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 isopenicillin N synthase (IPNS). In this study, an IPNS gene from Streptomyces lipmanii NRRL 3584 (slIPNS) was cloned via PCR-based homology cloning, sequenced and expressed in Escherichia coli. Soluble slIPNS was overexpressed up to 21% of total soluble protein, and verified to be functionally active when in an IPNS enzymatic assay. Sequence comparison of the slIPNS gene obtained (excluding the consensus primer sequences) with another cloned IPNS from S. lipmanii 16884.3, revealed one three-nucleotide deletion and three closely-spaced single nucleotide deletions. Furthermore, this paper also reports the first instance of the usage of PCR as an alternative and rapid strategy for IPNS cloning using consensus primers.
Loke
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Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore.
Ng
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Sim
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010406:Penicillins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*
D010406:Penicillins; D010088:Oxidoreductases; C038370:isopenicillin N synthetase