Over‐expression of ICE2 stabilizes cytochrome P450 reductase in Saccharomyces cerevisiae and Pichia pastoris. Issue 4 (23rd February 2015)

• Record Type: Journal Article
• Authors: Emmerstorfer, Anita; Wimmer‐Teubenbacher, Miriam; Wriessnegger, Tamara; Leitner, Erich; Müller, Monika; Kaluzna, Iwona; Schürmann, Martin; Mink, Daniel; Zellnig, Günther; Schwab, Helmut; Pichler, Harald
• Is Part Of: Biotechnology journal. Volume 10:Issue 4(2015:Apr.)
• Page Start: 623
• Page End: 635
• Publication Date: 2015-02-23
• Subjects: Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.201400780
• Languages: English
• ISSNs: 1860-6768

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p>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 <italic>Saccharomyces cerevisiae</italic> knockout collection for <italic>Hyoscyamus muticus</italic> premnaspirodiene oxygenase (HPO; CYP) and <italic>Arabidopsis thaliana</italic> CPR (<italic>At</italic>CPR) expression levels, as well as for activity towards (+)‐valencene. We found that in cells lacking the type III membrane protein Ice2p, <italic>At</italic>CPR was destabilized. Remarkably, over‐expression of <italic>ICE2</italic> 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 <italic>At</italic>CPR 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 <italic>ICE2</italic> over‐expression for two further CYP/CPR combinations and the alternative host <italic>Pichia pastoris</italic>. Thus, we propose Ice2 up‐regulation as a general tool for improving the applications of recombinant CYPs in yeasts.</p> </abstract>