Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

Palabıyık, Bedia; Karaer, Semian; Arda, Nazlı; Toker, Sıdıka Ertürk; Temizkan, Güler; Kelly, Steven L.; Sarikaya, Aysegul Topal

doi:10.2478/s11756-008-0069-0

Cited by 3 publications

(3 citation statements)

References 25 publications

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“…While P450 3A4 was also successfully expressed in other yeasts, [89,90] no preparative biotransformation with testosterone was performed with those catalysts. On the other hand, using S. pombe excellent human P450 expression and biocatalysis was exemplified by Drăgan et al, but no direct comparison is possible as a different human P450 converting another drug was reported for their preparative synthesis.…”

Section: Full Papermentioning

confidence: 99%

Preparative‐Scale Production of Testosterone Metabolites by Human Liver Cytochrome P450 Enzyme 3A4

Fessner

Srdič²,

Weber

et al. 2020

Adv Synth Catal

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Just like the drugs themselves, their metabolites have to be evaluated to succeed in a drug development and approval process. It is therefore essential to be able to predict drug metabolism and to synthesise sufficient metabolite quantities for further pharmacological testing. This study evaluates the possibility of using in vitro biotransformations to solve both these challenges in the case of testosterone as a representative component for steroids. The application of cells of Pichia pastoris with expressed membraneassociated human liver cytochrome P450 enzyme (P450) 3A4 in two cycles of a preparative-scale bioreactor experiment enabled the isolation of the common metabolites 6β-hydroxytestosterone and 6β-hydroxyandrostenedione on a 100 mg scale. Side-product formation caused by enzymes intrinsic to P. pastoris was reduced. In addition more polar testosterone metabolites formed by a P450 3A4-catalysed bioconversion, than the known mono-hydroxylated ones, are reported and 6-dehydro-15β-hydroxytestosterone as well as the di-hydroxylated steroids 6β,16β-dihydroxytestosterone, 6β,17β-dihydroxy-4-androstene-3,16-dione and 6β,12β-dihydroxyandrostenedione were isolated and verified by NMR analysis. Their respective biological significance remains to be investigated. Whole-cell P450 catalysts expressed in P. pastoris qualify as a tool for the preparative-scale synthesis of human metabolites. Biotransformation processes in combination with standard chemical procedures allow the isolation and characterisation even of minor drug metabolite products.

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Section: Full Papermentioning

confidence: 99%

Preparative‐Scale Production of Testosterone Metabolites by Human Liver Cytochrome P450 Enzyme 3A4

Fessner

Srdič²,

Weber

et al. 2020

Adv Synth Catal

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show abstract

“…For instance, among 120 CYP genes from P. chrsosporium , only 70 CYPs were successfully expressed based on a carbon monoxide binding assay [ 83 ]. Alternatively, unconventional non- Saccharomyces yeasts such as methylotrophic yeast Pichia pastoris [ 84 ], fission yeast Schizosaccharomyces pombe [ 85 ], dimorphic yeasts Yarrowia lipolytica [ 86 ] and Arxula adeninivorans [ 87 ], lactose-utilizing yeast Kluyveromyces lactis [ 88 ] and its thermophilic sister strain K. marxianus [ 88 ] can be employed to overcome the restrictions associated with heterologous CYP expression. For example, the expression of P. chrysosporium redox enzyme systems and CYPs ( CYP5136A3, CYP63A2 ) in P. pastoris enhanced active form of functional expression [ 53 , 89 ].…”

Section: Systematic Approach To Heterologous Expression Of Fungal Cypmentioning

confidence: 99%

Versatile biocatalysis of fungal cytochrome P450 monooxygenases

2016

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Cytochrome P450 (CYP) monooxygenases, the nature’s most versatile biological catalysts have unique ability to catalyse regio-, chemo-, and stereospecific oxidation of a wide range of substrates under mild reaction conditions, thereby addressing a significant challenge in chemocatalysis. Though CYP enzymes are ubiquitous in all biological kingdoms, the divergence of CYPs in fungal kingdom is manifold. The CYP enzymes play pivotal roles in various fungal metabolisms starting from housekeeping biochemical reactions, detoxification of chemicals, and adaptation to hostile surroundings. Considering the versatile catalytic potentials, fungal CYPs has gained wide range of attraction among researchers and various remarkable strategies have been accomplished to enhance their biocatalytic properties. Numerous fungal CYPs with multispecialty features have been identified and the number of characterized fungal CYPs is constantly increasing. Literature reveals ample reviews on mammalian, plant and bacterial CYPs, however, modest reports on fungal CYPs urges a comprehensive review highlighting their novel catalytic potentials and functional significances. In this review, we focus on the diversification and functional diversity of fungal CYPs and recapitulate their unique and versatile biocatalytic properties. As such, this review emphasizes the crucial issues of fungal CYP systems, and the factors influencing efficient biocatalysis.

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“…In the recent years, Schizosaccharomyces pombe could be successfully used for the expression of human P450 s (Bureik et al, 2002;Dragan et al, 2006a, Dragan et al, 2005Dragan et al, 2006b, Palabiyik et al, 2008Peters et al, 2009a;Peters et al, 2009b;Zöllner et al, 2009) and might become a powerful platform for the expression of these enzymes. Therefore, we have chosen S. pombe CAD 18 (Dragan et al, 2006a), functionally expressing CYP21 as model system to elucidate the bottlenecks during P450 catalyzed whole cell biotransformation.…”

Section: Introductionmentioning

confidence: 99%