Co-factor demand and regeneration in the enzymatic one-step reduction of carboxylates to aldehydes in cell-free systems

• Published in: Journal of biotechnology Vol. 307; pp. 202 - 207
• Main Authors: Strohmeier, Gernot A., Schwarz, Anna, Andexer, Jennifer N., Winkler, Margit
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 10-01-2020
• Subjects: Aldehyde; Carboxylate reductase; Cell-free ATP recycling; Multi-enzyme cascade; Polyphosphate; Multi-enzyme cascade; Carboxylate reductase; Polyphosphate; Cell-free ATP recycling; Aldehyde
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2019.10.016

•Polyphosphate (polyP) sources differ significantly in their capability to provide reactive phosphate groups for the polyphosphate kinase-based ATP regeneration•The concentration of ATP and NADP+ in the in vitro reduction of carboxylic acids can be reduced to 200 μM and 100 μM, respectively•Lower NADP+ concentrations allow reducing the formation of alcohol from the aldehyde products through side activities in the enzyme preparations
Addressing the challenges associated with the development of in vitro biocatalytic carboxylate reductions for potential applications, important aspects of the co-factor regeneration systems and strategies for minimizing over-reduction were investigated. The ATP recycling can be performed with similarly high efficiency exploiting the polyphosphate source by combining Meiothermus ruber polyphosphate kinase and adenylate kinase or with Sinorhizobium meliloti polyphosphate kinase instead of the latter. Carboxylate reductions with the enzyme candidates used in this work allow operating at co-factor concentrations of adenosine 5′-triphosphate and β-nicotinamide adenine dinucleotide 2′-phosphate of 100 μM and, thereby, reducing the amounts of alcohols formed by side activities in the enzyme preparations. This study confirmed the expected benefits of carboxylic acid reductases in chemoselectively reducing the carboxylates to the corresponding aldehydes while leaving reductively-sensitive nitro, ester and cyano groups intact.