Enhancing cytochrome P450-mediated conversions in P. pastoris

Sandra Moser, Tamara Wriessnegger, Anita Emmerstorfer-Augustin, Erich Leitner, Monika Müller, Iwona Kaluzna, Schürmann Martin, Mink Daniel

Publikation: KonferenzbeitragPosterBegutachtung

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 [1]. 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 [2].

[1] T. Wriessnegger, P. Augustin, M. Engleder, E. Leitner, M. Müller, I. Kaluzna, M. Schürmann, D. Mink, G. Zellnig, H. Schwab, H. Pichler, Metab. Eng. 2014, 24C, 18–29.
[2] T. Wriessnegger; S. Moser, A. Emmerstorfer-Augustin, E. Leitner; M. Müller, I. Kaluzna, M. Schürmann, D. Mink, H. Pichler, Fungal Genet. Biol 2016, 89, 114-25.
Originalspracheenglisch
PublikationsstatusVeröffentlicht - 23 Apr 2017
Veranstaltung9th Conference on Recombinant Protein Production (RPP9) - Dubrovnik, Kroatien
Dauer: 23 Apr 201725 Apr 2017

Konferenz

Konferenz9th Conference on Recombinant Protein Production (RPP9)
KurztitelRPP9
LandKroatien
OrtDubrovnik
Zeitraum23/04/1725/04/17

ASJC Scopus subject areas

  • Biochemie, Genetik und Molekularbiologie (insg.)

Fields of Expertise

  • Human- & Biotechnology

Treatment code (Nähere Zuordnung)

  • Application
  • Basic - Fundamental (Grundlagenforschung)
  • Experimental

Kooperationen

  • NAWI Graz

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