In this study, we present a significant advancement in the field of enzymatic asymmetric reductive amination (ARA) of ketones, a pivotal reaction for chiral amine synthesis. Through a combination of semirational enzyme design and bioprocess development, we achieve the dual activation and stabilization of amine dehydrogenase (AmDH) to meet industrial demands. The engineered AmDH exhibits remarkable catalytic efficiency (turnover number, TON >1,000,000) and exceptional stability (half-life >7 days at 50 °C), with a broadened substrate scope including various aryl alkyl ketones and fatty ketones. Leveraging biobased oleic acid as an activator and stabilizer, we achieved kilogram-scale synthesis of chiral amines. Furthermore, the integration of AmDH with chemical catalysts in chemoenzymatic cascades has enabled the synthesis of a wide array of pharmaceutically relevant amines from diverse substrates, demonstrating the enzyme’s versatility and potential to transform synthetic chemistry.

Fatty acids derived from renewable resources, such as vegetable oils, serve as essential feedstocks in various industries, including surfactants, cosmetics, lubricants, and polymers. Although current industrial applications of fatty acids are mainly centered around carboxylate group transformations, recent advancements in biocatalysis have opened new possibilities for converting fatty acids into valuable chemical building blocks. This contribution critically assesses novel biocatalytic transformations of fatty acids, including hydroxylation of the alkyl chain, epoxidation of C═C double bonds, reduction of the carboxylate group, and decarboxylation. These reactions hold great potential for producing important intermediates for chemical synthesis.

Practical Application: The enzymatic valorization of fatty acids offers transformative potential for sustainable oleochemical synthesis, presenting practical applications across various industries. Hydroxylated and epoxidized fatty acids are promising precursors for the production of polyesters, bio-based lubricants, and surfactants. Sophorolipids, derived from hydroxylated fatty acids, are gaining attraction as renewable biosurfactants with applications in detergents and cosmetics. Epoxidized fatty acids serve as intermediates for eco-friendly adhesives, coatings, and polymers. Furthermore, decarboxylation reactions yield alkanes, viable as biofuels, whereas reductions of carboxyl groups enable selective synthesis of aldehydes and alcohols for fragrances and pharmaceutical intermediates. The scalability of these biocatalytic transformations, combined with their mild operational conditions and high specificity, can substantially reduce environmental impact and production costs. These applications highlight biocatalysis as a pivotal technology for advancing the chemical industry toward sustainable practices.

Heme-containing unspecific peroxygenases (UPOs) have attracted significant attention as biocatalysts for oxidation reactions due to their ability to function without expensive nicotinamide cofactors. In the recent study, the UPO from aspergillus brasiliensis (AbrUPO) is found to catalyze the aromatic hydroxylation of substituted benzenes, a feature that distinguishes AbrUPO from other reported wild-type UPOs. To elucidate the underlying factors in the active site and substrate access channel of AbrUPO—which contains fewer phenylalanine residues compared to other UPOs that primarily catalyze benzylic hydroxylation—twenty two AbrUPO variants with single, double, triple, or quadruple amino acid substitutions were constructed to mimic the active sites or substrate access channels of other UPOs. A number of mutated variants exhibited altered activity and selectivity, and several positions were identified that influence enzyme chemoselectivity. Among them, substitution of alanine at position 186 with bulkier residues such as phenylalanine or leucine lead to a shift in chemoselectivity toward alkyl chain hydroxylation of substituted benzenes. Molecular docking studies indicated that the A186F mutation restricts the flexibility and reorientation of ethylbenzene in the active site of AbrUPO, thereby preventing oxidation at the aromatic ring while promoting benzylic hydroxylation.

