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

Research output: Contribution to conferencePoster

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.
Original languageEnglish
Publication statusPublished - 23 Apr 2017
Event9th Conference on Recombinant Protein Production (RPP9) - Dubrovnik, Croatia
Duration: 23 Apr 201725 Apr 2017

Conference

Conference9th Conference on Recombinant Protein Production (RPP9)
Abbreviated titleRPP9
CountryCroatia
CityDubrovnik
Period23/04/1725/04/17

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Fields of Expertise

  • Human- & Biotechnology

Treatment code (Nähere Zuordnung)

  • Application
  • Basic - Fundamental (Grundlagenforschung)
  • Experimental

Cooperations

  • NAWI Graz

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  • Cite this

    Moser, S., Wriessnegger, T., Emmerstorfer-Augustin, A., Leitner, E., Müller, M., Kaluzna, I., ... Daniel, M. (2017). Enhancing cytochrome P450-mediated conversions in P. pastoris. Poster session presented at 9th Conference on Recombinant Protein Production (RPP9) , Dubrovnik, Croatia.