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.

An artificial self‐sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio‐selectivity

“…Further studies are needed to better understand underlying structural and biochemical determinants such as enzyme kinetic parameters. Other than the fusion design, the formation of heterotrimeric protein‐mediated complex comprised of class I CYP and its redox partners and the stabilization of hetereologously expressed redox partners might be applied in creating efficient TxtE‐based nitration biocatalysts.…”

Stepwise engineering ofSaccharomyces cerevisiaeto produce (+)-valencene and its related sesquiterpenes

“…Usually raffinose is added to minimal medium containing galactose for protein expression induction. 27 As an organic compound, glycerol assimilation can occur in the presence of galactose in yeast. Earlier work indicated that the glycerol or raffinose addition might be favor to noscapine biosynthesis.…”

Heterologous Production of Flavour and Aroma Compounds in Saccharomyces cerevisiae

“…Production of trans -nootkatol was further improved by overexpression of the native yeast type III membrane associate protein Ice2p [ 77 ]. Introduction of this genetic modification into recombinant strain engineered earlier to produce (+)-valencene via expression of premnaspirodiene oxygenase (HPO; CYP) from Hyoscyamus muticus and cytochrome P450 from A. thaliana ( At CPR) [ 64 ], resulted in stabilizing the levels and enzymatic activity of the latter heterologous enzyme.…”