Browsing by Author "Hauer, Bernhard"

Now showing 1 - 13 of 13

Open Access
Active-site loop variations adjust activity and selectivity of the cumene dioxygenase
(2021) Heinemann, Peter M.; Armbruster, Daniel; Hauer, Bernhard
Active-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions along with a novel insertion approach result in up to 16-fold increases in activity and the formation of novel products and enantiomers. CAVER analysis suggests possible increases in the active pocket volume and formation of new active-site tunnels, suggesting additional degrees of freedom of the substrate in the pocket. The combination of identified hot-spots with the Linker In Loop Insertion approach proves to be a valuable addition to future loop engineering approaches for enhanced biocatalysts.
Open Access
Assembly of a Rieske non-heme iron oxygenase multicomponent system from Phenylobacterium immobile E DSM 1986 enables pyrazon cis-dihydroxylation in E. coli
(2021) Hunold, Andreas; Escobedo-Hinojosa, Wendy; Potoudis, Elsa; Resende, Daniela; Farr, Theresa; Syrén, Per-Olof; Hauer, Bernhard
Phenylobacterium immobile strain E is a soil bacterium with a striking metabolism relying on xenobiotics, such as the herbicide pyrazon, as sole carbon source instead of more bioavailable molecules. Pyrazon is a heterocyclic aromatic compound of environmental concern and its biodegradation pathway has only been reported in P. immobile. The multicomponent pyrazon oxygenase (PPO), a Rieske non-heme iron oxygenase, incorporates molecular oxygen at the 2,3 position of the pyrazon phenyl moiety as first step of degradation, generating a cis-dihydrodiendiol. The aim of this work was to identify the genes encoding for each one of the PPO components and enable their functional assembly in Escherichia coli. P. immobile strain E genome sequencing revealed genes encoding for RO components, such as ferredoxin-, reductase-, α- and β-subunits of an oxygenase. Though, P. immobile E displays three prominent differences with respect to the ROs currently characterized: (1) an operon-like organization for PPO is absent, (2) all the elements are randomly scattered in its DNA, (3) not only one, but 19 different α-subunits are encoded in its genome. Herein, we report the identification of the PPO components involved in pyrazon cis-dihydroxylation in P. immobile, its appropriate assembly, and its functional reconstitution in E. coli. Our results contributes with the essential missing pieces to complete the overall elucidation of the PPO from P. immobile.
Open Access
Biocatalytic stereocontrolled head-to-tail cyclizations of unbiased terpenes as a tool in chemoenzymatic synthesis
(2024) Schneider, Andreas; Lystbæk, Thomas B.; Markthaler, Daniel; Hansen, Niels; Hauer, Bernhard
Terpene synthesis stands at the forefront of modern synthetic chemistry and represents the state-of-the-art in the chemist’s toolbox. Notwithstanding, these endeavors are inherently tied to the current availability of natural cyclic building blocks. Addressing this limitation, the stereocontrolled cyclization of abundant unbiased linear terpenes emerges as a valuable tool, which is still difficult to achieve with chemical catalysts. In this study, we showcase the remarkable capabilities of squalene-hopene cyclases (SHCs) in the chemoenzymatic synthesis of head-to-tail-fused terpenes. By combining engineered SHCs and a practical reaction setup, we generate ten chiral scaffolds with >99% ee and de , at up to decagram scale. Our mechanistic insights suggest how cyclodextrin encapsulation of terpenes may influence the performance of the membrane-bound enzyme. Moreover, we transform the chiral templates to valuable (mero)-terpenes using interdisciplinary synthetic methods, including a catalytic ring-contraction of enol-ethers facilitated by cooperative iodine/lipase catalysis.
Open Access
Controlling monoterpene isomerization by guiding challenging carbocation rearrangement reactions in engineered squalene‐hopene cyclases
(2024) Ludwig, Julian; Curado‐Carballada, Christian; Hammer, Stephan C.; Schneider, Andreas; Diether, Svenja; Kress, Nico; Ruiz‐Barragán, Sergi; Osuna, Sílvia; Hauer, Bernhard
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.
Open Access
Enhanced semi‐preparative biotransformation of cumene dioxygenase : from analytical scale to product isolation
(2023) Schelle, Jona T.; Lepoittevin, William; Hauer, Bernhard
Scale‐up of oxygenase catalyzed reactions is often challenging due to the limited oxygen mass transfer in aqueous solutions. To overcome such limitation, we studied different scale‐up conditions using recombinant resting cells of E. coli JM109(DE3), harboring the cumene dioxygenase of Pseudomonas fluorescens IP01, for the dihydroxylation of naphthalene to (1R,2S)‐cis‐1,2‐dihydro‐1,2‐naphthalenediol. Thereby, vigorous stirring of the biotransformation in a 2 L round bottom flask in combination with an oxygen‐enriched headspace exhibited outstanding product formation after 1 h. Furthermore, the enhanced setup was used for the cumene dioxygenase catalyzed biosynthesis of 240 mg of valuable (+)‐trans‐carveol from (R)‐(+)‐limonene, demonstrating the application of our workflow for volatile compounds.
