Coevolution of the Activity and Thermostability of an ϵ-Keto Ester Reductase for Better Synthesis of an (R)-α-Lipoic Acid Precursor

Abstract

In this work, we have identified a significantly improved variant (S131Y/Q252I) of the natural ϵ-keto ester reductase CpAR2 from Candida parapsilosis for efficiently manufacturing (R)-8-chloro-6-hydroxyoctanoic acid [(R)-ECHO] through co-evolution of activity and thermostability. The activity of the variant CpAR2_S131Y/Q252I towards the ϵ-keto ester ethyl 8-chloro-6-oxooctanoate was improved to 214 U mg^-1 -from 120 U mg^-1 in the case of the wild-type enzyme (CpAR2_WT )-and the half-deactivating temperature (T₅₀ , for 15 min incubation) was simultaneously increased by 2.3 °C in relation to that of CpAR2_WT . Consequently, only 2 g L^-1 of lyophilized E. coli cells harboring CpAR2_S131Y/Q252I and a glucose dehydrogenase (GDH) were required in order to achieve productivity similar to that obtained in our previous work, under optimized reaction conditions (530 g L^-1 d^-1 ). This result demonstrated a more economical and efficient process for the production of the key (R)-α-lipoic acid intermediate ethyl 8-chloro-6-oxooctanoate.

Keywords: asymmetric synthesis; directed evolution; enzyme catalysis; keto ester reductases; lipoic acid.