bZIP transcription factors PcYap1 and PcRsmA link oxidative stress response to secondary metabolism and development in Penicillium chrysogenum.

BACKGROUND: Reactive oxygen species (ROS) trigger different morphogenic processes in filamentous fungi and have been shown to play a role in the regulation of the biosynthesis of some secondary metabolites. Some bZIP transcription factors, such as Yap1, AtfA and AtfB, mediate resistance to oxidative stress and have a role in secondary metabolism regulation. In this work we aimed to get insight into the molecular basis of this regulation in the industrially important fungus Penicillium chrysogenum through the characterization of the role played by two effectors that mediate the oxidative stress response in development and secondary metabolism. RESULTS: In P. chrysogenum, penicillin biosynthesis and conidiation are stimulated by the addition of H2O2 to the culture medium, and this effect is mediated by the bZIP transcription factors PcYap1 and PcRsmA. Silencing of expression of both proteins by RNAi resulted in similar phenotypes, characterized by increased levels of ROS in the cell, reduced conidiation, higher sensitivity of conidia to H2O2 and a decrease in penicillin production. Both PcYap1 and PcRsmA are able to sense H2O2-generated ROS in vitro and change its conformation in response to this stimulus. PcYap1 and PcRsmA positively regulate the expression of brlA, the first gene of the conidiation central regulatory pathway. PcYap1 binds in vitro to a previously identified regulatory sequence in the promoter of the penicillin gene pcbAB: TTAGTAA, and to a TTACTAA sequence in the promoter of the brlA gene, whereas PcRsmA binds to the sequences TGAGACA and TTACGTAA (CRE motif) in the promoters of the pcbAB and penDE genes, respectively. CONCLUSIONS: bZIP transcription factors PcYap1 and PcRsmA respond to the presence of H2O2-generated ROS and regulate oxidative stress response in the cell. Both proteins mediate ROS regulation of penicillin biosynthesis and conidiation by binding to specific regulatory elements in the promoters of key genes. PcYap1 is identified as the previously proposed transcription factor PTA1 (Penicillin Transcriptional Activator 1), which binds to the regulatory sequence TTAGTAA in the pcbAB gene promoter. This is the first report of a Yap1 protein directly regulating transcription of a secondary metabolism gene. A model describing the regulatory network mediated by PcYap1 and PcRsmA is proposed.

A natural short pathway synthesizes roquefortine C but not meleagrin in three different Penicillium roqueforti strains.

The production of mycotoxins and other secondary metabolites in Penicillium roqueforti is of great interest because of its long history of use in blue-veined cheese manufacture. In this article, we report the cloning and characterization of the roquefortine gene cluster in three different P. roqueforti strains isolated from blue cheese in the USA (the type strain), France, and the UK (Cheshire cheese). All three strains showed an identical roquefortine gene cluster organization and almost identical (98-99%) gene nucleotide sequences in the entire 16.6-kb cluster region. When compared with the Penicillium chrysogenum roquefortine/meleagrin seven-gene cluster, the P. roqueforti roquefortine cluster contains only four genes (rds, rdh, rpt, and gmt) encoding the roquefortine dipeptide synthetase, roquefortine D dehydrogenase, roquefortine prenyltransferase, and a methyltransferase, respectively. Silencing of the rds or rpt genes by the RNAi strategy reduced roquefortine C production by 50% confirming the involvement of these two key genes in roquefortine biosynthesis. An additional putative gene, orthologous of the MFS transporter roqT, is rearranged in all three strains as a pseudogene. The same four genes and a complete (not rearranged) roqT, encoding a MFS transporter containing 12 TMS domains, occur in the seven-gene cluster in P. chrysogenum although organized differently. Interestingly, the two "late" genes of the P. chrysogenum roquefortine/meleagrin gene cluster that convert roquefortine C to glandicoline B and meleagrin are absent in the P. roqueforti four-gene cluster. No meleagrin production was detected in P. roqueforti cultures grown in YES medium, while P. chrysogenum produces meleagrin in these conditions. No orthologous genes of the two missing meleagrin synthesizing genes were found elsewhere in the recently released P. roqueforti genome. Our data suggest that during evolution, the seven-gene cluster present in P. chrysogenum, and probably also in other glandicoline/meleagrin producing fungi, has been trimmed down to a short cluster in P. roqueforti leading to the synthesis of roquefortine C rather than meleagrin as a final product.

Autonomously replicating plasmids carrying the AMA1 region in Penicillium chrysogenum.

