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  • 1
    Electronic Resource
    Electronic Resource
    Calcium measurement in living filamentous fungi expressing codon-optimized aequorin (2004)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 52 (2004), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04066.x
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  • 2
    Electronic Resource
    Electronic Resource
    Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei (1997)
    Springer
    Molecular genetics and genomics 256 (1997), S. 446-455 
    Details
    ISSN: 1617-4623
    Keywords: Key words Gene cloning ; Protein secretion ; Filamentous fungi ; Small GTP binding protein ; Complementation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/PL00008613
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  • 3
    Electronic Resource
    Electronic Resource
    Regulation of expression of the Aspergillus niger benzoate para-hydroxylase cytochrome P450 system (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 601-609 
    Details
    ISSN: 1617-4623
    Keywords: Key words Cytochrome P450 ; Post-transcriptional regulation ; Benzoate ; Aspergillus niger
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380051207
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  • 4
    Electronic Resource
    Electronic Resource
    The effect of pre- and pro-sequences and multicopy integration on heterologous expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori (1996)
    Springer
    Applied microbiology and biotechnology 45 (1996), S. 755-763 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract  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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050759
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  • 5
    Electronic Resource
    Electronic Resource
    Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli (1998)
    Springer
    Applied microbiology and biotechnology 50 (1998), S. 447-454 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530051319
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  • 6
    Electronic Resource
    Electronic Resource
    Molecular genetic strain improvement for the overproduction of fungal proteins by Filamentous fungi (1995)
    Springer
    Applied microbiology and biotechnology 43 (1995), S. 195-205 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract  During the last 5 years, strain improvement by genetic engineering has been established as a good alternative for traditional methods of strain improvement such as mutagenesis and genetic recombination. Considerable success has been achieved in the overproduction of a variety of fungal proteins. However, from the available data a limitation at the level of transcription is evident. A more detailed analysis of this transcription limitation was carried out for the overproduction of glucoamylase in A. niger. This analysis revealed that both the site of integration of introduced gene copies and the available amount of trans-acting regulatory proteins were at the root of the observed limitation. Based on these results, approaches for the construction of a new generation of protein-overproducing strains are suggested.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050390
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  • 7
    Electronic Resource
    Electronic Resource
    Efficient production of secreted proteins by Aspergillus : progress, limitations and prospects (1997)
    Springer
    Applied microbiology and biotechnology 47 (1997), S. 1-11 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530050880
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  • 8
    Electronic Resource
    Electronic Resource
    Optimization of the benzoate-inducible benzoate p-hydroxylase cytochrome P450 enzyme system in Aspergillus niger (1996)
    Springer
    Applied microbiology and biotechnology 46 (1996), S. 360-364 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract  Introduction in the fungus Aspergillus niger of multiple copies of the A. niger bphA gene, encoding the cytochrome P450 enzyme benzoate p-hydroxylase, did not result in increased activities of this enzyme [Gorcom RFM van, et al. Mol Gen Genet (1990) 223: 192–197] probably because of low expression levels of the gene encoding the second component of the microsomal cytochrome P450 enzyme system, cytochrome P450 reductase. For improvement of this and other cytochrome-P450-dependent reactions, A. niger strains were constructed in which the copy number of the A. niger cprA gene (encoding cytochrome-P450 reductase) or the copy numbers of both cprA and the cytochrome-P450-encoding gene were increased. Expression of both genes was controlled by their own transcription control regions. Benzoate p-hydroxylase activity of different trans- formants was determined in microsomal fractions using a newly developed indirect in vitro assay. In trans- formants containing multiple copies of both genes, benzoate p-hydroxylase activity was significantly higher than in the wild-type strain or in transform- ants in which the copy number of only one of the genes was increased. These results clearly indicate the importance of co-expression of cytochrome-P450 reductase for achieving maximal cytochrome P450 activities in cytochrome-P450-overproducing filamentous fungi.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00166230
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