Identification of a Candida sp. reductase behind bicyclic exo-alcohol production

Stereoselective bakerʹs yeast-catalysed bioreduction of bicyclo [2.2.2]octane-2,6-dione generates (1R, 4S, 6S)-6-hydroxy-bicyclo [2.2.2]octane-2-one (endo-alcohol) with high enantiomeric and diastereomeric excess. In contrast, whole cells and crude membrane fractions of Candida sp. have been reported to produce the unusual (1R, 4S, 6S)-diastereomer (exo-alcohol) as a major product. Previous in silico screening has identified seven membrane or membrane-bound reductases in C. albicans as candidates for the exo-activity. In this work, purification of the corresponding exo-reductase(s) as well as the heterologous cloning of the seven candidate genes was attempted in C. tropicalis. The overexpression of IPF4033 (AYR1) gene generated an increased exo-to-endo ratio and exo-alcohol production in whole cells and membranes of C. tropicalis. In addition, a slight increased exo-to-endo ratio was observed when overexpressing IPF4033 in S. cerevisiae, although the reduction rate and exo-to-endo ratio were several fold lower compared to those obtained with C. tropicalis.