Enzymatic One‐Step Reduction of Carboxylates to Aldehydes with Cell‐Free Regeneration of ATP and NADPH

Abstract

The direct generation of aldehydes from carboxylic acids is often a challenging synthetic task but undoubtedly attractive in view of abundant supply of such feedstocks from nature. Though long known, biocatalytic carboxylate reductions are at an early stage of development, presumably because of their co‐factor requirement. To establish an alternative to whole‐cell‐based carboxylate reductions which are limited by side reactions, we developed an in vitro multi‐enzyme system that allows for quantitative reductions of various carboxylic acids with full recycling of all cofactors and prevention of undesired over‐reductions. Regeneration of adenosine 5′‐triphosphate is achieved through the simultaneous action of polyphosphate kinases from Meiothermus ruber and Sinorhizobium meliloti and β‐nicotinamide adenine dinucleotide 2′‐phosphate is reduced by a glucose dehydrogenase. Under these conditions and in the presence of the carboxylate reductases from Neurospora crassa or Nocardia iowensis , various aromatic, heterocyclic and aliphatic carboxylic acids were quantitatively reduced to the respective aldehydes.

In Vivo Reduction of Medium‐ to Long‐Chain Fatty Acids by Carboxylic Acid Reductase (CAR) Enzymes: Limitations and Solutions

“…The in vitro multi-enzyme system used was described to fully recycle ATP and NADPH and prevent undesired by-products (pyrophosphate, PP) in cell-free synthesis of aldehydes from acids. [31] Regeneration of ATP was achieved through the simultaneous action of polyphosphate kinases (PPK) from Meiothermus ruber and Sinorhizobium meliloti and NADPH was regenerated by a commercial glucose dehydrogenase (GDH-105). [31] Enzymes used for the in vitro recycling of ATP and NADPH are listed in Table S4.…”

“…[31] Regeneration of ATP was achieved through the simultaneous action of polyphosphate kinases (PPK) from Meiothermus ruber and Sinorhizobium meliloti and NADPH was regenerated by a commercial glucose dehydrogenase (GDH-105). [31] Enzymes used for the in vitro recycling of ATP and NADPH are listed in Table S4. All enzymes were expressed in E. coli MG1655 RARE (DE3) to keep background activities at a minimum.…”

“…Biphasic bioreduction set-up with in vitro ATP/NADPH regeneration system. Adapted from Strohmeier et al [31] recycling of ATP and NADPH. To optimize ratios of regeneration enzymes for cell-based application, equivalents of regeneration enzymes were compared to the standard set-up.…”

“…To optimize ratios of regeneration enzymes for cell-based application, equivalents of regeneration enzymes were compared to the standard set-up. [31] Product formation for 60 mM of 3 a, using the standard set-up, converted 24.4 mM of 3 a. Bioconversions with additional PPKs reached up to 40.9 mM conversion using twice the amount of PPKs for the regeneration of ATP (Nr. 9 and 15 in Figure S12).…”

“…For eliminating the CAR inhibitor pyrophosphate from the reaction, a pyrophosphatase from E. coli was also implemented. [31] In similar approaches, cascade reactions were investigated to pull the aldehyde intermediate from the reaction towards follow-up products. Because of impurities in enzyme preparations alcohol byproducts were observed.…”