Strain improvement of Penicillium chrysogenum by recombinant DNA techniques.

The penDE gene from Penicillium chrysogenum has been isolated; the gene is located in close vicinity of the pcbC gene. Amplification of the pcbC-penDE gene cluster in Penicillium chrysogenum Wis54-1255 leads to a significant increase in penicillin production. In selected transformants an increase of up to 40% is observed.     

Analysis of promoter activity by transformation of Acremonium chrysogenum.

Promoter activity was examined in the beta-lactam-producing fungus, Acremonium chrysogenum, by assessment of the properties of transformant isolates. Transformation was achieved using plasmid constructs specifying hygromycin B resistance (HyR) linked to the promoter elements of gpdA (the glucose-6-phosphate dehydrogenase-encoding gene of Aspergillus nidulans), and pcbC [the gene encoding the isopenicillin N synthetase (IPNS) enzyme of A. chrysogenum]. Transformation frequency, HyR levels, and Hy phosphotransferase (HPT) levels suggested that the transformants of constructs using the gpdA promoter showed a higher level of expression of the HyR gene than in transformants obtained using the pcbC promoter. The patterns of integration of the transforming DNA also differed in that pcbC promoter construct transformants appeared to have tandem repeats. All integrations of plasmid DNA occurred on a single chromosome which was different in four out of five transformants studied. Multiple copy transformants of constructs using the pcbC promoter did not show the regulated pattern of expression of HPT activity observed with IPNS in untransformed strains.     

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

A 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 amino-terminal 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 lambda 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.     

Quantitative analysis of Penicillium chrysogenum Wis54-1255 transformants overexpressing the penicillin biosynthetic genes

The low penicillin-producing, single gene copy strain Wis54-1255 was used to study the effect of overexpressing the penicillin biosynthetic genes in Penicillium chrysogenum. Transformants of Wis54-1255 were obtained with the amdS expression-cassette using the four combinations: pcbAB, pcbC, pcbC-penDE, and pcbAB-pcbC-penDE of the three penicillin biosynthetic genes. Transformants showing an increased penicillin production were investigated during steady-state continuous cultivations with glucose as the growth-limiting substrate. The transformants were characterized with respect to specific penicillin productivity, the activity of the two pathway enzymes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthetase (IPNS) and the intracellular concentration of the metabolites: delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV), isopenicillin N (IPN), glutathione (GSH), and glutathione disulphide (GSSG). Transformants with the whole gene cluster amplified showed the largest increase in specific penicillin productivity (r(p))-124% and 176%, respectively, whereas transformation with the pcbC-penDE gene fragment resulted in a decrease in r(p) of 9% relative to Wis54-1255. A marked increase in r(p) is clearly correlated with a balanced amplification of both the ACVS and IPNS activity or a large amplification of either enzyme activity. The increased capacity of a single enzyme occurs surprisingly only in the transformants where all the three biosynthetic genes are overexpressed but is not found within the group of pcbAB or pcbC transformants. The indication of the pcbAB and pcbC genes being closely regulated in fungi might explain why high-yielding strains of P. chrysogenum have been found to contain amplifications of a large region including the whole penicillin gene cluster and not single gene amplifications. Measurements of the total ACV concentration showed a large span of variability, which reflected the individual status of enzyme overexpression and activity found in each strain. The ratio ACV:bisACV remained constant, also at high ACV concentrations, indicating no limitation in the capacity of the thioredoxin-thioredoxin reductase (TR) system, which is assumed to keep the pathway intermediate LLD-ACV in its reduced state. The total GSH pool was at a constant level of approx. 5.7 mM in all cultivations.     

An efficient fungal RNA-silencing system using the DsRed reporter gene

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.     

The upstream region of the IPNS gene determines expression during secondary metabolism in Aspergillus nidulans

We have constructed a translational fusion between the isopenicillin-N-synthetase-encoding gene (IPNS) of Aspergillus nidulans and the lacZ gene of Escherichia coli. Recombinant strains carrying a single copy of the fusion integrated at the IPNS locus produced beta-galactosidase (beta Gal) during secondary metabolism. Integration of the fusion at the argB locus results in a situation in which the only 5'-flanking sequences of the IPNS gene upstream from the chimeric fused gene are those included in the transforming plasmid. Such a strain still expresses beta Gal activity during secondary metabolism, showing that a DNA fragment including sequences of the IPNS gene from nt -2000 to +35 (relative to the translation start codon) still contains sufficient information to drive expression of the fusion gene during secondary metabolism.     

Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase.