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.

Heterologous expression in Aspergillus nidulans of a Trichoderma longibrachiatum endoglucanase of enological relevance.

An Aspergillus nidulans transformant expressing the Trichoderma longibrachiatum endoglucanase 1 gene (egl1) has been constructed. The extracellular production of EGL1 in different culture media has been studied, and a medium has been found in which EGL1 is the predominant extracellular protein produced. The enzymatic properties of the heterologously produced EGL1 are very similar to those of the native enzyme. Grape maceration in the presence of culture filtrate enriched in EGL1 resulted in increased release of aroma precursors, particularly in the case of aromatic grapes. Cryoscanning electron microscopy of the flesh of grapes treated with EGL1-enriched culture filtrate revealed degradation of the cell wall matrix.

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.

Apparent mRNA instability in Aspergillus nidulans and Aspergillus terreus of a heterologous cDNA encoding the major capsid antigen of Rotavirus.

Two expression plasmids designed to produce the rotaviral VP6 protein in Aspergillus nidulans and Aspergillus terreus have been constructed. In one of these plasmids the inducible A. terreus Gla1 glucoamylase gene promoter and Gla1 signal sequence are fused to the VP6 cDNA to enable induction and extracellular secretion of the final protein product; in the other, the strong, constitutive A. nidulans gpdA gene promoter has been employed. A. nidulans and A. terreus transformants containing intact copies of these plasmids have been obtained but neither intra- nor extra-cellular VP6 protein was detectable. Northern analysis indicated specific degradation of the VP6 mRNA. This lack of VP6 mRNA stability may be related to fundamental differences between the general structure of Aspergillus mRNA and that of rotavirus, including codon usage and AU/GC ratio.

Identification, cloning and sequence of the Aspergillus niger areA wide domain regulatory gene controlling nitrogen utilisation.

The gene encoding the positive-acting regulator of nitrogen metabolite repression (AREA) has been cloned and characterised from the industrially important filamentous fungus Aspergillus niger. The deduced amino acid sequence has an overall level of identity with its homologues from other fungal species which varies between 32 and 72%. This gene (areAnig) complements the A. nidulans areAr-18 loss-of-function mutation. Sequences upstream of the structural gene contain several putative GATA-type zinc finger protein-binding motifs. Northern analysis indicates the synthesis of multiple transcripts, the major species being approximately 2.95 kb and 3.1 kb. Maximal expression of areAnig is observed under conditions of nitrogen starvation and is mainly due to an increase in the level of the shorter transcript.

Molecular cloning and transcriptional regulation of the Aspergillus nidulans xlnD gene encoding a beta-xylosidase.

The xlnD gene encoding the 85-kDa beta-xylosidase was cloned from Aspergillus nidulans. The deduced primary structure of the protein exhibits considerable similarity to the primary structures of the Aspergillus niger and Trichoderma reesei beta-xylosidases and some similarity to the primary structures of the class 3 beta-glucosidases. xlnD is regulated at the transcriptional level; it is induced by xylan and D-xylose and is repressed by D-glucose. Glucose repression is mediated by the product of the creA gene. Although several binding sites for the pH regulatory protein PacC were found in the upstream regulatory region, it was not clear from a Northern analysis whether PacC is involved in transcriptional regulation of xlnD.

Opposite patterns of expression of two Aspergillus nidulans xylanase genes with respect to ambient pH.

The Aspergillus nidulans xylanase genes xlnA and xlnB are subject to regulation by ambient pH via the zinc finger transcription factor PacC. In the presence of D-xylose, xlnA is expressed under conditions of alkaline ambient pH while xlnB is expressed at acidic ambient pH. These data have been confirmed for acidity- and alkalinity-mimicking A. nidulans mutants.

Identification, isolation and sequence of the Aspergillus nidulans xlnC gene encoding the 34-kDa xylanase.

The xlnC gene encoding the 34-kDa xylanase (X34) of Aspergillus nidulans (An) has been cloned and sequenced, as has its corresponding cDNA. xlnC contains nine introns and shows considerable similarity to the xynA and xylP xylanase-encoding genes of A. kawachii (Ak) and Penicillium chrysogenum (Pc), respectively. Analysis of xylanase production in An multicopy transformants showed elevated levels of X34 and increased total xylanase activity, but no elevated production of other xylanases. Northern analysis demonstrated transcriptional induction by xylan and repression by glucose.

Regulation of acid phosphatases in an Aspergillus niger pacC disruption strain.

An Aspergillus niger strain has been constructed in which the pH-dependent regulatory gene, pacC, was disrupted. The pacC gene of A. niger, like that of A. nidulans, is involved in the regulation of acid phosphatase expression. Disruptants were identified by a reduction in acid phosphatase staining of colonies. Southern analysis demonstrated integration of the disruption plasmid at the pacC locus and Northern analysis showed that the disruption strain produced a truncated pacC mRNA of 2.2 kb (as compared to 2.8 kb in the wild type). The strain carrying the pacC disruption was used to assign the pacC gene to linkage group IV; this was confirmed by CHEF electrophoresis and Southern analysis. This strain further allowed us to determine which extracellular enzyme and transport systems are under the control of pacC in A. niger. Expression of the A. niger pacC wild-type gene and the truncated pacC gene showed that, in contrast to the auto-regulated wild-type expression, which was elevated only at alkaline pH, the truncated pacC gene was deregulated, as high-level expression occurred regardless of the pH of the culture medium. Analysis of the phosphatase spectrum by isoelectric focussing and enzyme activity staining both in the wild-type and the pacC disruptant showed that at least three acid phosphatases are regulated by the pacC. For the single alkaline phosphatase no pH regulation was observed.

