Protein engineering for bioreduction of carboxylic acids

Carboxylic acids (CAs) are widespread in Nature. A prominent example is fatty acids, a major constituent of lipids. CAs are potentially economical precursors for bio-based products such as bio-aldehydes and bio-alcohols. However, carboxylate reduction is a challenging chemical transformation due to the thermodynamic stability of carboxylate. Carboxylic acid reductases (CARs), found in bacteria and fungi, offer a good solution to this challenge. These enzymes catalyse the NADPH- and ATP-dependent reduction of aliphatic and aromatic CAs. This review summarised all the protein engineering work that has been done on these versatile biocatalysts to date. The intricate catalytic mechanism and structure of CARs prompted us to first examine their domain architecture to facilitate the subsequent discussion of various protein engineering strategies. This then led to a survey of assays to detect aldehyde formation and to monitor aldenylation activity. Strategies for NADPH and ATP regeneration were also incorporated, as they are deemed vital to developing preparative-scale biocatalytic process and high-throughput screening systems. The objectives of the review are to consolidate CAR engineering research, stimulate interest, discussion or debate, and advance the field of bioreduction.