Dörte Rother

Engineering stereoselectivity of ThDP-dependent enzymes

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 carboligation and carbolyase reaction. Both structurally different enzyme families differ also in stereoselectivity: enzymes from the decarboxylase family are predominantly R‐selective, whereas those from the transketolase family are S‐selective. In recent years a key focus of our studies has been on stereoselective benzoin condensation‐like 1,2‐additions. Meanwhile, several S‐selective variants of pyruvate decarboxylase, benzoylformate decarboxylase and 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate (SEPHCHC) synthase as well as R‐selective transketolase variants were created that allow access to a broad range of enantiocomplementary α‐hydroxyketones and α,α′‐dihydroxyketones. This review covers recent studies and the mechanistic understanding of stereoselective C–C bond forming thiamine diphosphate‐dependent enzymes, which has been guided by structure–function analyses based on mutagenesis studies and from influences of different substrates and organic co‐solvents on stereoselectivity.

Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale

Recent advances in biocatalysis have strongly boosted its recognition as a valuable addition to traditional chemical synthesis routes. As for any catalytic process, catalysts costs and stabilities are of highest relevance for the economic application in chemical manufacturing. Employing biocatalysts as whole cells circumvents the need of cell lysis and enzyme purification and hence strongly cuts on cost. At the same time, residual cell wall components can shield the entrapped enzyme from potentially harmful surroundings and aid to enable applications far from natural enzymatic environments. Further advantages are the close proximity of reactants and catalysts as well as the inherent presence of expensive cofactors. Here, we review and comment on benefits and recent advances in whole cell biocatalysis.

S‐Selective Mixed Carboligation by Structure‐Based Design of the Pyruvate Decarboxylase from Acetobacter pasteurianus

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 to a resolution of 2.75 Å, and biochemical data, we mapped the active site. This enabled us to design variants with tailor‐made catalytic activities by modifications of the residues E469 and W388. Although the exchange of W388 by smaller amino acids yields variants with higher carboligase activity due to an increased access to the active site, the exchange of E469 to glycine opens the so‐called S‐pocket in ApPDC for aromatic aldehydes and thus alters the stereoselectivity. The variant ApPDC‐E469G provides access to (S)‐phenylacetylcarbinol derivatives by enzymatic carboligation with a good stereoselectivity of up to 89 % enantiomeric excess. The variant nicely complements the toolbox of ThDP‐dependent enzymes, which now gives access to all stereo‐ and regioisomers of the asymmetric aliphatic–aromatic cross‐benzoin‐like condensation. We prove that optimal stabilization of both aldehydes in the active site is essential to gain high yields and high selectivities.

Efficient 2-step biocatalytic strategies for the synthesis of all nor(pseudo)ephedrine isomers

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)-selective ω-transaminase resulted in the formation of (1R,2S)-NE or (1R,2R)-NPE in excellent optical purities (ee >99% and de >98%). For the synthesis of (1R,2R)-NPE, space–time yields up to ∼26 g L−1 d−1 have been achieved. Since a highly (S)-selective carboligase is currently not available for this reaction, another strategy was followed to complement the nor(pseudo)ephedrine platform. Here, the combination of an (S)-selective transaminase with an (S)-selective alcohol dehydrogenase yielded (1S,2S)-NPE with an ee >98% and a de >99%. Although lyophilized whole cells are cheap to prepare and were shown to be appropriate for use as biocatalysts, higher optical purities were observed with purified enzymes. These synthetic enzyme cascade reactions render the N(P)E-products accessible from inexpensive, achiral starting materials in only two reaction steps and without the isolation of the reaction intermediates.

Stereoselective synthesis of bulky 1,2-diols with alcohol dehydrogenases

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 showed good activities along with excellent enantio- (ee > 99%) and diastereoselectivities (de > 99%). Due to their differences in substrate preferences and stereoselectivity a broad range of diastereomerically pure 1,2-diols is now accessible via biotransformation. Best results were obtained using the alcohol dehydrogenase from Ralstonia sp. (Cupriavidus sp.) (RADH), which showed a broad substrate range, especially for sterically demanding compounds. Araliphatic 2-hydroxy ketones, like (R)-2-hydroxy-1-phenylpropan-1-one ((R)-2-HPP), were reduced much faster than aliphatic or aromatic aldehydes (e.g. benzaldehyde) under the applied conditions. Additionally (R)- as well as (S)-2-hydroxy ketones were converted with high diastereoselectivities (de > 99%). RADH, which was up to now only studied as a whole cell biocatalyst overexpressed in E. coli, was purified and thoroughly characterised concerning its catalytic properties.