Enzymatic processes for the remediation of wastewater containing organic pollutants are a promising alternative to advanced treatment processes that are often energy intensive and/or generate waste or by-products. For antibiotics, enzyme systems studied to date have been limited by substrate scope, pH tolerance, and stability. In this work, the remediation potential of two promiscuous H₂O₂-dependent enzymes is explored: the unspecific peroxygenase from Agrocybe aegerita (AaeUPO) and the chloroperoxidase from Curvularia inaequalis (CiVCPO), for the removal of four antibiotics commonly found in WWTP effluents and surface waters. While both enzymes showed a high removal potential for sulfamethoxazole (SMX) as a model antibiotic, CiVCPO was inactive in municipal wastewater, likely due to the presence of phosphate and nitrate. In contrast, AaeUPO remained active and stable within a suitable pH and temperature range. The transformation products showed decreased antibiotic activity against a susceptible strain of E. coli and decreased phytotoxicity, as indicated by the increased root length of Daucus carota. Peroxygenases are known to be sensitive to excess H₂O₂, and AaeUPO displays significant catalase activity at low substrate concentrations. To minimise H₂O₂-mediated inactivation, experiments were conducted at various H₂O₂ dosing rates in batch mode. Optimal conditions for the operation of a continuous enzymatic membrane reactor were then investigated, achieving over 95 % removal of SMX. This lays the groundwork for continuous operation and paves the way for efficient reactor design.

Plasma-generated H2O2 can be used to fuel biocatalytic reactions that require H2O2 as a cosubstrate, such as the conversion of ethylbenzene to (R)-1-phenylethanol ((R)-1-PhOl) catalyzed by unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). Immobilization is recently shown to protect biocatalysts from inactivation by highly reactive plasma-produced species; however, H2O2 supply by the employed plasma sources (μAPPJ and DBD) is limiting for rAaeUPO performance. This study evaluates a recently introduced capillary plasma jet for suitability to supply H2O2 in situ. H2O2 production is modulated by varying the water concentration in the feed gas, providing a greater operating window for applications in plasma-driven biocatalysis. In a static system after 80 min of biocatalysis, a turnover number of 44,199 mol(R)-1-PhOl mol−1rAaeUPO is achieved without significant enzyme inactivation. By exchanging the reaction solution every 5 min, a total product yield of 122 μmol (R)-1-PhOl is achieved in 700 min run time, resulting in a total turnover number of 174,209 mol(R)-1-PhOl mol−1rAaeUPO. This study concludes that the capillary plasma jet, due to its flexibility regarding feed gas, admixtures, and power input, is well suited for in situ H2O2 generation for plasma-driven biocatalysis tailoring to enzymes with high H2O2 turnover.

Unspecific peroxygenases (UPOs) are highly versatile biocatalysts capable of removing various persistent environmental contaminants and performing sustainable chemical transformations. These oxidoreductases contain heme b as their prosthetic group. As all classical peroxidases, they are activated by the molecules of hydrogen peroxide to incorporate the oxygen atom into numerous organic molecules. Alternatively, they can use ascorbate as a cosubstrate. In sequence databases an ever-increasing number of their DNA and protein sequences occurs. Reconstructed molecular phylogeny of the corresponding peroxidase-peroxygenase superfamily reveals a high diversity of gene distribution for UPOs in the whole kingdom of fungi. A majority of identified UPO sequences stems from numerous species of Dikarya. Although members of this superfamily were recently detected also in early diverging fungal lineages, UPOs from the phyla of Mucoromycota, Glomeromycota and Chytridiomycota remain not sufficiently investigated. Moreover, newly discovered genes coding for UPOs were recently identified also among early diverging eukaryotic lineages of amoebas and green algae in various biotops. With a large palette of potential substrates these oxidoreductases serve as a versatile tool in enzyme catalysed synthetic reactions, but their real physiological substrates need to be recognized in the future. Most important among self-sufficient UPO-catalysed reactions are oxyfunctionalizations of various aliphatic and aromatic molecules. In this critical review an outlook is given for investigation and engineering of novel UPO variants including products of directed evolution. Future research on UPOs shall be mainly focused on basal fungal and emerging non-fungal sources for their promising applications in environmentally friendly technologies.

Composed of various biosourced metabolites, NaDES offer significant economic, health, and environmental benefits. Their remarkable ability to interact with target compounds through non-covalent bonds enhances their versatility. As solvents, excipients, cofactors, catalysts, solubilisation promoters, stabilisers, and absorption agents, NaDES provide distinct advantages over conventional substances and can even act as active compounds themselves. Furthermore, their role in advancing innovative synthesis and formulation strategies, particularly in nanotechnology and biotechnology, is driving research in these areas. This review is the first to explore all the potential applications of NaDES in the pharmaceutical industry, while taking a comprehensive look at the theory behind them. It gives a precise definition of NaDES and describes their composition, characteristics, molecular interactions, preparation, stability and recovery. It presents detailed applications in pharmaceutical synthesis, extraction and formulation, and discusses roles as active compounds or tools for innovation. Using green metrics, the efficiency of routes including NaDES is compared to that of conventional processes. Lastly, this review addresses often overlooked points such as toxicity and process limitations.

