Synthesis of (R)-mandelic acid and (R)-mandelic acid amide by recombinant E. coli strains expressing a (R)-specific oxynitrilase and an arylacetonitrilase

Erik Müller,Janosch Alexander David Gröning,Olga Sosedov,Andreas Stolz

doi:10.1007/s10529-020-02998-8

Erik Müller, Janosch Alexander David Gröning + Show 2 more

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https://doi.org/10.1007/s10529-020-02998-8

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Abstract

ObjectivesChiral 2-hydroxycarboxylic acids and 2-hydroxycarboxamides are valuable synthons for the chemical industry.ResultsThe biocatalytic syntheses of (R)-mandelic acid and (R)-mandelic acid amide by recombinant Escherichia coli clones were studied. Strains were constructed which simultaneously expressed a (R)-specific oxynitrilase (hydroxynitrile lyase) from the plant Arabidopsis thaliana together with the arylacetonitrilase from the bacterium Pseudomonas fluorescens EBC191. In addition, recombinant strains were constructed which expressed a previously described acid tolerant variant of the oxynitrilase and an amide forming variant of the nitrilase. The whole cell catalysts which simultaneously expressed the (R)-specific oxynitrilase and the wild-type nitrilase transformed in slightly acidic buffer systems benzaldehyde plus cyanide preferentially to (R)-mandelic acid with ee-values > 95%. The combination of the (R)-specific oxynitrilase with the amide forming nitrilase variant gave whole cell catalysts which converted at pH-values ≤ pH 5 benzaldehyde plus cyanide with a high degree of enantioselectivity (ee > 90%) to (R)-mandelic acid amide. The acid and the amide forming catalysts also converted chlorinated benzaldehydes with cyanide to chlorinated mandelic acid or chlorinated mandelic acid amides.ConclusionsEfficient systems for the biocatalytic production of (R)-2-hydroxycarboxylic acids and (R)-2-hydroxycarboxamides were generated.

Highlights

Organic nitriles are natural products and are synthesized in huge amounts by the chemical industry
Sosedov Biochem Labor fur chemische Analytik GmbH, Daimlerstr. 5B, 76185 Karlsruhe, Germany the (R)-specific oxynitrilase and the wild-type nitrilase transformed in slightly acidic buffer systems benzaldehyde plus cyanide preferentially to (R)-mandelic acid with ee-values [ 95%
The combination of the (R)-specific oxynitrilase with the amide forming nitrilase variant gave whole cell catalysts which converted at pH-values B pH 5 benzaldehyde plus cyanide with a high degree of enantioselectivity to (R)-mandelic acid amide

Summary

Introduction

Organic nitriles are natural products and are synthesized in huge amounts by the chemical industry. The combination of (S)-specific oxynitrilases with nitrilases or nitrile hydratases has been described for the synthesis of chiral (S)-2-hydroxycarboxylic acids or (S)-2-hydroxycarboxamides In these systems, the (S)-specific oxynitrilase from the cassava plant (Manihot esculenta) was combined with nitrilases or nitrile hydratases either in-vitro (often in the form of cross-linked enzyme aggregates-‘‘CLEAs’’) or in-vivo by using recombinant organisms (Escherichia coli or Pichia pastoris) which simultaneously expressed oxynitrilase and nitrilase activities (Fig. 1). The (S)-specific oxynitrilase from the cassava plant (Manihot esculenta) was combined with nitrilases or nitrile hydratases either in-vitro (often in the form of cross-linked enzyme aggregates-‘‘CLEAs’’) or in-vivo by using recombinant organisms (Escherichia coli or Pichia pastoris) which simultaneously expressed oxynitrilase and nitrilase activities (Fig. 1) These systems allowed the efficient synthesis of chiral 2-hydroxycarboxylic acids and 2-hydroxycarboxamides from nonchiral aldehydes (or ketones) and cyanide (van Rantwijk and Stolz 2015)

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