Biotechnological Development of a Practical Synthesis of Ethyl (S)-2-Ethoxy-3-(p-methoxyphenyl)propanoate (EEHP): Over 100-Fold Productivity Increase from Yeast Whole Cells to Recombinant Isolated Enzymes

Abstract

The coupling of the enantioselective reduction catalyzed by Old Yellow Enzymes (OYEs), together with the in situ substrate feeding product removal (SFPR) concept, significantly improved the productivity of the g-scale preparation of ethyl (S)-2-ethoxy-3-(p-methoxyphenyl)propanoate (EEHP), an important precursor of several PPAR-α/γ agonists, such as Tesaglitazar. The OYEs and the glucose dehydrogenase for cofactor regeneration were cloned, overexpressed in Escherichia coli, and purified. The synthetic sequence was completed by a NaClO2 oxidation employing cheap and environmentally friendly conditions. The product was obtained in 94% yield and with an ee of 98% over the two steps.

Ene‐reductases and their Applications

“…Typically, ER-catalyzed reactions are performed at 5 mM substrate concentration since these enzymes are plagued by substrate and/or product inhibition [24,65,66]. The implementation of an in situ substrate feeding product removal (SFPR) technology was successfully applied to the OYE-catalyzed reaction of highly enantiopure ethyl (S)-2-ethoxy-3-(p-methoxyphenyl)propanoate (EEHP; 29c), an important precursor of several PPAR-α/γ agonists, like tesaglitazar [65,67]. SFPR works on the adsorption of substrate and products on a hydrophobic resin, maintaining soluble concentrations at levels that are not toxic for the microorganism or enzyme.…”

Unusual reactions mediated by FMN-dependent ene- and nitro-reductases

“…17), where hydride delivery on Cβ is sterically impeded (compare with Section 2.5). In turn, this leads to two competing reactions, i.e.…”

“…This technique allowed elevated substrate loading and gave a productivity of 56 g L −1 d −1 on the preparative scale. 17 The use of ene-reductases on a large scale was also reported for the reduction of citraconic acid dimethyl ester. The reaction was successfully scaled up to 70 g in a biphasic system, using cell paste containing the biocatalyst, in conjunction with a coupled-enzyme recycling system based on i-propanol and carbonyl reductase.…”

Stereochemical Outcome of the Biocatalysed Reduction of Activated Tetrasubstituted Olefins by Old Yellow Enzymes 1–3

“…All the enzymes employed were overexpressed in Escherichia coli BL21 (DE3) strains harbouring a specific plasmid prepared according to standard molecular biology techniques: pET30a-OYE1 from the original plasmid kindly provided by Neil C. Bruce, [15] pET30a-OYE2 and pET30a-OYE3 from Saccharomyces cerevisiae BY4741 and pKTS-GDH from Bacillus megaterium DSM509 (detailed steps reported in ref. [16] ).…”