PCR cloning, heterologous expression, and characterization of isopenicillin N synthase from Streptomyces lipmanii NRRL 3584

A key step which involves the cyclization of delta-(L-alpha-aminoadipyl)-L- cysteinyl-D-valine to the bicyclic ring structure of isopenicillin N in the penicillin and cephalosporin biosynthetic pathway, is catalyzed by isopeni cillin N synthase (IPNS). In this study, an IPNS gene from Streptomyces lip manii NRRL 3584 (slIPNS) was cloned via PCR-based homology cloning, sequenc ed and expressed in Escherichia coli. Soluble slIPNS was overexpressed up t o 21% of total soluble protein, and verified to be functionally active when in an IPNS enzymatic assay. Sequence comparison of the slIPNS gene obtaine d (excluding the consensus primer sequences) with another cloned IPNS from S. lipmanii 16884.3, revealed one three-nucleotide deletion and three close ly-spaced single nucleotide deletions. Futhermore, this paper also reports the first instance of the usage of PCR as an alternative and rapid strategy for IPNS cloning using consensus primers.