Peroxygenases represent a class of versatile heme-thiolate enzymes capable of catalysing highly selective oxyfunctionalisation reactions, particularly the hydroxylation of non-activated C-H bonds. This transformation, which poses substantial challenges in conventional organic synthesis, underscores the potential of peroxygenases in green chemistry applications. While cytochrome P450 monooxygenases have long been the primary focus for such biocatalytic transformations, their industrial adoption has been limited due to complex electron transfer chains and cofactor requirements. In contrast, peroxygenases bypass these limitations by directly utilising hydrogen peroxide (H2O2) to activate the catalytic heme site, thereby circumventing the oxygen dilemma typically encountered in P450 catalysis. Key milestones in peroxygenase research include the identification of chloroperoxidase from Caldariomyces fumago and the subsequent discovery of unspecific peroxygenases, such as those from Agrocybe aegerita, which exhibit broad substrate specificity and high catalytic efficiency. Here, we explore the mechanistic pathway of peroxygenase-catalysed reactions, emphasising the formation and decay of Compound I and the catalytic cycle’s various functional outcomes. Critical aspects such as in situ H2O2 generation to mitigate enzyme inactivation, substrate loading strategies for practical applications, and the role of enzyme and reaction engineering in enhancing regio- and stereoselectivity are examined. Additionally, we address challenges in reaction scalability and operational stability for preparative-scale applications, offering insights into innovative protocols involving immobilised enzymes and non-aqueous reaction media. This review highlights recent advancements in the peroxygenase field and underscores the enzyme’s promising role in sustainable oxyfunctionalisation reactions.

Peroxygenases are promising biocatalysts for selective oxyfunctionalization reactions including hydroxylation, epoxidation, and sulfoxidation. In this study, we explore the activity of two recently reported peroxygenases from Collariella virescens (CviUPO) and Daldinia caldariorum (DcaUPO) in a range of synthetically relevant transformations. Both enzymes were heterologously expressed in Escherichia coli and tested for various oxidative reactions. DcaUPO generally demonstrated higher activity compared to CviUPO across several substrates, showing significant conversions in al-cohol and arene oxidations as well as enantioselective epoxidations of styrene derivatives. Notably, the enzymes exhibited complementary selectivities in several reactions including allylic hydroxylation and benzylic oxidation. These results broaden the substrate scope of CviUPO and DcaUPO and highlight their potential for industrial applications. However, challenges with enzyme expression in E. coli remain, necessitating future work on alternative expression systems such as Pichia pastoris to improve yields.

Unspecific peroxygenase from Agrocybe aegerite (AaeUPO) is a remarkable catalyst for the oxyfunctionalization of non-activated C−H bonds under mild conditions. It exhibits comparable activity to P450 monooxygenase but offers the advantage of using H₂O₂ instead of a complex electron transport chain to reductively activate O₂. Here, we demonstrate the successful oxidation of cyclohexane to cyclohexanol/cyclohexanone (KA-oil) using sol-gel encapsulated AaeUPO. Remarkably, cyclohexane serves both as a solvent and a substrate in this system, which simplifies product isolation. The ratio of cyclohexanone to cyclohexanol using this approach is remarkably higher compared to the oxidation using free AaeUPO in aqueous media using acetonitrile as a cosolvent. The utilization of sol-gel encapsulated AaeUPO offers a promising approach for oxyfunctionalization reactions and improves the chances for this enzyme to be incorporated in the same pot with other chemical transformations.

