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Solid-state fermentation (SSF) withAspergillus oryzaeresults in high levels of secreted protein. However, control mechanisms of gene expression in SSF have been only poorly studied. In this study we show that both glucoamylase (glaB) and protease (alpA,nptB) genes are highly expressed during surface cultivation on wheat-based solid medium, and even higher during cultivation on wheat kernels. In wheat-based liquid medium, low levels of gene expression are observed. Typical SSF cultivation conditions, such as low water activity and the formation of aerial hyphae, did not contribute to the high-level gene expression on wheat-based solid medium. Analysis of wheat-based solid and liquid cultivations showed differences in carbon and nitrogen utilisation and external pH. The results presented show that the difference in regulation of transcription of thealpAandnptBgenes in wheat-based liquid and solid medium could be pH dependent, involving a pH-dependent transcription regulator. The results obtained suggest that the difference in regulation of transcription of theglaBgene in wheat-based liquid and solid medium is caused by a difference in carbohydrate degradation and consumption under the different culture conditions. [ABSTRACT FROM AUTHOR]

The Aspergillus nidulans amdS selection marker was used for the identification of multicopy T-DNA insertions in Agrobacterium-mediated transformation of Asp. awamori. The selection of transformants on agar plates containing acetamide as sole nitrogen source and hygromycin resulted in a six-fold decrease in the transformation frequency, compared with the transformation frequency obtained after hygromycin selection alone. However, it was found that 47% of the transformants obtained after hygromycin and acetamide double selection contained multiple T-DNA integrations. Furthermore, it was found that the multicopy transformants could easily be identified based on their growth rate on agar plates containing acetamide medium. Based on these data, it can be concluded that the amdS marker can also be used as a selection marker in Agrobacterium-mediated transformation of Asp. awamori and that it is a very useful marker to identify those transformants containing multiple T-DNA integrations. [ABSTRACT FROM AUTHOR]

Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in Neurospora crassa, Aspergillus niger and Aspergillus awamori by codon optimization of the aequorin gene. Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external calcium) were found transiently to increase [Ca2+]c. Each of the calcium signatures associated with these physico-chemical treatments was unique, suggesting the involvement of three distinct calcium-mediated signal transduction pathways. The fungal calcium channel blocker KP4 inhibited the [Ca2+]c responses to hypo-osmotic shock and high external calcium, but not to mechanical perturbation. The divalent cation chelator BAPTA inhibited [Ca2+]c responses to mechanical perturbation and hypo-osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca2+]c levels. [ABSTRACT FROM AUTHOR]

Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l-1. Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (Km) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation). [ABSTRACT FROM AUTHOR]

A well-known industrial fungus for enzyme production, Aspergillus niger, was selected to produce the feruloyl esterase FAEA by homologous overexpression for pulp bleaching application. The gpd gene promoter was used to drive FAEA expression. Changing the nature and concentration of the carbon source nature (maltose to glucose; from 2.5 to 60 g l-1), improved FAEA activity 24.5-fold and a yield of 1 g l-1 of the corresponding protein in the culture medium was achieved. The secreted FAEA was purified 3.5-fold to homogeneity in a two-step purification procedure with a recovery of 69%. The overproduced protein was characterised and presented properties in good agreement with those of native FAEA. The recombinant FAEA was tested for wheat straw pulp bleaching, with or without a laccase mediator system and xylanase. Best results were obtained using a bi-sequential process with a sequence including xylanase, FAEA and laccase, and yielded very efficient delignification--close to 75%--and a kappa number of 3.9. This is the first report on the potential application of recombinant FAEA in the pulp and paper sector. [ABSTRACT FROM AUTHOR]

