Ana T. Marcos

Expression of the cefG gene is limiting for cephalosporin biosynthesis in Acremonium chrysogenum

Abstract The conversion of deacetylcephalosporin C to cephalosporin C is inefficient in most Acremonium chrysogenum strains. The cefG gene, which encodes deacetylcephalosporin C acetyltransferase, is expressed very poorly in A. chrysogenum as compared to other genes of the cephalosporin pathway. Introduction of additional copies of the cefG gene with its native promoter (in two different constructions with upstream regions of 1056 bp and 538 bp respectively) did not produce a significant increase of the steady-state level of the cefG transcript. Expression of the cefG gene from the promoters of (i) the glyceraldehyde-3-phosphate dehydrogenase (gpd ) gene of Aspergillus nidulans, (ii) the glucoamylase (gla) gene of Aspergillus niger, (iii) the glutamate dehydrogenase (gdh) and (iv) the isopenicillin N synthase ( pcbC ) genes of Penicillium chrysogenum, led to very high steady-state levels of cefG transcript and to increased deacetylcephalosporin-C acetyltransferase protein concentration (as shown by immunoblotting) and enzyme activity in the transformants. Southern analysis showed that integration of the new constructions occurred at sites different from that of the endogenous cefG gene. Cephalosporin production was increased two- to threefold in A. chrysogenum C10 transformed with constructions in which the cefG gene was expressed from the gdh or gpd promoters as a result of a more efficient acetylation of deacetylcephalosporin C.

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 δ-(L-α-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.

Transcription of the pcbAB, pcbC and penDE genes of Penicillium chrysogenum AS-P-78 is repressed by glucose and the repression is not reversed by alkaline pHs.

Glucose repressed transcription of the penicillin biosynthesis genes pcbAB, pcbC and penDE when added at inoculation time to cultures of Penicillium chrysogenum AS-P-78 but it had little repressive effect when added at 12 h and no effect when added at 24 or 36 h. A slight increase in the expression of pcbC and penDE (and to a smaller extent of pcbAB) was observed in glucose-grown cultures at pH 6.8, 7.4 and 8.0 as compared with pH 6.2, but alkaline pHs did not override the strong repression exerted by glucose. Transcription of the actin gene used as control was not significantly affected by glucose or alkaline pHs. Repression by glucose of the three penicillin biosynthetic genes was also observed using the lacZ reporter gene coupled to each of the three promoters in monocopy transformants with the constructions integrated at the pyrG locus. Glucose repression of the three genes encoding enzymes of penicillin biosynthesis therefore appears to be exerted by a regulatory mechanism independent from pH regulation.

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 andpcbC 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.

The Histone Acetyltransferase GcnE (GCN5) Plays a Central Role in the Regulation of Aspergillus Asexual Development

Acetylation of histones is a key regulatory mechanism of gene expression in eukaryotes. GcnE is an acetyltransferase of Aspergillus nidulans involved in the acetylation of histone H3 at lysine 9 and lysine 14. Previous works have demonstrated that deletion of gcnE results in defects in primary and secondary metabolism. Here we unveil the role of GcnE in development and show that a ∆gcnE mutant strain has minor growth defects but is impaired in normal conidiophore development. No signs of conidiation were found after 3 days of incubation, and immature and aberrant conidiophores were found after 1 week of incubation. Centroid linkage clustering and principal component (PC) analysis of transcriptomic data suggest that GcnE occupies a central position in Aspergillus developmental regulation and that it is essential for inducing conidiation genes. GcnE function was found to be required for the acetylation of histone H3K9/K14 at the promoter of the master regulator of conidiation, brlA, as well as at the promoters of the upstream developmental regulators of conidiation flbA, flbB, flbC, and flbD (fluffy genes). However, analysis of the gene expression of brlA and the fluffy genes revealed that the lack of conidiation originated in a complete absence of brlA expression in the ∆gcnE strain. Ectopic induction of brlA from a heterologous alcA promoter did not remediate the conidiation defects in the ∆gcnE strain, suggesting that additional GcnE-mediated mechanisms must operate. Therefore, we conclude that GcnE is the only nonessential histone modifier with a strong role in fungal development found so far.

Nitric oxide synthesis by nitrate reductase is regulated during development in Aspergillus

Nitric oxide (NO) is a signalling molecule involved in many biological processes in bacteria, plants and mammals. However, little is known about the role and biosynthesis of NO in fungi. Here we show that NO production is increased at the early stages of the transition from vegetative growth to development in Aspergillus nidulans. Full NO production requires a functional nitrate reductase (NR) gene (niaD) that is upregulated upon induction of conidiation, even under N‐repressing conditions in the presence of ammonium. At this stage, NO homeostasis is achieved by balancing biosynthesis (NR) and catabolism (flavohaemoglobins). niaD and flavohaemoglobin fhbA are transiently upregulated upon induction of conidiation, and both regulators AreA and NirA are necessary for this transcriptional response. The second flavohaemoglobin gene fhbB shows a different expression profile being moderately expressed during the early stages of the transition phase from vegetative growth to conidiation, but it is strongly induced 24 h later. NO levels influence the balance between conidiation and sexual reproduction because artificial strong elevation of NO levels reduced conidiation and induced the formation of cleistothecia. The nitrate‐independent and nitrogen metabolite repression‐insensitive transcriptional upregulation of niaD during conidiation suggests a novel role for NR in linking metabolism and development.

