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J Bacteriol. 1994 August; 176(16): 4941-4948

research-article

Molecular characterization of three loss-of-function mutations in the isopenicillin N-acyltransferase gene (penDE) of Penicillium chrysogenum.

Department of Ecology, Genetics and Microbiology, Faculty of Biology, University of León, Spain.

ABSTRACT

Five mutants of Penicillium chrysogenum blocked in penicillin biosynthesis (npe) which are deficient in isopenicillin N-acyltransferase were isolated previously. Three of these mutants, npe6, npe7, and npe8, have been characterized at the molecular level and compared with npe10, a deletion mutant. Transcripts of normal size (1.15 kb) of the penDE genes, which encode isopenicillin N-acyltransferase, and also of the pcbAB (11.5 kb) and pcbC (1.1 kb) genes were observed in all mutants except for the npe10 mutant. Immunoblotting studies using antibodies against isopenicillin N-acyltransferase showed that all mutants (except npe10) formed the 40-kDa (unprocessed) protein and the 29-kDa subunit of the isopenicillin N-acyltransferase. The 11-kDa subunit could not be observed in the immunoblots. The mutant penDE genes of strains npe6, npe7, and npe8 were cloned and sequenced. These three strains showed a mutation in the penDE genes which results in a single amino acid change in each modified isopenicillin N-acyltransferase. The mutation in npe6 resulted in a change of Gly-150 to Val, whereas the mutation in both npe7 and npe8 introduced a change of Glu-258 to Lys. Replacement of the Val-150 and Lys-258 mutations by constructing hybrid isopenicillin N-acyltransferase molecules led to the recovery of the isopenicillin N-acyltransferase activity. The mutations in npe6, npe7, and npe8 do not affect the ability of the 40-kDa isopenicillin N-acyltransferase to be processed into the component subunits.

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