A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways.

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.

Learning from Protein Engineering by Deconvolution of Multi-Mutational Variants.

This review analyzes a development in biochemistry, enzymology and biotechnology that originally came as a surprise. As part of directed evolution of stereoselective enzymes in organic chemistry, the concept of partial or complete deconvolution of selective multi-mutational variants was established and refined during the past 15 years. Early deconvolution experiments of stereoselective variants led to the finding that mutations can interact cooperatively or antagonistically with one another, not just additively. Later, this phenomenon was shown to be general. 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 more mechanistic insight than 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.

Repertoire of Computationally Designed Peroxygenases for Enantiodivergent C-H Oxyfunctionalization Reactions.

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.

Enhancing the ROS Sensitivity of a Responsive Supramolecular Hydrogel Using Peroxizyme Catalysis.

Hydrogels that can disintegrate upon exposure to reactive oxygen species (ROS) have the potential for targeted drug delivery to tumor cells. In this study, we developed a diphenylalanine (FF) derivative with a thioether phenyl moiety attached to the N-terminus that can form supramolecular hydrogels at neutral and mildly acidic pH. The thioether can be oxidized by ROS to the corresponding sulfoxide, which makes the gelator hydrolytically labile. The resulting oxidation and hydrolysis products alter the polarity of the gelator, leading to disassembly of the gel fibers. To enhance ROS sensitivity, we incorporated peroxizymes in the gels, namely, chloroperoxidase CiVCPO and the unspecific peroxygenase rAaeUPO. Both enzymes accelerated the oxidation process, enabling the hydrogels to collapse with 10 times lower H2O2 concentrations than those required for enzyme-free hydrogel collapse. These ROS-responsive hydrogels could pave the way toward optimized platforms for targeted drug delivery in the tumor microenvironment.

Biocatalysis making waves in organic chemistry.

Biocatalysis has an enormous impact on chemical synthesis. The waves in which biocatalysis has developed, and in doing so changed our perception of what organic chemistry is, were reviewed 20 and 10 years ago. Here we review the consequences of these waves of development. Nowadays, hydrolases are widely used on an industrial scale for the benign synthesis of commodity and bulk chemicals and are fully developed. In addition, further enzyme classes are gaining ever increasing interest. Particularly, enzymes catalysing selective C-C-bond formation reactions and enzymes catalysing selective oxidation and reduction reactions are solving long-standing synthetic challenges in organic chemistry. Combined efforts from molecular biology, systems biology, organic chemistry and chemical engineering will establish a whole new toolbox for chemistry. Recent developments are critically reviewed.

Contribution of Enzyme Catalysis to the Achievement of the United Nations' Sustainable Development Goals.

In September 2015, the United Nations General Assembly established the 2030 Agenda for Sustainable Development, which includes 17 Sustainable Development Goals (SDGs) [...].

Peroxygenase-Catalysed Selective Oxidation of Silanes to Silanols.

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.

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids.

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L-1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.

A Photochemoenzymatic Hunsdiecker-Borodin-Type Halodecarboxylation of Ferulic Acid.

A photochemoenzymatic halodecarboxylation of ferulic acid was achieved using vanadate-dependent chloroperoxidase as (bio)catalyst and oxygen and organic solvent as sole stoichiometric reagents in a biphasic system. Performance and selectivity were improved through a phase transfer catalyst, reaching a turnover number of 660.000 for the enzyme.

A Peroxygenase-Alcohol Dehydrogenase Cascade Reaction to Transform Ethylbenzene Derivatives into Enantioenriched Phenylethanols.

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.

Immobilization and Application of Fatty Acid Photodecarboxylase in Deep Eutectic Solvents.

Since its discovery in 2017, the fatty acid decarboxylase (FAP) photoenzyme has been the focus of extensive research, given its ability to convert fatty acids into alka(e)nes using merely visible blue light. Unfortunately, there are still some drawbacks that limit the applicability of this biocatalyst, such as poor solubility of the substrates in aqueous media, poor photostability, and the impossibility of reusing the catalyst for several cycles. In this work, we demonstrate the use of FAP in non-conventional media as a free enzyme and an immobilized preparation. Namely, its applicability in deep eutectic solvents (DESs) and a proof-of-concept immobilization using a commercial His-tag selective carrier, a thorough study of reaction and immobilization conditions in each case, as well as reusability studies are shown. We observed an almost complete selectivity of the enzyme towards C18 decarboxylation over C16 when used in a DES, with a product analytical yield up to 81 % when using whole cells. Furthermore, when applying the immobilized enzyme in DES, we obtained yields >10-fold higher than the ones obtained in aqueous media.

Stabilisation of the Fatty Acid Decarboxylase from Chlorella variabilis by Caprylic Acid.

