Vector-borne diseases pose a rising global health challenge, necessitating the development of safe and effective pest protection agents. Here, we report a highly selective biocatalytic direct Prins cyclohydration for the synthesis of (1R)-cis-p-menthane-3,8-diol (PMD), a natural insect repellent with high efficacy. By strategically engineering squalene-hopene cyclases (SHCs), we achieved >96% diastereomeric excess, surpassing previous synthetic methods. Structural and mechanistic analyses suggest direct Prins cyclohydration and a precisely positioned water molecule within the enzyme's active pocket adjacent to the final carbocation that drives hydration and catalytic efficiency. Fine-tuning the biocatalytic setup enabled preparative scale production, without losing much product selectivity. Moreover, we demonstrate access to the other naturally occurring PMD isomers from (R)- and (S)-citronellal, as well as a one-pot cascade starting from E/Z-citral. This study paves the way for highly selective access to stereodefined terpene-derived repellents and establishes engineered squalene-hopene cyclases as a tool for direct asymmetric Prins cyclohydration.

To drastically reduce the carbon footprint of the food production chain, a major shift towards alternatives to conventional meat and dairy products is required. The use of plant-based proteins is a promising route, but it also comes with challenges: Plant-based proteins often contain antinutritional factors and off-flavors, which can negatively impact consumer acceptance. Fermentation is broadly used to improve the quality of these products. However, how these unwanted molecules are synthesized and degraded is poorly understood, but this knowledge is essential for fermentation-based strategies to improve the sensory and nutritional value of plant-based products. This review provides a comprehensive overview of synthesis and degradation pathways of key antinutritional factors and off-flavor compounds in plant-based substrates, including aldehydes, furans, sulfur compounds, pyrazines, glycoalkaloids (GAs), pyrimidine glycosides, polyphenols, saponins, glucosinolates (GSLs), phytic acid (PA), oxalates, lectins, and protease and amylase inhibitors. With this we identified the research gaps in the field, which can be divided into three types: (i) degradation pathways that are unknown (furans, alkyl-methoxypyrazines, and dimethyl trisulfide), (ii) well-characterized pathways but typically not found in food-grade organisms (dimethyl sulfide, dimethyl disulfide, and isothiocyanates derived from GSLs), and (iii) pathways that are only described partially (GAs, saponins, polyphenols, PA, and pyrimidine glycosides). Other molecule classes, like aldehydes, alcohols, and oxalate, have well-characterized degradation pathways in food-grade organisms. Focusing future research on compounds with poorly understood degradation pathways will help to accelerate the development of more rationally designed cultures for producing healthy and sustainable plant-based foods.

In Cell Reports Physical Science, Maier et al. unveil a biocatalytic approach for synthesizing N-hydroxy compounds by integrating the flavin-dependent monooxygenase GorA into an enzymatic cascade with the decarboxylase GorB and a formate dehydrogenase-driven cofactor recycling system. This work showcases GorA's substrate scope and establishes a biocatalytic synthetic route for valuable N-hydroxy compounds.

(R)-Citronellal is one of the key chiral intermediates in the synthesis of the isomer (−)-menthol, one of the most commercialised terpenoid flavours worldwide. Enzymatic approaches could represent a less energy-demanding alternative for its synthesis, such as a previously reported bienzymatic cascade starting from inexpensive, commercially available geraniol. A copper radical oxidase (CgrAlcOx) followed by a flavin-dependent ene reductase (OYE2) were used to obtain (R)-citronellal. Here, we used a metal-affinity immobilisation strategy on the His-tagged enzymes for the cascade and studied enzyme recovery and reusability as well as increased solvent tolerance. After screening a panel of resins for enzyme immobilisation and water-immiscible co-solvents, we successfully obtained 95% conversion to (R)-citronellal with 96.9% enantiomeric excess (ee) in a concurrent cascade after 7 h of reaction time, starting from 10 mM of geraniol.

The development of synthetic routes to produce enantiopure (R)-citronellal as a key intermediate for the synthesis of (–)-menthol and other valuable terpenoids is highly relevant in the pharmaceutical, flavor, and fragrance industries. Herein, we showcase a cascade with two consecutive biocatalytic steps performed separately using the inherent selectivity of a short-chain alcohol dehydrogenase (SDR) and an ene reductase (ERED) from the Old Yellow Enzyme (OYE) family. The first reaction involves the AaSDR1-catalyzed oxidation of relatively inexpensive geraniol in a biphasic system, providing geranial as an intermediate. The organic phase containing geranial is then extracted and transferred to the second step, where the ERED variant OYE2_Y83V catalyzes the asymmetric reduction of geranial to produce (R)-citronellal, achieving >90% conversion and >99% enantiomeric excess. The use of n-heptane in a two-liquid phase system not only facilitates substrate and product solubilization but also minimizes geranial isomerization. This biocatalytic cascade therefore enables the synthesis of enantiopure (R)-citronellal.

