Identification, heterologous expression and characterization of a new unspecific peroxygenase from Marasmius fiardii PR-910

Abstract

Unspecific peroxygenases (UPOs) are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide (H₂O₂) as the oxygen donor. However, their poor heterologous expression has hindered their practical application. Here, a novel UPO from Marasmius fiardii PR910 (MfiUPO) was identified and heterologously expressed in Pichia pastoris. By employing a two-copy expression cassette, the protein titer reached 1.18 g L⁻¹ in a 5 L bioreactor, marking the highest record. The glycoprotein rMfiUPO exhibited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation, epoxidation and alcohol oxidation. Moreover, the peroxidative activity was enhanced by 150% after exposure to 50% (v/v) acetone for 40 h. A semi-preparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2% isolated yield with 95% purity. With its high expression level, rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution, thereby meeting the necessary criteria for practical application.

Keywords

Unspecific peroxygenase / Marasmius fiardii / Heterologous expression / Characterization / Semi-preparative

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Funding

National Key Research and Development Program of China(2021YFC2102700)

Science and Technology Program of Hubei Province(2021BAD001)