Structure‐Guided Engineering of α‐Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature. Issue 18 (31st July 2018)

• Record Type: Journal Article
• Authors: Sutiono, Samuel; Carsten, Jörg; Sieber, Volker
• Is Part Of: ChemSusChem. Volume 11:Issue 18(2018)
• Page Start: 3335
• Page End: 3344
• Publication Date: 2018-07-31
• Subjects: alcohols -- decarboxylation -- enzyme catalysis -- enzymes -- high-throughput screening; Green chemistry -- Periodicals; Sustainable engineering -- Periodicals; Chemistry -- Periodicals; Chemical engineering -- Periodicals; 660
• DOI: 10.1002/cssc.201800944
• Languages: English
• ISSNs: 1864-5631

Abstract

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 T 1 h 50 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. Abstract : Engineered stability : A keto acid decarboxylase is evolved by structure‐guided engineering for higher thermal stability. A variant with seven amino acid substitutions shows an improvement in the temperature at which 50 % of activity remains after incubation for 1 h by 15 °C and tolerates an almost three times higher isobutanol concentration at 50 °C. The substitutions do not compromise substrate scope.