Benchmarking of laboratory evolved unspecific peroxygenases for the synthesis of human drug metabolites

Santos, Patricia Gómez de; Cervantes, Fadia V.; Tieves, Florian; Plou, Francisco J.; Hollmann, Frank; Alcalde, Miguel

doi:10.1016/j.tet.2019.02.013

Cited by 41 publications

(35 citation statements)

References 17 publications

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“…Although substantial work has been invested into the heterologous expression of the firstly discovered Agrocybe aegerita UPO ( Aae UPO) using the yeast Saccharomyces cerevisiae , sufficient protein amounts of 8 mg/L were obtained as the result of an intensive directed evolution campaign 10 . This fundamental work led to several successful UPO application studies on the conversion of a range of substrates from agrochemicals to pharmaceuticals 11 – 14 . The yeast secretion variant PaDa-I (hereinafter: AaeU PO*) was adapted subsequently for large-scale protein production by utilising the methylotrophic yeast Pichia pastoris (syn.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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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“…Apart from cytochrome c and P450s a number of reports on UPO engineering have been published during recent years (Gomez de Santos et al, ). Directed evolution approaches by random mutagenesis and in vivo shuffling did target expression, secretion, and stability of UPOs in yeast but also improved enzyme promiscuity and activity.…”

Section: Protein Engineering and Directed Evolution Approaches To Expmentioning

confidence: 99%

Recent advances in heme biocatalysis engineering

Schmitz

Rosenthal

Lütz

2019

Biotech & Bioengineering

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Heme enzymes have the potential to be widely used as biocatalysts due to their capability to perform a vast variety of oxidation reactions. In spite of their versatility, the application of heme enzymes was long time-limited for the industry due to their low activity and stability in large scale processes. The identification of novel natural biocatalysts and recent advances in protein engineering have led to new reactions with a high application potential. The latest creation of a serine-ligated mutant of BM3 showed an efficient transfer of reactive carbenes into C═C bonds of olefins reaching total turnover numbers of more than 60,000 and product titers of up to 27 g/L −1 . This prominent example shows that heme enzymes are becoming competitive to chemical syntheses while being already advantageous in terms of high yield, regioselectivity, stereoselectivity and environmentally friendly reaction conditions. Advances in reactor concepts and the influencing parameters on reaction performance are also under investigation resulting in improved productivities and increased stability of the heme biocatalytic systems. In this mini review, we briefly present the latest advancements in the field of heme enzymes towards increased reaction scope and applicability.

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Benchmarking of laboratory evolved unspecific peroxygenases for the synthesis of human drug metabolites

Cited by 41 publications

References 17 publications

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

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

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

Recent advances in heme biocatalysis engineering

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