Simultaneous expression of an arylacetonitrilase from Pseudomonas fluorescens and a (S)-oxynitrilase from Manihot esculenta in Pichia pastoris for the synthesis of (S)-mandelic acid.
The arylacetonitrilase of Pseudomonas fluorescens EBC191 catalyzes the conversion of (S)-mandelonitrile to (S)-mandelic acid and (S)-mandeloamide. This biotransformation is optimally performed under acidic pH values because (S)-mandelonitrile rapidly decomposes under neutral conditions. Therefore, the gene encoding the arylacetonitrilase of P. fluorescens EBC191 was integrated and expressed under the control of the AOX1 promoter in the methylotrophic yeast Pichia pastoris which was supposed to act as an acidotolerant expression system. These recombinant strains hydrolyzed (R,S)-mandelonitrile at pH values >or=3 to mandelic acid and mandeloamide and were more acidotolerant than previously constructed Escherichia coli whole cell catalysts synthesizing the same nitrilase activity. Subsequently, recombinant P. pastoris strains were constructed which simultaneously expressed the (S)-oxynitrilase of Manihot esculenta and the arylacetonitrilase of P. fluorescens EBC191 each under the control of individual AOX1 promoters in order to obtain a whole cell catalyst for the synthesis of (S)-mandelic acid from benzaldehyde and cyanide. Resting cells of the recombinant strains converted under acidic conditions benzaldehyde and cyanide initially to mandelonitrile which was immediately converted to mandelic acid and mandeloamide. The chiral analysis of the products formed revealed a high enantiomeric excess for the (S)-enantiomers.
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, Stuttgart, Germany.
Sven
S
Rustler
Hassan
H
Motejadded
Josef
J
Altenbuchner
Andreas
A
Stolz
D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D000619:Aminohydrolases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001547:Benzaldehydes / Q000378:metabolism; D003485:Cyanates / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D008333:Mandelic Acids / Q000378:metabolism*; D002365:Manihot / Q000201:enzymology*; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology*
D001426:Bacterial Proteins; D001547:Benzaldehydes; D003485:Cyanates; D008333:Mandelic Acids; D010940:Plant Proteins; D000619:Aminohydrolases; D000446:Aldehyde-Lyases; C021541:mandelonitrile lyase; C024835:potassium cyanate; C037938:mandelic acid; C032175:benzaldehyde