Purification and characterization of benzoate-para-hydroxylase, a cytochrome P450 (CYP53A1), from Aspergillus niger.

Benzoate-para-hydroxylase (CYP51A or BpH) and NADPH:cytochrome P450 reductase from the filamentous fungus Aspergillus niger were purified to apparent homogeneity, using an overproducing A. niger strain. This is the first membrane-bound fungal cytochrome P450 to be isolated and characterized. Combining BpH with NADPH:cytochrome P450 oxidoreductase in the presence of the phospholipid dilauryl phosphatidylcholine restored the BpH activity, although to only a minor extent. Spectral analysis of BpH showed characteristic spectra for a cytochrome P450. Substrate binding studies with purified BpH as a function of temperature and as a function of pH were performed. Temperature-dependent studies, at pH 8.0, showed that the simplified spin equilibrium model originally proposed for camphor binding to cytochrome P450cam (M. T. Fisher and S. G. Sligar, 1987, Biochemistry 26, 4797-4803) also applies to the benzoate-BpH system. Two equilibrium constants were determined, K(1) for substrate binding without a spin change and K(2) for the spin change of the benzoate-BpH complex. pH-dependent binding studies showed that both K(1) and K(2) increase with pH, indicative of a higher affinity. As K(1) decreases more strongly with pH than K(2), we suggest that benzoate first binds to a binding site on the outside of the protein in a pH-dependent way, followed by transfer to the inside of the protein causing a spin change at the heme iron. The strong pH dependence of K(1) could be the result of the need to break salt bridges at the binding site on the outside of the protein. pH-dependent kinetic studies with microsomes showed that the apparent K(M) values followed the trend observed for benzoate binding to purified BpH, while k(cat) values were virtually constant between pH 6.6 and 8.0 and decreased above pH 8, probably due to loss of productive interaction between BpH and NADPH:cytochrome P450 oxidoreductase. Research into the substrate specificity of BpH showed that BpH can only use benzoic acid and some of its derivatives. Monosubstitution on the phenyl ring is allowed but only at certain positions with specific, not too large groups. Substitution always leads to a lower affinity of the substrate. With one exception, all substrates were converted to their 4-hydroxy derivative. The exception, 3-methoxybenzoate, was demethylated to yield 3-hydroxybenzoate only. The restricted number of substrates and the specificity in catalysis suggest that BpH is not a general-purpose hydroxylase but that its role is confined to benzoate hydroxylation in the beta-ketoadipate pathway of A. niger.

Constitutive and inducible hydroxylase activities involved in the degradation of naphthalene by Cunninghamella elegans.

The non-ligninolytic fungus Cunninghamella elegans was investigated for its ability to produce naphthalene hydroxylase (NAH) and naphthol hydroxylase (NOH) activities under various conditions. When the organism was cultivated on a rich growth medium, the mycelia exhibited significant constitutive NAH activity in the late exponential growth phase, but not in the early-exponential-growth-phase. On incubating the early-exponential-growth-phase mycelia with naphthalene, NAH activity was increased five-fold; however, this increase did not occur in the presence of the protein synthesis inhibitor cycloheximide. Since incubation of the late-phase mycelia with naphthalene did not lead to a higher degradation rate of naphthalene, mycelia in this physiological state have apparently lost the ability to induce synthesis of the enzyme exhibiting NAH activity. This is not due to an overall inability to perform de novo protein synthesis, since NOH activity, non-constitutive at all growth phases, could be induced by incubating late-phase mycelia with naphthalene. Whether inducible and constitutive NAH activity originate from one and the same enzyme remains to be elucidated. It is suggested that naphthalene oxidizing enzyme(s) may also oxidize pyrene, but not anthracene or benzo[a]pyrene, although the latter are degradable by C. elegans.

Regulation of expression of the Aspergillus niger benzoate para-hydroxylase cytochrome P450 system.

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 (cprAbeta) were larger then the constitutively expressed cprAalpha transcript. The difference in size between the cprAalpha and cprAbeta transcript is caused by differential promoter use. As the longer cprAbeta 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.

Cytochrome P450 enzyme systems in fungi.

The involvement of cytochrome P450 enzymes in many complex fungal bioconversion processes has been characterized in recent years. Accordingly, there is now considerable scientific interest in fungal cytochrome P450 enzyme systems. In contrast to S. cerevisiae, where surprisingly few P450 genes have been identified, biochemical data suggest that many fungi possess numerous P450 genes. This review summarizes the current information pertaining to these fungal cytochrome P450 systems, with emphasis on the molecular genetics. The use of molecular techniques to improve cytochrome P450 activities in fungi is also discussed.

An improved colony-PCR method for filamentous fungi for amplification of PCR-fragments of several kilobases.

A method was developed to perform PCR directly on mycelial pellets or colonies treated with NOVOzym 234. The method allows rapid screening of large numbers of transformants of both sporulating and non-sporulating fungi for the presence of (co)transforming plasmid copies or for specific genetic modifications such as gene disruption and site specific integration. PCR fragments of at least 3.2 kb can be obtained. Using this method the identification of specific disruption mutants from Aspergillus niger and Beauveria bassiana was carried out.

