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A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases.

ABSTRACT: Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once-enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations.

SUBMITTER: Pullmann P

PROVIDER: S-EPMC8115255 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases.

Püllmann Pascal P Knorrscheidt Anja A Münch Judith J Palme Paul R PR Hoehenwarter Wolfgang W Marillonnet Sylvestre S Alcalde Miguel M Westermann Bernhard B Weissenborn Martin J MJ

Communications biology 20210512 1

Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once-enabled by an intensive directed evolution campaign. We developed and applied a modu ...[more]

PMID: 33980981

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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.

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.

Dataset Information

A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases.

Publications

A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases.

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