Synthetic biohybrid systems by coupling artificial system with nature's machinery may offer a disruptive solution to address the global energy crisis. We developed a versatile electroenzymatic pathway for the continuous synthesis of valuable chemicals, facilitated by formate-driven NADH regeneration. Utilizing a bismuth electrocatalyst, we achieved stable CO2 reduction to formate with approximately 90 % Faraday efficiency at a current density of 150 mA cm−2. The generated formate acts as a mediator to regenerate NADH, which is then coupled with immobilized redox enzymes—alcohol dehydrogenase (ADH), L-lactate dehydrogenase (LDH), and L-glutamate dehydrogenase (GDH)—to produce targeted chemicals at significant rates and exceptionally high turnover numbers (1.8×106 to 3.1×106). These achievements not only underscore the efficiency of the system but also its practical applicability in industrial settings. By leveraging in situ generated formate, this innovative approach demonstrates the potential of integrating electrocatalysis with enzymatic reactions for sustainable and efficient chemical production on a practical scale.

Lignin is the most abundant renewable and sustainable source of aromatic compounds to replace fossil resources, causing environmental issues. However, most lignin generated from pulping and biorefinery processes is combusted or discarded as waste. In this work, we first propose a photocatalytic artificial wood platform consisting of lignin and cellulose, inspired by woody cell walls, where lignin is arranged around cellulose fibers. The wood-mimetic particles were fabricated through reconstitution after the complete dissolution of lignin and cellulose. Intensive characterization revealed that the introduction of cellulose suppressed lignin’s self-aggregation, which limits its practical photocatalytic applications. As a result, the artificial wood photocatalyst exhibited a 3.2 times higher production rate of H2O2 from oxygen and water than lignin under solar light. The wood-mimetic photocatalyst was further coupled with an unspecific peroxygenase (UPO) biocatalyst to catalyze the selective oxygenation of inert C–H bonds using H2O2 as an oxidant. The wood-UPO hybrid catalyst demonstrated a universal photobiocatalytic oxyfunctionalization of various hydrocarbons with a higher total turnover number and turnover frequency than the lignin-UPO catalyst. This work suggests a practical strategy to utilize waste lignin as a photocatalyst and further demonstrates sustainable biosolar oxygenation reactions using sunlight and water as green energy and electron sources, respectively.

Chemoenzymatic cascade catalysis has emerged as a revolutionary tool for streamlining traditional retrosynthetic disconnections, creating new possibilities for the asymmetric synthesis of valuable chiral compounds. Here we construct a one-pot concurrent chemoenzymatic cascade by integrating organobismuth-catalyzed aldol condensation with ene-reductase (ER)-catalyzed enantioselective reduction, enabling the formal asymmetric α-benzylation of cyclic ketones. To achieve this, we develop a pair of enantiocomplementary ERs capable of reducing α-arylidene cyclic ketones, lactams, and lactones. Our engineered mutants exhibit significantly higher activity, up to 37-fold, and broader substrate specificity compared to the parent enzyme. The key to success is due to the well-tuned hydride attack distance/angle and, more importantly, to the synergistic proton-delivery triade of Tyr28-Tyr69-Tyr169. Molecular docking and density functional theory (DFT) studies provide important insights into the bioreduction mechanisms. Furthermore, we demonstrate the synthetic utility of the best mutants in the asymmetric synthesis of several key chiral synthons.

Engineering nonribosomal peptide synthetases (NRPSs) has been a “holy grail” in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the “gate-keeper” adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, MabCAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency (kcat/KM) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.

This review analyzes a development in biochemistry, enzymology and biotechnology that originally came as a surprise. Following the establishment of directed evolution of stereoselective enzymes in organic chemistry, the concept of partial or complete deconvolution of selective multi-mutational variants was introduced. Early deconvolution experiments of stereoselective variants led to the finding that mutations can interact cooperatively or antagonistically with one another, not just additively. During the past decade, this phenomenon was shown to be general. In some studies, molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) computations were performed in order to shed light on the origin of non-additivity at all stages of an evolutionary upward climb. Data of complete deconvolution can be used to construct unique multi-dimensional rugged fitness pathway landscapes, which provide mechanistic insights different from traditional fitness landscapes. Along a related line, biochemists have long tested the result of introducing two point mutations in an enzyme for mechanistic reasons, followed by a comparison of the respective double mutant in so-called double mutant cycles, which originally showed only additive effects, but more recently also uncovered cooperative and antagonistic non-additive effects. We conclude with suggestions for future work, and call for a unified overall picture of non-additivity and epistasis.

