Expression of Cytochromes P450 in Yeast: Practical Aspects
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.
KeywordsTestosterone Pyrene Chitin Galactose Epoxide
Unable to display preview. Download preview PDF.
- Bairn SB, Sherman F (1988) MRNA structures influencing translation in the yeast Saccharomyces cerevisiae. Mol Cell Biol 8: 1591–1601Google Scholar
- 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: 859–865Google Scholar
- 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)Google Scholar
- 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)Google Scholar
- 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: 1028–1033Google Scholar
- 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)Google Scholar
- 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 169–190Google Scholar
- 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: 8388Google Scholar