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.

The selective chemical dehydration leading to CC double bond formation is a challenging reaction that harbors great potential for industrial applications. The cofactor independent bifunctional linalool dehydratase isomerase (LinD) from Castellaniella defragrans catalyzes the reversible dehydration of (S)-linalool to myrcene, as well as its isomerization to geraniol. We previously reported that LinD is able to convert the small alkenol 2-methyl-3-buten-2-ol to the valuable product isoprene. To foster the LinD-catalyzed production of isoprene in a novel recombinant E. coli whole-cell two-phase system, we targeted the active site and a flexible α-helix near the putative substrate channel via enzyme engineering. Interestingly, none of the active site variants exhibited an increased product formation. In contrast, saturation mutagenesis of the 10 amino acids forming the α-helix, identified the variants K103N, R104G, G107T and D112T, which exhibited a 1.73 ± 0.05, 1.56 ± 0.12, 2.08 ± 0.12 and 1.93 ± 0.06-fold increase in product formation compared to the wild-type enzyme, respectively. Notably, a combinatorial approach targeting these four variants led to decreased activity in all cases, compared to the corresponding single-point variants, indicating negative epistatic interactions. Thus, employing the most catalytically efficient single point variant G107T, which exhibited a 28-fold higher kcat (app) compared to the wild-type, a total of 2.8 ± 0.2 mM isoprene was obtained utilizing the whole-cell two-phase system. Crystallographic analysis of G107T revealed only minor structural changes; however, molecular dynamic simulations uncovered striking conformational differences relative to the LinD wild-type, emphasizing the role of altered substrate channel in variant G107T.

Squalene-hopene cyclases (SHCs) are a highly valuable and attractive class of membrane-bound enzymes as sustainable biotechnological tools to produce aromas and bioactive compounds at industrial scale. However, their application as whole-cell biocatalysts suffer from the outer cell membrane acting as a diffusion barrier for the highly hydrophobic substrate/product, while the use of purified enzymes leads to dramatic loss of stability. Here we present an unexplored strategy for biocatalysis: the application of SHC spheroplasts. By removing the outer cell membrane, we produced stable and substrate-accessible biocatalysts. SHC spheroplasts exhibited up to 100-fold higher activity than their whole-cell counterparts for the biotransformations of squalene, geranyl acetone, farnesol, and farnesyl acetone. Their catalytic ability was also higher than the purified enzyme for all high molecular weight terpenes. In addition, we introduce a new concept for the carrier-free immobilization of spheroplasts via crosslinking, CLS (crosslinked spheroplasts). The CLS maintained the same catalytic activity of the spheroplasts, offering additional advantages such as recycling and reuse. These timely solutions contribute not only to harness the catalytic potential of the SHCs, but also to make biocatalytic processes even greener and more cost-efficient.

The regioselective hydration of carbon-carbon double bonds to generate alcohols is a fundamental reaction in synthetic organic chemistry, offering pathways to valuable secondary and tertiary alcohols. Biocatalysis using hydratase enzymes, which add water to a double bond, provides a selective and sustainable alternative to traditional chemical methods. This study investigates the potential of Escherichia coli to hydrate the monoterpene geraniol in whole-cell biotransformation systems. Through a targeted knockout approach using the Keio collection, fumarases were identified as key contributors to geraniol hydration. Overexpression studies further revealed that FumA and FumB overexpression substantially enhanced geraniol hydration activity at the terminal alkene, suggesting promiscuity towards this non-native substrate. This result indicates an expanded substrate scope of class I fumarases beyond their established role in fumarate metabolism. By establishing a link between geraniol hydration and specific genes, we aim to extend the enzymatic toolbox for monoterpene transformations. Utilizing the inherent regioselectivity and atom economy of fumarases, the potential of fumarases as efficient biocatalysts in terpene modification could open new avenues to advance applications in green chemistry and biocatalysis.

The interconversion of monoterpenes is facilitated by a complex network of carbocation rearrangement pathways. Controlling these isomerization pathways is challenging when using common Brønsted and Lewis acid catalysts, which often produce product mixtures that are difficult to separate. In contrast, natural monoterpene cyclases exhibit high control over the carbocation rearrangement reactions but are reliant on phosphorylated substrates. In this study, we present engineered squalene-hopene cyclases from Alicyclobacillus acidocaldarius (AacSHC) that catalyze the challenging isomerization of monoterpenes with unprecedented precision. Starting from a promiscuous isomerization of (+)-β-pinene, we first demonstrate noticeable shifts in the product distribution solely by introducing single point mutations. Furthermore, we showcase the tuneable cation steering by enhancing (+)-borneol selectivity from 1 % to >90 % (>99 % de) aided by iterative saturation mutagenesis. Our combined experimental and computational data suggest that the reorganization of key aromatic residues leads to the restructuring of the water network that facilitates the selective termination of the secondary isobornyl cation. This work expands our mechanistic understanding of carbocation rearrangements and sets the stage for target-oriented skeletal reorganization of broadly abundant terpenes.

