1. Over-expression of ICE2 stabilizes cytochrome P450 reductase in Saccharomyces cerevisiae and Pichia pastoris.
- Author
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Emmerstorfer A, Wimmer-Teubenbacher M, Wriessnegger T, Leitner E, Müller M, Kaluzna I, Schürmann M, Mink D, Zellnig G, Schwab H, and Pichler H
- Subjects
- Arabidopsis Proteins genetics, Basic Helix-Loop-Helix Transcription Factors genetics, Cytochrome P-450 Enzyme System chemistry, Cytochrome P-450 Enzyme System genetics, Fungal Proteins genetics, Pichia genetics, Protein Stability, Saccharomyces cerevisiae genetics, Sesquiterpenes metabolism, Up-Regulation genetics, Arabidopsis Proteins metabolism, Basic Helix-Loop-Helix Transcription Factors metabolism, Cytochrome P-450 Enzyme System metabolism, Fungal Proteins metabolism, Pichia metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Membrane-anchored cytochrome P450 enzymes (CYPs) are a versatile and interesting class of enzymes for industrial applications, as they are capable of regio- and stereoselectively hydroxylating hydrophobic molecules. However, CYP activity requires sufficient levels of suitable cytochrome P450 reductases (CPRs) for regeneration of catalytic capacity, which is a bottleneck in many industrial applications. Searching for positive effectors of membrane-anchored CYP/CPR function, we transformed and screened selected strains from a Saccharomyces cerevisiae knockout collection for Hyoscyamus muticus premnaspirodiene oxygenase (HPO; CYP) and Arabidopsis thaliana CPR (AtCPR) expression levels, as well as for activity towards (+)-valencene. We found that in cells lacking the type III membrane protein Ice2p, AtCPR was destabilized. Remarkably, over-expression of ICE2 improved (+)-valencene hydroxylation to trans-nootkatol by 40-50%, both in resting cells and in vivo. Time-resolved immunoblot analysis and cytochrome c reductase activity assays revealed that Ice2 up-regulation stabilized AtCPR levels and activity over extended periods of bioconversion. To underscore that we had identified a novel positive effector of recombinant CYP/CPR function, we confirmed the beneficial effect of ICE2 over-expression for two further CYP/CPR combinations and the alternative host Pichia pastoris. Thus, we propose Ice2 up-regulation as a general tool for improving the applications of recombinant CYPs in yeasts., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2015
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