Production of Streptomyces clavuligerus isopenicillin N synthase in Escherichia coli using two-cistron expression systems.
Streptomyces clavuligerus isopenicillin N synthase (IPNS) gene expression was achieved in Escherichia coli by the construction of two-cistron expression systems formed in the high copy number plasmid vector pUC119. These two-cistron constructions were composed of the IPNS gene and its flanking sequences which encoded an upstream open reading frame (ORF), the IPNS ribosome binding site and a putative transcription terminator. No E. coli- like Streptomyces promoter motif was present upstream of the IPNS gene therefore transcriptional regulation of the two-cistron system was provided by the lac promoter of pUC119. Enzymatically active IPNS was detected in E. coli cells harboring the recombinant plasmids thereby providing evidence for the activity of the IPNS ORF and for the feasibility of production of S. clavuligerus IPNS in E. coli. These results indicate that simple two-cistron constructions involving foreign gene flanking sequences may be used to express foreign proteins in E. coli.
Doran
J L
JL
Department of Microbiology, University of Alberta, Edmonton, Canada.
Leskiw
B K
BK
Petrich
A K
AK
Westlake
D W
DW
Jensen
S E
SE
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D013302:Streptomyces / Q000201:enzymology*
D004269:DNA, Bacterial; D010088:Oxidoreductases; C038370:isopenicillin N synthetase