A Novel Unspecific Peroxygenase from Galatian marginata for Biocatalytic Oxyfunctionalization Reactions

Ma, Yunjian; Liang, Hongjing; Zhao, Zexin; Wu, Bin; Lan, Dongming; Hollmann, Frank; Wang, Yonghua

doi:10.1016/j.mcat.2022.112707

Cited by 10 publications

(6 citation statements)

References 57 publications

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“…marginata, which exhibits 94% sequence identity to the one we selected, was shown recently (after the start of our work) to be functionally expressed in P. pastoris . Throughout this work, we use the name Gma UPO referring to the first published Gma UPO …”

Section: Resultsmentioning

confidence: 99%

“…18 Another UPO derived from G. marginata, which exhibits 94% sequence identity to the one we selected, was shown recently (after the start of our work) to be functionally expressed in P. pastoris. 14 Throughout this work, we use the name GmaUPO referring to the first published GmaUPO. 18 CciUPO has so far only been produced in Aspergillus oryzae 16,42 and not in any fast-growing microbe but holds substantial scientific interest for its relatively high activity levels.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…So far, the main limitation to using UPOs has been their poor and difficult heterologous production. Although databases contain sequence information on >4000 putative UPOs, , only about 50 have been heterologously produced since the discovery of this family in 2004. − Thus, selecting UPOs for fundamental and applied research is dominated by considerations of heterologous production, limiting the phylogenetic and functional scope of enzymes that have been subjected to research.…”

Section: Introductionmentioning

confidence: 99%

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Functionally Diverse Peroxygenases by AlphaFold2, Design, and Signal Peptide Shuffling

Münch,

Dietz,

Barber-Zucker

et al. 2024

ACS Catal.

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Unspecific peroxygenases (UPOs) are fungal enzymes that attract significant attention for their ability to perform versatile oxyfunctionalization reactions using H 2 O 2 . Unlike other oxygenases, UPOs do not require additional reductive equivalents or electron transfer chains that complicate basic and applied research. Nevertheless, UPOs generally exhibit low to no heterologous production levels and only four UPO structures have been determined to date by crystallography limiting their usefulness and obstructing research. To overcome this bottleneck, we implemented a workflow that applies PROSS stability design to AlphaFold2 model structures of 10 unique and diverse UPOs followed by a signal peptide shuffling to enable heterologous production. Nine UPOs were functionally produced in Pichia pastoris, including the recalcitrant CciUPO and three UPOs derived from oomycetes�the first nonfungal UPOs to be experimentally characterized. We conclude that the high accuracy and reliability of new modeling and design workflows dramatically expand the pool of enzymes for basic and applied research.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Functionally Diverse Peroxygenases by AlphaFold2, Design, and Signal Peptide Shuffling

Münch,

Dietz,

Barber-Zucker

et al. 2024

ACS Catal.

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“… 2021 ), Gma UPO from Galerina marginata (Ma et al. 2022 ), among others. Therefore, it is crucial to access more enzymes to gain further insight into the natural function of UPOs.…”

Section: Introductionmentioning

confidence: 99%

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

Fu,

Lin,

Zhang

et al. 2024

Bioresour. Bioprocess.

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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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“…Although a vast diversity of UPO genes are available, only a limited number of UPOs have been purified and experimentally characterized to date, including Aae UPO, Coprinopsis cinerea UPO ( Cci UPO), Marasmius rotula UPO ( Mro UPO), Chaetomium globosum UPO ( Cgl UPO), Collariella virescens UPO ( Cvi UPO), Daldinia caldariorum UPO ( Dca UPO), Psathyrella aberdarensis UPO ( Pab UPO), Hypoxylon sp. EC38 UPO ( Hsp UPO), Galerina marginata UPO ( Gma UPO), and Aspergillus brasiliensis UPO ( Abr UPO) . In addition, the number of peroxygenase variants with tailored selectivity for a broader range of starting materials is also limited due to the lack of an efficient engineering strategy.…”

Section: Introductionmentioning

confidence: 99%

Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in Escherichia coli

Yan,

Zhang,

et al. 2024

JACS Au

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Unspecific peroxygenases (UPOs), secreted by fungi, demonstrate versatility in catalyzing challenging selective oxyfunctionalizations. However, the number of peroxygenases and corresponding variants with tailored selectivity for a broader substrate scope is still limited due to the lack of efficient engineering strategies. In this study, a new unspecific peroxygenase from Coprinopsis marcescibilis (CmaUPO) is identified and characterized. To enhance or reverse the enantioselectivity of wildtype (WT) CmaUPO catalyzed asymmetric hydroxylation of ethylbenzene, CmaUPO was engineered using an efficient superfolder-green-fluorescent-protein (sf GFP)-mediated secretion system in Escherichia coli. Iterative saturation mutagenesis (ISM) was used to target the residual sites lining the substrate tunnel, resulting in two variants: T125A/A129G and T125A/A129V/A247H/T244A/F243G. The two variants greatly improved the enantioselectivities [21% ee (R) for WT], generating the (R)-1-phenylethanol or (S)-1-phenylethanol as the main product with 99% ee (R) and 84% ee (S), respectively. The sf GFP-mediated secretion system in E. coli demonstrates applicability for different UPOs (AaeUPO, CciUPO, and PabUPO-I). Therefore, this developed system provides a robust platform for heterologous expression and enzyme engineering of UPOs, indicating great potential for their sustainable and efficient applications in various chemical transformations.

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A Novel Unspecific Peroxygenase from Galatian marginata for Biocatalytic Oxyfunctionalization Reactions

Cited by 10 publications

References 57 publications

Functionally Diverse Peroxygenases by AlphaFold2, Design, and Signal Peptide Shuffling

Functionally Diverse Peroxygenases by AlphaFold2, Design, and Signal Peptide Shuffling

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

Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in Escherichia coli

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