteria , antibiotics , antibiotic resistance , computer science , programming language
Specifically designed PCR primers were applied to amplify a segment of dTDP‐glucose synthase gene from six actinomycete strains. About 300‐bp or 580‐bp DNA fragments were obtained from all the organisms tested. By DNA sequence analysis, seven amplified fragments showed high homology with dTDP‐glucose synthase genes that participate in the biosynthesis of secondary metabolites or in deoxy‐sugar moieties in lipopolysaccharides. In addition, we have cloned a 45‐kb region of DNA from Streptomyces spectabilis ATCC27741, a spectinomycin producer which contained the dTDP‐glucose synthase and dTDP‐glucose 4,6‐dehydratase genes named spcD and spcE , respectively. The spcE gene was expressed in Escherichia coli and the activity was assayed in cell extracts. The enzyme showed substrate specificity only to dTDP‐glucose.