Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene

An industrial Penicillium chrysogenum strain was transformed using two dominant selection markers, namely the bacterial gene for phleomycin resistance (ble) fused to a fungal promoter, and the acetamidase (amdS) gene from Aspergillus nidulans. Transformation frequencies of up to 20 transformants per microgram of DNA were obtained with the ble system. With the amdS marker the frequency was up to 120 transformants. Cotransformation was very efficient when using amdS as a selection marker. The introduction of pAN5-41B, a plasmid carrying the Escherichia coli lacZ gene fused to the strong glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter from A. nidulans, resulted in the formation of blue colonies on XGal plates indicating expression of the lacZ fusion gene in P. chrysogenum. A more detailed analysis of expression levels in several transformants showed that up to 6% of the total amount of soluble protein consists of the beta-galactosidase fusion protein.     

Transformation of Penicillium chrysogenum to sulfonamide resistance

Penicillium chrysogenum has been transformed to sulfonamide resistance by vectors containing the dihydropteroate synthetase gene from plasmid R388 controlled by the promoter and terminator sequences of the P. chrysogenum trpC gene. Transformation frequencies of four to ten transformants per microgram of vector DNA were obtained.     

High-level expression of the Streptomyces clavuligerus isopenicillin N synthase gene in Escherichia coli.

The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein.     

High-level expression of the Streptomyces clavuligerus isopenicillin N synthase gene in Escherichia coli.

The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein.     

Characterization of a loss-of-function mutation in the isopenicillin N synthetase gene of Acremonium chrysogenum

The N-2 strain of Acremonium chrysogenum accumulates the beta-lactam precursor tripeptide delta-(L-alpha-amino-adipoyl)-L-cysteinyl-D-valine and has no discernible activity for three of the cephalosporin C (Ce) biosynthetic enzymes. This phenotype is consistent with a mutation either within pcbC [the isopenicillin N synthetase (IPNS)-encoding gene] or in a pathway-regulator gene. To distinguish these possibilities we have cloned and sequenced pcbC from strain N-2. There is a single C----T mutation at nt 854 within the coding sequence, changing aa 285 from proline to leucine. An IPNS-specific monoclonal antibody recognises a catalytically inactive IPNS protein in extracts of N-2 cells. These findings suggest that strain N-2 carries a simple IPNS mutation and that IPNS or its biosynthetic product isopenicillin N is involved in regulation of the later stages of the Ce biosynthetic pathway.     

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.     

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.     

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.     

Cloning and use of sC as homologous marker for Aspergillus niger transformation

The sC sequence from Aspergillus niger was cloned and developed into a homologous marker system for genetic transformation. The coding region of the sC gene amplified by PCR from the A. niger genome was provided with Aspergillus nidulans expression signals (gpdA promoter and trpC terminator). This chimeric construct was used to successfully transform a spontaneous sC- isolate of A. niger to prototrophy. The transformants analyzed by Southern analysis showed integration of multiple copies of the transforming DNA. They also exhibited much higher ATP sulfurylase activity than the wild-type A. niger strain reinforcing the molecular data. This demonstrates the usefulness of the sCniger construct, driven by PgpdA, as a marker for A. niger transformation.     

Structure of the cutinase gene and detection of promoter activity in the 5''-flanking region by fungal transformation.

The cutinase gene from Fusarium solani f. sp. pisi (Nectria hematococa) was cloned and sequenced. Sau3A fragments of genomic DNA from the fungus were cloned in a lambda Charon 35 vector. When restriction fragments generated from the inserts were screened with 5' and 3' probes from cutinase cDNA, a 5.5-kilobase SstI fragment hybridized with both probes, suggesting the presence of the entire cutinase gene. A 2,818-base pair segment was sequenced, revealing a 690-nucleotide open reading frame that was identical to that found in the cutinase cDNA with a single 51-base pair intron. Transformation vectors were constructed containing a promoterless gene for hygromycin resistance, which was translationally fused to flanking sequences of the cutinase gene. When protoplasts and mycelia were transformed with these vectors, hygromycin-resistant transformants were obtained. Successful transformation was assessed by Southern blot analysis by using radiolabeled probes for the hygromycin resistance gene and the putative promoter. The results of Southern blot analysis indicated that the plasmid had integrated into the Fusarium genome and that the antibiotic resistance was a manifestation of the promoter activity of the cutinase flanking sequences. Transformation of Colletotrichum capsici with the same construct confirmed the promoter activity of the flanking region and the integration of the foreign DNA. Transformation and deletion analysis showed that promoter activity resided within the 360 nucleotides immediately 5' to the cutinase initiation codon.