Optimization of the benzoate-inducible benzoate p-hydroxylase cytochrome P450 enzyme system in Aspergillus niger.

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.

Increased resistance to 14 alpha-demethylase inhibitors (DMIs) in Aspergillus niger by coexpression of the Penicillium italicum eburicol 14 alpha-demethylase (cyp51) and the A. niger cytochrome P450 reductase (cprA) genes.

In this paper we describe the effects of over-expression of the Penicillium italicum gene encoding eburicol 14 alpha-demethylase (cyp51), in Aspergillus niger strains with one or multiple copies of the gene encoding cytochrome P450 reductase (cprA), on the eburicol 14 alpha-demethylase activity. Eburicol 14 alpha-demethylase activity was determined by measuring the resistance of transformants against some eburicol 14 alpha-demethylase inhibitors (DMIs). DMIs are widely used as fungicides in crop protection and human and veterinarian health care. DMI resistance in a transformant overexpressing both CPR and CYP51 was increased 5-30-fold compared to DMI resistance in the wild type strain, depending on the test compound used. Resistance in this strain was approximately 2-5-fold increased compared to DMI resistance in a transformant that was overexpressing the cyp51 gene but had only the wild type copy of the cprA gene and approximately 3-12-fold increased compared to a strain overexpressing the cprA gene (and having only the wild type copy of the cyp51 gene). These results show the importance of CPR overexpression for increasing cytochrome P450 activities in filamentous fungi.

Isolation and molecular characterisation of the gene encoding eburicol 14 alpha-demethylase (cYP51) from Penicillium italicum.

The CYP51 gene encoding eburicol 14 alpha-demethylase (P450(14DM)) was cloned from a genomic library of the filamentous fungal plant pathogen Penicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14 alpha-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 P450(14DM) protein and the P450(14DM) 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 Pl italicum containing the cloned P450 gene were introduced into Aspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P450(14DM) activity, indicating that the cloned gene encodes a functional eburicol 14 alpha-demethylase.

Cloning and characterization of the NADPH cytochrome P450 oxidoreductase gene from the filamentous fungus Aspergillus niger.

In this paper, we describe the cloning and molecular characterization of the Aspergillus niger cytochrome P450 reductase (CPR) gene, cprA. Attempts to clone the cprA gene by heterologous hybridization techniques were unsuccessful. Using the polymerase chain reaction (PCR) with degenerate primers based on conserved regions found in cpr genes from other organisms, we were able to isolate a fragment that contained part of the gene. With the aid of this fragment, a genomic fragment containing the entire coding region and 5' and 3' untranslated ends of the cprA gene was isolated and sequenced. The cprA gene was introduced in multiple copies in A. niger strain N402 using the amdS transformation system. One of the resulting transformants, AB2-2, showed a 14-fold increase in CPR activity, indicating that the cloned cprA gene is functional. We analyzed the induction of cprA gene expression by several generally used cytochrome P450 inducers but did not find any induction of cprA gene expression. However, A. niger cprA gene expression could be induced by benzoic acid, which is the substrate of the highly inducible A. niger cytochrome P450 gene, bphA (cyp53). On the basis of a comparison of the deduced protein sequence of the A. niger cprA gene with CPR proteins isolated from other organisms, the structure-function relationship of some conserved regions is discussed.

Isolation and characterization of a 1,4-beta-endoxylanase gene of A. awamori.

An enzyme with a particular 1,4-beta-xylanase activity was identified and purified from wheat-bran culture medium of an Aspergillus awamori strain. With oligonucleotides based on the N-terminal amino-acid sequence of the enzyme, the exlA gene of A. awamori, encoding 1,4-beta-xylanase A, has been cloned. Based on the deduced amino-acid sequence, 1,4-beta-xylanase A is produced as a 211 amino-acid-residue-long precursor, which is converted post-translationally into a 184-aa residue-long mature protein. Transformation of the original A. awamori strain with multiple copies of the exlA gene resulted in a 40-fold overproduction of 1,4-beta-xylanase A. The overproduced enzyme has the same biochemical and enzymological properties as the wild-type enzyme.

Cloning, characterization and overexpression of the phytase-encoding gene (phyA) of Aspergillus niger.

Phytase catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. A gene (phyA) of Aspergillus niger NRRL3135 coding for extracellular, glycosylated phytase was isolated using degenerate oligodeoxyribonucleotides deduced from phytase amino acid (aa) sequences. Nucleotide (nt) sequence analysis of the cloned region revealed the presence of an open reading frame coding for 467 aa and interrupted once by an intron of 102 bp in the 5' part of the gene. The start codon is followed by a sequence coding for a putative signal peptide. Expression of phyA is controlled at the level of mRNA accumulation in response to inorganic phosphate levels. After cell growth in low-phosphate medium, a transcript of about 1.8 kb was visualized. Transcription of phyA initiates at at least seven start points within a region located 45-25 nt upstream from the start codon. In transformants of A. niger, expression of multiple copies of phyA resulted in up to more than tenfold higher phytase levels than in the wild-type strain.

Development of a new transformant selection system for Penicillium chrysogenum: isolation and characterization of the P. chrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous selection marker.

