Abstract
The enzyme isopenicillin N synthetase (IPS) catalyses the oxidative condensation of δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N, which is a central reaction in the pathway to clinically important penicillins and cephalosporins. Here we report the cloning, characterization and expression in Escherichia coli of the gene encoding the IPS protein in Cephalosporium acremonium. The IPS gene was identified by purifying IPS protein, determining the first 23 amino-terminal amino acids, preparing a set of synthetic oligonucleotides encoding a portion of the determined amino-acid sequence, and probing a cosmid genome library with the mixed oligonucleotides. A cosmid hybridizing with the probe was isolated and the IPS gene was localized and sequenced. The IPS gene encodes a polypeptide of relative molecular mass (Mr) 38,416. When this open reading frame was cloned into an E. coli expression vector and inserted into E. coli, the recombinant E. coli produced a new protein co-migrating with authentic IPS as the major protein of the cell (∼20% of cell protein). Crude cell extracts condensed LLD-ACV to a penicillinase-sensitive molecule whose antibacterial activity indicated that it was isopenicillin N.
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References
Abraham, E. P., Huddlestone, J. A., Jayatilake, G. S., O'Sullivan, J. & White, R. L. Spec. Publs chem. Soc. 38, 125–134 (1981).
Pang, C.-C. et al. Biochem. J. 222, 789–795 (1984).
Hollander, I.J., Shen, Y.-Q., Heim, J., Demain, A.L. & Wolfe, S. Science 224, 610–612 (1984).
Bahadur, G. A. et al. J. Am. chem. Soc. 103, 7650–7651 (1981).
Baldwin, J. E. et al. JCS chem. Commun. 1317–1319 (1983).
Baldwin, J. E. et al. JCS chem. Commun. 1225–1227 (1984).
Wolfe, S., Hollander, I. J., Demain, A. L. Biotechnology 2, 635–636 (1984).
Maxam, A. M. & Gilbert, W. Proc. natn. Acad. Sci. U.S.A. 74, 560 (1977).
Bibb, M. J., Findlay, P. R. & Johnson, M. W. Gene 30, 157–166 (1984).
Richardson, M. A., Mabe, J. A., Beerman, N. E., Nakatsukasa, W. M. & Fayerman, J. T. Gene 20, 451 (1982).
Rao, R. N. et al. Antimicrob. Ag. Chemother. 24, 689–695 (1983).
Vieira, J. & Messing, J. Gene 19, 259–268 (1982).
Schoner, B. E., Hsiung, H. M., Belagaje, R. M., Mayne, N. G. & Schoner, R. G. Proc. natn. Acad. Sci. U.S.A. 81, 5403–5407 (1984).
Queener, S. W., Ingolia, T.D., Skatrud, P.L., Chapman, J.L. & Kaster, K.R. in Microbiology 1985 (ed. Leive, L.) (American Society of Microbiology, in the press).
Grunstein, M. & Hogness, D. Proc. natn. Acad. Sci. U.S.A. 72, 3961–3965 (1975).
Ingolia, T. D. & Craig, E. A. Nucleic Acids Res. 9, 1627–1642 (1981).
Maniatis, T., Fritsch, E. F. & Sambrook, J. Molecular Cloning: A Laboratory Manual 326–328 (Cold Spring Harbor Laboratory, New York, 1982).
Laemmli, U. K. Nature 227, 680–685 (1970).
Shen, Y.-Q., Wolfe, S. & Demain, A.L. J. Antibiot. 37, 1044–1048 (1984).
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Samson, S., Belagaje, R., Blankenship, D. et al. Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium. Nature 318, 191–194 (1985). https://doi.org/10.1038/318191a0
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DOI: https://doi.org/10.1038/318191a0
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