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

• Main Authors: Marina Schramm (Author), Stephanie Friedrich (Author), Kai-Uwe Schmidtke (Author), Jan Kiebist (Author), Paul Panzer (Author), Harald Kellner (Author), René Ullrich (Author), Martin Hofrichter (Author), Katrin Scheibner (Author)
• Format: Book
• Published: MDPI AG, 2022-01-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 10.3390/antiox11020284; 2076-3921
• Keywords: unspecific peroxygenase; EC 1.11.2.1; monooxygenase; cell-free protein synthesis; in vitro translation; Therapeutics. Pharmacology; RM1-950
• Source: Antioxidants, Vol 11, Iss 2, p 284 (2022)

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 <i>Neurospora crassa</i> and <i>Aspergillus niger</i>, respectively, was lysed by French press and tested for translational activity with a luciferase reporter enzyme. The <i>upo1</i> gene from <i>Cyclocybe (Agrocybe) aegerita</i> (encoding the main peroxygenase, <i>Aae</i>UPO) 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 (cf<i>Aae</i>UPO) 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 (wt<i>Aae</i>UPO). 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.