Cloning of a Streptomyces clavuligerus DNA fragment encoding the cephalosporin 7 alpha-hydroxylase and its expression in Streptomyces lividans.
A 26-mer DNA probe was designed from N-terminal sequence data for the cephalosporin 7 alpha-hydroxylase (CH) of Streptomyces clavuligerus NRRL 3585 and used to screen a DNA library from this organism. The library was constructed in the lambda GEM-11 phage system. After plaque purification and reprobing, positive recombinant phages were chosen for further analysis. Characterization of the cloned DNA by restriction mapping and Southern hybridization showed that a 1.5-kb SalI fragment hybridized to the probe. Polymerase chain reaction assays using this fragment as a template and the probe as a primer indicated that the fragment carries the entire putative CH gene (cmcI). This was confirmed through the expression of CH enzymatic activity when the fragment was introduced into Streptomyces lividans. A putative beta-lactamase activity was detected in S. lividans.
Xiao
X
X
Department of Biology, Northeastern University, Boston, Massachusetts 02115.
Hintermann
G
G
Häusler
A
A
Barker
P J
PJ
Foor
F
F
Demain
A L
AL
Piret
J
J
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000378:metabolism; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D015342:DNA Probes; D004269:DNA, Bacterial / Q000096:biosynthesis*; D015870:Gene Expression; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D013302:Streptomyces / Q000201:enzymology / Q000378:metabolism*
D015342:DNA Probes; D004269:DNA, Bacterial; D009841:Oligonucleotides; D006899:Mixed Function Oxygenases; C071965:cephalosporin 7-alpha-hydroxylase