A 24-kb region of Cephalosporium acremonium C10 DNA was cloned by hybridization with the pcbAB and pcbC genes of Penicillium chrysogenum. A 3.2-kb BamHI fragment of this region complemented the mutation in the structural pcbC gene of the C. acremonium N2 mutant, resulting in cephalosporin production. A functional alpha-aminoadipyl-cysteinyl-valine (ACV) synthetase was encoded by a 15.6-kb EcoRI-BamHI DNA fragment, as shown by complementation of an ACV synthetase-deficient mutant of P. chrysogenum. Two transcripts of 1.15 and 11.4 kb were found by Northern (RNA blot) hybridization with probes internal to the pcbC and pcbAB genes, respectively. An open reading frame of 11,136 bp was located upstream of the pcbC gene that matched the 11.4-kb transcript initiation and termination regions. It encoded a protein of 3,712 amino acids with a deduced Mr of 414,791. The nucleotide sequence of the gene showed 62.9% similarity to the pcbAB gene encoding the ACV synthetase of P. chrysogenum; 54.9% of the amino acids were identical in both ACV synthetases. Three highly repetitive regions occur in the deduced amino acid sequence of C. acremonium ACV synthetase. Each is similar to the three repetitive domains in the deduced sequence of P. chrysogenum ACV synthetase and also to the amino acid sequence of gramicidin synthetase I and tyrocidine synthetase I of Bacillus brevis. These regions probably correspond to amino acid activating domains in the ACV synthetase protein. In addition, a thioesterase domain was present in the ACV synthetases of both fungi. A similarity has been found between the domains existing in multienzyme nonribosomal peptide synthetases and polyketide and fatty acid synthetases. The pcbAB gene is linked to the pcbC gene, forming a cluster of early cephalosporin-biosynthetic genes.

The isopenicillin-N acyltransferase of Penicillium chrysogenum catalyzes the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin-N acyltransferase copurified with the acyl-CoA :6-aminopenicillanic acid (6-APA) acyltransferase activity which transfers an acyl residue from acyl-CoA derivatives (e.g. phenylacetyl-CoA, phenoxyacetyl-CoA) to 6-APA. Other thioesters of phenylacetic acid were also used as substrates. An amino acid sequence similar to that of the active site of thioesterases was found in the isopenicillin-N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenylacetyl thioesters. Purified isopenicillin-N acyltransferase also showed isopenicillin-N amidohydrolase, penicillin transacylase and penicillin amidase activities. The isopenicillin-N amidohydrolase (releasing 6-APA) showed a much lower specific activity than the isopenicillin-N acyltransferase of the same enzyme preparation, suggesting that in the isopenicillin-N acyltransferase reaction the 6-APA is not released and is directly converted into benzylpenicillin. Penicillin transacylase exchanged side chains between two hydrophobic penicillin molecules ; or between one penicillin molecule and 6-APA. The penicillin amidase activity is probably the reverse of the biosynthetic acyl-CoA :6-APA acyltransferase. Four F? chrysogenum mutants deficient in acyl-CoA :6-APA acyltransferase lacked the other four related activities. Transformation of these mutants with the penDE gene restored all five enzyme activities.The characteristic penam nucleus of penicillin, consisting of condensed B-lactam and thiazolidine rings, is formed by cyclization of the tripeptide 6-(L-a-aminoadipy1)-L-cysteinyl-D-valine to form isopenicillin N (IPN) (an antibiotically active intermediate in the biosynthetic pathway) which contains an L-a-aminoadipyl side chain [l, 21. Some fungi including Penicillium chrysogenum and Aspergillus nidulans, but not others (e.g. Cephalosporium acremonium, syn. Acremonium chrysogenum), have the ability to replace the aaminoadipyl side chain of IPN with non-polar side chains such as phenylacetyl and phenoxyacetyl groups when the corresponding acids are added to the culture medium. In the absence of exogenous precursors of the side chain the aaminoadipyl side chain of IPN can be removed to form 6-aminopenicillanic acid (6-APA) [3] which is accumulated by some penicillin-producing strains [4].

The organization of the genes of the penicillin cluster has been studied in three different mutants of P. chrysogenum impaired in penicillin biosynthesis. The three blocked mutants (derived from the parental strain P. chrysogenum Bb-1) lacked the genes pcbAB, pcbC and penDE of the penicillin biosynthetic pathway and were unable to form isopenicillin N synthase and isopenicillin N acyltransferase. All strains were identified as P. chrysogenum derivatives by fingerprinting analysis with (GTG)n as a probe. The borders of the deleted region were cloned and sequenced, showing the same junction point in the three mutants. The deleted DNA region was found to be identical to that described in P. chrysogenum npe10. The frequent deletion of the pen gene cluster at this point may indicate that this cluster is located in an unstable genetic region, flanked by hot spots of recombination, that is easily lost by mutagen-induced recombination.

The penDE gene encoding acyl-CoA:6-amino penicillanic acid acyltransferase (AAT), the last enzyme of the penicillin biosynthetic pathway, has been cloned from the DNA of Aspergillus nidulans. The gene contains three introns which are located in the 5' region of the open reading frame. It encodes a protein of 357 amino acids with a molecular weight of 39,240 Da. The penDE gene of A. nidulans shows 73% similarity at the nucleotide level with the penDE gene of Penicillium chrysogenum. The A. nidulans gene was expressed in P. chrysogenum and complemented the AAT deficiency of the non-producer mutants of P. chrysogenum, npe6 and npe8. The penDE gene of A. nidulans is linked to the pcbC gene, which encodes the isopenicillin N synthase, as also occurs in P. chrysogenum. Both genes show the same orientation and are separated by an intergenic region of 822 nucleotides.

Saccharomyces cerevisiae cultures exposed to 110 mT and 220 mT steady magnetic fields (SMF) were studied to observe eventual induced growth alterations and changes in metabolic activity. Cell mass (biomass) growth was evaluated by light spectrometry, and metabolic alterations were estimated on the basis of the CO2 pressure produced and by the culture media pH changes, measured at the beginning and the end of the observation. The yeast strain DAUFPE-1012, cultivated in a nonaerated liquid agar Sabouraud glucose medium, was exposed to SMF generated by NdFeBr magnets. Results showed alterations induced by 220 mT SMF as an increment in cell proliferation (1.84%) and an increased CO2 production (36.1%) as compared to control groups. Furthermore, the initial-to-final pH difference in 220 mT SMF exposed cultures was higher than the 110 mT SMF and the control values. The whole acidification and the rise in CO2 production observed after 220 mT SMF exposure did not correspond to the biomass growth values, as compared to the other cultures, and was apparently provoked by a enhancement in the cellular metabolic rate. This technique becomes very promising for future biotechnological applications in fermentative processes.