title: Over-expression of ICE2 stabilizes cytochrome P450 reductase in Saccharomyces cerevisiae and Pichia pastoris.
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.
authors: ACIB-Austrian Centre of Industrial Biotechnology, Graz, Austria.
authors: Anita
authors: A
authors: Emmerstorfer
authors: Miriam
authors: M
authors: Wimmer-Teubenbacher
authors: Tamara
authors: T
authors: Wriessnegger
authors: Erich
authors: E
authors: Leitner
authors: Monika
authors: M
authors: Müller
authors: Iwona
authors: I
authors: Kaluzna
authors: Martin
authors: M
authors: Schürmann
authors: Daniel
authors: D
authors: Mink
authors: Günther
authors: G
authors: Zellnig
authors: Helmut
authors: H
authors: Schwab
authors: Harald
authors: H
authors: Pichler
mesh_terms: D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012717:Sesquiterpenes / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D051792:Basic Helix-Loop-Helix Proteins; D003577:Cytochrome P-450 Enzyme System; D005656:Fungal Proteins; D012717:Sesquiterpenes; C538823:ICE2 protein, Arabidopsis; C506706:valencene
keywords: Cytochrome P450 reductase; Membrane protein expression; Protein stability; Whole-cell conversion; Yeast