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Biocatalytic Reduction of Carboxylic Acids for Industrial Chemical Synthesis

Levi Kramer, University of Nebraska - Lincoln


Biocatalysts are robust tools for the conversion of renewable feedstock chemicals to value-added products. Carboxylic acids are abundant chemicals in Nature with the potential for conversion to numerous value-added products via aldehyde intermediacy. However, reduction of carboxylic acids to aldehydes is technically challenging due to the thermodynamic stability of carboxylic acids and instability of aldehydes. Carboxylic Acid Reductase (CAR) enzymes have surfaced as biocatalysts for selective reduction of carboxylic acids to the corresponding aldehydes. Although CARs exhibit remarkable substrate promiscuity, previously characterized enzymes display limited activity on short-chain aliphatic carboxylates that are common microbial metabolites (e.g., lactic acid). Herein we report the discovery and characterization of three putative CAR enzymes. New CARs were characterized in steady-state kinetic studies on short-chain dicarboxylates and hydroxy carboxylates to elucidate the structure-activity relationship on such substrates. Enzyme engineering was subsequently pursued using systematic construction of hybrid CARs. Chimeric CARs were characterized in steady-state and transient kinetic studies, revealing a rational approach for hybrid CAR engineering. CARs were also engineered using a whole-cell growth-coupled NADPH recycling strategy. This strategy was successfully employed for the identification of CAR variants exhibiting improved kinetic properties on target substrates. In addition to CAR engineering efforts, variants of a CoA-dependent aldehyde dehydrogenase with improved catalytic properties on lactoyl-CoA substrates were selected using a novel microbial 1,2-propanediol biosensor. Enzyme variants exhibiting appreciable kinetic properties on target substrates were implemented in whole-cell biocatalytic cascades for the production of value-added chemicals. This work highlights the potential of CARs in biocatalysis, but further enzyme engineering is required for industrial feasibility.

Subject Area

Chemical engineering|Cellular biology|Microbiology|Industrial engineering|Organic chemistry

Recommended Citation

Kramer, Levi, "Biocatalytic Reduction of Carboxylic Acids for Industrial Chemical Synthesis" (2020). ETD collection for University of Nebraska - Lincoln. AAI28258861.