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.

Protein tyrosine phosphatases (PTPs) are promising drug targets for treating a wide range of diseases such as diabetes, cancer, and neurological disorders, but their conserved active sites have complicated the design of selective therapeutics. This study examines the allosteric inhibition of PTP1B by amorphadiene (AD), a terpenoid hydrocarbon that is an unusually selective inhibitor. Molecular dynamics (MD) simulations carried out in this study suggest that AD can stably sample multiple neighboring sites on the allosterically influential C-terminus of the catalytic domain. Binding to these sites requires a disordered α7 helix, which stabilizes the PTP1B−AD complex and may contribute to the selectivity of AD for PTP1B over TCPTP. Intriguingly, the binding mode of AD differs from that of the most well-studied allosteric inhibitor of PTP1B. Indeed, biophysical measurements and MD simulations indicate that the two molecules can bind simultaneously. Upon binding, both inhibitors destabilize the α7 helix by disrupting interactions at the α3−α7 interface and prevent the formation of hydrogen bonds that facilitate closure of the catalytically essential WPD loop. These findings indicate that AD is a promising scaffold for building allosteric inhibitors of PTP1B and illustrate, more broadly, how unfunctionalized terpenoids can engage in specific interactions with protein surfaces.

Rapid evolution of enzyme activities is often hindered by the lack of efficient and affordable methods to identify beneficial mutants. We report the development of a new growth-coupled selection method for evolving NADPH-consuming enzymes based on the recycling of this redox cofactor. The method relies on a genetically modified Escherichia coli strain, which overaccumulates NADPH. This method was applied to the engineering of a carboxylic acid reductase (CAR) for improved catalytic activities on 2-methoxybenzoate and adipate. Mutant enzymes with up to 17-fold improvement in catalytic efficiency were identified from single-site saturated mutagenesis libraries. Obtained mutants were successfully applied to whole-cell conversions of adipate into 1,6-hexanediol, a C6 monomer commonly used in polymer industry.

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1,2-propanediol (1,2-PDO) is an industrial chemical with a broad range of applications, such as the production of alkyd and unsaturated polyester resins. It is currently produced as a racemic mixture from nonrenewable petroleum-based feedstocks. We have reported a novel artificial pathway for the biosynthesis of 1,2-PDO via lactic acid isomers as the intermediates. The pathway circumvents the cytotoxicity issue caused by methylglyoxal intermediate in the naturally existing pathway. Successful E. coli bioconversion of lactic acid to 1,2-PDO was shown in previous report. Here, we demonstrated the engineering of E. coli host strains for the de novo biosynthesis of 1,2-PDO through this pathway. Under fermenter-controlled conditions, the R-1,2-PDO was produced at 17.3 g/L with a molar yield of 42.2% from glucose, while the S-isomer was produced at 9.3 g/L with a molar yield of 23.2%. The optical purities of the two isomers were 97.5% ee (R) and 99.3% ee (S), respectively. To the best of our knowledge, these are the highest titers of 1,2-PDO biosynthesized by either natural producer or engineered microbial strains that are published in peer-reviewed journals.