This study explores a chemoenzymatic cascade to synthesise chiral β-hydroxy ketones by integrating the selective oxyfunctionalisation capabilities of peroxygenases with the carbon-carbon bond-forming progress of organocatalysts. Initial results with simple organocatalysts demonstrated poor performance due to mutual inactivation of the biocatalyst and organocatalyst. However, the use of more complex prolinamide derivatives improved the reaction efficiency and enantioselectivity, enabling a one-pot, one-step synthesis process. This methodology was further optimised to produce high yields of enantiomerically pure aldol products and was shown to be extendable to other substituted toluenes and aldol donors.

The biocatalytic oxidative deamination of β-amino alcohols holds significant practical potential in kinetic resolution and/or deracemization process to access (R)-β-amino alcohols. This study exemplifies a notable instance of acquisition and utilization of this valuable oxidative deamination activity. Initially, the mutation N261M (M0) was identified to endow a native valine dehydrogenase with oxidative deamination activity toward a few (S)-β-amino alcohols. Subsequently, a phylogenetic analysis-guided, double-code saturation mutagenesis strategy was proposed to engineer M0's side-chain binding site. This strategy facilitated the substrate-specific evolution of M0, resulting in the creation of a panel of mutants (M1-M4) with noteworthy oxidative deamination activity toward structurally diverse (S)-β-amino alcohols. Using these engineered amine dehydrogenases, termed as β-amino alcohol dehydrogenases (β-AADHs), the complete kinetic resolution and even deracemization of a range of β-amino alcohols have been achieved. This work reports distinct biocatalysts and a synthetic strategy for the synthesis of enantiopure (R)-β-amino alcohols and offers an innovative approach for substrate-specificity engineering of enzymes.

This study presents a three-step one pot enzymatic cascade for the synthesis of a δ-lactone. Utilising acetaldehyde, combining 2-deoxyribose-5-phosphate aldolase (DERA) with an alcohol dehydrogenase (ADH) and a cofactor regeneration system this δ-lactone is synthesised with the same stereochemistry as the statin side chain precursor. The initial stage in this cascade involves the double aldol reaction, catalysed by DERA to produce the chiral lactone precursor from the achiral substrate acetaldehyde. The main challenge at this stage is the instability of DERA in the presence of high acetaldehyde concentrations. Therefore, Lactobacillus brevis DERA with a high natural acetaldehyde tolerance was genetically engineered to further improve this property. LbDERA C42M E78K exhibited improved activity and stability (no activity loss over 2 h) compared to the wild type (20% activity loss). In the second stage of the cascade, the aldol product is selectively oxidised to the lactone. A commercially available ADH was identified to selectively catalyse this oxidation using NADP⁺ as electron acceptor. NADP⁺ regeneration was achieved using O₂ as substrate in two different ways: using either photo-activated flavin or NADPH oxidase (NOX). The lactone was successfully purified from the enzymatic cascades from a preparative scale reaction in 97% purity with an optical rotation [α]_D = +34.2° (c = 0.7), proving the feasibility in a multi-enzyme three-step one-pot cascade.

Oleate hydratases open a biocatalytic access to hydroxy fatty acids by hydration of unsaturated fatty acids. Their practical applicability, however, is hampered by their low stability. In this study we report the immobilization of the oleate hydratase from Rhodococcus erythropolis PR4 on functionalized porous, spherical polymer beads. Different carrier materials promoting covalent, hydrophobic, ionic and his-tag affinity were screened and immobilization yields typically >95 % were observed. The highest activity recovery of 32 % was achieved by immobilization via ionic interaction with quaternary ammonium functionalized beads. Biochemical properties of the enzyme immobilized via ionic interaction remain unchanged upon immobilization. The immobilized enzyme was applied for synthesis of 10-hydroxystearic acid remaining stable under process conditions. Conversion of up to 100 mM oleic acid gave 10-hydroxystearic acid achieving a TON of up to 19,000. Successful recycling of the biocatalyst for up to ten cycles further demonstrate its potential for the synthesis of 10-hydroxystearic acid.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.