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

Fu, Xin; Lin, Kexin; Zhang, Xiaodong; Guo, Zhiyong; Kang, Lixin; Li, Aitao

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.

ALCOHOL oxidation; PICHIA pastoris; HYDROGEN peroxide; EPOXIDATION; HYDROXYLATION

Bioresources & Bioprocessing, 2024, Vol 11, Issue 1, p1

2197-4365

Academic Journal

10.1186/s40643-024-00751-x