YEAST CATALYZED REDUCTION OF BETA-KETO-ESTERS .1. FACTORS AFFECTING WHOLE-CELL CATALYTIC ACTIVITY AND STEREOSELECTIVITY

Six yeasts were studied for their ability to reduce ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) stereoselectively. Five species reduced the substrate to ethyl (S)-4-chloro-3-hydroxybutanoate of high (92-99%) optical purity. With glucose-grown cells, substrate reduction could only be demonstrated when growth was oxygen-limited, whereas xylose-grown Pichia capsulata could be grown under conditions of oxygen excess without losing its reducing ability. Zygosaccharomyces rouxii exhibited high enantioselectivity (greater than or equal to 98% ee (S)-enantiomer) under all conditions tested, whilst in P. capsulata, a novel switch was observed from producing mainly the (S)-enantiomer using glucose as co-substrate to producing mainly the (R)-enantiomer using 2-propanol as co-substrate. This switch was correlated with a change in reduction predominantly from an NADPH-dependent dehydrogenase system to an NADH-dependent system. In the production of ethyl (R)-4-chloro-3-hydroxybutanoate with P. capsulata, the enantioselectivity was also found to depend upon growth conditions. With glucose-grown cells, higher enantioselectivity was observed using cells harvested in stationary phase (93-94% ee) compared with cells harvested in exponential phase (43-60% ee). Growing P. capsulata with xylose rather than glucose as the major source of carbon for growth resulted in an eight-fold increase in the specific rate of ethyl (R)-4-chloro-3-hydroxybutanoate production using 2-propanol as co-substrate, although enantioselectivity was slightly reduced (65-81% ee). compared with the maximum achieved with glucose-grown cells. The effect of growth on xylose could also be correlated with enhanced activity of an NADH-dependent (R)-selective dehydrogenase system.