Identification, cloning and analysis of the Aspergillus niger gene pacC, a wide domain regulatory gene responsive to ambient pH.

A wide domain regulatory gene implicated in modulating gene expression in response to ambient pH has been cloned and sequenced from the industrially useful filamentous fungus Aspergillus niger. This gene, pacC, is able to restore a pacC+ phenotype to A. nidulans pacCc11 and pacCc14 mutants with respect to extent of conidiation, conidial pigment intensity and acid phosphatase regulation. The pacC gene of A. niger comprises three exons, encodes a three-zinc-finger protein of 677 amino acids, and shows pH-dependent regulation of expression: mRNA levels are elevated under alkaline conditions and considerably reduced under acidic conditions. The occurrence of PacC consensus binding targets within the sequences upstream of pacC may indicate autoregulation.

Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. Molecular characterization of the acvA gene encoding the first enzyme of the penicillin biosynthetic pathway.

The Aspergillus nidulans gene (acvA) encoding the first catalytic steps of penicillin biosynthesis that result in the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), has been positively identified by matching a 15-amino acid segment of sequence obtained from an internal CNBr fragment of the purified amino-terminally blocked protein with that predicted from the DNA sequence. acvA is transcribed in the opposite orientation to ipnA (encoding isopenicillin N synthetase), with an intergenic region of 872 nucleotides. The gene has been completely sequenced at the nucleotide level and found to encode a protein of 3,770 amino acids (molecular mass, 422,486 Da). Both fast protein liquid chromatography and native gel estimates of molecular mass are consistent with this predicted molecular weight. The enzyme was identified as a glycoprotein by means of affinity blotting with concanavalin A. No evidence for the presence of introns within the acvA gene has been found. The derived amino acid sequence of ACV synthetase (ACVS) contains three homologous regions of about 585 residues, each of which displays areas of similarity with (i) adenylate-forming enzymes such as parsley 4-coumarate-CoA ligase and firefly luciferase and (ii) several multienzyme peptide synthetases, including bacterial gramicidin S synthetase 1 and tyrocidine synthetase 1. Despite these similarities, conserved cysteine residues found in the latter synthetases and thought to be essential for the thiotemplate mechanism of peptide biosynthesis have not been detected in the ACVS sequence. These observations, together with the occurrence of putative 4'-phosphopantetheine-attachment sites and a putative thioesterase site, are discussed with reference to the reaction sequence leading to production of the ACV tripeptide. We speculate that each of the homologous regions corresponds to a functional domain that recognizes one of the three substrate amino acids.

Identification and expression of the ACV synthetase gene.

The gene coding for ACV synthetase has recently been identified and cloned. Analysis of its structure and expression, along with similar studies of other genes involved in beta-lactam biosynthesis, should lead to a better understanding of the molecular basis of regulation of the pathway and the possibility of modifying yield and diversity of fungal antibiotics.

The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis.

Clones of Aspergillus nidulans genomic DNA spanning 20 kb have been isolated and shown by a combination of classical and molecular genetic means to represent the npeA locus, previously found to be one of four loci (npeA, npeB, npeC and npeD) involved in the synthesis of penicillin. As well as containing the gene encoding the second enzyme for penicillin biosynthesis, namely isopenicillin N synthetase (IPNS) (designated ipnA), our results show that these clones (pSTA200, pSTA201 and pSTA207) contain two more genes to form a cluster of three contiguous penicillin biosynthetic genes. Our evidence suggests that these genes encode delta (L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and acyl transferase (ACYT) (designated acvA and acyA respectively), the first and third enzymes required for penicillin biosynthesis, with the gene order being acvA-ipnA-acyA. Transcripts have been identified for the three genes and their approximate sizes determined--acvA 9.5 kb, ipnA 1.4 kb and acyA 1.6 kb. All three mRNA species are observed in cells grown in fermentation medium but not in cells grown in minimal medium, suggesting that the control of penicillin biosynthesis is, in part, at the level of mRNA accumulation. Finally our results show that acvA and ipnA genes are divergently transcribed, whilst acyA is transcribed in the same orientation as ipnA.

Structural and transcriptional properties associated with a member of a new family of conserved short dispersed repeated elements in human DNA.

The sequence of a 500-bp segment of human DNA containing a member of the 'A3' family of dispersed repeated elements, a family hitherto uncharacterized in human or rodent DNAs, is presented. The repeated element maps to a region of 270 bp, within which the most notable features are 17-mer direct repeats separated by 190 bp. A probe containing the repeated element, together with flanking single-copy DNA illuminates a series of multiple bands in blot transfers of restriction digests of human, mouse and hamster DNAs. However, the flanking single-copy DNA hybridizes to human, but not to rodent DNA. The same probe illuminates homologous transcripts occurring in a single size class of 0.8 kb in total RNA from a variety of human and mouse cells, levels of which vary approx. 100-fold in the tissues analyzed. Poly(A)+ RNAs from EJ-transformed 3T3 cells and mouse fibrosarcomas have been analysed and found also to contain an homologous 0.8-kb transcript.