Evaluation of Saccharomyces cerevisiae reductases as practical catalysts for ketone reductions

Optically pure alcohols are valuable chiral building blocks for a variety of commercially important compounds, such as carnitine and Taxol. Baker's yeast reductions can provide inexpensive access to these materials. Saccharomyces cerevisiae is the most popular and well-characterized whole cell biocatalyst for asymmetric reduction of carbonyl compounds. Whole cell catalysis is convenient since it does not require an external source of cofactors; however, the yeast genome has over 40 suspected carbonyl reductases and competition among these dehydrogenases often leads to a mixture of stereoisomeric products. This problem has restricted the utility of baker's yeast for stereoselective synthesis of beta-hydroxy esters and alpha-substituted beta-hydroxy esters.; The use of purified yeast reductases avoids problems associated with competing enzymes. Identifying the substrate and stereoselectivities of the proteins is much quicker with this method as compared to previous ones. The problem lies in how to obtain the purified reductases quickly and easily. Out of almost fifty potential carbonyl reductases present in the yeast genome, twenty-three were chosen based on sequence similarity to known reductases with broad substrate specificities. These proteins were expressed in E. coli, fused to a DNA sequence encoding the 26 kDa glutathione S-transferase protein, which serves as a tag to purify the fusion proteins. Escherichia coli strains overexpressing each of the twenty-three yeast reductases have been constructed and the corresponding fusion proteins have also been purified.; The construction of the library of the fusion proteins has allowed further characterization of each one of them and we have also explored applications of recombinant proteins for novel reductions. A representative set of alpha- and beta-keto esters was tested as substrates for each purified fusion protein. The stereoselectivities of beta-keto ester reductions depended both on the identity of the enzyme and the substrate structure, and some reductases yielded both L- and D-alcohols with high stereoselectivities. While alpha-keto esters were generally reduced with lower enantioselectivities, it was possible in all but one case to identify pairs of yeast reductases that delivered both alcohol antipodes in optically pure form. We also examined a series of alpha-chloro- and alpha-fluoro-beta-keto esters as potential substrates for individual purified yeast reductases. In a majority of cases, exclusive formation of L-alcohols was observed, although formation of the opposite D-isomer was also found. (Abstract shortened by UMI.)...