The enzyme isopenicillin N synthetase (IPS) catalyses the oxidative condensation of delta-(L-alpha-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 (approximately 20% of cell protein). Crude cell extracts condensed LLD-ACV to a penicillinase-sensitive molecule whose antibacterial activity indicated that it was isopenicillin N.

No abstract

No abstract

,-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the ifiamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution.The penicillin, cephalosporin, and cephamycin classes of P-lactam antibiotics share two common steps in their respective biosynthetic pathways: the joining of three amino acids to form 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) and the conversion of LLD-ACV to isopenicillin N. These two reactions are carried out in filamentous fungi and streptomycetes, including Penicillium chrysogenum and Aspergillus nidulans (producers of penicillin), Cephalosporium acremonium (producer of cephalosporin C), and Streptomyces lipmanii and Streptomyces clavuligerus (producers of cephamycins) (16). We have studied the biochemical and genetic basis of the conversion of LLD-ACV to isopenicillin N by cloning and characterizing the gene encoding the isopenicillin N synthetase (IPNS) enzyme from C. acremonium and P. chrysogenum (6, 23). The IPNS genes from these two fungi show a high degree of nucleotide and amino acid sequence identity. The 77% identity of the predicted amino acid sequences of the IPNSs from these fungi is so extensive that it is difficult to identify regions of potential functional importance. We therefore decided to extend the comparison to IPNS genes from other organisms which might be more distant evolutionarily.The IPNS gene from A. nidulans was of particular interest because of the well-developed genetic techniques available for this organism. Also, mutant A. nidulans strains with altered ,-lactam-biosynthetic ability have been described (10) and characterized (20

No abstract