Cytochrome P450 enzyme systems are found throughout nature and are involved in many different, often complex, bioconversions. In the endoplasmic reticulum of the filamentous fungus Aspergillus niger a cytochrome P450 enzyme system is present that is capable of the para-hydroxylation of benzoate. The expression of the two genes encoding the components of this system, the cytochrome P450 gene encoding benzoate para-hydroxylase ( bphA) and the gene encoding cytochrome P450 reductase ( cprA), is inducible by benzoate. The BPH system was used as a model system to study the mechanisms that result in co-regulation of both components of an eukaryote cytochrome P450 enzyme system. Deletion analysis of the transcription control regions of cprA and bphA resulted in the identification of a region that was involved in benzoate induction of gene expression. The functional competence of the cprA Benzoate Responsive Region thus defined was demonstrated directly by cloning this fragment upstream of a constitutively expressed mini-promoter and analysing expression of the hybrid transcription control region in a lacZ reporter system. Further analysis of cprA gene expression revealed a clear quantitative discrepancy between induction at the protein level (approximately 4-fold) and at the transcription level (>20-fold). The majority of the transcripts observed after benzoate induction ( cprAβ) were larger then the constitutively expressed cprAα transcript. The difference in size between the cprAα and cprAβ transcript is caused by differential promoter use. As the longer cprAβ transcript carries a small uORF we propose that post-transcriptional regulation of CPR expression underlies the discrepancy in the degree of induction at the protein and transcriptional level. Our results show that regulation of CPR expression is particularly complex, involving regulatory promoter elements, differential promoter use and regulation at the post-transcriptional level. [ABSTRACT FROM AUTHOR]

The Aspergillusniger and Trichodermareesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene ( sarA) contains five introns, whereas the T. reesei gene ( sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70–80% identity to the SAR1 protein. Complementation of S. cerevisiaesar1 and sec12 mutants by expression vectors carrying the A. nigersarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. nigersarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger. [ABSTRACT FROM AUTHOR]

Filamentous fungi are widely used for the production of homologous and heterologous proteins but, compared to homologous proteins, the levels of production of heterologous proteins are usually low. During the last 5 years, the levels of production of heterologous proteins have been drastically improved by fusing the corresponding gene to the 3' end of a homologous gene, encoding a well-secreted protein such as glucoamylase. Nevertheless, little research has been carried out to determine the limitations that hamper heterologous protein production. Recently we have carried out a detailed analysis of the levels of production of several proteins and glucoamylase fusion proteins in defined recombinant Aspergillus awamori strains. In this review we will focus on the use of filamentous fungi for the production of heterologous, especially non-fungal, proteins. In particular, the effect of gene-fusion strategies will be reviewed. Furthermore, the remaining limitations in heterologous protein production and suggestions for improvement strategies for overproduction of these protein will be discussed. [ABSTRACT FROM AUTHOR]

A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II ( exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus. Transformants containing a construct encoding a direct, in-frame fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase pro-peptide. The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains with different numbers of a cutinase construct containing its own pre-prosequence. The multicopy strains showed a 6- to 12-fold increased production of extracellular cutinase relative to the single-copy strains. No linear dose response relation to the number of expression cassettes present in the strains was observed. The amount of active enzyme produced by the strains correlated with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation or secretion. [ABSTRACT FROM AUTHOR]

The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bipA, the BiP-encoding gene from Aspergillus niger and Aspergillus awamori. As this result could imply that BiP plays a role in protein overproduction, the effect of modulation of bipA gene expression on protein secretion was studied in several recombinant strains expressing glucoamylase ( glaA) fusion genes. For overproduction of BiPA in these strains, extra copies of the bipA gene under the control of an inducible promoter were introduced. To allow analysis of the effect of a decreased bipA expression level on protein secretion, replacement of the wild-type gene for a bipA gene driven by the glaA promoter was attempted. However, this endeavour failed because of the lethality of this replacement. Although the final amount of secreted recombinant protein did not change significantly in strains with increased BiPA levels, increased levels of unprocessed fusion protein were detected in the total protein extracts of these strains. [ABSTRACT FROM AUTHOR]