Plasmid vectors containing the AMA1 sequence transformed with high efficiency and replicated autonomously in Penicillium chrysogenum. The efficiency of transformation of P. chrysogenum was related to the length of the AMA1 fragment used for constructing the different autonomously replicating plasmids. One of the two palindromic inverted repeats of AMA1 (the 2.2-kb SalI-HindIII fragment) is sufficient to confer autonomous replication and a high transformation efficiency. Deletion of the 0.6-kb central fragment located between the inverted repeats did not affect either the ability of the plasmids to replicate autonomously or the efficiency of transformation, but did alter the mitotic stability and the plasmid copy number. Deletion of any fragment of the 2.2-kb repeat caused the loss of the ability to replicate autonomously and reduced the transformation efficiency. Most of the transformants retained the original plasmid configuration, as multimers and without reorganization, after several rounds of autonomous replication. The AMA1 region works as an origin of replication in P. chrysogenum and A. nidulans but not apparently in Acremonium chrysogenum.

A moderate amplification of the mecB gene encoding cystathionine-gamma-lyase stimulates cephalosporin biosynthesis in Acremonium chrysogenum.

L-cysteine is a precursor of the penicillin, cephalosporin and cephamycin families of beta-lactam antibiotics. Cystathionine-gamma-lyase (encoded by the mecB gene), an enzyme that splits cystathionine releasing cysteine, is required for high-level cephalosporin production in methionine-supplemented medium. By amplification of the mecB gene in Acremonium chrysogenum C10, several transformants were obtained that produced 10-40% higher levels of cephalosporin. All selected transformants contained at least two or three copies of the mecB gene as shown by Southern hybridization with a probe internal to mecB. Two of these transformants, A. chrysogenum T27 and A. chrysogenum T58, showed 4- to 10-fold higher cystathionine-gamma-lyase activity than the control strain. Northern hybridizations indicated that the levels of the two mecB transcripts of 1.7 and 1.5 kb were greatly increased in transformants T27 and T58. Fermentor studies using controlled conditions confirmed that transformant T27 was a cephalosporin overproducer, reaching titers of nearly 2000 microg/ml of cephalosporin in Shen-defined medium that correlated with two- to fourfold higher cystathionine-gamma-lyase levels than in the control strain. Transformant T58 containing five- to sixfold higher levels of cystathionine-gamma-lyase in fermentor cultures showed a reduced growth rate and a slow cephalosporin accumulation rate. In conclusion, moderately increased levels of cystathionine-gamma-lyase stimulated cephalosporin production but very high levels of this enzyme were deleterious for growth and cephalosporin biosynthesis.

Penicillin and cephalosporin biosynthesis: mechanism of carbon catabolite regulation of penicillin production.

Penicillins and cephalosporins are synthesized by a series of enzymatic reactions that form the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and convert this tripeptide into the final penicillin or cephalosporin molecules. One of the enzymes, isopenicillin N synthase has been crystallyzed and its active center identified. The three genes pcbAB, pcbC and penDE involved in penicillin biosynthesis are clustered in Penicillium chrysogenum, Aspergillus nidulans and Penicillium nalgiovense. Carbon catabolite regulation of penicillin biosynthesis is exerted by glucose and other easily utilizable carbon sources but not by lactose. The glucose effect is enhanced by high phosphate concentrations. Glucose represses the biosynthesis of penicillin by preventing the formation of the penicillin biosynthesis enzymes. Transcription of the pcbAB, pcbC and penDE genes of P. chrysogenum is strongly repressed by glucose and the repression is not reversed by alkaline pHs. Carbon catabolite repression of penicillin biosynthesis in A. nidulans is not mediated by CreA and the same appears to be true in P. chrysogenum. The first two genes of the penicillin pathway (pcbAB and pcbC) are expressed from a bidirectional promoter region. Analysis of different DNA fragments of this bidirectional promoter region revealed two important DNA sequences (boxes A and B) for expression and glucose catabolite regulation of the pcbAB gene. Using protein extracts from mycelia grown under carbon catabolite repressing or derepressing conditions DNA-binding proteins that interact with the bidirectional promoter region were purified to near homogeneity.

Expression of genes and processing of enzymes for the biosynthesis of penicillins and cephalosporins.