The galE gene encoding the UDP-galactose 4-epimerase of Brevibacterium lactofermentum is coupled transcriptionally to the dmdR gene

The galE gene of Brevibacterium lactofermentum, encoding UDP-galactose 4-epimerase (EC 5.1.3.2), has been identified by DNA sequencing downstream from the orf1-sigB-dmdR region. The arrangement of the sigB-dtxR-galE cluster is also conserved in Corynebacterium diphtheriae. The deduced galE product was a protein of 329 aa residues (35.4 kDa) that shared a high degree of identity to known UDP-galactose 4-epimerase proteins from Gram-positive microorganisms (Streptomyces lividans and Streptococcus thermophilus). Transcriptional analysis of the dmdR and galE genes in nutrient-rich medium showed that these genes are part of an operon, that is actively transcribed as a bicistronic mRNA during the exponential growth phase, but transcription of the operon is decreased during the stationary growth phase. In addition, the dmdR gene was also expressed as a monocistronic 0.7-kb transcript during the active growth phase.

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

The genespcbAB,pcbC andpenDE encoding the enzymes (α-aminoadipyl-cysteinyl-valine synthetase, isopenicillin N synthase and isopenicillin N acyltransferase, respectively) involved in the biosynthesis of penicillin have been cloned fromPenicillium chrysogenum andAspergillus nidulans. They are clustered in chromosome I (10.4 Mb) ofP. chrysogenum, in chromosome II ofPenicillium notatum (9.6 Mb) and in chromosome VI (3.0 Mb) ofA. 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 thepenDE 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. InC. acremonium the genes involved in cephalosporin biosynthesis are separated in at least two clusters. Cluster I (pcbAB-pcbC) encodes the first two enzymes (α-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 thecefD gene encoding isopenicillin epimerase is linked to any of these two clusters. Methionine stimulates cephalosporin biosynthesis in cultures of three different strains ofA. chrysogenum. Methionine increases the levels of enzymes (isopenicillin N synthase and deacetylcephalosporin C acetyltransferase) expressed from genes (pcbC andcefG 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 thepcbAB,pcbC,cefEF genes and to a lesser extent ofcefG than cells grown in absence of methionine. The levels of thecefG transcript were very low as compared to those ofpcbAB,pcbC andcefEF. The induction by methionine of transcription of the four cephalosporin biosynthesis genes and the known effect of this amino acid on differentiation ofA. chrysogenum indicates that methionine exerts a pleiotropic effect that regulates coordinately cephalosporin biosynthesis and differentiation.

Structural and Phylogenetic Analysis of the γ-Actin Encoding Gene from the Penicillin-Producing Fungus Penicillium chrysogenum

The nucleotide sequence of a 2994-bp genomic fragment, including the γ-actin encoding gene from Penicillium chrysogenum, has been determined, showing an open reading frame (ORF) of 1756 bp interrupted by five introns with fungal consensus splice-site junctions. The 5′ untranslated region contains a consensus TATA box, five CAAT motifs, and two large pyrimidine stretches. The predicted protein (375 amino acids) revealed high identity to γ-actins from fungi (>90%), and gene phylogenies support the grouping of P. chrysogenum actin close to those from the majority of the filamentous fungi. The actA gene is present as a single copy in the genome of P. chrysogenum, and its expression is constitutive during penicillin fermentation, showing a single 1.4-kb transcript.

KdmA, a histone H3 demethylase with bipartite function, differentially regulates primary and secondary metabolism in Aspergillus nidulans

Aspergillus nidulans kdmA encodes a member of the KDM4 family of jumonji histone demethylase proteins, highly similar to metazoan orthologues both within functional domains and in domain architecture. This family of proteins exhibits demethylase activity towards lysines 9 and 36 of histone H3 and plays a prominent role in gene expression and chromosome structure in many species. Mass spectrometry mapping of A. nidulans histones revealed that around 3% of bulk histone H3 carried trimethylated H3K9 (H3K9me3) but more than 90% of histones carried either H3K36me2 or H3K36me3. KdmA functions as H3K36me3 demethylase and has roles in transcriptional regulation. Genetic manipulation of KdmA levels is tolerated without obvious effect in most conditions, but strong phenotypes are evident under various conditions of stress. Transcriptome analysis revealed that – in submerged early and late cultures – between 25% and 30% of the genome is under KdmA influence respectively. Transcriptional imbalance in the kdmA deletion mutant may contribute to the lethal phenotype observed upon exposure of mutant cells to low‐density visible light on solid medium. Although KdmA acts as transcriptional co‐repressor of primary metabolism genes, it is required for full expression of several genes involved in biosynthesis of secondary metabolites.