Martina Pohl

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• Prof. Dr.
• Group Leader at Forschungszentrum Jülich

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 o...

C−C bond forming enzymes, such as 2‐deoxyribose‐5‐phosphate aldolase (DERA), are an important class of enzymes in synthesis. The use of DERA, with its reaction routes to the double aldol product, is an established method for producing statin side chains in industry. Herein, the selective synthesis of 3‐hydroxyaldehydes as single aldol products of D...

Fe(II)/α-ketoglutarate-dependent dioxygenases (KDOs) catalyze a broad range of selective C–H oxidation reactions. However, the difficult production of KDOs in recombinant E. coli strains and their instability in purified form have so far limited their application in preparative biotransformations. Here, we investigated the immobilization of three K...

Inspired by the modular architecture of natural signaling proteins, ligand binding proteins are equipped with two fluorescent proteins (FPs) in order to obtain Förster resonance energy transfer (FRET)-based biosensors. Here, we investigated a glucose sensor where the donor and acceptor FPs were attached to a glucose binding protein using a variety...

Background In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escheri...

The synthesis of aldol adduct (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose, a precursor of the interesting dietary supplement, iminosugar d-fagomine, was studied in a cascade reaction with three enzymes starting from Cbz-N-3-aminopropanol. This system was studied previously using a statistical optimization method which enabled a 79%...

The enantioselective synthesis of α-hydroxy ketones and vicinal diols is an intriguing field because of the broad applicability of these molecules. Although, butandiol dehydrogenases are known to play a key role in the production of 2,3-butandiol, their potential as biocatalysts is still not well studied. Here, we investigate the biocatalytic prope...

The front cover picture, designed by Thomas Classen from the group of Jörg Pietruszka, illustrates an efficient closed‐loop cofactor recycling in continuous flow biocatalysis. This was achieved by using phase separation techniques in combination with an immobilized alcohol dehydrogenase as biocatalyst and nicotinamide adenine dinucleotide phosphate...

α-hydroxy ketones (HK) and 1,2-diols are important building blocks for fine chemical synthesis. Here, we describe the R-selective 2,3-butanediol dehydrogenase from B. clausii DSM 8716T (BcBDH) that belongs to the metal-dependent medium chain dehydrogenases/reductases family (MDR) and catalyzes the selective asymmetric reduction of prochiral 1,2-dik...

Biocatalytic redox reactions regularly depend on expensive cofactors that require recycling. For continuous conversions in flow chemistry, this is often an obstacle since the cofactor is washed away. Here, we present a quasi‐stationary recycling system for nicotinamide adenine dinucleotide phosphate utilizing an immobilized alcohol dehydrogenase. F...

Background: In most microbial cultivations D-glucose is the main carbon and energy source. However, quantification of D-glucose especially in small scale is still challenging. Therefore, we developed a FRET-based glucose biosensor, which can be applied in microbioreactor-based cultivations. This sensor consists of a glucose binding protein sandwic...

Thiamine diphosphate‐dependent decarboxylases catalyze both cleavage and formation of CC bonds in various reactions, which have been assigned to different homologous sequence families. This work compares 53 ThDP‐dependent decarboxylases with known crystal structures. Both sequence and structural information were analyzed synergistically and data we...

Alcohol dehydrogenases are of high interest for stereoselective syntheses of chiral building blocks such as 1,2‐diols. As this class of enzymes requires nicotinamide cofactors, their application in biotechnological synthesis reactions is economically only feasible with appropriate cofactor regeneration. Therefore, a co‐substrate is oxidized to the...

Effects of molecular crowding on structural and dynamical properties of biological macromolecules do depend on the concentration of crowding agents, but also on the molecular mass and the structural compactness of the crowder molecules. By employing fluorescence correlation spectroscopy (FCS) we investigated the translational mobility of several bi...

Optimal performance of multi‐step enzymatic one‐pot cascades requires a facile balance between enzymatic activity and stability of multiple enzymes under the employed reaction conditions. We here describe the optimization of an exemplary two‐step one‐pot recycling cascade utilizing the thiamine diphosphate (ThDP)‐dependent benzaldehyde lyase from P...

