Abstract
Heterologous expression systems must ideally feature high, stable, reproducible and low-cost synthesis of any P450 under a native folding state, including full saturation by the hemin prosthetic group. Additionally, the host cell must be free of any interfering endogenous P450 activity and must offer a suitable P450 environment mimicking as closely as possible the natural microsomal membrane of the organism from which the P450 originates. Production of large amounts of metabolites further requires the reconstitution of an in vivo self-sufficient system. The natural presence or the coexpression of P450-associated electron transfer proteins, particularly NADPH-dependent reductase and cytochrome b 5, is thus useful. In contrast, the presence of endogenous phase II activities in the host system could be deleterious because it sometimes results in conjugation reactions that mask the formation of reactive intermediates. Nevertheless, some P450-dependent activation reactions involve an obligatory association with a phase II enzyme, such as microsomal epoxide hydrolase, to generate the intermediates then used as substrates by the same or other P450 to produce the final activated metabolites. Optional coexpression of phase II activity is thus an interesting feature for a more accurate simulation of drug and pollutant metabolism. Among the numerous organisms in which expression tools are available, yeast is rather unique in meeting all the different criteria previously listed.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
References
Bairn SB, Sherman F (1988) MRNA structures influencing translation in the yeast Saccharomyces cerevisiae. Mol Cell Biol 8: 1591–1601
Barnes HJ, Arlotto MP, Waterman MR (1991) Expression and enzymatic activity of recombinant cytochrome P450 17a-hydroxylase in Escherichia coli. Proc Natl Acad Sci USA 88: 5597–5601
Briza P, Breitenbach M, Ellinger A, Segal J (1990) Isolation of two developmentally regulated genes involved in spore wall maturation in Saccharomyces cerevisiae. Gene Dev 4: 1775–1789
Fisher CW, Caudle DL, Martin-Wixtron C, Quattrochi LC, Tukey RH, Waterman MR, Estabrook RW (1992) High-level expression of a functional human cytochrome P450 1A2 in Escherichia coli. FASEB J 6: 759–764
Gauner JC, Urban P, Beaune P, Pompon D (1993) Engineered yeast cells as model to study coupling between human metabolizing enzymes. Eur J Biochem 211: 63–72
Hata S, Nishino T, Katsuki H, Aoyama Y, Yoshida Y (1983) Two species of cytochrome P450 involved in ergosterol biosynthesis of yeast. Biochem Biophys Res Commun 116: 162–166
Kalb VF, Woods CW, Turi TG, Dey CR, Sutter TR, Loper JC (1987) Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae. DNA 6: 529–537
Kelly SL, Kelly DE, King DJ, Wiseman A (1985) Interaction between yeast cytochrome P450 and chemical carcinogens. Carcinogenesis 6: 1321–1325
Murakami H, Yabusaki Y, Ohkawa H (1986) Expression of rat NADPH cytoch- rome P450 reductase CDNA in Saccharomyces cerevisiae. DNA 5: 1–10
Murakarni H, Yabusaki Y, Sakaki T, Shibata M, Ohkawa H (1990) Expression of cloned yeast NADPH-cytochrome P450 reductase gene in Saccharomyces cerevisiae. J Biochem (Tokyo) 108: 859–865
Oeda K, Sakaki T, Ohkawa H (1985) Expression of rat liver cytochrome P450MC CDNA in Saccharomyces cerevisiae. DNA 4: 203–210
Peyronneau MA, Renaud JP, Truan G, Urban P, Pompon D, Mansuy D (1992) Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5. Eur J Biochem 207: 109–116
Pompon D (1988) CDNA cloning and functional expression in yeast Saccharomyces cerevisiae of (3-naphtoflavone-induced rabbit liver P450 LM4 and LM6. Eur J Biochem 177: 285–293
Pompon D, Truan G, Bellamine A, Kazmaier M, Urban P (1994) Coexpression of mammalian, plant or yeast P450s and P450 reductases in Saccharomyces cerevisiae as cloning and bioconversion tools. In: Lechner MC (ed) Cytochrome P450, biochemistry and biophysics. Elsevier, Amsterdam (in press)
Shibata M, Sakaki T, Yabusaki Y, Murakami H, Ohkawa H (1990) Genetically engineered P450 monooxygenases: construction of bovine P450 c17/yeast reductase fused enzyme. DNA Cell Biol 9: 27–36
Truan G, Cullin C, Reisdorf P, Urban P, Pompon D (1993) Enhanced in vivo monooxygenase activties of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and cytochrome b5. Gene 125: 49–55
Truan G, Epinat JC, Rougeulle C, Cullin C, Pompon D (1994) Cloning and characterisation of a yeast cytochrome b5 gene which suppresses ketoconazole hypersensitivity in NADPH P450 reductase disrupted strain. Gene (in press)
Urban P, Cullin C, Pompon D (1990) Maximizing the expression of mammalian cytochrome P450 monooxygenase activities in yeast cells. Biochimie 72: 463–472
Urban P, Truan G, Gauner JC, Pompon D (1993) Xenobiotic metabolism in humanized yeast: engineered yeast cells producing human NADPH-cytochrome P450 reductase, cytochrome b5, epoxide hydrolase and P450s. Biochem Soc Transact 21: 1028–1033
Urban P, Werck-Reichhart D, Teutsch H, Durst F, Regnier S, Kazmaier M, Pompon D (1994) Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast: kinetic and spectral properties of the major plant P450 of the phenylpropanoid pathway. Eur J Biochem (submitted)
Wiseman A, Woods LFJ (1978) Regulation of the biosynthesis of cytochrome P450 in yeast. Biochim Biophys Acta 544: 615–623
Yabusaki Y, Ohkawa H (1991) Genetic engineering on cytochrome P-450 monooxygenases. In: Ruckpaul K, Rein H (eds) Frontiers in biotransformations, vol 4. Akademie, Berlin, pp 169–190
Yamano S, Aoyama T, McBride OW, Hardwick JP, Gelboin HV, Gonzalez FJ (1989) Human NADPH-P450 oxidoreductase: complementary DNA cloning, sequence and vaccinia virus-mediated expression and localization of the CYPOR gene to chromosome VII. Mol Pharmacol 35: 8388
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1994 Springer-Verlag Berlin Heidelberg
About this paper
Cite this paper
Pompon, D., Truan, G., Bellamine, A., Urban, P. (1994). Expression of Cytochromes P450 in Yeast: Practical Aspects. In: Waterman, M.R., Hildebrand, M. (eds) Assessment of the Use of Single Cytochrome P450 Enzymes in Drug Research. Ernst Schering Research Foundation Workshop, vol 13. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-03019-6_6
Download citation
DOI: https://doi.org/10.1007/978-3-662-03019-6_6
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-662-03021-9
Online ISBN: 978-3-662-03019-6
eBook Packages: Springer Book Archive