Margarita Orejas

Signaling of Ambient pH in Aspergillus Involves a Cysteine Protease

In Aspergillus nidulans, the regulation of gene expression in response to changes in ambient pH is mediated by the PacC zinc finger transcriptional regulator. At alkaline ambient pH, PacC is proteolytically processed to a functional form serving as an activator of alkaline-expressed genes and a repressor of acid-expressed genes. This activation of PacC occurs in response to a signal mediated by the products of the pal genes. Thus, the products of the palA, -B, -C, -F, -H, and -I genes constitute an alkaline ambient pH signal transduction pathway. How the pal signal transduction pathway senses ambient pH and transduces a signal to trigger PacC processing is a fascinating unresolved problem. We have cloned and sequenced the palB gene. The predicted palB gene product has similarity to the catalytic domain of the calpain family of calcium-activated cysteine proteases. We have shown, however, that the PalB protein does not catalyze the final step of proteolytic processing of PacC.

CreA mediates repression of the regulatory gene xlnR which controls the production of xylanolytic enzymes in Aspergillus nidulans

The Aspergillus nidulans xlnR gene encodes a Zn(2)Cys(6) transcription activator necessary for the synthesis of the main xylanolytic enzymes, i.e. endo-xylanases X(22), X(24) and X(34), and beta-xilosidase XlnD. Expression of xlnR is not sufficient for induction of genes encoding the xylanolytic complex, the presence of xylose is absolutely required. It has been established previously that the wide-domain carbon catabolite repressor CreA indirectly represses xlnA (encodes X(22)) and xlnB (encodes X(24)) genes as well as exerting direct repression on xlnA. This work provides evidence that CreA-mediated indirect repression occurs through repression of xlnR: (i) the xlnR gene promoter is repressed by glucose and this repression is abolished in creA(d)30 mutant strains and (ii) deregulated expression of xlnR completely relieves glucose repression of xlnA and xlnB. Thus, CreA and XlnR form a transcriptional cascade regulating A. nidulans xylanolytic genes.

Specificity determinants of proteolytic processing of Aspergillus PacC transcription factor are remote from the processing site, and processing occurs in yeast if pH signalling is bypassed.

ABSTRACT The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of “neutrality-mimicking” pacC mutations having aspects of both acidity- and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues ∼252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur inSaccharomyces cerevisiae.

Carbon catabolite repression of the Aspergillus nidulans xlnA gene

Expression of the Aspergillus nidulans 22 kDa endoxylanase gene, xlnA, is controlled by at least three mechanisms: specific induction by xylan or xylose; carbon catabolite repression (CCR); and regulation by ambient pH. Deletion analysis of xlnA upstream sequences has identified two positively acting regions: one that mediates specific induction by xylose; and another that mediates the influence of ambient pH and contains two PacC consensus binding sites. The extreme derepressed mutation creAd30 results in considerable, although not total, loss of xlnA glucose repressibility, indicating a major role for CreA in its CCR. Three consensus CreA binding sites are present upstream of the structural gene. Point mutational analysis using reporter constructs has identified a single site, xlnA.C1, that is responsible for direct CreA repression in vivo. Using the creAd30 derepressed mutant background, our results indicate the existence of indirect repression by CreA.

Molecular characterization of a fungal secondary metabolism promoter: transcription of the Aspergillus nidulans isopenicillin N synthetase gene is modulated by upstream negative elements

