Engineering an Alcohol Dehydrogenase for Balancing Kinetics in NADPH Regeneration with 1,4-Butanediol as a Cosubstrate

Cofactor regeneration using diols as “smart cosubstrates” is one of the most promising approaches, due to the thermodynamic preference and 0.5-equiv requirement. In order to establish an efficient NADPH regeneration system with 1,4-butanediol (1,4-BD), a NADP + -dependent alcohol dehydrogenase from Kluyveromyces polysporus ( Kp ADH) was engineered to solve the kinetic imbalance. Several hotspots were identified through molecular dynamic simulation and subjected to saturation and combinatorial mutagenesis. Variant Kp ADH V84I/Y127M exhibited a lower K M of 15.1 mM and a higher k cat of 30.1 min –1 than WT Kp ADH. The oxidation of 1,4-BD to 4-hydroxybutanal was found to be the rate-limiting step, for which the k cat / K M value of double mutant Kp ADH V84I/Y127M was 2.00 min –1 ·mM –1, 11.6-fold higher than that of WT Kp ADH. Kp ADH V84I/Y127M preferred diols with a longer chain length (C5–C6). The ratio of k cat / K M toward 2-hydroxytetrahydrofuran (2-HTHF), in comparison to 1,4-BD, in Kp ADH V84I/Y127M was dramatically reduced by almost 100-fold compared to WT Kp ADH, which was advantageous for NADPH regeneration. As much as 100 mM phenylpyruvic acid could be reduced into d -phenylalanine with 99.2% conversion in 6 h using merely 0.5 equiv of 1,4-BD. Both the improved catalytic efficiency toward 1,4-BD and the balanced k cat / K M between 1,4-BD and 2-HTHF contributed to the higher NADPH regeneration efficiency. This study provides guidance for engineering alcohol dehydrogenases for cosubstrate specificity toward diols and its application in NADPH regeneration for the preparation of chiral compounds of pharmaceutical relevance.