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

IF 4.3 3区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Bioresources and Bioprocessing Pub Date : 2024-03-27 DOI:10.1186/s40643-024-00751-x

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

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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.

Graphical Abstract

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一种新的非特异性过氧化氢酶的鉴定、异源表达和特征描述--来自 Marasmius fiardii PR-910

非特异性过氧化氢酶（UPOs）是一种糖基化酶，它提供了一种仅使用过氧化氢（H2O2）作为氧供体对多种底物进行氧官能化的有效方法。然而，它们的异源表达能力较差，阻碍了它们的实际应用。在此，研究人员从 Marasmius fiardii PR910（MfiUPO）中发现了一种新型 UPO，并在 Pichia pastoris 中进行了异源表达。通过使用双拷贝表达盒，蛋白质滴度在 5 L 生物反应器中达到了 1.18 g L-1，创下了最高记录。糖蛋白 rMfiUPO 显示出 40 至 55 kDa 范围内的斑点带，并表现出羟基化、环氧化和醇氧化作用。此外，在 50%（v/v）丙酮中暴露 40 小时后，过氧化活性增强了 150%。在 100 mL 规模上半制备 4-OH-β-ionone 的分离产率为 54.2%，纯度为 95%。rMfiUPO 的表达水平很高，有望成为通过定向进化提高稳定性和选择性等理想性状的优良亲本模板，从而满足实际应用的必要标准。

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来源期刊

Bioresources and Bioprocessing BIOTECHNOLOGY & APPLIED MICROBIOLOGY-

CiteScore

7.20

自引率

8.70%

发文量

118

审稿时长

13 weeks

期刊介绍： Bioresources and Bioprocessing (BIOB) is a peer-reviewed open access journal published under the brand SpringerOpen. BIOB aims at providing an international academic platform for exchanging views on and promoting research to support bioresource development, processing and utilization in a sustainable manner. As an application-oriented research journal, BIOB covers not only the application and management of bioresource technology but also the design and development of bioprocesses that will lead to new and sustainable production processes. BIOB publishes original and review articles on most topics relating to bioresource and bioprocess engineering, including: -Biochemical and microbiological engineering -Biocatalysis and biotransformation -Biosynthesis and metabolic engineering -Bioprocess and biosystems engineering -Bioenergy and biorefinery -Cell culture and biomedical engineering -Food, agricultural and marine biotechnology -Bioseparation and biopurification engineering -Bioremediation and environmental biotechnology