Characterization of Carboxylic Acid Reductases for Biocatalytic Synthesis of Industrial Chemicals

Abstract

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids into aldehydes, which can serve as common biosynthetic precursors to many industrial chemicals. This work presents the systematic biochemical characterization of five carboxylic acid reductases from different microorganisms, including two known and three new ones, by using a panel of short-chain dicarboxylic acids and hydroxy acids, which are common cellular metabolites. All enzymes displayed broad substrate specificities. Higher catalytic efficiencies were observed when the carbon chain length, either of the dicarboxylates or of the terminal hydroxy acids, was increased from C to C . In addition, when substrates of the same carbon chain length are compared, carboxylic acid reductases favor hydroxy acids over dicarboxylates as their substrates. Whole-cell bioconversions of eleven carboxylic acid substrates into the corresponding alcohols were investigated by coupling the CAR activity with that of an aldehyde reductase in Escherichia coli hosts. Alcohol products were obtained in yields ranging from 0.5 % to 71 %. The de novo stereospecific biosynthesis of propane-1,2-diol enantiomer was successfully demonstrated with use of CARs as the key pathway enzymes. E. coli strains accumulated 7.0 mm (R)-1,2-PDO (1.0 % yield) or 9.6 mm (S)-1,2-PDO (1.4 % yield) from glucose. This study consolidates carboxylic acid reductases as promising enzymes for sustainable synthesis of industrial chemicals.

Characterization of Type IV Carboxylate Reductases (CARs) for Whole Cell‐Mediated Preparation of 3‐Hydroxytyrosol

“…Overall, Tv CAR showed activity in a rather broad range from pH 6.0‐8.0 without losing activity considerably (Figure A). This observation was consistent with the biophysical characterizations of other CARs . The pH optimum determined for Tv2 CAR and Ds CAR showed the highest relative activity at pH 7.5 in 100 mM bicine‐ NaOH buffer and showed a preference for neutral to basic aqueous environment (Figure B–C).…”

“…Consequently, it can be concluded that type IV fungal CARs showed no significantly reduced activity up to 35 °C and Tv CAR even remained active up to 50 °C when incubated for 1 h at the specific temperatures. Up to date only the bacterial Mav CAR was described to be more thermostable and active at temperatures higher than 40 °C . Hence, Tv CAR would be a good fungal candidate when increased thermostability is needed.…”

“…This observation was consistent with the biophysical characterizations of other CARs. [10,24] The pH optimum determined for Tv2CAR and DsCAR showed the highest relative activity at pH 7.5 in 100 mM bicine-NaOH buffer and showed a preference for neutral to basic aqueous environment ( Figure 4B-C).…”

“…Up to date only the bacterial MavCAR was described to be more thermostable and active at temperatures higher than 40°C. [10] Hence, TvCAR would be a good fungal candidate when increased thermostability is needed. The broad substrate scope of TvCAR, its improved heterologous expression, the good specific activity of DOPAC 3 a (~0.43 U mg À 1 ) and the acceptance of acidic pH conditions made TvCAR a promising candidate for the overreduction of DOPAC to 3-HT.…”

Site-directed mutagenesis and stability of the carboxylic acid reductase MAB4714 from Mycobacterium abscessus

“…Currently, several biological routes to carboxylic acid reduction are known including direct reduction, catalyzed by a single enzyme or two-enzyme cascades with thioester or phosphoester intermediates (Kramer et al, 2018;Napora-Wijata et al, 2014).…”