Background Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobil...

The development of process steps catalyzed by immobilized enzymes usually encompasses the screening of enzyme variants, as well as the optimization of immobilization protocols and process parameters. Direct immobilization of biocatalysts by physical entrapment into hydrogels can be applied to reduce the effort required for immobilization, as the en...

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching...

We have recently demonstrated that fusions of different target enzymes to the coiled-coil domain TDoT induced the formation of catalytically active inclusion bodies CatIBs in E. coli (doi:10.1016/j.jbiotec.2017.04.033). Here we show that the CatIB properties can be tailored to the requirements of different reaction systems using two different coile...

A wide range of thiamine diphosphate (ThDP) dependent enzymes catalyze benzoin‐type carboligation of pyruvate with aldehydes. Few ThDP‐dependent enzymes, such as YerE from Yersinia pseudotuberculosis (YpYerE), are known to accept ketones as acceptor substrates. Catalysis by YpYerE gives access to chiral tertiary alcohols, a group of products diffic...

In nature, enzymatic reaction cascades, i.e. realized in metabolic networks, operate with unprecedented efficacy, with the reactions often being spatially and temporally orchestrated. The principle of “learning from nature” has in recent years inspired the setup of synthetic reaction cascades combining biocatalytic reaction steps to artificial casc...

Genetically encoded Förster resonance energy transfer (FRET)-based biosensors for the quantification of ligand molecules change the magnitude of FRET between two fluorescent proteins upon binding a target metabolite. When highly sensitive sensors are being designed, extensive sensor optimization is essential. However, it is often difficult to verif...

Sustainable and eco-efficient alternatives for the production of platform chemicals, fuels and chemical building blocks require the development of stable, reusable and recyclable biocatalysts. Here we present a novel concept for the biocatalytic production of 1,5-diaminopentane (DAP, trivial name: cadaverine) using catalytically active inclusion bo...

A structural model for thiamine‐diphosphate (ThDP)‐dependent transketolase (TK) was developed to analyse the effect of amino acid exchanges on the stereoselectivity of this synthetically important class of enzymes. In this study the carboligation of 3‐hydroxypyruvate as a donor and propanal as well as pentanal was studied. Based on literature data...

A strategy for biocatalyst immobilization in flow directly from the crude cell extract is described. The efficiency and the stability of the immobilized enzyme were demonstrated during the asymmetric reduction of a range of ketones. The cascade two-step chemo-enzymatic preparation of chiral epoxides was possible through the initial ketone bioreduct...

A strategy for biocatalyst immobilization in flow directly from the crude cell extract is described.

Compartmentalization of biocatalysts is an effective tool to integrate biocatalytic steps in continuous (chemo)enzymatic cascades. Therefore, efficient covalent immobilization techniques are of utmost importance, which enable a fast and selective immobilization of the enzyme directly from crude cell extracts. Here we demonstrate that the HaloTagTM...

The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33-43%) and exhibited some uncharted peculiarities. An N-terminally StrepI...

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) is in general a very good catalyst for the amination of fluoro- and chloro-cinnamic acid derivatives yielding halogenated (S)-phenylalanine derivatives with ≥85% conversion and excellent ee values >99%. We have studied the application of this enzyme as whole cell biocatalyst and i...

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) was comparatively characterized to the well-studied enzyme from parsley (PcPAL1) and Rhodosporidium toruloides (RtPAL) with respect to kinetic parameters for the deamination and the amination reaction, pH- and temperature optima and the substrate range of the amination reaction. W...

Bacterial inclusion bodies (IBs) consist of unfolded protein aggregates and represent inactive waste products often accumulating during heterologous overexpression of recombinant genes in Escherichia coli. This general misconception has been challenged in recent years by the discovery that IBs, apart from misfolded polypeptides, can also contain su...

Easy, fast and gentle immobilization for the efficient reuse of important biocatalysts is highly demanded. We used the commercially available HaloTag™ technology (Promega), so far relatively unknown in the context of biocatalysis, to immobilize the benzaldehyde lyase from P. fluorescence (PfBAL). Immobilization mediated by this fusion tag proceeds...

Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially...

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The catalytic asymmetric synthesis of chiral 2-hydroxy ketones by using different thiamine diphosphate dependent enzymes, namely benzaldehyde lyase from Pseudomonas fluorescens (PfBAL), a variant of benzoylformate decarboxylase from Pseudomonas putida (PpBFD-L461A), branched-chain 2-keto acid decarboxylase from Lactococcus lactis (LlKdcA) and a var...

The development of novel enzymes for biocatalytic processes requires knowledge on substrate profile and selectivity which can be derived from separate databases and publications. Often, these data sources lack the time courses of substrate or product, and an unambiguous link between experiment and enzyme sequence. The lack of integrated, original d...

Enzymatic parameter determination is an essential step in biocatalytic process development. Therefore higher throughput in miniaturized devices is urgently needed. An ideal microfluidic device should combine easy immobilization and retention of a minimal amount of biocatalyst with a well-mixed reaction volume. Together, all criteria are hardly met...

Silicate nanoparticles with immobilized FRET-based biosensors were developed for the detection of glucose and maltose. Immobilization of the protein biosensor in the nanoparticle was achieved through specific interaction between the hexa-histidine tag of the protein and a calcium-silicate complex of the silica matrix. Encapsulation of the biosensor...

(S)-Phenylacetylcarbinol [(S)-PAC] and its derivatives are valuable intermediates for the synthesis of various active pharmaceutical ingredients (APIs), but their selective synthesis is challenging. As no highly selective enzymes or chemical catalysts were available, we used semi-rational enzyme engineering to tailor a potent biocatalyst to be >97%...

The increasing number of biocatalytic reactions implemented in chemical synthesis routes raises the urgent need for large amounts of enzymes. Hence, new generic methods are required for their simple and cost-efficient production. Here, we describe a generally applicable method based on the production of catalytically active inclusion bodies (CatIBs...

An enantioselective preparation of O-acetylcyanohydrins has been accomplished by a three-step telescoped continuous process. The modular components enabled accurate control of two sequential biotransformations, safe handling of an in situ generated hazardous gas, and in-line stabilization of products. This method proved to be advantageous over the...

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Tedious, time- and labor-intensive protein purification and immobilization procedures still represent a major bottleneck limiting the widespread application of enzymes in synthetic chemistry and industry. We here exemplify a simple strategy for the direct site-specific immobilization of proteins from crude cell extracts by fusion of a family 2 carb...

The introduction of aromatic residues connected by a C-C bond into the non-reducing end of carbohydrates is highly significant for the development of innovative structures with improved binding affinity and selectivity (e.g., C-aril-sLex). In this work, an expedient asymmetric “de novo” synthetic route to new aryl carbohydrate derivatives based on...

This chapter gives a broad overview of different thiamine diphosphate (ThDP) dependent enzymes and their applicability in organic synthesis as a practical alternative to tradition- al cross-coupling reactions. Complementary to known nonenzymatic umpolung reac- tions, enzymatic versions of the benzoin condensation, the asymmetric cross-benzoin conde...

Thiamindiphosphat-abhängige Enzyme sind bekannt für die Katalyse der asymmetrischen Synthese von chiralen α-Hydroxyketonen ausgehend von einfachen prochiralen Substraten. Dabei wird das Produktspektrum von den sterischen und chemischen Eigenschaften des aktiven Zentrums des Enzyms festgelegt. Enzyme, die die Carboligation von aromatischen Aldehyden...

A broad range of genetically-encoded fluorescence biosensors has been developed, allowing the detection of signaling intermediates and metabolites in real time. Many of these biosensors are based on Foerster Resonance Energy Transfer (FRET). The two biosensors of the well-known "Venus-flytrap" type exemplarily studied in this work are composed of a...

The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in...

Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to (S)-benzoins have not so far been identifi...

Chiral 1,2-amino alcohols are important building blocks for chemistry and pharmacy. Here, we developed two different biocatalytic 2-step cascades for the synthesis of all four nor(pseudo)ephedrine (N(P)E) stereoisomers. In the first one, the combination of an (R)-selective thiamine diphosphate (ThDP)-dependent carboligase with an (S)- or (R)-select...