The asymmetric mixed carboligation of aldehydes catalyzed by thiamine diphosphate (ThDP)-dependent enzymes provides a sensitive system for monitoring changes in activity, chemo-, and enantioselectivity. While previous studies have shown that organic cosolvents influence these parameters, we now demonstrate that similar effects occur upon addition of water-miscible ionic liquids (ILs). In this study, six ThDP-dependent enzymes were analyzed in the presence of 14 ILs under comparable conditions to assess their influence on enzymatic carboligation reactions yielding 2-hydroxy ketones. ILs exerted a moderate to strong influence on activity and, more notably, altered enantioselectivity. (R)-selective reactions were generally stable upon IL addition, while (S)-selective reactions frequently showed reduced selectivity or even inversion to the (R)-enantiomer. The most significant change was observed for the ApPDC_E469G variant of pyruvate decarboxylase from Acetobacter pasteurianus, where the enantiomeric excess shifted from 86 % (S) to 60 % (R) in the presence of 9 % (w/v) Ammoeng 102. Control experiments indicated that this shift was primarily due to the Ammoeng cation rather than the anion. To explore the molecular basis of this phenomenon, all-atom molecular dynamics (MD) simulations were performed on wild-type ApPDC and the E469G variant in Ammoeng 101 and Ammoeng 102. The simulations revealed that hydrophobic and hydrophilic regions of the Ammoeng cations interact with the (S)-selective binding pocket, thereby favoring formation of the (R)-product. These results highlight the potential of solvent engineering for modulating enzyme selectivity and demonstrate that MD simulations can capture functionally relevant enzyme–solvent interactions at the atomic level.

Reductive amination is one of the most synthetically direct routes to access chiral amines. Several Imine Reductases (IREDs) have been discovered to catalyze reductive amination (Reductive Aminases or RedAms), yet they are dependent on the expensive phosphorylated nicotinamide adenine dinucleotide cofactor NADPH and usually more active at basic pH. Here, we describe the discovery and synthetic potential of an IRED from Rhodococcus erythropolis (RytRedAm) that catalyzes reductive amination between a series of medium to large carbonyl and amine compounds with conversions of up to >99% and 99% enantiomeric excess at neutral pH. RytRedAm catalyzes the formation of a substituted γ-lactam and N-methyl-1-phenylethanamine with stereochemistry opposite to that of fungal RedAms, giving the (S)-enantiomer. This enzyme remarkably uses both NADPH and NADH cofactors with K_M values of 15 and 247 μM and turnover numbers k_cat of 3.6 and 9.0 s^-1, respectively, for the reductive amination of hexanal with allylamine. The crystal structure obtained provides insights into the flexibility to also accept NADH, with residues R35 and I69 diverging from that of other IREDs/RedAms in the otherwise conserved Rossmann fold. RytRedAm thus represents a subfamily of enzymes that enable synthetic applications using NADH-dependent reductive amination to access complementary chiral amine products.

Cyclopropane fatty acid synthases (CFAS) are a class of S-adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene-based approaches. This work describes the characterisation of E. coli CFAS (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with up to 58 % conversion and 73 % ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ecCFAS was found to be correlated with the electronic properties of phospholipid headgroups and for the first time ecCFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ecCFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH₃I and CD₃I respectively.

The unmatched chemo-, regio-, and stereoselectivity of enzymes renders them powerful catalysts in the synthesis of chiral active pharmaceutical ingredients (APIs). Inspired by the discovery route toward the LPA₁-antagonist BMS-986278, access to the API building block (1S,3R)-3-hydroxycyclohexanecarbonitrile was envisaged using an ene reductase (ER) and alcohol dehydrogenase (ADH) to set both stereocenters. Starting from the commercially available cyclohexene-1-nitrile, a C-H oxyfunctionalization step was required to introduce the ketone functional group, yet several chemical allylic oxidation strategies proved unsuccessful. Enzymatic strategies for allylic oxidation are underdeveloped, with few examples on selected substrates with cytochrome P450s and unspecific peroxygenases (UPOs). In this case, UPOs were found to catalyze the desired allylic oxidation with high chemo- and regioselectivity, at substrate loadings of up to 200 mM, without the addition of organic cosolvents, thus enabling the subsequent ER and ADH steps in a three-step one-pot cascade. UPOs even displayed unreported enantioselective oxyfunctionalization and overoxidation of the substituted cyclohexene. After screening of enzyme panels, the final product was obtained at titers of 85% with 97% ee and 99% de, with a substrate loading of 50 mM, the ER being the limiting step. This synthetic approach provides the first example of a three-step, one-pot UPO-ER-ADH cascade and highlights the potential for UPOs to catalyze diverse enantioselective allylic hydroxylations and oxidations that are otherwise difficult to achieve.

Contemporary Biocatalysis heavily relies on enzyme engineering as natural enzymes frequently lack the requisite attributes for effective organic synthesis. The inherent limitations in stability, catalytic activity, and selectivity of wild-type enzymes often hinder their suitability for chemical synthesis. Over the past 25 years, there has been an unprecedented advancement in protein engineering tools, empowering enzymologists to customise enzymes to precisely meet the demands of organic synthesis. In this discussion, we delineate some of the most crucial techniques in enzyme engineering and their significance in facilitating chemical synthesis.

Biocatalytic asymmetric reduction of alkenes in organic solvent is attractive for enantiopurity and product isolation, yet remains under developed. Herein we demonstrate the robustness of an ene reductase immobilised on Celite for the reduction of activated alkenes in micro-aqueous organic solvent. Full conversion was obtained in methyl t-butyl ether, avoiding hydrolysis and racemisation of products. The immobilised ene reductase showed reusability and a scale-up demonstrated its applicability.

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.