Co-evolving the Activity and Thermostability of an ε-Ketoester Reductase for Better Synthesis of (R)-α-Lipoic Acid Precursor. 
In this work, we identified a significantly improved variant S131Y/Q252I of natural ε-ketoester reductase CpAR2 from Candida parapsilosis for efficient manufacturing (R)-ECHO [(R)-8-chloro-6-hydroxyoctanoate] by co-evolving the activity and thermostability.  The activity of variant CpAR2S131Y/Q252I towards ε-ketoester ethyl 8-chloro-6-oxooctanoate was improved to 214 U mg-1 from 120 U mg-1 of the wild-type enzyme (CpAR2WT), and the half-deactivating temperature (T50, for 15 min incubation) was simultaneously increased by 2.3°C compared with CpAR2WT.  Consequently, only 2 g L-1 of lyophilized E. coli cells harboring CpAR2S131Y/Q252I and glucose dehydrogenase (GDH) were required to achieve a similar productivity (530 g L-1 d-1) of our previous work under the optimized reaction conditions.  This result demonstrated a more economical and efficient process for the production of the key (R)-α-lipoic acid intermediate.