Large-scale preparation of (S)-ethyl 3-hydroxybutanoate with a high enantiomeric excess through baker's yeast-mediated bioreduction

Strain engineering for stereoselective bioreduction of dicarbonyl compounds by yeast reductases

“… using activators and inhibitors of specific ( R )‐ or ( S )‐reductases: allyl bromide, sulfur compounds (dimethyl sulfoxide, thioacetamide), magnesium or calcium salts, aliphatic carboxylic acids, adenine and ethylchloroacetate [2,47]; modifying the temperature to inactivate competing enzymes by exploiting differences in thermal stability [47,48]; lowering the substrate concentration with adsorbing resins [49], fed‐batch systems [50] or organic–water two‐phase systems [51,52] to take advantage of the higher K m values of competing reductases; using osmotic and oxidative stress, heat shock and diauxic shift to induce specific reductases in S. cerevisiae , as shown with the induction of the reductase genes GRE2 and GRE3 under hyperosmotic conditions [53]; exploring the physiological state of the cell by choosing harvest time, pH, aeration, growing or non‐growing cells, substrate concentration, and co‐substrate to modify the reductase level, as shown for the reduction of β‐keto esters by S. cerevisiae [54] and the reduction of ketosulfone by stationary phase Rhodoturula rubra cells [41]; choosing an alternative carbon source for growth, for instance with galactose‐grown cells that gave higher ee in the reduction of β‐keto esters compared to cells grown on glucose, due to the up‐regulation of the GCY1 gene [20]. …”

“…lowering the substrate concentration with adsorbing resins [49], fed‐batch systems [50] or organic–water two‐phase systems [51,52] to take advantage of the higher K m values of competing reductases;…”

Influence of the ethanol and glucose supply rate on the rate and enantioselectivity of 3‐oxo ester reduction by baker's yeast

“…These numbers are significantly higher than 0.05 mol glucose.kg dw −1 .h −1 or 0.15 mol EtOH.kg dw −1 .h −1 , which is sufficient according to our results. In an industrial reduction process, not only the electron donor supply must be controlled, it is also recommended to feed the substrate ethyl 3-oxobutanoate to keep its concentration low and thus achieve high enantiomenc purities of the product (Chen et al, 1984;Kometani, 1993Kometani, , 1994Rohner et al, 1984;Wipf et al, 1983). For an efficient industrial process the use of higher yeast concentrations and also its re-use is recommended to speed up the reduction and to minimize as much as possible the environmental burden.…”

“…To obtain high q EOB values at otherwise comparable conditions, electron donor supply rates in optimized literature procedures for ethyl 3-oxobutanoate were calculated to be ∼1.0 mol EtOH.kg dw −1 .h −1 (Kometani et al, 1993) and, using a combined electron donor supply, ∼0.14 mol glucose.kg dw −1 .h −1 plus ∼0.9 mol EtOH.kg dw −1 .h −1 (Wipf et al, 1983). These numbers are significantly higher than 0.05 mol glucose.kg dw −1 .h −1 or 0.15 mol EtOH.kg dw −1 .h −1 , which is sufficient according to our results.…”

“…Even if such protocols are described in sufficient detail to be reproduced, quantitative understanding of the results and scaling-up is difficult because several process parameters change simultaneously. In only a few publications, optimization of the reduction of ethyl 3-oxobutanoate while controlling and measuring the most important param- eters has been reported (Kometani et al, 1993(Kometani et al, , 1994Rohner et al, 1990;Wipf et al, 1983). From these publications a useful protocol for controlled EOB addition, leading to high enantiomeric excess values of the product can be obtained.…”