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Tandem-yeast expression system for engineering and producing unspecific peroxygenase

Paper ID Volume ID Publish Year Pages File Format Full-Text
16920 42622 2015 5 PDF Available
Title
Tandem-yeast expression system for engineering and producing unspecific peroxygenase
Abstract

•Unspecific peroxygenases (UPOs) are promising biocatalysts for organic synthesis.•Directed evolution and large scale production are required for UPO applications.•A compatible tandem-yeast expression system for UPO engineering and production was developed.•Evolved UPO in S. cerevisiae is readily over-produced with high yields in P. pastoris.

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 (∼8 mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217 mg/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.

Keywords
Unspecific peroxygenase (UPO); Directed evolution; Functional expression; Saccharomyces cerevisiae; Pichia pastorisABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); CPO, chloroperoxidase; DMP, 2,6-dimethoxyphenol; DO, dissolved oxygen; HTP, he
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Tandem-yeast expression system for engineering and producing unspecific peroxygenase
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volumes 73–74, June 2015, Pages 29–33
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering