Martina Pohl

Improved biocatalysts by directed evolution and rational protein design

The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity. However, wild-type enzymes often need to be optimized to fulfill these requirements. Two rather contradictory tools can be used on a molecular level to create tailor-made biocatalysts: directed evolution and rational protein design.

Benzoylformate Decarboxylase from Pseudomonas putida as Stable Catalyst for the Synthesis of Chiral 2-Hydroxy Ketones

The thiamin diphosphate- and Mg2+-dependent enzyme benzoylformate decarboxylase (BFD) from Pseudomonas putida was characterized with respect to its suitability to catalyze the formation of chiral 2-hydroxy ketones in a benzoin-condensation type reaction. Carboligation constitutes a side reaction of BFD, whereas the predominant physiological task of the enzyme is the non-oxidative decarboxylation of benzoylformate. For this purpose the enzyme was obtained in sufficient purity from Pseudomonas putida cells in a one-step purification using anion-exchange chromatography. To facilitate the access to pure BFD for kinetical studies, stability investigations, and synthetical applications, the coding gene was cloned into a vector allowing the expression of a hexahistidine fusion protein. The recombinant enzyme shows distinct activity maxima for the decarboxylation and the carboligation beside a pronounced stability in a broad pH and temperature range. The enzyme accepts a wide range of donor aldehyde substrates which are ligated to acetaldehyde as an acceptor in mostly high optical purities. The enantioselectivity of the carboligation was found to be a function of the reaction temperature, the substitution pattern of the donor aldehyde and, most significantly, of the concentration of the donor aldehyde substrate. Our data are consistent with a mechanistical model based on the X-ray crystallographic data of BFD. Furthermore we present a simple way to increase the enantiomeric excess of (S)-2-hydroxy-1-phenyl-propanone from 90% to 95% by skillful choice of the reaction parameters. Enzymatic synthesis with BFD are performed best in a continuously operated enzyme membrane reactor. Thus, we have established a new enzyme tool comprising a vast applicability for stereoselective synthesis.

Thiamin-Diphosphate-Dependent Enzymes: New Aspects of Asymmetric CC Bond Formation

Starting from a thorough investigation of mechanistic aspects of ThDP-dependent (ThDP = thiamin diphosphate) enzymes in combination with mutagenesis studies and a detailed substrate screening, new general synthetic methods have been developed based on Umpolung reactions by thiamin catalysis. A selective donor-acceptor concept was established leading to the first asymmetric cross-benzoin condensation, and a kinetic racemic resolution through C-C bond cleavage was developed. With these tools and in combination with protein engineering, we approached the synthesis of new chiral building blocks on a preparative scale. An outlook is given with respect to the potential of other ThDP-dependent enzymes as catalysts in asymmetric synthesis.

Enantioselective Synthesis of α-Hydroxy Ketones via Benzaldehyde Lyase-Catalyzed C−C Bond Formation Reaction

(R)-Benzoins and (R)-2-hydroxypropiophenone derivatives are formed on a preparative scale by benzaldehyde lyase (BAL)-catalyzed C−C bond formation from aromatic aldehydes and acetaldehyde in aqueous buffer/DMSO solution with remarkable ease in high chemical yield and high optical purity. The substrate range of this thiamin diphosphate-dependent enzyme was examined with respect to a broad applicability of this benzoin condensation-type reaction in stereoselective synthesis.

Effect of Oxygen Limitation and Medium Composition on Escherichia coli Fermentation in Shake-Flask Cultures

Shake‐flask cultures are widely used for screening of high producing strains. To select suitable strains for production scale, cultivation parameters should be applied that provide optimal growth conditions. A novel method of measuring respiratory activity in shake‐flask cultures was employed to analyze Escherichia coli fermentation under laboratory conditions. Our results suggest that the length of fermentation, choice of medium, and aeration do not normally satisfy the requirements for unlimited growth in shake flasks. Using glycerol rather than glucose as a carbon source greatly reduced the accumulation of overflow and fermentative metabolites when oxygen supply was unlimited. A rich buffered medium, Terrific Broth (TB), yielded 5 times more biomass compared to LB medium but also caused oxygen limitation in standard shake‐flask cultures at shaking frequencies below 400 rpm. These results were used to optimize the production of benzoylformate decarboxylase from Pseudomonas putida in E. coli SG13009, resulting in a 10‐fold increase in volumetric enzyme production. This example demonstrates how variation of medium composition and oxygen supply can be evaluated by the measurement of the respiratory activity. This can help to efficiently optimize screening conditions for E. coli.

Thiamin diphosphate in biological chemistry: exploitation of diverse thiamin diphosphate-dependent enzymes for asymmetric chemoenzymatic synthesis

Thiamin diphosphate‐dependent enzymes participate in numerous biosynthetic pathways and catalyse a broad range of reactions, mainly involving the cleavage and formation of C–C bonds. For example, they catalyse the nonoxidative and oxidative decarboxylation of 2‐keto acids, produce 2‐hydroxy ketones and transfer activated aldehydes to a variety of acceptors. Moreover, they can also catalyse C–N, C–O and C–S bond formation. Because of their substrate spectra and different stereospecificity, these enzymes extend the synthetic potential for asymmetric carboligations appreciably. Different strategies have been developed to identify new members of this promiscuous enzyme class and the reactions they catalyse. This enabled us to introduce solutions for longstanding synthetic problems, such as asymmetric cross‐benzoin condensation. Moreover, through a combination of protein structure analysis, enzyme and substrate engineering, and screening methods we explored additional stereochemical routes that have not been described previously for any of these interesting enzymes.

Application of α-keto acid decarboxylases in biotransformations

The advantages of using enzymes in the synthesis of organic compounds relate to their versatility, high reaction rates, and regio- and stereospecificity and the relatively mild reaction conditions involved. Stereospecificity is especially important in the synthesis of bioactive molecules, as only one of the enantiomeric forms usually manifests bioactivity, whereas the other is often toxic. Although enzymes which catalyze asymmetric carbon-carbon bond formation are of great importance in bioorganic chemistry, only a few examples are known for thiamin diphosphate (ThDP)-dependent enzymes, whereas transformations using e.g. aldolases, lipases and lyases are well documented already. The present review surveys recent work on the application of pyruvate decarboxylase and benzoylformate decarboxylase in organic synthesis. These enzymes catalyze the synthesis of chiral alpha-hydroxy ketones which are versatile building blocks for organic and pharmaceutical chemistry. Besides the substrate spectra of both enzymes amino acid residues relevant for substrate specificity and enantioselectivity of pyruvate decarboxylase have been investigated by site-directed mutagenesis.

Alteration of the Substrate Specificity of Benzoylformate Decarboxylase from Pseudomonas putida by Directed Evolution

Alteration of the substrate specificity of thiamin diphosphate (ThDP)‐dependent benzoylformate decarboxylase (BFD) by error‐prone PCR is described. Two mutant enzymes, L476Q and M365L‐L461S, were identified that accept ortho‐substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2‐hydroxy ketones. Both variants, L476Q and M365L‐L461S, selectively catalyze the formation of enantiopure (S)‐2‐hydroxy‐1‐(2‐methylphenyl)propan‐1‐one with excellent yields, a reaction which is only poorly catalyzed by the wild‐type enzyme. Different ortho‐substituted benzaldehyde derivatives, such as 2‐chloro‐, 2‐methoxy‐, or 2‐bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding (S)‐2‐hydroxy‐1‐phenylpropan‐1‐one derivatives. As deduced from modeling studies based on the 3D structure of wild‐type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho‐substituted benzaldehyde derivatives to the cofactor ThDP.

Asymmetric benzoin reaction catalyzed by benzoylformate decarboxylase

Abstract Aromatic aldehydes are converted into benzoins by benzoylformate decarboxylase catalyzed C–C bond formation. The reaction affords ( R )-benzoins with high enantiomeric excess and in good chemical yields. A broad range of aromatic aldehydes can be used as substrates in aqueous buffer or buffer/DMSO-solutions.

Characterization of phenylpyruvate decarboxylase, involved in auxin production of Azospirillum brasilense.

Azospirillum brasilense belongs to the plant growth-promoting rhizobacteria with direct growth promotion through the production of the phytohormone indole-3-acetic acid (IAA). A key gene in the production of IAA, annotated as indole-3-pyruvate decarboxylase (ipdC), has been isolated from A. brasilense, and its regulation was reported previously (A. Vande Broek, P. Gysegom, O. Ona, N. Hendrickx, E. Prinsen, J. Van Impe, and J. Vanderleyden, Mol. Plant-Microbe Interact. 18:311-323, 2005). An ipdC-knockout mutant was found to produce only 10% (wt/vol) of the wild-type IAA production level. In this study, the encoded enzyme is characterized via a biochemical and phylogenetic analysis. Therefore, the recombinant enzyme was expressed and purified via heterologous overexpression in Escherichia coli and subsequent affinity chromatography. The molecular mass of the holoenzyme was determined by size-exclusion chromatography, suggesting a tetrameric structure, which is typical for 2-keto acid decarboxylases. The enzyme shows the highest kcat value for phenylpyruvate. Comparing values for the specificity constant kcat/Km, indole-3-pyruvate is converted 10-fold less efficiently, while no activity could be detected with benzoylformate. The enzyme shows pronounced substrate activation with indole-3-pyruvate and some other aromatic substrates, while for phenylpyruvate it appears to obey classical Michaelis-Menten kinetics. Based on these data, we propose a reclassification of the ipdC gene product of A. brasilense as a phenylpyruvate decarboxylase (EC 4.1.1.43).