title: Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris.
abstract: The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208mg L(-1) cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria. Electronic address: tamara.wriessnegger@acib.at.
authors.forename: Tamara
authors.initials: T
authors.lastname: Wriessnegger
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria. Electronic address: peter.augustin@tugraz.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Augustin
authors.affiliation: Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14/2, 8010 Graz, Austria. Electronic address: matthias.engleder@student.TUGraz.at.
authors.forename: Matthias
authors.initials: M
authors.lastname: Engleder
authors.affiliation: Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz, Austria. Electronic address: erich.leitner@tugraz.at.
authors.forename: Erich
authors.initials: E
authors.lastname: Leitner
authors.affiliation: DSM Innovative Synthesis B.V., Urmonderbaan 22, 6167 RD Geleen, The Netherlands. Electronic address: monika.muller@dsm.com.
authors.forename: Monika
authors.initials: M
authors.lastname: Müller
authors.affiliation: DSM Innovative Synthesis B.V., Urmonderbaan 22, 6167 RD Geleen, The Netherlands. Electronic address: iwona.kaluzna@dsm.com.
authors.forename: Iwona
authors.initials: I
authors.lastname: Kaluzna
authors.affiliation: DSM Innovative Synthesis B.V., Urmonderbaan 22, 6167 RD Geleen, The Netherlands. Electronic address: martin.schurmann@dsm.com.
authors.forename: Martin
authors.initials: M
authors.lastname: Schürmann
authors.affiliation: DSM Innovative Synthesis B.V., Urmonderbaan 22, 6167 RD Geleen, The Netherlands. Electronic address: daniel.mink@dsm.com.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mink
authors.affiliation: Institute of Plant Sciences, University of Graz, Schubertstrasse 51, 8010 Graz, Austria. Electronic address: guenther.zellnig@uni-graz.at.
authors.forename: Günther
authors.initials: G
authors.lastname: Zellnig
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14/2, 8010 Graz, Austria. Electronic address: helmut.schwab@tugraz.at.
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14/2, 8010 Graz, Austria. Electronic address: harald.pichler@tugraz.at.
authors.forename: Harald
authors.initials: H
authors.lastname: Pichler
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000235:genetics; D029781:Cupressus / Q000201:enzymology / Q000235:genetics; D032304:Hyoscyamus / Q000201:enzymology / Q000235:genetics; D060847:Metabolic Engineering; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; C050302:nootkatone
keywords: Cytochrome P450; Membrane protein; Metabolic engineering; Nootkatone; Pichia pastoris; Terpenoid