Jesus Manuel Cantoral

Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78

SummaryA gene (ips) encoding the isopenicillin N synthase of Penicillium chrysogenum AS-P-78 was cloned in a 3.9 kb SalI fragment using a probe corresponding to the aminoterminal end of the enzyme. The SalI fragment was trimmed down to a 1.3 kb NcoI-BglII fragment that contained an open reading frame of 996 nucleotides encoding a polypeptide of 331 amino acids with an Mr of 38012 dalton. The predicted polypeptide encoded by the ips gene of strain AS-P-78 contains a tyrosine at position 195, whereas the gene of the high penicillin producing strain 23X-80-269-37-2 shows an isoleucine at the same position. The ips gene is expressed in Escherichia coli minicells using the λ phage PL promoter. Some similar sequence motifs were found in the upstream region of the ips gene of P. chrysogenum when compared with the upstream sequences of the ips genes of Cephalosporium acremonium and Aspergillus nidulans. Primer extension studies indicated that the start of the mRNA coincides with a T in position-11 which is located in a conserved pyrimidine-rich sequence, near two CAAG boxes. Clones of P. chrysogenum Wis 54–1255 transformed with the ips gene showed a five-fold higher isopenicillin N synthase activity than the untransformed cultures.

Selection and characterization of pyrG mutants of Penicillium chrysogenum lacking orotidine-5′-phosphate decarboxylase and complementation by the pyr4 gene of Neurospora crassaa

SummaryPyrimidine auxotrophs of Penicillium chrysogenum have been isolated at a high frequency among mutants resistant to 5-fluoroorotic acid (5.2 mM). Some of the pyrimidine auxotrophs (e.g. strain pyrG1) showed no reversion. A radiometric assay based on the conversion of (6-14C)orotidine 5′-monophosphate (OMP) into (6-14C)uridine 5′-monophosphate (UMP) was developed to determine OMP-decarboxylase activity. One of the pyrimidine auxotrophs (P. chrysogenum pyrGl) was studied in detail. It was deficient in OMP-decarboxylase activity, whereas the parental strain (P. chrysogenum Wis. 54-1255) showed a normal enzyme activity. A five-fold higher OMP-decarboxylase activity was found in a P. chrysogenum pyrGI clone transformed with plasmids containing the Neurospora crassa pyr4 gene (which codes for the same enzyme).

Two genes involved in penicillin biosynthesis are linked in a 5.1 kb SalI fragment in the genome of Penicillium chrysogenum

SummaryTwo genes, pcbC and penDE (also named ips and aat, respectively) encoding the enzymes isopenicillin N synthase and acyl-CoA:6-amino penicillanic acid (6-APA) acyltransferase, which are involved in the penicillin biosynthetic pathway in Penicillium chrysogenum, were cloned. Both genes are clustered together in a 5.1 kb SalI DNA fragment and are separated by a nontranscribed intergenic region of 1.5 kb. These genes are transcribed from different promoters in two separate transcripts of about 1.15 kb each. The penDE gene complements mutants of P. chrysogenum deficient in acyltransferase and the pcbC gene increases the level of isopenicillin N synthase in strains containing low levels of this enzyme. The clustering of penicillin biosynthetic genes is of great interest in the light of previous claims of horizontal transfer of the pcbC gene from β-lactam producing Streptomyces to filamentous fungi.

Formation of bulges associated with penicillin production in high-producing strains ofPenicillium chrysogenum

Bulges were formed in the hyphae of a high penicillin-producing strain ofPenicillium chrysogenum AS-P-78 when penicillin was accumulated in the broth. The mycelium of cultures grown in the presence of 50 mM lysine, which specifically inhibits penicillin formation, showed a greatly reduced number of bulges. The size and number of bulges increased during the fermentation in parallel with penicillin accumulation. A smaller number of bulges was formed in the mycelium of the low-producing strain Wis 54-1255 than in the high-producing mutant. Three mutants blocked in penicillin biosynthesis did not form these globose structures. Bulges were not osmotically sensitive, although some of them burst out. They may be formed by weakening of the cell wall of hyphae following accumulation of high concentrations of penicillin.