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

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.