A basic requirement for the efficiency of reductive whole-cell biotransformations is the reducing capacity of the host. Here, the pentose phosphate pathway (PPP) was applied for NADPH regeneration with glucose as the electron-donating co-substrate using Escherichia coli as host. Reduction of the prochiral β-keto ester methyl acetoacetate to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) served as a model reaction, catalyzed by an R-specific alcohol dehydrogenase. The main focus was maximization of the reduced product per glucose yield of this pathway-coupled cofactor regeneration with resting cells. With a strain lacking the phosphoglucose isomerase, the yield of the reference strain was increased from 2.44 to 3.78 mol MHB/mol glucose. Even higher yields were obtained with strains lacking either phosphofructokinase I (4.79 mol MHB/mol glucose) or phosphofructokinase I and II (5.46 mol MHB/mol glucose). These results persuasively demonstrate the potential of NADPH generation by the PPP in whole-cell biotransformations.