Cloning and expression of rat CYP2E1 inSaccharomyces cerevisiae: Detection of genotoxicity ofN-alkylformamides

Carratore, M.R. Del; Mezzatesta, Caterina; Hidestrand, Mats; Neve, P.; Amato, Gianluca; Gervasi, Pier Giovanni

doi:10.1002/1098-2280(2000)36:2<97::aid-em3>3.0.co;2-4

Cited by 8 publications

(6 citation statements)

References 40 publications

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“…Low-level functional expression of CYP2E1, in combination with human NADPH cytochrome reductase, was achieved in Escherichia coli , and was significantly improved by the application of different expression plasmid/host combinations (Cheng et al 2004). Other hosts for the functional expression of these mammalian enzymes are S. cerevisiae (Del Carratore et al 2000), and Chinese hamster ovary cells (Nakagawa et al 1996;Bernauer et al 2003). To solubilize the membrane-bound human CYP2E1, and therefore to enhance its possible application in drug metabolism studies, the so-called molecular LEGO approach was developed.…”

Section: Microsomal Cyp52 Cyp2e and Cyp4bmentioning

confidence: 95%

Alkane activation by P450 oxygenases

Funhoff¹,

Beilen²

2007

Biocatalysis and Biotransformation

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The hydroxylation of alkane molecules, especially at terminal positions, is a challenging reaction. Enzymes that catalyze this reaction could be used to produce high-value compounds from aliphatic and alkyl-substituted substrates. However, until a few years ago, all known alkane hydroxylating enzymes were membrane-bound, and difficult to use. Recently, three bacterial P450 enzymes of the (soluble) CYP101 and CYP102 families were engineered to hydroxylate alkanes, but even after extensive efforts hydroxylation was mainly at sub-terminal positions. More recently, a new soluble P450 family (CYP153) was identified and characterized, which activates the terminal position of alkanes and alkyl-substituted compounds with very high regio-selectivity. The use of CYP153s in biotechnological applications is now being explored.

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Section: Microsomal Cyp52 Cyp2e and Cyp4bmentioning

confidence: 95%

Alkane activation by P450 oxygenases

Funhoff¹,

Beilen²

2007

Biocatalysis and Biotransformation

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“…However, metabolites can be too reactive or hydrophilic to penetrate into the cells, as has been described for the epoxide of benzo[a]pyrene [53]. In a direct comparison between the two test systems, the number of DNA mutations induced by N-nitrosodimethylamine metabolism outside the cell by S9-fractions was about three-fold lower than that obtained by metabolism inside CYP2E1-expressing yeast cells [33].…”

Section: Models Using Exogenous Activation Systemsmentioning

confidence: 99%

“…Yeast strains expressing CYP1A1 or CYP1A2 were also used to examine the mutagenicity of benzo [a]pyrene-trans-7,8-dihydrodiol, 3-amino-1-methyl-5H-pyrido [4,3-b]indole, benzo[a]pyrene and 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline [34]. Additionally, homologous mitotic recombination was highly increased in yeast cells expressing rat CYP2E1 incubated with N-nitrosodimethylamine, N-methylformamide and N-ethylformamide compared to cells transformed with an empty plasmid or CYP2E1 expressing cells co-incubated with a CYP2E1-inhibitor [33].…”

Section: Cytochrome P450-related Toxicity In Yeastmentioning

confidence: 99%

“…Various bioactivationdependent toxicity studies in yeast describe the genotoxicity of the natural toxin aflatoxin B 1 after bioactivation by CYP1A1, CYP1A2 or CYP3A4 [24][25][26][27][28][29][30]. Yeast cells expressing mammalian CYP1A1, CYP1A2, CYP2B1, CYP2E1 or CYP3A4 were also used in genotoxicity assays for a wide range of other environmental or food contaminants and drugs, including N-nitrosodimethylamine, benzo [a]pyrene and the anticancer drug cyclophosphamide [31][32][33][34][35]. First, we will focus on the various other cellular bioactivation models before discussing in more detail the properties of yeast as eukaryotic model in biotransformation-related toxicity studies.…”