A two-step biocatalytic cascade in micro-aqueous medium: using whole cells to obtain high concentrations of a vicinal diol

Although single- and multi-step biocatalytic approaches show persuasive advantages for the synthesis of especially chiral compounds (e.g. high chemo- and stereoselectivity), their application often suffers from low substrate loads and hence low space-time-yields. We herein present a synthetic cascade approach in which lyophilised, recombinant whole cells are applied in micro-aqueous reaction systems yielding extremely high space-time-yields. As an example we investigated the two-step synthesis of 1-phenylpropane-1,2-diol starting from cheap aldehydes and achieved high selectivities (ee/de > 99%) and high product concentrations. The new concept of running biocatalytic cascades in a mixture of high substrate loads and organic solvents under addition of small amounts of highly concentrated buffer is not only very easy-to-apply, but also exhibits several economic and ecologic advantages. On the one hand the usage of whole, lyophilised cells circumvents time-consuming enzyme purification as well as addition of expensive cofactors (here ThDP and NADPH). Additionally, catalyst and product workup is facilitated by the application of organic solvents (here MTBE). On the other hand, the employment of whole cells very effectively circumvents stability problems of biocatalysts in unconventional media enabling the addition of extremely high substrate loads (up to 500 mM in our example) and is therefore an easy and effective approach for multi-step biocatalysis. After optimisation, the combination of a carboligation step followed by a second oxidoreduction step with whole cell catalysts afforded an efficient two-step cascade for the production of 1-phenylpropane-1,2-diol with space-time yields up to 327 g L−1 d−1 and an E-factor of 21.3 kgwaste kgproduct−1.

TTC-based screening assay for ω-transaminases: a rapid method to detect reduction of 2-hydroxy ketones.

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 of a wide range of various aliphatic, aliphatic-aromatic and aromatic-aromatic 2-hydroxy ketones can be determined by the decrease of the red colouration due to substrate consumption. The conversion can be quantified spectrophotometrically at 510 nm based on reactions, e.g. with crude cell extracts in 96-well plates. Since the assay is independent of the choice of diverse amine donors a panel of ω-transaminases was screened to detect conversion of 2-hydroxy ketones with three different amine donors: l-alanine, (S)-α-methylbenzylamine and benzylamine. The results could be validated using HPLC and GC analyses, showing a deviation of only 5-10%. Using this approach enzymes were identified demonstrating high conversions of acetoin and phenylacetylcarbinol to the corresponding amines. Among these enzymes three novel wild-type ω-transaminases have been identified.

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

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 comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site.

(S)-Selective MenD variants from Escherichia coli provide access to new functionalized chiral α-hydroxy ketones.

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% ee.

Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp.

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 this enzyme indicated outstanding potential for the reduction of various alpha‐hydroxy ketones, but were performed with crude cell extract, which hampered its detailed characterization. We have established a procedure for the purification and storage of RADH and found a significantly stabilizing effect by addition of CaCl2. Detailed analysis of the pH‐dependent activity and stability yielded a broad pH‐optimum (pH 6–9.5) for the reduction reaction and a sharp optimum of pH 10–11.5 for the oxidation reaction. The enzyme exhibits highest stability at pH 5.5–8 and 8–15°C; nevertheless, biotransformations can also be carried out at 25°C (half‐life 80 h). Under optimized reaction parameters a thorough study of the substrate range of RADH including the reduction of different aldehydes and ketones and the oxidation of a broad range of alcohols was conducted. In contrast to most other known alcohol dehydrogenases, RADH clearly prefers aromatic and cyclic aliphatic compounds, which makes this enzyme unique for conversion of space demanding substrates. Further, reductions are catalyzed with extremely high stereoselectivity (>99% enantio‐ and diastereomeric excess). In order to identify appropriate substrate and cofactor concentrations for biotransformations, kinetic parameters were determined for NADP(H) and selected substrates. Among these, we studied the reduction of both enantiomers of 2‐hydroxypropiophenone in more detail. Biotechnol. Bioeng. 2013; 110: 1838–1848.