Structure-Guided Engineering of α-Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature.

Abstract

Branched-chain keto acid decarboxylases (KDCs) are a class of enzymes that catalyze the decarboxylation of α-keto acids. They are key enzymes for production of higher alcohols in vivo and in vitro. However, the two most active KDCs (KivD and KdcA) have only moderate thermostability (<55 °C), which hinders the production of alcohols at high temperatures. Herein, structure-guided engineering toward improved thermostability of KdcA is outlined. Strategies such as stabilization of the catalytic center, surface engineering, and optimization of dimer interactions were applied. With seven amino acid substitutions, variant 7M.D showed an increase of the temperature at which 50 % of activity remains after one-hour incubation by 14.8 °C without compromising its substrate specificity. 7M.D exhibited greater than 400-fold improvement of half-life at 70 °C and greater than 600-fold increase in process stability in the presence of 4 % isobutanol at 50 °C. 7M.D is more promising for the production of higher alcohols in thermophiles (>65 °C) and in cell-free applications.