The genes pcbAB, pcbC and penDE encoding the enzymes (alpha-aminoadipyl-cysteinyl-valine synthetase, isopenicillin N synthase and isopenicillin N acyltransferase, respectively) involved in the biosynthesis of penicillin have been cloned from Penicillin chrysogenum and Aspergillus nidulans. They are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of Penicillium notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. Each gene is expressed as a single transcript from separate promoters. Enzyme regulation studies and gene expression analysis have provided useful information to understand the control of genes involved in penicillin biosynthesis. The enzyme isopenicillin N acyltransferase encoded by the penDE gene is synthesized as a 40 kDa protein that is (self)processed into two subunits of 29 and 11 kDa. Both subunits appear to be required for acyl-CoA 6-APA acyltransferase activity. The isopenicillin N acyltransferase was shown to be located in microbodies, whereas the isopenicillin N synthase has been reported to be present in vesicles of the Golgi body and in the cell wall. A mutant in the carboxyl-terminal region of the isopenicillin N acyltransferase lacking the three final amino acids of the enzymes was not properly located in the microbodies and failed to synthesize penicillin in vivo. In C. acremonium the genes involved in cephalosporin biosynthesis are separated in at least two clusters. Cluster I (pcbAB-pcbC) encodes the first two enzymes (alpha-aminoadipyl-cysteinyl) valine synthetase and isopenicillin N synthase) of the cephalosporin pathway which are very similar to those involved in penicillin biosynthesis. Cluster II (cefEF-cefG), encodes the last three enzymatic activities (deacetoxycephalosporin C synthetase/hydroxylase and deacetylcephalosporin C acetyltransferase) of the cephalosporin pathway. It is unknown, at this time, if the cefD gene encoding isopenicillin epimerase is linked to any of these two clusters. Methionine stimulates cephalosporin biosynthesis in cultures of three different strains of A. chrysogenum. Methionine increases the levels of enzymes (isopenicillin N synthase and deacetylcephalosporin C acetyltransferase) expressed from genes (pcbC and cefG respectively) which are separated in the two different clusters of cephalosporin biosynthesis genes. This result suggests that both clusters of genes have regulatory elements which are activated by methionine. Methionine-supplemented cells showed higher levels of transcripts of the pcbAB, pcbC, cefEF genes and to a lesser extent of cefG than cells grown in absence of methionine. The levels of the cefG transcript were very low as compared to those of pcbAB, pcbC and cefEF.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel heptameric sequence (TTAGTAA) is the binding site for a protein required for high level expression of pcbAB, the first gene of the penicillin biosynthesis in Penicillium chrysogenum.

The first two genes pcbAB and pcbC of the penicillin biosynthesis pathway are expressed from a 1.01-kilobase bidirectional promoter region. A series of sequential deletions were made in the pcbAB promoter region, and the constructions with the modified promoters coupled to the lacZ reporter gene were introduced as single copies at the pyrG locus in Penicillium chrysogenum npe10. Three regions, boxes A, B, and C, produced a significant decrease in expression of the reporter gene when deleted. Protein-DNA complexes were observed by using the electrophoretic mobility shift assay with boxes A and B (complexes AG1, BG1, BG2, and BL1) but not with box C. Uracil interference assay showed that a protein in P. chrysogenum cell extracts interacts with the thymines in a palindromic heptanucleotide TTAGTAA. Point mutations and deletion of the entire TTAGTAA sequence supported the involvement of this sequence in the binding of a transcriptional activator named penicillin transcriptional activator 1 (PTA1). In vivo studies using constructions carrying point mutations in the TTAGTAA sequence (or a deletion of the complete heptanucleotide) confirmed that this intact sequence is required for high level expression of the pcbAB gene. The TTAGTAA sequence resembles the target sequence of BAS2 (PHO2), a factor required for expression of several genes in yeasts.

Characterization of the reverse transsulfuration gene mecB of Acremonium chrysogenum, which encodes a functional cystathionine-gamma-lyase.

In Acremonium chrysogenum, biosynthesis of cysteine for the formation of cephalosporin has been proposed to occur through the reverse transsulfuration pathway. A gene, named mecB, has been cloned from an A. chrysogenum C10 genomic library in lambdaEMBL3-ble. The cloned DNA fragment encodes a protein of 423 amino acids with a deduced molecular mass of 45 kDa that shows great similarity to cystathionine-gamma-lyases from Saccharomyces cerevisiae and other eukaryotic organisms. The protein was shown to be functional because it restores growth on methionine to A. nidulans C47 (mecB10), a mutant that is known to be defective in cystathionine-gamma-lyase. The cloned gene did not complement A. nidulans mecA or metG mutants. Enzyme activity assays confirmed that the cloned mecB gene encodes a cystathionine-gamma-lyase activity. The mecB gene is present in a single copy in the wild-type A. chrysogenum (Brotzu's strain) and also in the A. chrysogenum strain C10, a high cephalosporin producer. The gene is localized on chromosome VIII (5.3 Mb), as shown by hybridization to A. chrysogenum chromosomes resolved by pulsed-field gel electrophoresis. Transcription of the mecB gene gives rise to a major transcript of 1.5 kb and a minor one of 1.7 kb. The transcript levels were not significantly affected by addition of DL-methionine to the culture, indicating that expression of this gene is not regulated by methionine. The availability of this gene provides a very useful tool for understanding the proposed role of cystathionine-gamma-lyase in splitting cystathionine to supply cysteine for cephalosporin biosynthesis.