Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S-adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli. Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.

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.

Lignin peroxidase (LiP) and its natural substrate veratryl alcohol (VA) play a crucial role in lignin degradation by white-rot fungi. Understanding the molecular determinants for the interaction of this enzyme with its substrates is essential in the rational design of engineered peroxidases for biotechnological application. Here, we combine computational and experimental approaches to analyze the interaction of Phanerochaete chrysosporium LiP (isoenzyme H8) with VA and its radical cation (VA^•+, resulting from substrate oxidation by the enzyme). Interaction energy calculations at semiempirical quantum mechanical level (SQM) between LiP and VA/VA^•+ enabled to identify those residues at the acidic environment of catalytic Trp171 involved in the main interactions. Then, a battery of variants, with single and multiple mutations at these residues (Glu168, Asp165, Glu250, Asp264, and Phe267), was generated by directed mutagenesis, and their kinetics parameters were estimated on VA and two additional substrates. The experimental results show that Glu168 and Glu250 are crucial for the binding of VA, with Glu250 also contributing to the turnover of the enzyme. The experimental results were further rationalized through new calculations of interaction energies between VA/VA^•+ and LiP with each of the single mutations. Finally, the delocalization of spin density was determined with quantum mechanics/molecular mechanics calculations (QM/MM), further supporting the contribution of Glu250 to VA oxidation at Trp171.

Peroxygenases require a controlled supply of H₂O₂ to operate efficiently. Here, we propose a photocatalytic system for the reductive activation of ambient O₂ to produce H₂O₂ which uses the energy provided by visible light more efficiently based on the combination of wavelength-complementary photosensitizers. This approach was coupled to an enzymatic system to make formate available as a sacrificial electron donor. The scope and current limitations of this approach are reported and discussed.

The selective oxidation of trans-2-hexen-1-ol to the corresponding aldehyde using a recombinant aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) is reported. Especially using the two liquid phase system to overcome solubility and product inhibition issues enabled to achieve more than 2.200.000 catalytic turnovers for the production enzyme as well as molar product concentrations, pointing towards an economic feasible reaction.

The E-factor has become an important measure for the environmental impact of (bio)chemical reactions. However, summing up the obvious wastes generated in the laboratory neglects energy-related wastes (mostly greenhouse gases) which are generated elsewhere. To estimate these wastes, we propose to extend the E-factor by an energy-term (E⁺-factor). At the example of a lab-scale enzyme fermentation, we demonstrate that the E⁺-factor can constitute a multiple of the classical E-factor and therefore must not be neglected striving for a holistic estimation of the environmental impact.

The biocatalytic preparation of trans-hex-2-enal from trans-hex-2-enol using a novel aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) is reported. As O₂-dependent enzyme PeAAOx-dependent reactions are generally plagued by the poor solubility of O₂ in aqueous media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by conducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency (up to 38 s^-1) and turnover numbers (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme.

A photoenzymatic NADH regeneration system was established. The combination of deazariboflavin as a photocatalyst with putidaredoxin reductase enabled the selective reduction of NAD⁺ into the enzyme-active 1,4-NADH to promote an alcohol dehydrogenase catalysed stereospecific reduction reaction. The catalytic turnover of all the reaction components was demonstrated. Factors influencing the efficiency of the overall system were identified.

Peroxidases are considered essential agents of lignin degradation by white-rot basidiomycetes. However, low-molecular-weight oxidants likely have a primary role in lignin breakdown because many of these fungi delignify wood before its porosity has sufficiently increased for enzymes to infiltrate. It has been proposed that lignin peroxidases (LPs, EC 1.11.1.14) fulfill this role by oxidizing the secreted fungal metabolite veratryl alcohol (VA) to its aryl cation radical (VA), releasing it to act as a one-electron lignin oxidant within woody plant cell walls. Here, we attached the fluorescent oxidant sensor BODIPY 581/591 throughout beads with a nominal porosity of 6 kDa and assessed whether peroxidase-generated aryl cation radical systems could oxidize the beads. As positive control, we used the 1,2,4,5-tetra-methoxybenzene (TMB) cation radical, generated from TMB by horseradish peroxidase. This control oxidized the beads to depths that increased with the amount of oxidant supplied, ultimately resulting in completely oxidized beads. A reaction-diffusion computer model yielded oxidation profiles that were within the 95% confidence intervals for the data. By contrast, bead oxidation caused by VA and the LPA isozyme of Phanerochaete chrysosporium was confined to a shallow shell of LP-accessible volume at the bead surface, regardless of how much oxidant was supplied. This finding contrasted with the modeling results, which showed that if the LP/VA system were to release VA, it would oxidize the bead interiors. We conclude that LPA releases insignificant quantities of VA and that a different mechanism produces small ligninolytic oxidants during white rot.

Peroxygenases offer an attractive means to address challenges in selective oxyfunctionalization chemistry. Despite this, their application in synthetic chemistry remains challenging due to their facile inactivation by the stoichiometric oxidant H₂O₂. Often atom-inefficient peroxide generation systems are required, which show little potential for large-scale implementation. Here, we show that visible-light-driven, catalytic water oxidation can be used for in situ generation of H₂O₂ from water, rendering the peroxygenase catalytically active. In this way, the stereoselective oxyfunctionalization of hydrocarbons can be achieved by simply using the catalytic system, water and visible light.

There is an increasing interest in the application of peroxygenases in biocatalysis, because of their ability to catalyse the oxyfunctionalisation reaction in a stereoselective fashion and with high catalytic efficiencies, while using hydrogen peroxide or organic peroxides as oxidant. However, enzymes belonging to this class exhibit a very low stability in the presence of peroxides. With the aim of bypassing this fast and irreversible inactivation, we study the use of a gradual supply of hydrogen peroxide to maintain its concentration at stoichiometric levels. In this contribution, we report a multienzymatic cascade for in situ generation of hydrogen peroxide. In the first step, in the presence of NAD⁺ cofactor, formate dehydrogenase from Candida boidinii (FDH) catalysed the oxidation of formate yielding CO₂. Reduced NADH was reoxidised by the reduction of the flavin mononucleotide cofactor bound to an old yellow enzyme homologue from Bacillus subtilis (YqjM), which subsequently reacts with molecular oxygen yielding hydrogen peroxide. Finally, this system was coupled to the hydroxylation of ethylbenzene reaction catalysed by an evolved peroxygenase from Agrocybe aegerita (rAaeUPO). Additionally, we studied the influence of different reaction parameters on the performance of the cascade with the aim of improving the turnover of the hydroxylation reaction.

In this study, we combined photo-organo redox catalysis and biocatalysis to achieve asymmetric C–H bond functionalization of simple alkane starting materials. The photo-organo catalyst anthraquinone sulfate (SAS) was employed to oxyfunctionalise alkanes to aldehydes and ketones. We coupled this light-driven reaction with asymmetric enzymatic functionalisations to yield chiral hydroxynitriles, amines, acyloins and α-chiral ketones with up to 99 % ee. In addition, we demonstrate functional group interconversion to alcohols, esters and carboxylic acids. The transformations can be performed as concurrent tandem reactions. We identified the degradation of substrates and inhibition of the biocatalysts as limiting factors affecting compatibility, due to reactive oxygen species generated in the photocatalytic step. These incompatibilities were addressed by reaction engineering, such as applying a two-phase system or temporal and spatial separation of the catalysts. Using a selection of eleven starting alkanes, one photo-organo catalyst and 8 diverse biocatalysts, we synthesized 26 products and report for the model compounds benzoin and mandelonitrile > 97 % ee at gram scale.