The generation of enantiodivergent biocatalysts for C-H oxyfunctionalizations is ever more important in modern synthetic chemistry. Here, we have applied the FuncLib algorithm based on phylogenetic and Rosetta calculations to design a diverse repertoire of active, stable, and enantiodivergent fungal peroxygenases. 24 designs, each carrying 4-5 mutations in the catalytic core, were expressed functionally in yeast and benchmarked against characteristic model compounds. Several designs were active and stable in a range of temperature and pH, displaying unprecedented enantiodivergence, changing regioselectivity from alkyl to aromatic hydroxylation, and increasing catalytic efficiencies up to 10-fold, with 15-fold improvements in total turnover numbers over the parental enzyme. We find that this dramatic functional divergence stems from beneficial epistasis among the mutations and an extensive reorganization of the heme channel. Our work demonstrates that FuncLib can rapidly design highly functional libraries enriched in enantioselective peroxygenases not seen in nature for a range of biotechnological applications.

Unspecific peroxygenases (UPOs) are promising biocatalysts for oxyfunctionalisation reactions, owing to their simplicity of handling, stability and robustness. A limitation of using UPOs on a large scale is their deactivation in the presence of even rather modest concentrations of H₂O₂, requiring a constant and controlled supply of low amount of H₂O₂. Herein, we report an organometallic complex [Cp*Ir(pica)NO₃] {pica=picolinamidate=κ²-pyridine-2-carboxamide ion (−1)} 1 capable of efficiently regenerating FMNH₂ from FMN (TOF=350 h⁻¹, 298 K), driven by NaHCOO; FMNH₂, in turn, spontaneously reacts with O₂ leading to H₂O₂. After having studied the compatibility of 1 with the UPO from Agrocybe aegerita (rAaeUPO PaDa-I) and individuated the best experimental conditions, we applied such a hybrid catalytic tandem in some hydroxylation, epoxidation and sulfoxidation reactions. Best performances were obtained by using a 1/rAaeUPO molar ratio of 50. TONs for the biocatalyst of up to 18933 were obtained for the transformation of ethylbenzene derivatives into (R)-1-phenylethanols (ee>99 %). 1/rAaeUPO was found to oxidise also cis-methyl styrene (TON=13488), leading exclusively (1R,2S)-cis-methyl styrene oxide (ee>99 %), cyclohexane (TON=1634) and thioanisole (TON=1369).

Biocatalytic oxidation reactions of toluene derivates to the corresponding aldehydes are typically challenged by regio- and chemoselectivity issues. In this contribution we address both challenges by a combined reactant- and reaction engineering approach. We demonstrate that the peroxygenase-catalysed transformation of ring-substituted toluenes proceeds highly regioselectively in benzylic position. Furthermore, neat reaction conditions not only enable attractive product concentrations (up to 185 mM) but also result in highly chemoselective oxidations to the aldehyde level.

Mol-scale oxyfunctionalization of cyclohexane to cyclohexanol/cyclohexanone (KA-oil) using an unspecific peroxygenase is reported. Using AaeUPO from Agrocybe aegerita and simple H2O2 as an oxidant, cyclohexanol concentrations of more than 300 mM (>60% yield) at attractive productivities (157 mM h-1, approx. 15 g L-1 h-1) were achieved. Current limitations of the proposed biooxidation system have been identified paving the way for future improvements and implementation.

A peroxygenase-catalysed hydroxylation of organosilanes is reported. The recombinant peroxygenase from Agrocybe aegerita (AaeUPO) enabled efficient conversion of a broad range of silane starting materials in attractive productivities (up to 300 mM h⁻¹), catalyst performance (up to 84 s⁻¹ and more than 120 000 catalytic turnovers). Molecular modelling of the enzyme-substrate interaction puts a basis for the mechanistic understanding of AaeUPO selectivity.

