Metabolic control analysis of xylose catabolism in Aspergillus.

A kinetic model for xylose catabolism in Aspergillus is proposed. From a thermodynamic analysis it was found that the intermediate xylitol will accumulate during xylose catabolism. Use of the kinetic model allowed metabolic control analysis (MCA) of the xylose catabolic pathway to be carried out, and flux control was shown to be dependent on the metabolite levels. Due to thermodynamic constraints, flux control may reside at the first step in the pathway, i.e., at the xylose reductase, even when the intracellular xylitol concentration is high. On the basis of the kinetic analysis, the general dogma specifying that flux control often resides at the step following an intermediate present at high concentrations was, therefore, shown not to hold. The intracellular xylitol concentration was measured in batch cultivations of two different strains of Aspergillus niger and two different strains of Aspergillus nidulans grown on media containing xylose, and a concentration up to 30 mM was found. Applying MCA showed that the first polyol dehydrogenase (XDH) in the catabolic pathway of xylose exerted the main flux control in the two strains of A. nidulans and A. niger NW324, but the flux control was exerted mainly at the first enzyme of the pathway (XR) of A. niger NW 296.

Regulation of the alpha-glucuronidase-encoding gene ( aguA) from Aspergillus niger.

The alpha-glucuronidase gene aguA from Aspergillus niger was cloned and characterised. Analysis of the promoter region of aguA revealed the presence of four putative binding sites for the major carbon catabolite repressor protein CREA and one putative binding site for the transcriptional activator XLNR. In addition, a sequence motif was detected which differed only in the last nucleotide from the XLNR consensus site. A construct in which part of the aguA coding region was deleted still resulted in production of a stable mRNA upon transformation of A. niger. The putative XLNR binding sites and two of the putative CREA binding sites were mutated individually in this construct and the effects on expression were examined in A. niger transformants. Northern analysis of the transformants revealed that the consensus XLNR site is not actually functional in the aguA promoter, whereas the sequence that diverges from the consensus at a single position is functional. This indicates that XLNR is also able to bind to the sequence GGCTAG, and the XLNR binding site consensus should therefore be changed to GGCTAR. Both CREA sites are functional, indicating that CREA has a strong influence on aguA expression. A detailed expression analysis of aguA in four genetic backgrounds revealed a second regulatory system involved in activation of aguA gene expression. This system responds to the presence of glucuronic and galacturonic acids, and is not dependent on XLNR.

Isolation of UV-induced mutations in the areA nitrogen regulatory gene of Aspergillus niger, and construction of a disruption mutant.

The areA gene of filamentous fungi encodes a positive-acting transcriptional factor required for the expression of genes involved in the utilisation of nitrogen sources other than ammonium and glutamine. In Aspergillus niger we have isolated three UV-induced areA mutants and constructed a well-defined disruption allele of the areA gene. The areA gene was genetically localised on Linkage Group III, 3.6 map units (m.u.) from bioA1 and 4.5 m.u. from lysA7. Analysis of the expression of the nitrate reductase encoding gene and of nitrate reductase activities show that the mutated areA strains behave as loss-of-function mutants and can be classified as areAr type. In addition, growth tests were performed using several nitrogen sources in combination with glucose. The results suggest that, unlike the case in A. nidulans, in A. niger the AreA protein also plays a role in the presence of ammonium. Furthermore, the spectrum of protease activities secreted by A. niger differs from that produced by A. nidulans, as only A. niger is able to degrade elastin.

Characterization of the kexin-like maturase of Aspergillus niger.

Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein. The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction. We have cloned the kexin-encoding gene of Aspergillus niger (kexB). We constructed strains that either overexpressed KexB or lacked a functional kexB gene. Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB. In contrast, no kexin-specific activity was detected in the similar protein fractions of the kexB disruptant. Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein. The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway of A. niger.

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.

Disruption of three acid proteases in Aspergillus niger--effects on protease spectrum, intracellular proteolysis, and degradation of target proteins.

Three acid protease genes encoding two extracellular proteases (PEPA and PEPB) and one intracellular protease (PEPE) were disrupted in Aspergillus niger. Northern-blot analysis showed the absence of wild-type protease mRNAs in the disruptants while western-blot analysis proved the absence of the encoded proteases. Characterization of the residual proteolytic spectra in the disruptants indicated that the extracellular protease activity was reduced to 16% and 94% for the delta pepA and the delta pepB disruptants, repectively. In the delta pepE disruptant, the total intracellular proteolytic activity was reduced to 32%. Apart from the reduced intracellular pepstatin-inhibitable aspartyl protease activity, serine protease and serine carboxypeptidase activities were also significantly reduced in the delta pepE strain. This may indicate the presence of a cascade activation mechanism for several vacuolar proteases, triggered by the PEPE protein, similar to the situation in Saccharomyces cerevisiae. Disruption of a single protease gene had no effects on the transcription of other non-disrupted protease genes in A. niger. In supernatants of the disruptants, reduced degradation of a proteolytically very susceptible tester protein (PELB) was observed. By recombination, we also constructed delta pepA delta pepB, delta pepB delta pepE and delta pepA delta pepE double disruptants as well as a delta pepA delta pepB delta pepE triple disruptant, lacking all three acid protease activities. The in vitro residual PELB activity was the highest in the triple disruptant and the delta pepA delta pepB recombinant.

Aspergillus as a host for heterologous protein production: the problem of proteases.

Homologous and heterologous protein production by filamentous fungi is often limited by the expression of proteases at high levels. By eliminating specific protease activities, protein production in Aspergillus niger can be improved considerably. Both classical mutagenesis and gene disruption techniques have yielded strains with reduced protease expression. Combinations of these mutations and disruptions result in a further reduction of protease activity. The coupling of efficient promoters to target genes allows their expression under conditions that repress the expression of several proteases, which further improves product yields. The strategies used have led to the development of a set of tester strains from which the appropriate genetic background for production can be selected.