A new transformation system for the filamentous fungus Penicillium chrysogenum is described, based on the use of the homologous acetyl-coenzyme A synthetase (facA) gene as a selection marker. Acetate-non-utilizing (Fac-) strains of P. chrysogenum were obtained by positive selection for spontaneous resistance to fluoroacetate. Among these fac mutants putative facA strains were selected for a loss of acetyl-coenzyme A (CoA) synthetase activity. The facA gene, coding for the enzyme acetyl-CoA synthetase, was isolated from a P. chrysogenum genomic library using synthetic oligonucleotides derived from conserved regions from the corresponding genes of Aspergillus nidulans and Neurospora crassa. Vector pPC2-3, comprising a genomic 6.5 kb PstI fragment, was able to complement P. chrysogenum facA strains with frequencies up to 27 transformants.micrograms-1 DNA. Direct selection of transformants was accomplished using acetate and low amounts (0.001%) of glucose as carbon sources. About 50% of the transformants arose by integration of pPC2-3 DNA at the homologous facA locus and 50% by integration elsewhere in the genome. Determination of the nucleotide sequence of part of the cloned fragment showed the presence of an open reading frame of 2007 nucleotides, interrupted by five putative introns. Comparison of the nucleotide and the amino acid sequence of the facA gene of P. chrysogenum with the facA gene of A. nidulans reveals similarities of 80% and 89%, respectively. The putative introns present in the P. chrysogenum facA gene appear at identical positions as those in the A. nidulans facA gene, but show no significant sequence similarity.

Cloning of the nitrate-nitrite reductase gene cluster of Penicillium chrysogenum and use of the niaD gene as a homologous selection marker.

A new homologous transformation system for the filamentous fungus Penicillium chrysogenum is described. The system is based on complementation of niaD mutants using the nitrate reductase structural gene (niaD) of P. chrysogenum. Spontaneous niaD mutants were identified after selection for chlorate resistance, in growth tests and subsequent complementation with the niaD gene of Aspergillus oryzae. The P. chrysogenum niaD gene was isolated from a genomic library using the Aspergillus nidulans niaD gene as a probe. After subcloning of the hybridizing fragment, the vector obtained, pPC1-1, was capable of transforming a P. chrysogenum niaD mutant at an average of 40 transformants per micrograms of circular DNA. Southern analysis of genomic DNA from a number of transformants showed that pPC1-1 DNA was integrated predominantly at sites other than the niaD locus. Using hybridization analysis it was shown that the niaD gene of P. chrysogenum is clustered with the nitrite reductase gene (niiA). From analysis of the nucleotide sequences of parts of the niaD and niiA genes of P. chrysogenum and comparison of these sequences with nucleotide sequences of the corresponding A. nidulans genes it was deduced that the P. chrysogenum genes are divergently transcribed.

Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function.

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.

Isolation and molecular characterisation of the benzoate-para-hydroxylase gene (bphA) of Aspergillus niger: a member of a new gene family of the cytochrome P450 superfamily.

The gene coding for benzoate-para-hydroxylase (bphA) of Aspergillus niger was cloned using differential hybridisation techniques and complementation of mutants deficient in this enzyme activity. The nucleotide sequence of the gene was determined, the presence of two introns was shown and the transcription start and termination sites were determined. The structure of the mRNA upstream from the long open reading frame (ORF) is unusual. It contains two small, overlapping ORFs whose function is unknown. Comparison of the deduced amino acid sequence of the protein with the sequences present in the databanks, indicated a significant similarity of BPH to the superfamily of cytochrome P450 enzymes. Further analysis revealed that this protein is a member of a new P450 gene family designated P450LIII. The gene is designated CYP53. To increase the BPH activity of A. niger, multiple copies of the bphA gene were introduced into the genome of a recipient strain by transformation. Although increased intracellular levels of the BPH protein could be detected, the BPH enzyme activity was decreased, suggesting titration of another essential component.

A system for the analysis of expression signals in Aspergillus.

To analyse gene expression signals in Aspergillus we have constructed a set of integration vectors each of which contains in front of the Escherichia coli 'lacZ gene a unique BamHI site in one of the three possible translational reading frames and the A. nidulans argB gene as a selection marker. The vectors allow in-phase translational fusion of any gene to 'lacZ. After transformation of an A. nidulans argB strain, the vectors integrate with a high percentage at the argB locus of the genome, as a single copy. The insertion of the fusion genes at the argB locus assures the constancy of influences of the chromosomal environment on gene expression.

Expression of an Escherichia coli beta-galactosidase fusion gene in Aspergillus nidulans.

We inserted in frame the Escherichia coli lacZ gene into the protein-coding region of the Aspergillus nidulans trpC gene and introduced the resultant fused gene into the A. nidulans genome. A functional beta Gal fusion protein was produced. Removal of the trpC transcription and translation initiation sequences from the fusion gene abolished production of the fusion protein, showing that expression is dependent on these sequences. Thus, lacZ fusions should be of use for estimating gene activity in a. nidulans.

Gene amplification in Aspergillus nidulans by transformation with vectors containing the amdS gene.

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 microgram 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.