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  • 1
    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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  • 2
    Electronic Resource
    Electronic Resource
    Genetic analysis of benzoate metabolism in Aspergillus niger (1990)
    Springer
    Applied microbiology and biotechnology 34 (1990), S. 225-228 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The benzoate metabolism of Aspergillus niger was studied as part of a design to clone the benzoate-4-hydroxylase gene of this fungus on the basis of complementation. Filtration enrichment techniques yielded mutants defective for different steps of benzoate degradation: bph (benzoate-4-hydroxylase), phh (4-hydroxybenzoate-3-hydroxylase) and prc (protocatechuate ring cleavage) mutants. In this way the degradation pathway for benzoate, involving the formation of 4-hydroxybenzoate and 3,4-dihydroxybenzoate has been confirmed. In addition a mutant sensitive to benzoate has been found. Complementation tests in somatic diploids showed that the bph mutants belonged to two complementation groups. The major group is probably defective in the structural gene (bphA). All phh mutants tested belonged to one complementation group. The prc mutants could be divided into several groups on the basis of their growth on different aromatic substrates and on the basis of the complementation test. The phh and both bph mutations are shown to be located on different chromosomes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00166785
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    Paper (German National Licenses)
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  • 3
    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 transformants was determined in microsomal fractions using a newly developed indirect in vitro assay. In transformants containing multiple copies of both genes, benzoate p-hydroxylase activity was significantly higher than in the wild-type strain or in transformants 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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    Paper (German National Licenses)
    Fulltext
  • 4
    Electronic Resource
    Electronic Resource
    Gene amplification in Aspergillus nidulans by transformation with vectors containing the amdS gene (1985)
    Springer
    Current genetics 9 (1985), S. 361-368 
    Details
    ISSN: 1432-0983
    Keywords: Transformation of Aspergillus ; Conidial protoplasts ; Multicopy/tandem integration ; Gene amplification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Conidial protoplasts of an A. nidulans amdS deletion strain (MH1277) have been transformed to the AmdS+ phenotype with a plasmid carrying the wild type gene (p3SR2). Optimalisation of transformation and plating conditions now has resulted in frequencies of 300–400 transformants per μg of DNA. Analysis of DNA from AmdS+ transformants of MH1277 showed that transformation had occurred by integration of vector DNA sequences into the genome. In virtually all these transformants multiple copies of the vector were present in a tandemly repeated fashion, not preferentially at the resident, partially deleted amdS gene. It is suggested that the observed integration phenomena are dependent on the genetic background of the A. nidulans strain, used for transformation. A model to explain the tandem type of integration is proposed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00421606
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  • 5
    Electronic Resource
    Electronic Resource
    Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function (1991)
    Springer
    Current genetics 19 (1991), S. 261-264 
    Details
    ISSN: 1432-0983
    Keywords: Benzoate conversion ; Mutant isolation ; Transformants ; Aspergillus niger
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This study was prompted by the observation that an Aspergillus niger transformant with a multicopy bphA (benzoate-4-hydroxylase gene) insert did not grow on benzoate, whereas a transformant with only one extra copy could grow. Therefore, an extensive survey has been made for other genes involved in the conversion of benzoate into 4-hydroxy-benzoate. A transformant with two copies of the bphA gene was used in part of the mutation experiments in order to avoid the isolation of many bphA mutants. Filtration enrichment was used to isolate mutants defective in the conversion of benzoate. The Bph mutants that have been isolated belong to six complementation groups. Mutants with a defected structural gene (bphA) were again predominantly found but, in addition, five other groups of mutants that could not grow on benzoate were isolated. Genetic analysis of the mutants showed that the six genes were localized in different parts of the genome. This was used as an additional proof that some mutants involved different genes. Diploids with seven copies of the bphA gene and heterozygous for one of the other bph genes were constructed. No indication has been obtained that any one of the mutant classes is responsible for the growth-limiting factor in bphA multicopy transformants. This study shows that the p-hydroxylation of benzoate is very complex, although the metabolic pathway is straight forward.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00355052
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  • 6
    Electronic Resource
    Electronic Resource
    Isolation and molecular characterisation of the gene encoding eburicol 14α-demethylase (CYP51) from Penicillium italicum (1996)
    Springer
    Molecular genetics and genomics 250 (1996), S. 725-733 
    Details
    ISSN: 1617-4623
    Keywords: Key words Penicillium italicum ; Lanosterol 14α-demethylase ; Fungicides ; Sterol biosynthesis inhibitors ; Cytochrome P450
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The CYP51 gene encoding eburicol 14α-demethylase (P45014DM) was cloned from a genomic library of the filamentous fungal plant pathogen Penicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14α-demethylase from the yeast Candida tropicalis. The nucleotide sequence of a 1739-bp genomic fragment and the corresponding cDNA clone comprises an open reading frame (ORF) of 1545 bp, encoding a protein of 515 amino acids with a predicted molecular mass of 57.3 kDa. The ORF is interrupted by three introns of 60, 72 and 62 bp. The C-terminal part of the protein includes a characteristic haem-binding domain, HR2, common to all P450 genes. The deduced P. italicum P45014DM protein and the P45014DM proteins from Candida albicans, C. tropicalis and Saccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of the CYP51 family. Multiple copies of a genomic DNA fragment of P. italicum containing the cloned P450 gene were introduced into Aspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P45014DM activity, indicating that the cloned gene encodes a functional eburicol 14α-demethylase.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02172984
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  • 7
    Electronic Resource
    Electronic Resource
    Isolation and molecular characterisation of the gene encoding eburicol 14α-demethylase (CYP51) fromPenicillium italicum (1996)
    Springer
    Molecular genetics and genomics 250 (1996), S. 725-733 
    Details
    ISSN: 1617-4623
    Keywords: Penicillium italicum ; Lanosterol 14α-demethylase ; Fungicides ; Sterol biosynthesis inhibitors ; Cytochrome P450
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract TheCYP51 gene encoding eburicol 14α-demethylase (P45014DM) was cloned from a genomic library of the filamentous fungal plant pathogenPenicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14α-demethylase from the yeastCandida tropicalis. The nucleotide sequence of a 1739-bp genomic fragment and the corresponding cDNA clone comprises an open reading frame (ORF) of 1545 bp, encoding a protein of 515 amino acids with a predicted molecular mass of 57.3 kDa. The ORF is interrupted by three introns of 60, 72 and 62 bp. The C-terminal part of the protein includes a characteristic haem-binding domain, HR2, common to all P450 genes. The deducedP. italicum P45014DM protein and the P45014DM proteins fromCandida albicans, C. tropicalis andSaccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of theCYP51 family. Multiple copies of a genomic DNA fragment ofP. italicum containing the cloned P450 gene were introduced intoAspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P45014DM activity, indicating that the cloned gene encodes a functional eburicol 14α-demethylase.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02172984
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    Paper (German National Licenses)
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  • 8
    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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