Utilisation of fatty acids generally relies on pre-existing functional groups such as the carboxylate group or C=C-double bonds. Addition of new functionalities into the hydrocarbon part opens up new possibilities for fatty acid valorisation. In this contribution we demonstrate the synthetic potential of a peroxygenase mutant AaeUPO−Fett for selective fatty acid oxyfunctionalisation. The ω-1 hydroxy fatty acid (esters) produced are further transformed into lactones, alcohols, esters and amines via multi-enzyme cascades thereby paving the way for new fatty acid valorisation pathways.

The selective insertion of oxygen into non-activated organic molecules has to date been considered of utmost importance to synthesize existing and next generation industrial chemicals or pharmaceuticals. In this respect, the minimal requirements and high activity of fungal unspecific peroxygenases (UPOs) situate them as the jewel in the crown of C–H oxyfunctionalization biocatalysts. Although their limited availability and development has hindered their incorporation into industry, the conjunction of directed evolution and computational design is approaching UPOs to practical applications. In this review, we will address the most recent advances in UPO engineering, both of the long and short UPO families, while discussing the future prospects in this fast-moving field of research.

In this study, we developed a new bienzymatic reaction to produce enantioenriched phenylethanols. In a first step, the recombinant, unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) was used to oxidise ethylbenzene and its derivatives to the corresponding ketones (prochiral intermediates) followed by enantioselective reduction into the desired (R)- or (S)-phenylethanols using the (R)-selective alcohol dehydrogenase (ADH) from Lactobacillus kefir (LkADH) or the (S)-selective ADH from Rhodococcus ruber (ADH-A). In a one-pot two-step cascade, 11 ethylbenzene derivatives were converted into the corresponding chiral alcohols at acceptable yields and often excellent enantioselectivity.

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L-1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L-1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).

Oxyfunctionalisation reactions in neat substrate still pose a challenge for biocatalysis. Here, we report an alginate-confined peroxygenase-CLEA to catalyse the enantioselective epoxidation of cis-β-methylstyrene in a solvent-free reaction system achieving turnover numbers of 96 000 for the biocatalyst and epoxide concentrations of 48 mM.

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.

Peroxygenases are an emerging new class of enzymes allowing selective oxyfunctionalisation reactions in a cofactor-independent way different from well-known P450 monooxygenases. Herein, we focused on recent developments from organic synthesis, molecular biotechnology and reaction engineering viewpoints that are devoted to bring these enzymes in industrial applications. This covers natural diversity from different sources, protein engineering strategies for expression, substrate scope, activity and selectivity, stabilisation of enzymes via immobilisation, and the use of peroxygenases in low water media. We believe that peroxygenases have much to offer for selective oxyfunctionalisations and we have much to study to explore the full potential of these versatile biocatalysts in organic synthesis.

Aromatic hydroxylation reactions catalyzed by heme-thiolate enzymes proceed via an epoxide intermediate. These aromatic epoxides could be valuable building blocks for organic synthesis giving access to a range of chiral trans-disubstituted cyclohexadiene synthons. Here, we show that naphthalene epoxides generated by fungal peroxygenases can be subjected to nucleophilic ring opening, yielding non-racemic trans-disubstituted cyclohexadiene derivates, which in turn can be used for further chemical transformations. This approach may represent a promising shortcut for the synthesis of natural products and APIs.

Peroxyzymes simply use H2O2 as a cosubstrate to oxidize a broad range of inert C-H bonds. The lability of many peroxyzymes against H2O2 can be addressed by a controlled supply of H2O2, ideally in situ. Here, we report a simple, robust, and water-soluble anthraquinone sulfonate (SAS) as a promising organophotocatalyst to drive both haloperoxidase-catalyzed halogenation and peroxygenase-catalyzed oxyfunctionalization reactions. Simple alcohols, methanol in particular, can be used both as a cosolvent and an electron donor for H2O2 generation. Very promising turnover numbers for the biocatalysts of up to 318 »000 have been achieved.

