Production ofStreptomyces clavuligerus isopenicillin N synthase inEscherichia coli using two-cistron expression systems

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Abstract
Streptomyces clavuligerus isopenicillin N synthase (IPNS) gene expression was achieved inEscherichia 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. NoE. coli- likeStreptomyces promoter motif was present upstream of the IPNS gene therefore transcriptional regulation of the two-cistron system was provided by thelac promoter of pUC119. Enzymatically active IPNS was detected inE. coli cells harboring the recombinant plasmids thereby providing evidence for the activity of the IPNS ORF and for the feasibility of production ofS. clavuligerus IPNS inE. coli. These results indicate that simple two-cistron constructions involving foreign gene flanking sequences may be used to express foreign proteins inE. coli.