Section: Introductionmentioning

confidence: 99%

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Yeast as a Humanized Model Organism for Biotransformation-Related Toxicity

Leeuwen

Vermeulen

Vos

2012

CDM

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High drug attrition rates due to toxicity, the controversy of experimental animal usage, and the EU REACH regulation demanding toxicity profiles of a high number of chemicals demonstrate the need for new, in vitro toxicity models with high predictivity and throughput. Metabolism by cytochrome P450s (P450s) is one of the main causes of drug toxicity. As some of these enzymes are highly polymorphic leading to large differences is metabolic capacity, isotype-specific test systems are needed. In this review, we will discuss the use of yeast expressing (mammalian) P450s as a powerful, additional model system in drug safety. We will discuss the various cellular model systems for bioactivation-related toxicity and subsequently describe the properties of yeast as a model system, including the endogenous bioactivation enzymes present, the heterologous expression of (mammalian) P450s and the application of yeasts expressing heterologous P450s and/or other biotransformation enzymes in toxicity studies. All major human drug-metabolizing P450s have been successfully expressed in yeast and various mutagenicity tests have been performed with these humanized yeast strains. The few examples of non-mutagenic toxicity studies with these strains and of the combination of P450s with phase II or other human enzymes show the potential of yeast as a model system in metabolism-related toxicity studies. The wide variety of genome-wide screens available in yeast, combined with its well-annotated genome, also facilitate follow-up studies on the genes involved in toxicity. Unless indicated otherwise "yeast" will refer to baker's yeast Saccharomyces cerevisiae.

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“…Proteins were isolated based on adapted procedures of Bèki et al (2003) andDel Carratore et al (2000). A 10 mL SC Àura yeast preculture was grown overnight at 30 1C and inoculated into 100 mL SC Àura medium and grown overnight at 30 1C.…”

Section: Isolation Of Membrane-associated and Cell Wallassociated Promentioning

confidence: 99%

Heterologous expression of aClostridiumminicellulosome inSaccharomyces cerevisiae

Lilly

Fierobe

Zyl

et al. 2009

FEMS Yeast Research

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The yeast Saccharomyces cerevisiae was genetically modified to assemble a minicellulosome on its cell surface by heterologous expression of a chimeric scaffoldin protein from Clostridium cellulolyticum under the regulation of the phosphoglycerate kinase 1 (PGK1) promoter and terminator regulatory elements, together with the beta-xylanase 2 secretion signal of Trichoderma reesei and cell wall protein 2 (Cwp2) of S. cerevisiae. Fluorescent microscopy and Far Western blot analysis confirmed that the Scaf3p is targeted to the yeast cell surface and that the Clostridium thermocellum cohesin domain is functional in yeast. Similarly, functionality of the C. thermocellum dockerin domain in yeast is shown by binding to the Scaf3 protein in Far Western blot analysis. Phenotypic evidence for cohesin-dockerin interaction was also established with the detection of a twofold increase in tethered endoglucanase enzyme activity in S. cerevisiae cells expressing the Scaf3 protein compared with the parent strain. This study highlights the feasibility to future design of enhanced cellulolytic strains of S. cerevisiae through emulation of the cellulosome concept. Potentially, Scaf3p-armed yeast could also be developed into an alternative cell surface display strategy with various tailor-made applications.

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Cloning and expression of rat CYP2E1 inSaccharomyces cerevisiae: Detection of genotoxicity ofN-alkylformamides

Cited by 8 publications

References 40 publications

Alkane activation by P450 oxygenases

Alkane activation by P450 oxygenases

Yeast as a Humanized Model Organism for Biotransformation-Related Toxicity

Heterologous expression of aClostridiumminicellulosome inSaccharomyces cerevisiae

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