Fungal peroxygenases are deemed emergent biocatalysts for selective C-H bond oxyfunctionalization reactions. In this study, we have engineered a functional and stable self-sufficient chimeric peroxygenase-oxidase fusion. The bifunctional biocatalyst carried a laboratory-evolved version of the fungal peroxygenase fused to an evolved fungal aryl-alcohol oxidase that supplies H2O2 in situ. Enzyme fusion libraries with peptide linkers of different sizes and amino acid compositions were designed, while attached leader sequences favored secretion in yeast. The most promising functional enzyme fusions were characterized biochemically and further tested for the synthesis of dextrorphan, a metabolite of the antitussive drug dextromethorphan. This reaction system was optimized to control the aromatic alcohol transformation rate, and therefore the H2O2 supply, to achieve total turnover numbers of 62,000, the highest value reported for the biocatalytic synthesis of dextrorphan to date. Accordingly, our study opens an avenue for the use of peroxygenase-aryl alcohol oxidase fusions in the pharmaceutical and chemical sectors.

Peroxidases and peroxygenases are promising classes of enzymes for biocatalysis because of their ability to carry out one-electron oxidation reactions and stereoselective oxyfunctionalizations. However, industrial application is limited, as the major drawback is the sensitivity toward the required peroxide substrates. Herein, we report a novel biocatalysis approach to circumvent this shortcoming: in situ production of H₂O₂ by dielectric barrier discharge plasma. The discharge plasma can be controlled to produce hydrogen peroxide at desired rates, yielding desired concentrations. Using horseradish peroxidase, it is demonstrated that hydrogen peroxide produced by plasma treatment can drive the enzymatic oxidation of model substrates. Fungal peroxygenase is then employed to convert ethylbenzene to (R)-1-phenylethanol with an ee of >96 % using plasma-generated hydrogen peroxide. As direct treatment of the reaction solution with plasma results in reduced enzyme activity, the use of plasma-treated liquid and protection strategies are investigated to increase total turnover. Technical plasmas present a noninvasive means to drive peroxide-based biotransformations.

It is well-known that energy-rich radiation induces water splitting, eventually yielding hydrogen peroxide. Synthetic applications, however, are scarce and to the best of our knowledge, the combination of radioactivity with enzyme-catalysis has not been considered yet. Peroxygenases utilize H2O2 as an oxidant to promote highly selective oxyfunctionalization reactions but are also irreversibly inactivated in the presence of too high H2O2 concentrations. Therefore, there is a need for efficient in situ H2O2 generation methods. Here, we show that radiolytic water splitting can be used to promote specific biocatalytic oxyfunctionalization reactions. Parameters influencing the efficiency of the reaction and current limitations are shown. Particularly, oxidative inactivation of the biocatalyst by hydroxyl radicals influences the robustness of the overall reaction. Radical scavengers can alleviate this issue, but eventually, physical separation of the enzymes from the ionizing radiation will be necessary to achieve robust reaction schemes. We demonstrate that nuclear waste can also be used to drive selective, peroxygenase-catalyzed oxyfunctionalization reactions, challenging our view on nuclear waste in terms of sustainability.

The use of neat reaction media, that is the avoidance of additional solvents, is the simplest and the most efficient approach to follow in biocatalysis. Here, we show that unspecific peroxygenase from Agrocybe aegerita (AaeUPO) can hydroxylate the neat model substrate cyclohexane. H₂O₂ was photocatalytically generated in situ by nitrogen-doped carbon nanodots (N−CNDs) and UV LED illumination. AaeUPO entrapment in alginate beads increased enzyme stability and facilitated the reaction in neat cyclohexane. N−CNDs absorption in beads containing AaeUPO created a 2-in-1 heterogeneous photobiocatalyst that was active for up to seven days under reaction conditions and produced cyclohexanol, 2.5 mM. To increase productivity, the bead size and the photocatalyst-to-enzyme ratio have been identified as promising targets for optimisation.