Methoxylated polybrominated diphenyl ethers (MeO-BDEs) are a class of environmentally relevant halogenated natural products. The two most relevant isomers, 2'-MeO-BDE 68 and 6-MeO-BDE 47, were repeatedly detected at levels comparable with persistent organic pollutants in marine environmental and food samples. MeO-BDEs were suspected to be biosynthesized by bromoperoxidases through the merging of two bromophenol units, three of which (2,4-dibromophenol, 2,6-dibromophenol and 2,4,6-tribromophenol) are abundant in marine environments, followed by O-methylation to give MeO-BDEs. However, not all abundant MeO-BDEs can be explained by the direct coupling of bromophenols. Therefore, several bromophenols were incubated with CYP109B1 from Bacillus subtilis. Incubations of 2,4-dibromophenol with CYP109B1 resulted in both 2'-OH-BDE 68 and 6-OH-BDE 47. Mechanistic considerations indicated that both compounds emerged from an epoxidized intermediate. After subsequent methylation this leads to the two most relevant MeO-BDEs in marine environments. By contrast, incubations with 2,6-dibromophenol and 2,4,6-tribromophenol did not form any OH-BDEs but tri- or tetrabrominated dibenzo-p-dioxins (PBDDs), i.e. another known class of environmentally relevant halogenated natural products whose toxicity has shown to be similar to their chlorinated analogues. SYNOPSIS: Treatment of bromophenols with cytochrome P450 monooxygenase CYP109B1 led to the formation of hydroxylated polybrominated diphenyl ethers and polybrominated dibenzo-p-dioxins.

Terpenoids are built from isoprene building blocks and have numerous biological functions. Selective late-stage modification of their carbon scaffold has the potential to optimize or transform their biological activities. However, the synthesis of terpenoids with a non-natural carbon scaffold is often a challenging endeavor because of the complexity of these molecules. Herein we report the identification and engineering of (S)-adenosyl-l-methionine-dependent sterol methyltransferases for selective C-methylation of linear terpenoids. The engineered enzyme catalyzes selective methylation of unactivated alkenes in mono-, sesqui- and diterpenoids to produce C11 , C16 and C21 derivatives. Preparative conversion and product isolation reveals that this biocatalyst performs C-C bond formation with high chemo- and regioselectivity. The alkene methylation most likely proceeds via a carbocation intermediate and regioselective deprotonation. This method opens new avenues for modifying the carbon scaffold of alkenes in general and terpenoids in particular.

Cascade reactions appeared as a cutting-edge strategy to streamline the assembly of complex structural scaffolds from naturally available precursors in an atom-, as well as time, labor- and cost-efficient way. We herein report a strategy to control cationic cyclization cascades by exploiting the ability of anchoring dynamic substrates in the active site of terpene cyclases via designed hydrogen bonding. Thereby, it is possible to induce "directed" cyclizations in contrast to established "non-stop" cyclizations (99:1) and predestinate cascade termination at otherwise catalytically barely accessible intermediates. As a result, we are able to provide efficient access to naturally widely occurring apocarotenoids, value-added flavors and fragrances in gram-scale by replacing multi-stage synthetic routes to a single step with unprecedented selectivity (>99.5 % ee) and high yields (up to 89 %).

The Friedel‐Crafts alkylation constitutes one of the most important reactions for the formation of carbon‐carbon bonds. Here, we report the Friedel‐Crafts alkylation using squalene‐hopene cyclases, which provides a biological alternative to the traditional strategy. The squalene‐hopene cyclase from Alicyclobacillus acidocaldarius ( Aac SHC) and variants have been demonstrated to have a broad substrate and reaction scope, making them valuable for potential applications in biocatalysis. Notably, the Friedel‐Crafts alkylation of the substrate geranyl phenyl ether was found to be highly regioselective. Furthermore, squalene‐hopene cyclases exhibit promiscuous activity in catalyzing the hydration of geranyl phenyl ether in an aqueous buffer. Finally, we have analyzed the roles of various active‐site residues and studied their influence on the reaction and product specificity. These findings highlight the promise of enzymatic catalysis for enabling selective C−C bond formations.

Diclofenac one of the widespread xenobiotics, among other organic micropollutants, is persistent accumulating in different habitats like earth, water, plants and even mammalians. Natural microbial communities in soil and water play a key role in fundamental ecological processes such as regulating the fate of pollution released in the environment. As presented in this study varying concentrations of diclofenac between 0.1 and 1.0 g/L solubilized in a low salt medium could be aerobically degraded by forest soil within less than 10 days. In the course of full degradation, a carboxylated diclofenac intermediate could be isolated and identified by LC-MS/MS-TOF. The carboxylated diclofenac might be a key intermediate to enable a complete biodegradation of diclofenac via 2,6-dichloroaniline and carboxylated 2-hydroxyphenylacetic acid by a microbial consortium.

