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Cell-Free Protein Synthesis with Fungal Lysates for the Rapid Production of Unspecific Peroxygenases

  • Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal biocatalysts that have attracted considerable interest for application in chemical syntheses due to their ability to selectively incorporate peroxide-oxygen into non-activated hydrocarbons. However, the number of available and characterized UPOs is limited, as it is difficult to produce these enzymes in homologous or hetero-logous expression systems. In the present study, we introduce a third approach for the expression of UPOs: cell-free protein synthesis using lysates from filamentous fungi. Biomass of Neurospora crassa and Aspergillus niger, respectively, was lysed by French press and tested for translational activity with a luciferase reporter enzyme. The upo1 gene from Cyclocybe (Agrocybe) aegerita (encoding the main peroxygenase, AaeUPO) was cell-free expressed with both lysates, reaching activities of up to 105 U L−1 within 24 h (measured with veratryl alcohol as substrate). The cell-free expressed enzyme (cfAaeUPO) was successfully tested in a substrate screening thatUnspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal biocatalysts that have attracted considerable interest for application in chemical syntheses due to their ability to selectively incorporate peroxide-oxygen into non-activated hydrocarbons. However, the number of available and characterized UPOs is limited, as it is difficult to produce these enzymes in homologous or hetero-logous expression systems. In the present study, we introduce a third approach for the expression of UPOs: cell-free protein synthesis using lysates from filamentous fungi. Biomass of Neurospora crassa and Aspergillus niger, respectively, was lysed by French press and tested for translational activity with a luciferase reporter enzyme. The upo1 gene from Cyclocybe (Agrocybe) aegerita (encoding the main peroxygenase, AaeUPO) was cell-free expressed with both lysates, reaching activities of up to 105 U L−1 within 24 h (measured with veratryl alcohol as substrate). The cell-free expressed enzyme (cfAaeUPO) was successfully tested in a substrate screening that included prototypical UPO substrates, as well as several pharmaceuticals. The determined activities and catalytic performance were comparable to that of the wild-type enzyme (wtAaeUPO). The results presented here suggest that cell-free expression could become a valuable tool to gain easier access to the immense pool of putative UPO genes and to expand the spectrum of these sought-after biocatalysts.show moreshow less

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Author: Marina SchrammORCiD, Stephanie Friedrich, Kai-Uwe Schmidtke, Paul Panzer, Harald Kellner, René UllrichORCiD, Martin HofrichterORCiD, Katrin Scheibner
URL:https://www.mdpi.com/2076-3921/11/2/284
DOI:https://doi.org/10.3390/antiox11020284
ISSN:2076-3921
Title of the source (English):Antioxidants
Document Type:Scientific journal article peer-reviewed
Language:English
Year of publication:2022
Contributing Corporation:TU Dresden
Tag:cell-free protein synthesis; in vitro translation; monooxygenase; unspecific peroxygenase
Volume/Year:11
Issue number:2
First Page:1
Last Page:15
Article number:284
Faculty/Chair:Fakultät 2 Umwelt und Naturwissenschaften / FG Enzymtechnologie
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