Shuffling the Neutral Drift of Unspecific Peroxygenase in Saccharomyces cerevisiae

Martin-Diaz, Javier; Paret, Carmen; García-Ruiz, Eva; Molina‐Espeja, Patricia; Alcalde, Miguel

doi:10.1128/aem.00808-18

Cited by 27 publications

(29 citation statements)

References 41 publications

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“…Although substantial work has been invested into the heterologous expression of the first discovered Agrocybe aegerita UPO (AaeUPO) using the yeast Saccharomyces cerevisiae, sufficient protein amounts of 8 mg/L were obtained as the result of an intensive directed evolution campaign 6 . This fundamental work led to several successful UPO studies on a range of substrates from agrochemicals to pharmaceuticals [7][8][9][10] .…”

Section: Introductionmentioning

confidence: 99%

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

Puellmann

Knorrscheidt

Muench

et al. 2020

Preprint

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Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. 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 solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. 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 workflow from putative UPO gene to preparative scale enantioselective biotransformations.

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Section: Introductionmentioning

confidence: 99%

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

Puellmann

Knorrscheidt

Muench

et al. 2020

Preprint

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“…HPLC-and GC-dependent systems generally require long analysis time. The development of efficient screening methods to identify new UPOs of currently uncharacterized strains and to screen mutant libraries is thus -Espeja et al, 2015-Espeja et al, , 2019-Espeja et al, , 2016aGómez de Santos et al, 2018;Martin-Diaz et al, 2018;Ramirez-Escudero et al, 2018;Ramirez-Ramirez et al, 2020 needed. An outstanding approach includes a directed-evolution step coupled to a multiple injections in a single experimental run (MISER)-GC-MS-system (Knorrscheidt et al, 2019(Knorrscheidt et al, , 2020.…”

Section: Current Upo Screening Strategies and Analytical Methodsmentioning

confidence: 99%

Identification and Expression of New Unspecific Peroxygenases – Recent Advances, Challenges and Opportunities

Kinner

Rosenthal

Lütz

2021

Front. Bioeng. Biotechnol.

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In 2004, the fungal heme-thiolate enzyme subfamily of unspecific peroxygenases (UPOs) was first described in the basidiomycete Agrocybe aegerita. As UPOs naturally catalyze a broad range of oxidative transformations by using hydrogen peroxide as electron acceptor and thus possess a great application potential, they have been extensively studied in recent years. However, despite their versatility to catalyze challenging selective oxyfunctionalizations, the availability of UPOs for potential biotechnological applications is restricted. Particularly limiting are the identification of novel natural biocatalysts, their production, and the description of their properties. It is hence of great interest to further characterize the enzyme subfamily as well as to identify promising new candidates. Therefore, this review provides an overview of the state of the art in identification, expression, and screening approaches of fungal UPOs, challenges associated with current protein production and screening strategies, as well as potential solutions and opportunities.

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“…Abbreviations: ABTS, 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; ACN, acetonitrile; DMSO, dimethyl sulfoxide. a Mutants 4.7 and 6.1 were generated after 8 rounds of neutral genetic drift through purifying selection (Martin-Diaz et al, 2018). The improvement is defined as the ratio of the activity of the corresponding mutant under the conditions specified to that of the parental PaDa-I under the same conditions.…”

Section: Mutational Analysismentioning

confidence: 99%

Directed evolution of unspecific peroxygenase in organic solvents

Martin-Diaz

Molina‐Espeja

Hofrichter

et al. 2021

Biotech & Bioengineering

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Fungal unspecific peroxygenases (UPOs) are efficient biocatalysts that insert oxygen atoms into nonactivated C-H bonds with high selectivity. Many oxyfunctionalization reactions catalyzed by UPOs are favored in organic solvents, a milieu in which their enzymatic activity is drastically reduced. Using as departure point the UPO secretion mutant from Agrocybe aegerita (PaDa-I variant), in the current study we have improved its activity in organic solvents by directed evolution. Mutant libraries constructed by random mutagenesis and in vivo DNA shuffling were screened in the presence of increasing concentrations of organic solvents that differed both in regard to their chemical nature and polarity. In addition, a palette of neutral mutations generated by genetic drift that improved activity in organic solvents was evaluated by site directed recombination in vivo. The final UPO variant of this evolutionary campaign carried nine mutations that enhanced its activity in the presence of 30% acetonitrile (vol/vol) up to 23-fold over PaDa-I parental type, and it was also active and stable in aqueous acetone, methanol and dimethyl sulfoxide mixtures. These mutations, which are located at the surface of the protein and in the heme channel, seemingly helped to protect UPO from harmful effects of cosolvents by modifying interactions with surrounding residues and influencing critical loops.

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Shuffling the Neutral Drift of Unspecific Peroxygenase in Saccharomyces cerevisiae

Cited by 27 publications

References 41 publications

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

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

Identification and Expression of New Unspecific Peroxygenases – Recent Advances, Challenges and Opportunities

Directed evolution of unspecific peroxygenase in organic solvents

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