The thiamine diphosphate-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) catalyzes a Stetter-like 1,4-addition of α-ketoglutarate to isochorismate in the biosynthesis of menaquinone (vitamin K). Here, we describe the carboligation potential of MenD from Bacillus subtilis (BsMenD) for the nonphysiologi...

The synthesis of (S)-2-hydroxypropiophenone ((S)-2-HPP) from benzaldehyde and acetaldehyde catalyzed by benzoylformate decarboxylase (BFD) from Pseudomonas putida was studied in an enzyme ultrafiltration membrane reactor (UFMR) and in three different microreactors (MRs). The aim was to compare the volume productivity (Q(p)) as well as biocatalyst p...

S-selective EcMenD variants of the thiamine diphosphate (TDP)-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase from Escherichia coli (E.C.

The thiamine diphosphate (ThDP)-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase from Escherichia coli (EcMenD, E.C. 2.2.1.9) catalyzes the carboligation of α-ketoglutarate (α-KG) and various benzaldehyde derivatives with excellent chemo- as well as high R-selectivity (enantiomeric excess (ee) >93 %) to yield...

Stereoselective traffic engineering The cover picture shows how engineered S‐selective EcMenD variants catalyze the carboligation of α‐ketoglutarate and benzaldehyde derivatives with excellent enantioselectivities of up to 99 % ee S, in contrast to the wild‐type enzyme, which produces R‐enantiomers. In their Full Paper on p. 3587 ff., M. Pohl et al...

Thiamine diphosphate-dependent enzymes are broadly distributed in all organisms, and they catalyse a broad range of C-C bond forming and breaking reactions. Enzymes belonging to the structural families of decarboxylases and transketolases have been particularly well investigated concerning their substrate range, mechanism of stereoselective carboli...

Stereoselective reduction towards pharmaceutically potent products with multi chiral centers is an ongoing hot topic, but up to now catalysts for reductions of bulky aromatic substrates are rare. The NADPH-dependent alcohol dehydrogenase from Ralstonia sp. (RADH) is an exception as it prefers sterically demanding substrates. Recent studies with thi...

Zwei in einem: Eine synthetische Enzymkaskade liefert (1R,2R)‐Norpseudoephedrin und (1R,2S)‐Norephedrin durch Kombination einer Lyase und einer (R)‐ oder (S)‐selektiven ω‐Transaminase in zwei Schritten im selben Reaktionsgefäß. Die Produkte können ohne Isolierung des Zwischenprodukts mit hohen optischen Reinheiten generiert werden. Zudem war es mög...

Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) was fused to different fluorescent reporter proteins. Whereas all fusion constructs retained enzymatic activity and fluorescence in vivo and in vitro, significant differences in activity and pH stability were observed. In particular, flavin-based fluorescent reporter (FbFP) fusions showed almos...

Two steps in one pot: An enzyme cascade consisting of a lyase and an (R)- or (S)-selective ω-transaminase (TA) provides (1R,2R)-norpseudoephedrine and (1R,2S)-norephedrine in only two steps. The intermediate is not isolated in this one-pot reaction and the products are obtained in high enantio- and diastereomeric purity. Moreover, the by-product fr...

The ultimate ambition in cell biology, microbiology and biomedicine is to unravel complex physiological and pathophysiological processes within living organisms. To conquer this challenge, fluorescent proteins (FPs) are used as versatile in vivo reporters and biosensors to study gene regulation as well as the synthesis, localization and function of...

The present review summarizes recent achievements in enzymatic thiamine catalysis during the past three years. With well-established enzymes such as BAL, PDC and TK new reactions have been identified and respective variants were prepared, which enable access to stereoisomeric products. Further we highlight recent progress with 'new' ThDP-dependent...

The use of enzyme engineering for optimisation of an enzymatic reaction can significantly improve lifetime and performance of the biocatalyst. Reaction optimisation is a second powerful tool to enhance yield and enantiomeric excess (ee) of a desired product. In combination, an effective toolbox for biocatalytic process development is given. Previou...

