Enzymatic synthesis of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase in an aqueous-organic solvent system

- A newly synthesized SCR gene was expressed in E. coli for (S)-ET-5 production.
- The bioproduction of (S)-ET-5 in a biphasic solvent was first constructed.
- The (S)-ET-5 was achieved by bioconversion without extra addition of cofactor.
- The conversion of 99.1%, d.e. > 99.9% was achieved at 150 g L−1 ET-4.

(4S)-3-[(5S)-5-(4-Fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one ((S)-ET-5) is an important chiral intermediate in the synthesis of chiral side chain of ezetimibe. Recombinant Escherichia coli expressing carbonyl reductase (CBR) was successfully constructed in this study. The total E. coli biomass and the specific activity of recombinant CBR in 5 L fermenter culture were 10.9 g DCW L−1 and 14900.3 U g−1 DCW, respectively. The dual-enzyme coupled biocatalytic process in an aqueous-organic biphasic solvent system was first constructed using p-xylene as the optimal organic phase under optimized reaction conditions, and 150 g L−1 (4S)-3-[5-(4-fluorophenyl)-1,5-dioxophentyl]-4-phenyl-1,3-oxazolidin-2-one (ET-4) was successfully converted to (S)-ET-5 with a conversion of 99.1% and diastereomeric excess of 99% after 24-h, which are the highest values reported to date for the production of (S)-ET-5.

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