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.
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References
Ballard SA, Kelly SL, Ellis SW, Troke PF (1990) Interactions of microsomal cytochrome P450 isolated from Aspergillus fumigatus with fluconazole and itranazole. J Med Vet Mycol 28:327–334
Boschloo JG, Paffen A, Koot T, Tweel WJJ van den, Gorcom RFM van, Cordewener JHG, Bos CJ (1990) Genetic analysis of benzoate metabolism in A. niger. Appl Microbiol Biotechnol 34:225–228
Brink JM van den, Zeijl CMJ van, Brons JF, Hondel CAMJJ van den, Gorcom RFM van (1995) Cloning and characterization of the NADPH-cytochrome P450 oxidoreductase gene from the filamentous fungus Aspergillus niger. DNA Cell Biol 14:719–729
Cerniglia CE (1984) Microbial metabolism of polycyclic aromatic hydrocarbons. In: Laskin AI (ed) Advances in applied microbiology. Academic Press, New York, pp 31–71
Dutta D, Ghosh DK, Mishra AK, Samanta TB (1983) Induction of a benz[a]pyrene hydroxylase in Aspergillus ochraceus TS: evidence for multiple forms of cytochrome P450. Biochem Biophys Res Comm 115:692–699
Gerritse K, Gorcom RFM van, Fasbender M, Lange J, Schellekens MM, Zegers ND, Claassen E, Boersma W (1990) Specific immuno detection of benzoate-para-hydroxylase with antibodies raised against specific peptides. Biochem Biophys Res Commun 167:33–39
Ghosh DK, Dutta D, Samanta TB, Mishra AK (1983) Microsomal benzo[a]pyrene hydroxylase in Aspergillus ochraceus TS: assay and characterization of the enzyme system. Biochem Biophys Res Commun 113:497–505
Gorcom RFM van, Boschloo JB, Kuijvenhoven A, Lange J, Vark AJ van, Bos CJ, Balken JAM van, Pouwels PH, Hondel CAMJJ van den (1990) Isolation and molecular characterization of the benzoate-para-hydroxylase gene (bphA) of A. niger: a member of a new gene family of the cytochrome P450 superfamily. Mol Gen Genet 223:192–197
Hohn TM, Desjardins AE, McCormick SP (1995) The Tri4 gene of Fusarium sporotrichiodes encodes a cytochrome P450 monooxygenase involved in trichothecene biosynthesis. Mol Gen Genet 248:95–102
Holland HL (1992) Bioconversions. In: Finkelstein DB, Ball C (eds) Biotechnology of filamentous fungi. Technology and products. pp 157–187
Kizawa H, Tomura D, Oda M, Fukamizu A, Hoshino T, Goto O, Yasui T, Shoun H (1991) Nucleotide sequence of the unique nitrate/nitrite-inducible cytochrome P-450 cDNA from Fusarium oxysporum. J Biol Chem 266:10632–10637
Launen L, Pinto L, Wiebe C, Kiehlmann E, Moore M (1995) The oxidation of pyrene and benzo[a]pyrene by nonbasidiomycete soil fungi. Can J Microbiol 41:477–488
Madyastha KM, Coscia CJ (1979) Detergent solubilized NADPH-cytochrome c (P-450) reductase from the higher plant Catharanthus roseus. J Biol Chem 254:2419–2427
Nelson DR, Kamataki T, Waxman DJ, Guengerich FP, Estabrook RW, Feyereisen R, Gonzalez FJ, Coon MJ, Gunsalus IC, Gotoh O, Okuda K, Nebert DW (1993) The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol 12:1–51
Nistelrooij JGM van, Brink JM van den, Kan JAL van, Gorcom RFM van, Waard MA de (1995) Isolation and molecular characterization of the gene encoding eburicol 14α-demethylase (cyp51) from Penicillium italicum. Mol Gen Genet 250:725–733
Peterson DH, Murray HC (1952) Microbial oxygenation of steroids at carbon 11. J Am Chem Soc 74:1871–1872
Pontecorvo G, Roper JA, Hemmons LJ, Macdonald KD, Bufton AWJ (1953) The genetics of Aspergillus nidulans. Adv Genet 5:141–238
Punt PJ, Hondel CAMJJ van den (1993) Transformation of filamentous fungi based on hygromycine B and phleomycine resistance markers. Methods Enzymol 216:447–457
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, Hondel CAMJJ van den (1987) Transformation of Aspergillus based on the hygromycine B resistance marker from Escherichia coli. Gene 56:117–124
Sakaki T, Shibata M, Yabusaki Y, Murakami H, Ohkawa H (1990) Expression of bovine cytochrome P450c21 and its fused enzymes with yeast NADPH-cytochrome P450 reductase in Saccharomyces cerevisiae. DNA Cell Biol 9:603–614
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, cold Spring Harbor, NY
Schunck W-H, Vogel F, Gross B, Kaergel E, Mauersberger S, Koepke K, Gengnagel C, Mueller H-G (1991) Comparison of two cytochromes P450 from Candida maltosa: primary structures, substrate specificities and effect of their expression in Saccharomyces cerevisiae on the proliferation of the endoplasmic reticulum. Eur J Cell Biol 55:336–345
Truan G, Cullin C, Reisdorf P, Urban P, Pompon D (1993) Enhanced in vivo monooxygenase activities of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and human cytochrome b5. Gene 125:49–55
Weltring KM, Turgeon BG, Yoder OC, VanEtten HD (1988) Isolation of a phytoalexin-detoxification gene from the plant pathogenic fungus Nectria haematococca by detecting its expression in Aspergillus nidulans. Gene 68:335–344
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13mp18 and pUC19 vectors. Gene 33:103–119
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van der Brink, J.M., van den Hondel, C.A.M.J.J. & van Gorcom, R.F.M. Optimization of the benzoate-inducible benzoate p-hydroxylase cytochrome P450 enzyme system in Aspergillus niger . Appl Microbiol Biotechnol 46, 360–364 (1996). https://doi.org/10.1007/BF00166230
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DOI: https://doi.org/10.1007/BF00166230