Molecular cloning and expression of the glucose/xylose isomerase gene from Streptomyces sp. NCIM 2730 in Escherichia coli.
A partial genomic library of Streptomyces sp. NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the D-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes. Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E. coli mutants. The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed. GXI-deficient strains of E. coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27. E. coli JM105 (pMSG27) and E. coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter. Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M(r) 110. This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli.
Bhosale
S H
SH
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Ghatge
M S
MS
Deshpande
V V
VV
D019747:Aldose-Ketose Isomerases; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015139:Blotting, Southern; D015153:Blotting, Western; D002238:Carbohydrate Epimerases / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D005820:Genetic Testing; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase