An efficient approach for cloning the dNDP-glucose synthase gene from actinomycetes and its application in Streptomyces spectabilis, a spectinomycin producer.
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.
Hyun
C
C
Department of Biological Science, Institute of Bioscience and Biotechnology, Myong Ji University, Yongin, South Korea.
Kim
S S
SS
Sohng
J K
JK
Hahn
J
J
Kim
J
J
Suh
J
J
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D017931:DNA Primers; D003837:Deoxy Sugars / Q000096:biosynthesis; D015723:Gene Library; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics* / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D000198:Spectinomycin / Q000096:biosynthesis; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D017931:DNA Primers; D003837:Deoxy Sugars; D000198:Spectinomycin; D009713:Nucleotidyltransferases; C085243:dTDP-D-glucose synthase; D008359:Mannose-6-Phosphate Isomerase