Enzymes are nature's powerful catalytic proteins to perform reactions with often outstanding activity, selectivity and specificity. Moreover, the access to non-natural functions of biocatalysts can be facilitated by enzyme engineering. While rational approaches are often focused on an enzyme's active site, from random directed evolution we know that further functional hotspots must exist beyond the active site. Addressing flexible structural elements of these biocatalysts like loops and channels in enzyme engineering has the potential to fill this knowledge gap. The structural dynamics of enzyme catalysts are vital to promote their remarkable functions. This influences for example the access, recognition and orientation of substrates. Herein, we review recent examples of loop and channel engineering and classify them according to their use of simulation methodologies, predictions prior to engineering, the engineering methodologies themselves and discoveries found after the engineering. Thereby we highlight current possibilities and make suggestions to further unlock the potential of this yet underexplored strategy.

The reduction of activated C=C double bonds is an important reaction in synthetic chemistry owing to the potential formation of up to two new stereogenic centers. Artificial nicotinamide cofactors were recently presented as alternative suppliers of hydride equivalents needed for alkene reduction. To study the effect of cofactors on the reduction of activated alkenes, a set of N-substituted synthetic nicotinamide cofactors with differing oxidation potentials were synthesized and their electrochemical and kinetic behavior was studied. The effects of the synthetic cofactors on enzyme activity of four ene reductases are outlined in this study, where the cofactor mimic with an N-substituted 4-hydroxy-phenyl residue led to a sixfold higher v(max) relative to the natural cofactor NADH.

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.

A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids.

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.

Polymers based on renewable resources have become increasingly important. The natural functionalization of fats and oils enables an easy access to interesting monomeric building blocks, which in turn transform the derivative biopolymers into high-performance materials. Unfortunately, interesting building blocks of medium-chain length are difficult to obtain by traditional chemical means. Herein, a biotechnological pathway is established that could provide an environmentally suitable and sustainable alternative. A multiple enzyme two-step one-pot process efficiently catalyzed by a coupled 9S-lipoxygenase (St-LOX1, Solanum tuberosum) and 9/13-hydroperoxide lyase (Cm-9/13HPL, Cucumis melo) cascade reaction is proposed as a potential route for the conversion of linoleic acid into 9-oxononanoic acid, which is a precursor for biopolymers. Lipoxygenase catalyzes the insertion of oxygen into linoleic acid through a radical mechanism to give 9S-hydroperoxy-octadecadienoic acid (9S-HPODE) as a cascade intermediate, which is subsequently cleaved by the action of Cm-9/13HPL. This one-pot process afforded a yield of 73 % combined with high selectivity. The best reaction performance was achieved when lipoxygenase and hydroperoxide lyase were applied in a successive rather than a simultaneous manner. Green leaf volatiles, which are desired flavor and fragrance products, are formed as by-products in this reaction cascade. Furthermore, we have investigated the enantioselectivity of 9/13-HPLs, which exhibited a strong preference for 9S-HPODE over 9R-HPODE.

CYP153A from Marinobacter aquaeolei has been identified as a fatty acid ω-hydroxylase with a broad substrate range. Two hotspots predicted to influence substrate specificity and selectivity were exchanged. Mutant G307A is 2- to 20-fold more active towards fatty acids than the wild-type. Residue L354 is determinant for the enzyme ω-regioselectivity.

Immobilized cutinase HiC from the ascomycete Humicola insolens was applied as a novel biocatalyst for the synthesis of functionalized acryclic esters by transesterification. As a model reaction, transesterification of methyl acrylate with 6-mercapto-1-hexanol at a high molar ratio in a solvent free system was chosen. Besides two minor Michael-addition by-products, 6-mercaptohexyl acrylic ester was identified as the main product with the thiol as the functional end group. Reaction conditions were optimized regarding the influence of water (0–1.72 M), temperature (22–50 °C), product inhibition and addition of the radical inhibitor butylated hydroxytoluol (BHT; 0.14–0.71 M) on conversion and by-product formation. Highest conversion of 6-mercapto-1-hexanol to 6-mercaptohexyl acrylic ester (95.4 ± 0.3%) was achieved after 6 h at 40 °C in the presence of 0.025% (w/w) water without formation of by-products in a solvent free system. Applying methyl methacrylate, transesterification with 6-mercapto-1-hexanol was significantly lower (43.6 ± 0.1%) compared to transesterification of methyl acrylate with 6-mercapto-1-hexanol.

Masking toxic proteins: How can toxic proteins be expressed in natural systems without killing the host? Recently, a new in vivo approach has shown how to sequester large amounts of toxic proteins in a protein container by making use of electrostatic interactions.