The Aspergillus nidulans IPNS gene, encoding isopenicillin N synthetase, is a secondary metabolism gene. It is contiguous to, but divergently transcribed from, the ACVS gene at the penicillin gene cluster. The untranslated region between both ORFs is 872bp long. Here we present the physical and functional characterization of the IPNS transcriptional unit. Transcriptional start point (tsp) mapping reveals heterogeneity at the 5′‐end of the mRNA, with a major start at −106 relative to the initiation codon. This indicates that the actual length of the non‐transcribed intergenic region is 525bp. Functional elements in the IPNS upstream region have been defined by assaying β‐galactosidase activity in extracts from recombinant strains carrying deletion derivatives of the IPNS promoter fused to lacZ, integrated in single copy at the argB locus. Strains were grown in penicillin production broth under carbon catabolite repressing or derepressing conditions. The results of deletion analysis indicate that: (i) the IPNS promoter is mostly regulated by negative controls that act upon a high basal activity; (ii) sequential deletion of three of the negative cis‐acting elements results in a mutated promoter that is 40 times (sucrose broth) or 12 times (lactose broth) more active than the wild type; (iii) one of these negative cis‐acting elements is involved in sucrose repression. Strikingly, it is located outside the non‐transcribed 525 bp intergenic region and maps to the coding region of the divergently transcribed ACVS gene; (iv) a 5′‐del‐etion up to −56 (relative to the major tsp) contains information to provide almost half of the maximal promoter activity and allows initiation of transcription at the correct site. By using total‐protein extracts from mycelia grown under penicillin producing conditions we have detected a DNA‐binding activity that specifically shifts a promoter fragment located between −654 and −455(relative to IPNS tsp). Deletions covering this region partially abolish IPNS promoter activity. The fragment in question overlaps the ACVS tsp.

Enhanced production of a plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase catalytic domain in Saccharomyces cerevisiae.

ABSTRACT Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T73 was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount). The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a haploid laboratory strain, engineering of this key step also improved linalool yield.

Construction of a Genetically Modified Wine Yeast Strain Expressing the Aspergillus aculeatus rhaA Gene, Encoding an -L-Rhamnosidase of Enological Interest

ABSTRACT The Aspergillus aculeatus rhaA gene encoding anα -l-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain expressing a β-glucosidase, show increased content mainly of the aromatic compound linalool.

Purification and characterization of an α- l-rhamnosidase from Aspergillus nidulans

An enzyme exhibiting α‐ l‐rhamnosidase activity was purified by fractionating a culture filtrate of Aspergillus nidulans grown on l‐rhamnose as the sole carbon source. The α‐ l‐rhamnosidase was shown to be N‐glycosylated and had a molecular mass of 102 kDa, of which approximately 7% was contributed by carbohydrate. The enzyme, optimally active at pH 4·5–6 and 60 °C, had an isoelectric point of 5. With ρ‐nitrophenyl‐α‐ l‐rhamnopyranoside as the substrate it showed Km and Vmax values of 0·27 mmol l−1 and 64·6 U mg−1, respectively. The enzyme was competitively inhibited by l‐rhamnose (Ki 0·3 mmol l−1). Ca2+ (2 mmol l−1) stimulated the activity of the enzyme by 14%, whereas Mg2+ (2 mmol l−1) inhibited it by 63%. Substrate specificity studies showed the α‐ l‐rhamnosidase to be active both on α‐1,2 and α‐1,6 linkages to β‐ d‐glucosides.

Improving extracellular production of food-use enzymes from Aspergillus nidulans

Filamentous fungi, and particularly those of the genus Aspergillus, are major producers of enzymatic activities that have important applications in the food and beverage industries. Prior to the availability of transformation systems improvement of industrial production strains was largely restricted to the strategy of mutagenesis, screening and selection. Aspergillus nidulans is a genetically amenable filamentous fungus the ease of handling and analysis of which has led to its use as a model system for the investigation of eukaryotic gene regulation. Although not used industrially it is able to produce a wide variety of extracellular enzymatic activities. As a consequence of half a century of study a considerable resource of characterised mutants has been generated in conjunction with extensive genetic and molecular information on various gene regulatory systems in this micro-organism. Investigation of xylanase gene regulation in A. nidulans as a model for the production of food-use extracellular enzymes suggests strategies by which production of these enzymes in industrially useful species may be improved.

The wide-domain carbon catabolite repressor CreA indirectly controls expression of the Aspergillus nidulans xlnB gene, encoding the acidic endo-beta-(1,4)-xylanase X(24).

The Aspergillus nidulans xlnB gene, which encodes the acidic endo-beta-(1,4)-xylanase X(24), is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation creA(d)30 results in considerably elevated levels of xlnB mRNA indicates a role for the wide-domain repressor CreA in the repression of xlnB promoter (xlnBp) activity. Functional analyses of xlnBp::goxC reporter constructs show that none of the four CreA consensus target sites identified in xlnBp are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence xlnB expression by acting directly on xlnB.