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Broadening the Biocatalytic Toolbox-Screening and Expression of New Unspecific Peroxygenases.

ABSTRACT: Unspecific peroxygenases (UPOs) catalyze the selective transfer of single oxygen atoms from peroxides to a broad range of substrates such as un-activated hydrocarbons. Since specific oxyfunctionalizations are among the most-desired reactions in synthetic chemistry, UPOs are of high industrial interest. To broaden the number of available enzymes, computational and experimental methods were combined in this study. After a comparative alignment and homology modelling, the enzymes were expressed directly in P. pastoris. Out of ten initially selected sequences, three enzymes (one from Aspergillus niger and two from Candolleomyces aberdarensis) were actively expressed. Cultivation of respective expression clones in a bioreactor led to production titers of up to 300 mg L-1. Enzymes were purified to near homogeneity and characterized regarding their specific activities and pH-optima for typical UPO substrates. This work demonstrated that directed evolution is not necessarily required to produce UPOs in P. pastoris at respective titers. The heterologous producibility of these three UPOs will expand the toolbox of available enzymes and help to advance their synthetic application.

SUBMITTER: Bormann S

PROVIDER: S-EPMC8868357 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Broadening the Biocatalytic Toolbox-Screening and Expression of New Unspecific Peroxygenases.

Bormann Sebastian S Kellner Harald H Hermes Johanna J Herzog Robert R Ullrich René R Liers Christiane C Ulber Roland R Hofrichter Martin M Holtmann Dirk D

Antioxidants (Basel, Switzerland) 20220124 2

Unspecific peroxygenases (UPOs) catalyze the selective transfer of single oxygen atoms from peroxides to a broad range of substrates such as un-activated hydrocarbons. Since specific oxyfunctionalizations are among the most-desired reactions in synthetic chemistry, UPOs are of high industrial interest. To broaden the number of available enzymes, computational and experimental methods were combined in this study. After a comparative alignment and homology modelling, the enzymes were expressed dir ...[more]

PMID: 35204106

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Project description:Unspecific peroxygenases (UPOs) constitute a new family of fungal heme-thiolate enzymes in which there is high biotechnological interest. Although several thousand genes encoding hypothetical UPO-type proteins have been identified in sequenced fungal genomes and other databases, only a few UPO enzymes have been experimentally characterized to date. Therefore, gene screening and heterologous expression from genetic databases are a priority in the search for ad hoc UPOs for oxyfunctionalization reactions of interest. Very recently, Escherichia coli production of a previously described basidiomycete UPO (as a soluble and active enzyme) has been reported. Here, we explored this convenient heterologous expression system to obtain the protein products from available putative UPO genes. In this way, two UPOs from the ascomycetes Collariella virescens (syn., Chaetomium virescens) and Daldinia caldariorum were successfully obtained, purified, and characterized. Comparison of their kinetic constants for oxidation of model substrates revealed 10- to 20-fold-higher catalytic efficiency of the latter enzyme in oxidizing simple aromatic compounds (such as veratryl alcohol, naphthalene, and benzyl alcohol). Homology molecular models of these enzymes showed three conserved and two differing residues in the distal side of the heme (the latter representing two different positions of a phenylalanine residue). Interestingly, replacement of the C. virescens UPO Phe88 by the homologous residue in the D. caldariorum UPO resulted in an F88L variant with 5- to 21-fold-higher efficiency in oxidizing these aromatic compounds.IMPORTANCE UPOs catalyze regio- and stereoselective oxygenations of both aromatic and aliphatic compounds. Similar reactions were previously described for cytochrome P450 monooxygenases, but UPOs have the noteworthy biotechnological advantage of being stable enzymes requiring only H2O2 to be activated. Both characteristics are related to the extracellular nature of UPOs as secreted proteins. In the present study, the limited repertoire of UPO enzymes available for organic synthesis and other applications is expanded with the description of two new ascomycete UPOs obtained by Escherichia coli expression of the corresponding genes as soluble and active enzymes. Moreover, directed mutagenesis in E. coli, together with enzyme molecular modeling, provided relevant structure-function information on aromatic substrate oxidation by these two new biocatalysts.

Project description:BackgroundUnspecific peroxygenases (UPO) (EC 1.11.2.1) represent an intriguing oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity. With over 300 identified substrates, UPOs catalyze numerous oxidations including 1- or 2- electron oxygenation, selective oxyfunctionalizations, which make them most significant in organic syntheses and potentially attractive as industrial biocatalysts. There are very few UPOs available with distinct properties, notably, MroUPO which shows behavior ranging between UPO and another heme-thiolate peroxidase, called Chloroperoxidase (CPO). It prompted us to search for more UPOs in fungal kingdom which led us to studying their relationship with CPO.ResultsIn this study, we searched for novel UPOs in more than 800 fungal genomes and found 113 putative UPO-encoding sequences distributed in 35 different fungal species (or strains), amongst which single sequence per species were subjected to phylogeny study along with CPOs. Our phylogenetic study show that the UPOs are distributed in Basidiomycota and Ascomycota phyla of fungi. The sequence analysis helped to classify the UPOs into five distinct subfamilies: classic AaeUPO and four new subfamilies with potential new traits. We have also shown that each of these five subfamilies (supported by) have their own signature motifs. Surprisingly, some of the CPOs appeared to be a type of UPOs indicating that they were previously identified incorrectly. Selection pressure was observed on important motifs in UPOs which could have driven their functional divergence. Furthermore, the sites having different evolutionary rates caused by the functional divergence were also identified on some motifs along with the other relevant amino acid residues. Finally, we predicted critical amino acids responsible for the functional divergence in the UPOs and identified some sequence differences among UPOs, CPOs, and MroUPO to predict it's ranging behavior.ConclusionThis study discovers new UPOs, provides a glimpse of their evolution from CPOs, and presents new insight on their functional divergence. We present a new classification of UPOs and shed new light on its phylogenetics. These different UPOs may exhibit a wide range of characteristics and specificities which may help in various fields of synthetic chemistry and industrial biocatalysts, and may as well lead to an advancement towards the understanding of physiological role of UPOs in fungi.

Dataset Information

Broadening the Biocatalytic Toolbox-Screening and Expression of New Unspecific Peroxygenases.

Publications

Broadening the Biocatalytic Toolbox-Screening and Expression of New Unspecific Peroxygenases.

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