The fatty acid photodecarboxylase from Chlorella variabilis NC64 A (CvFAP) catalyses the light-dependent decarboxylation of fatty acids. Photoinactivation of CvFAP still represents one of the major limitations of this interesting enzyme en route to practical application. In this study we demonstrate that the photostability of CvFAP can easily be improved by the administration of medium-chain length carboxylic acids such as caprylic acid indicating that the best way of maintaining CvFAP stability is 'to keep the enzyme busy'.

Directed evolution of unspecific peroxygenase in organic solvents.

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.

Novel oleate hydratases and potential biotechnological applications.

Oleate hydratase catalyses the addition of water to the CC double bond of oleic acid to produce (R)-10-hydroxystearic acid. The enzyme requires an FAD cofactor that functions to optimise the active site structure. A wide range of unsaturated fatty acids can be hydrated at the C10 and in some cases the C13 position. The substrate scope can be expanded using 'decoy' small carboxylic acids to convert small chain alkenes to secondary alcohols, albeit at low conversion rates. Systematic protein engineering and directed evolution to widen the substrate scope and increase the conversion rate is possible, supported by new high throughput screening assays that have been developed. Multi-enzyme cascades allow the formation of a wide range of products including keto-fatty acids, secondary alcohols, secondary amines and α,ω-dicarboxylic acids. KEY POINTS: • Phylogenetically distinct oleate hydratases may exhibit mechanistic differences. • Protein engineering to improve productivity and substrate scope is possible. • Multi-enzymatic cascades greatly widen the product portfolio.

Choline Chloride-Based DES as Solvents/Catalysts/Chemical Donors in Pharmaceutical Synthesis.

DES are mixtures of two or more compounds, able to form liquids upon mixing, with lower freezing points when compared to the individual constituents (eutectic mixtures). This attitude is due to the specific hydrogen-bond interactions network between the components of the mixture. A notable characteristic of DES is the possibility to develop tailor-made mixtures by changing the components ratios or a limited water dilution, for special applications, making them attractive for pharmaceutical purposes. In this review, we focused our attention on application of ChCl-based DES in the synthesis of pharmaceutical compounds. In this context, these eutectic mixtures can be used as solvents, solvents/catalysts, or as chemical donors and we explored some representative examples in recent literature of such applications.

Biocatalytic Reduction Reactions from a Chemist's Perspective.

Reductions play a key role in organic synthesis, producing chiral products with new functionalities. Enzymes can catalyse such reactions with exquisite stereo-, regio- and chemoselectivity, leading the way to alternative shorter classical synthetic routes towards not only high-added-value compounds but also bulk chemicals. In this review we describe the synthetic state-of-the-art and potential of enzymes that catalyse reductions, ranging from carbonyl, enone and aromatic reductions to reductive aminations.

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications.

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

Metals in Biotechnology: Cr-Driven Stereoselective Reduction of Conjugated C=C Double Bonds.

Elemental metals are shown to be suitable sacrificial electron donors to drive the stereoselective reduction of conjugated C=C double bonds using Old Yellow Enzymes as catalysts. Both direct electron transfer from the metal to the enzyme as well as mediated electron transfer is feasible, although the latter excels by higher reaction rates. The general applicability of this new chemoenzymatic reduction method is demonstrated, and current limitations are outlined.

Solar-Assisted eBiorefinery: Photoelectrochemical Pairing of Oxyfunctionalization and Hydrogenation Reactions.

Inspired by natural photosynthesis, biocatalytic photoelectrochemical (PEC) platforms are gaining prominence for the conversion of solar energy into useful chemicals by combining redox biocatalysis and photoelectrocatalysis. Herein, we report a dual biocatalytic PEC platform consisting of a molybdenum (Mo)-doped BiVO4 (Mo:BiVO4 ) photoanode and an inverse opal ITO (IO-ITO) cathode that gives rise to the coupling of peroxygenase and ene-reductase-mediated catalysis, respectively. In the PEC cell, the photoexcited electrons generated from the Mo:BiVO4 are transferred to the IO-ITO and regenerate reduced flavin mononucleotides to drive ene-reductase-catalyzed trans-hydrogenation of ketoisophrone to (R)-levodione. Meanwhile, the photoactivated Mo:BiVO4 evolves H2 O2 in situ via a two-electron water-oxidation process with the aid of an applied bias, which simultaneously supplies peroxygenases to drive selective hydroxylation of ethylbenzene into enantiopure (R)-1-phenyl-1-hydroxyethane. Thus, the deliberate integration of PEC systems with redox biocatalytic reactions can simultaneously produce valuable chemicals on both electrodes using solar-powered electrons and water.

Whole-Cell Photoenzymatic Cascades to Synthesize Long-Chain Aliphatic Amines and Esters from Renewable Fatty Acids.

Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engineered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octylnonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer-Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g-1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.