Open Access
Harnessing the structure and dynamics of the squalene‐hopene cyclase for (-)‐ambroxide production
(2023) Schneider, Andreas; Curado, Christian; Lystbaek, Thomas B.; Osuna, Sílvia; Hauer, Bernhard
Terpene cyclases offer enormous synthetic potential, given their unique ability to forge complex hydrocarbon scaffolds from achiral precursors within a single cationic rearrangement cascade. Harnessing their synthetic power, however, has proved to be challenging owing to their generally low catalytic performance. In this study, we unveiled the catalytic potential of the squalene‐hopene cyclase (SHC) by harnessing its structure and dynamics. First, we synergistically tailored the active site and entrance tunnel of the enzyme to generate a 397‐fold improved (-)‐ambroxide synthase. Our computational investigations explain how the introduced mutations work in concert to improve substrate acquisition, flow, and chaperoning. Kinetics, however, showed terpene‐induced inactivation of the membrane‐bound SHC to be the major turnover limitation in vivo. Merging this insight with the improved and stereoselective catalysis of the enzyme, we applied a feeding strategy to exceed 10 5 total turnovers. We believe that our results may bridge the gap for broader application of SHCs in synthetic chemistry.
Open Access
Loops und Tunnel : unterschätzte Elemente in Enzymen
(2020) Heinemann, Peter M.; Rapp, Lea R.; Hauer, Bernhard
In enzymes, the active site is the location where substrates are chemically converted. If this site is deeply buried within the protein, substrates must pass not only through the body of the protein via a tunnel, but also flexible, site decorating loops to access the active site. These elements can act as filters that influence on both substrate specificity and activity. Identifying and understanding how they exert such control has been of growing interest over the past several years.
Open Access
Methylation of unactivated alkenes with engineered methyltransferases to generate non‐natural terpenoids
(2023) Aberle, Benjamin; Kowalczyk, Daniel; Massini, Simon; Egler‐Kemmerer, Alexander‐N.; Gergel, Sebastian; Hammer, Stephan C.; Hauer, Bernhard
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.
Open Access
Modifizierte Enzyme ermöglichen die selektive N‐Alkylierung von Pyrazolen unter Verwendung einfacher Halogenalkane
(2021) Bengel, Ludwig L.; Aberle, Benjamin; Egler‐Kemmerer, Alexander‐N.; Kienzle, Samuel; Hauer, Bernhard; Hammer, Stephan C.
Die selektive Alkylierung von Pyrazolen ist eine Herausforderung in der Chemie und könnte die Synthese wichtiger Moleküle vereinfachen. In dieser Arbeit berichten wir über eine katalysatorgesteuerte Alkylierung von Pyrazolen durch eine cyclische Kaskadenreaktion mit zwei Enzymen. In diesem enzymatischen System nutzt ein promiskuitives Enzym Halogenalkane als Ausgangsstoffe, um nicht-natürliche Analoga des Cosubstrats S-Adenosyl-l-Methionin zu synthetisieren. Ein zweites engineertes Enzym überträgt die Alkylgruppen in einer hochselektiven C-N-Bindungsknüpfung auf das Pyrazol-Substrat. Das Cosubstrat wird regeneriert und nur in katalytischen Mengen eingesetzt. Für das Enzym-Engineering wurde eine computerbasierte Methode verwendet, um eine Mutantenbibliothek in silico zu entwickeln. In einer Runde von Mutagenese und Screening wurde somit eine promiskuitive Methyltransferase in eine kleine Pyrazol-alkylierende Enzymfamilie umgewandelt. Mit diesem bienzymatischen System konnte die Alkylierung von Pyrazolen (Methylierung, Ethylierung, Propylierung) mit bislang unerreichter Regioselektivität (>99 %), Regiodivergenz und in einem ersten Beispiel in präparativem Maßstab gezeigt werden.
Open Access
Regioselective hydration of terpenes with cofactor‐independent carotenoid 1,2‐hydratase
(2025) Horz, Philip; Härterich, Natalie; Schneider, Andreas; Travnicek, Nicolas D.; Nestl, Bettina M.; Kahler, Ursula; Hauer, Bernhard
Terminally hydrated terpenes are highly sought‐after compounds in the flavor and fragrance industries. However, their selective synthesis remains a considerable challenge in catalysis. Regioselective hydration of non‐activated C-C double bonds is typically hindered by poor selectivity and low atom efficiency in conventional methods. In this study, we harness the underexplored potential of the acyclic carotenoid 1,2‐hydratase from Rubrivivax gelatinosus IL144, employing it as a whole‐cell biocatalyst for cofactor‐independent terminal hydration of a diverse range of terpenes. This enzyme demonstrates exceptional activity across more than 20 C12-C20 terpenes and shows notable tolerance to various functional groups, establishing it as a valuable tool for sustainable organic synthesis. We emphasize the critical influence of expression system choice in maximizing enzymatic performance, enabling high‐yield transformations on the gram scale. Through a combination of homology modeling, consensus analysis, and targeted mutagenesis, essential residues involved in catalytic activity were identified. Notably, enhanced catalytic efficiency was only achievable through the epistatic effect of three specific mutations. These findings highlight the biocatalytic potential of acyclic carotenoid hydratase, offering a green and efficient route to the production of valuable tertiary alcohols.
