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.

Evolution of E. coli Phytase for Increased Thermostability Guided by Rational Parameters

“…As reported previously by Manfred [28] and Samuel [29], a reduction of the B-values can increase a protein's heat stability. In this approach, a reduction of the protein flexibility (entropy) and modification of the surface amino acids can increase the protein's thermostability to some extent [29]. Thus, the method of protein crystal structure analysis may have a more considerable role in enhancing the stability of thermophilic proteins.…”

“…In highresolution X-ray crystallographic studies of the protein, the temperature factor (B-value) is used to reflect the blurring of atomic electron densities and ambiguity of the atomic spatial state in the crystal structure [27]. As reported previously by Manfred [28] and Samuel [29], a reduction of the B-values can increase a protein's heat stability. In this approach, a reduction of the protein flexibility (entropy) and modification of the surface amino acids can increase the protein's thermostability to some extent [29].…”

To beat the heat – engineering of the most thermostable pyruvate decarboxylase to date

“…Our previous work on evolving thermostable, branchedchain a-keto acid decarboxylase from Lactococcus lactis (LlKdcA) showed that improved thermostability was also translated to increased stability against isobutanol. 24 The direct relationship between thermostability and organic solvent stability has also been described in other enzyme classes. 21,25,26 Therefore, to save time and expense in nding PDC variants with increased thermostability and butanol-stability, we developed a simple screening platform to screen only for improved thermostability.…”

“…7M.D). 24 As ApPDC and LlKdcA belong to the same a-keto acid decarboxylase family, we intended to (Fig. S1).…”

“…S3 †). 24 To check the stability of PDC-Var. 2 in comparison with WT ApPDC for ethanol fermentation applications via thermophilic microorganisms, both enzymes were incubated at 65 C, 70 C and 75 C (Fig.…”