Production of the homologous pectin lyase B protein in six genetically defined protease-deficient Aspergillus niger mutant strains.

An expression cassette has been transformed into six protease-deficient (prt) mutant strains of Aspergillus niger. Transformants were tested for improved production of the proteolytically susceptible PELB tester protein. In four complementation groups (prtA, B, D and F) distinct improvement of PELB yield was observed. These in vivo experiments in single prt mutants confirmed earlier in vitro PELB degradation data and demonstrated how the use of protease-deficient mutants can significantly improve protein production in A. niger. The strong effects of several prt alleles on the stability of the PELB tester protein have initiated a more detailed genetical and molecular characterization of the prt mutations. Mapping of the cloned protease genes pepA [I], B [II], C [IV], D [I], E [IV] and F [IV] indicated that none of the prt mutations represent alleles of the presently cloned protease (pep) genes from A. niger. Analysis of the expression of the pep genes in prt strains demonstrated that the strongly reduced protease activities observed in several prt mutants are not reflected by reduced transcription levels for a number of extracellular proteases. These results indicate that the mode of action of the prt genes constitute an interesting group of new genetic functions which severely affect protease production, and as such improve protein production, in A. niger.

Identification of regulatory mutants of Aspergillus aculeatus affected in rhamnogalacturonan hydrolase expression.

Rhamnogalacturonan hydrolase expression in A. aculeatus can be induced by pectin, but also by a combination of two constituent monosaccharides of pectin, rhamnose and galacturonic acid. The rhgA promoter was fused to the A. niger glucose oxidase coding sequence and a single copy of the hybrid gene was integrated at the rhgA locus in the genome of A. aculeatus. The gene product was subsequently used as reporter in a screening assay for the selection of rhamnogalacturonan hydrolase-overproducing mutant strains. At least four of the mutations were recessive and could be assigned to different loci. One mutation (rgr25) showed linkage with the rhgA locus. Inducible rhamnogalacturonan hydrolase expression levels of about 5-10 times that in the wild-type were found in the mutants rgr48, rgr25 and rgr34 after growth on a combination of rhamnose and galacturonic acid with or without fructose as a carbon source. In mutant rgr48 elevated levels of rhgA transcription were found.

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.

New protease mutants in Aspergillus niger result in strongly reduced in vitro degradation of target proteins; genetical and biochemical characterization of seven complementation groups.

Several mutants of Aspergillus niger, deficient in extracellular protease expression, have been isolated and characterized both genetically and biochemically. The mutant strains, obtained after in vivo UV-mutagenesis of conidiospores and selected by haloscreening on a new dual-substrate plate assay, belong to at least seven different complementation groups. These seven prt loci were assigned to linkage groups using master strains with marked chromosomes. One prt locus (prtC) could be assigned to linkage group I, three (prtB, prtE and prtG) to linkage group III, one (prtF) to linkage group V and the two remaining loci (prtA and prtD) to linkage group VIII. Extracellular proteolytic activities varied from 2 to 3% up to 80% of the protease activity of the parental strain. Assigning the different prt mutants to structural or regulatory genes is difficult since only one structural gene, pepA, has been mapped unambiguously on linkage group I but is not identical to prtC. All prt mutants except for prtC are likely to be regulatory mutants or else belong to a proteolytic cascade because residual activities showed that more proteolytic activities were affected simultaneously. Double mutants were constructed both by recombination and by a second round of mutagenesis. In both cases mutants with further reduced extracellular proteolytic activities were isolated. A sensitive in vitro degradation assay, based on the homologous pectin lyase B (PELB) protein to analyze proteolytic degradation in A. niger, was developed and used to show extremely reduced proteolytic PELB degradation in the culture media of some of these mutants.

Induction of glucose oxidase, catalase, and lactonase in Aspergillus niger.

The induction of glucose oxidase, catalase, and lactonase activities was studied both in wild-type and in glucose oxidase regulatory and structural mutants of Aspergillus niger. The structural gene for glucose oxidase was isolated and used for Northern analysis and in transformation experiments using various gox mutations. Wild-type phenotype could be restored in the glucose oxidase-negative mutant (goxC) by transformation with the structural gene. We conclude, therefore, that the goxC marker which is located on chromosome 2 represents the structural gene of glucose oxidase. Glucose and a high oxygen level are necessary for the induction of all three enzyme activities in the wild-type strain and it was shown that both glucose and oxygen effects reflect regulation at the transcriptional level. The goxB mutation results in constitutive expression of all three activities although modulated to some extent by the carbon source. The goxE mutation only has an effect on lactonase and glucose oxidase expression and does not relieve the necessity for a high oxygen level. Catalase and lactonase could not be induced in the glucose oxidase-negative strain (goxC). Addition of H2O2 resulted in the induction of all three enzymes in the wild-type without glucose being present. The H2O2 induction is probably mediated by the goxB product. Besides the H2O2 induction there is still an effect of the carbon source on the induction. A model for induction of glucose oxidase, catalase, and lactonase in A. niger is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic localization of a series of genes affecting glucose oxidase levels in Aspergillus niger.

A number of mutants of Aspergillus niger, affected in glucose oxidase (GOX) expression, are described. The overproducing mutants could be classified into seven complementation groups whereas two glucose oxidase-negative complementation groups were recognized. These nine gox loci were assigned to linkage groups using master strains with marked chromosomes. Three gox loci are in linkage group II, one is in III, two are in V and two are in linkage group VII. One weak glucose oxidase-overproducing mutant could not be assigned to one of the linkage groups. These genetically well characterized mutants will be used in a strain improvement program based on genetic recombination.