title: Tandem-yeast expression system for engineering and producing unspecific peroxygenase.
abstract: Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae, the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (∼8mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217mg/L). The P. pastoris UPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production.
authors.affiliation: Department of Biocatalysis, Institute of Catalysis, CSIC, 28049 Madrid, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Molina-Espeja
authors.affiliation: Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU-University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
authors.forename: Su
authors.initials: S
authors.lastname: Ma
authors.affiliation: DWI-Leibniz-Institute for Interactive Materials, Forckenbeckstraβe 50, 52056 Aachen, Germany.
authors.forename: Diana M
authors.initials: DM
authors.lastname: Mate
authors.affiliation: Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU-University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Ludwig
authors.affiliation: Department of Biocatalysis, Institute of Catalysis, CSIC, 28049 Madrid, Spain. Electronic address: malcalde@icp.csic.es.
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcalde
mesh_terms: D055362:Agrocybe / Q000201:enzymology* / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D005800:Genes, Fungal; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D006899:Mixed Function Oxygenases; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; C022333:peroxygenase
keywords: Directed evolution; Functional expression; Pichia pastoris; Saccharomyces cerevisiae; Unspecific peroxygenase (UPO)