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

• Published in: Antioxidants Vol. 11; no. 2; p. 284
• Main Authors: Schramm, Marina, Friedrich, Stephanie, Schmidtke, Kai-Uwe, Kiebist, Jan, Panzer, Paul, Kellner, Harald, Ullrich, René, Hofrichter, Martin, Scheibner, Katrin
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 30.01.2022; MDPI
• Subjects: Agrocybe; alcohols; Aspergillus niger; Biocatalysts; biomass; catalytic activity; cell-free protein synthesis; drugs; E coli; EC 1.11.2.1; Enzymes; Fungi; genes; Genomes; Glycerol; in vitro translation; luciferase; Lysates; monooxygenase; Neurospora crassa; Pharmaceuticals; Protein biosynthesis; Protein synthesis; Proteins; unspecific peroxygenase; Veratryl alcohol; Yeast; England; United Kingdom > UK; Switzerland; Germany; cell-free protein synthesis; unspecific peroxygenase; in vitro translation; EC 1.11.2.1; monooxygenase
• ISSN: 2076-3921; 2076-3921
• DOI: 10.3390/antiox11020284

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 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.