We report the first rationally designed (S)-selective MenD from E. coli for the synthesis of functionalized α-hydroxy ketones. By mutation of two amino acids in the active site stereoselectivity of the (R)-selective EcMenD (ee > 93%) was inverted giving access to (S)-5-hydroxy-4-oxo-5-phenylpentanoate derivatives with stereoselectivities up to 97%...

Background Standard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented f...

Comparison of alignments generated using the numbering method and T-Coffee.

Reference alignment for the standard numbering method.

The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a compar...

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identifie...

Synthesis of chiral cyanohydrins is performed in a monophasic micro-aqueous reaction system using whole recombinant Escherichia coli cells expressing the Arabidopsis thaliana hydroxynitrile lyase (AtHNL). Microscopy studies employing a fusion of AtHNL with a flavin-based fluorescent protein (FbFP) reveal that the cells remain intact in the reaction...

Umpolung Reactions in Chemistry and BiologyAcyloin CondensationsBenzoin CondensationsMiscellaneous Acyloin CondensationsReferences

The R-selective hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) cannot be applied for stereoselective cyanohydrin syntheses in aqueous media because of its limited stability at pH<5, which is required in order to suppress the uncatalyzed racemic cyanohydrin formation. To stabilize AtHNL we designed a surface-modified variant incorporating 11...

Although biotransformations implementing alcohol dehydrogenases (ADHs) are widespread, enzymes which catalyse the reduction and oxidation of sterically demanding substrates, especially 2-hydroxy ketones, are still rare. To fill this gap eight ADHs were investigated concerning their potential to reduce bulky 2-hydroxy ketones. All of these enzymes s...

A rapid TTC-based screening assay for ω-transaminases was developed to determine the conversion of substrates with a 2-hydroxy ketone motif. Oxidation of the compounds in the presence of 2,3,5-triphenyltetrazolium chloride (TTC) results in a reduction of the colourless TTC to a red-coloured 1,3,5-triphenylformazan. The enzymatic reductive amination...

The thiamine diphosphate (ThDP)-dependent pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC) catalyzes the carboligation of aldehydes that yields (R)-2-hydroxy ketones with high chemoselectivity in mixed carboligations of aliphatic donor and aromatic acceptor aldehydes. On the basis of the crystal structure of ApPDC, which was determined...

The (R)-selective hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) is a promising biocatalyst for the synthesis of a broad range of chiral cyanohydrins. However, the enantiomeric excess of the reaction is strongly compromised by a non-catalyzed side reaction resulting in racemic cyanohydrins besides the chiral product obtained by enzymatic ca...

We present an efficient method for the production of D- and L-allo-threonine (allo-Thr) with very high purity by enzymatic isomerization of L- or D-threonine (Thr) and simultaneous crystallization. Isomerization of Thr to allo-Thr is catalyzed by a purified amino acid racemase (AArac12996) from Pseudomonas putida NBRC12996, which can easily be obta...

Stereoselective reduction of 2-hydroxy ketones should in principle give access to syn- and anti-1,2-diols. anti-1,2-Diols are accessible in a highly selective way using zinc borohydride [Zn(BH4)2] under chelation control (dr>20:1). Diastereoselective reduction of unprotected or even protected 2-hydroxy ketones towards syn-1,2-diols could be achieve...

Table S2: Periodically measured DLS data of differently concentrated BSL-B samples.

Table S3: PCR Primers QuikChange-PCR-primer sequences for site-directed mutagenesis of each lysine residue in BSL-B for alanine and arginine, respectively. Each primer pair (e.g. lipB-K25R-fw und lipB-K25A-fw) differs only in the mutagenesis sequence (bold and underlined).

Figure S1: Tributyrine plate assay of BSL-B wild type and BSL-B point variants. Tributyrine plate assay of BSL-B wild type as well as BSL-B point variants in which each lysine residue is substituted by alanine and arginine, respectively. -: E. coli BL21(DE3) carrying the empty vector pET19b. WT: E. coli BL21(DE3) expressing BSL-B wild type enzyme (...