Open Access
Semi‐rational engineering of toluene dioxygenase from Pseudomonas putida F1 towards oxyfunctionalization of bicyclic aromatics
(2021) Wissner, Julian L.; Schelle, Jona T.; Escobedo‐Hinojosa, Wendy; Vogel, Andreas; Hauer, Bernhard
Toluene dioxygenase (TDO) from Pseudomonas putida F1 was engineered towards the oxyfunctionalization of bicyclic substrates. Single and double mutant libraries addressing 27 different positions, located at the active site and entrance channel were generated. In total, 176 different variants were tested employing the substrates naphthalene, 1,2,3,4‐tetrahydroquinoline, and 2‐phenylpyridine. Introduced mutations in positions M220, A223 and F366, exhibited major influences in terms of product formation, chemo‐, regio‐ and enantioselectivity. By semi‐rational evolution, we lighted up the TDO capability to convert bulkier substrates than its natural substrate, at unprecedented reported conversions. Thus, the most active TDO variants were applied to biocatalytic oxyfunctionalizations of 1,2,3,4‐tetrahydroquinoline, and 2‐phenylpyridine, enabling the production of substantial amounts of (+)‐(R)‐1,2,3,4‐tetrahydroquinoline‐4‐ol (71% isolated yield, 94% ee) and (+)‐(1S,2R)‐3‐(pyridin‐2‐yl)cyclohexa‐3,5‐diene‐1,2‐diol (60% isolated yield, 98% ee), respectively. Here, we provide a set of novel TDO‐based biocatalysts useful for the preparation of oxyfunctionalized bicyclic scaffolds, which are valuable to perform downstream synthetic processes.
Open Access
Structure‐guided engineering of oleate hydratase for the synthesis of small chiral alcohols without decoy molecule
(2025) Härterich, Natalie; Schneider, Andreas; Horz, Philip; Travnicek, Nicolas D.; Hauer, Bernhard
Small and chiral secondary alcohols are sought‐after compounds that are frequently used in the synthesis of biologically active compounds. However, their stereochemically correct synthesis remains a challenge for the chemical industry. Synthetic routes are restrained by multiple steps and thus low atom efficiency. Here we employ engineered variants from the oleate hydratase from E. meningoseptica to produce chiral C5‐C8 secondary alcohols within a single step. First, we established a two‐phase system (2‐PS) to trap the volatile substrates/products which facilitated semi‐rational mutagenesis in the active site and tunnel of the enzyme. These efforts led to variants with an up to 20‐fold increase in catalytic activity and >99% ee , notably, without the use of a decoy molecule. Computational analysis indicated structural changes in the tunnel radius, ligand transport and energy profiles, which gave us hints to explain the enhanced performance of the variants. Interestingly, scaling up the reaction volume demonstrated significant increases in product concentrations, leading up to a 100‐fold increase in comparison to the current benchmark. This new 2‐PS and the engineered enzyme variants offer a promising approach for a scalable, asymmetric hydration of small unactivated alkenes, which would drastically ease the access to chiral alcohol‐based pheromones or drugs.
Open Access
Surfactant monolayer bending elasticity in lipase containing bicontinuous microemulsions
(2021) Engelskirchen, Sandra; Wellert, Stefan; Holderer, Olaf; Frielinghaus, Henrich; Laupheimer, Michaela; Richter, Sven; Nestl, Bettina; Nebel, Bernd; Hauer, Bernhard
Lipase-catalyzed reactions offer many advantages among which a high degree of selectivity combined with the possibility to convert even non-natural substrates are of particular interest. A major drawback in the applicability of lipases in the conversion of synthetically interesting, non-natural substrates is the substantial insolubility of such substrates in water. The conversion of substrates, natural or non-natural, by lipases generally involves the presence of a water-oil interface. In the present paper, we exploit the fact that the presence of lipases, in particular the lipase from Candida antarctica B (CalB), changes the bending elastic properties of a surfactant monolayer in a bicontinuous microemulsion consisting of D2O/NaCl -n-(d)-octane-pentaethylene glycol monodecyl ether (C10E5) in a similar manner as previously observed for amphiphilic block-copolymers. To determine the bending elastic constant, we have used two approaches, small angle neutron scattering (SANS) and neutron spin echo (NSE) spectroscopy. The time-averaged structure from SANS showed a slight decrease in bending elasticity, while on nanosecond time scales as probed with NSE, a stiffening has been observed, which was attributed to adsorption/desorption mechanisms of CalB at the surfactant monolayer. The results allow to derive further information on the influence of CalB on the composition and bending elasticity of the surfactant monolayer itself as well as the underlying adsorption/desorption mechanism.