title: Enhancing cytochrome P450-mediated conversions in P. pastoris through RAD52 over-expression and optimizing the cultivation conditions.
abstract: Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6.
authors.affiliation: ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Petersgasse 14/2, 8010 Graz, Austria.
authors.forename: Tamara
authors.initials: T
authors.lastname: Wriessnegger
authors.affiliation: ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Petersgasse 14/2, 8010 Graz, Austria.
authors.forename: Sandra
authors.initials: S
authors.lastname: Moser
authors.affiliation: ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Anita
authors.initials: A
authors.lastname: Emmerstorfer-Augustin
authors.affiliation: Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, NAWI Graz, Stremayrgasse 9, 8010 Graz, Austria.
authors.forename: Erich
authors.initials: E
authors.lastname: Leitner
authors.affiliation: DSM Chemical Technology R&D B.V., Innovative Synthesis, Urmonderbaan 22, 6167 RD Geleen, The Netherlands.
authors.forename: Monika
authors.initials: M
authors.lastname: Müller
authors.affiliation: DSM Chemical Technology R&D B.V., Innovative Synthesis, Urmonderbaan 22, 6167 RD Geleen, The Netherlands.
authors.forename: Iwona
authors.initials: I
authors.lastname: Kaluzna
authors.affiliation: DSM Chemical Technology R&D B.V., Innovative Synthesis, Urmonderbaan 22, 6167 RD Geleen, The Netherlands.
authors.forename: Martin
authors.initials: M
authors.lastname: Schürmann
authors.affiliation: DSM Chemical Technology R&D B.V., Innovative Synthesis, Urmonderbaan 22, 6167 RD Geleen, The Netherlands.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mink
authors.affiliation: ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Petersgasse 14/2, 8010 Graz, Austria. Electronic address: harald.pichler@tugraz.at.
authors.forename: Harald
authors.initials: H
authors.lastname: Pichler
mesh_terms: D001711:Biotransformation; D003470:Culture Media; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D006863:Hydrogen-Ion Concentration; D036142:Mentha piperita / Q000201:enzymology; D010084:Oxidation-Reduction; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics* / Q000378:metabolism; D012717:Sesquiterpenes / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D003470:Culture Media; D051721:Rad52 DNA Repair and Recombination Protein; D012717:Sesquiterpenes; D013729:Terpenes; C041991:Nootkatol; D003577:Cytochrome P-450 Enzyme System; C120915:limonene-3-hydroxylase; D019389:Cytochrome P-450 CYP2D6
keywords: Cultivation conditions; Cytochrome P450 enzyme; Pichia pastoris; RAD52; Terpenoid; Yeast