tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is an important chiral intermediate for the synthesis of rosuvastatin. The biotechnological production of (3R,5S)-CDHH is catalyzed from tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) by a carbonyl reductase, and this synthetic pathway is becoming a primary route for (3R,5S)-CDHH production due to its high enantioselectivity, mild reaction conditions, low cost, process safety, and environmental friendship. However, the requirement of the pyridine nucleotide cofactors, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) limits its economic flexibility. In the present study, a recombinant Escherichia coli strain harboring carbonyl reductase R9M and glucose dehydrogenase (GDH) was constructed with high carbonyl reduction activity and cofactor regeneration efficiency. The recombinant E. coli cells were applied for the efficient production of (3R,5S)-CDHH with a substrate conversion of 98.8%, a yield of 95.6% and an enantiomeric excess (e.e.) of >99.0% under 350 g/L of (S)-CHOH after 12 hr reaction. A substrate fed-batch strategy was further employed to increase the substrate concentration to 400 g/L resulting in an enhanced product yield to 98.5% after 12 hr reaction in a 1 L bioreactor. Meanwhile, the space-time yield was 1,182.3 g L −1 day −1 , which was the highest value ever reported by a coupled system of carbonyl reductase and glucose dehydrogenase.
K E Y W O R D Scarbonyl reductase, co-expression, glucose dehydrogenase, tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate
Ezetimibe is a top-selling hypolipidemic drug for the treatment of cardiovascular diseases. Biosynthesis of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one ((S)-ET-5) using carbonyl reductase has shown advantages including high catalytic efficiency, excellent stereoselectivity, mild reaction conditions, and environmental friendness, and was considered as the key step for ezetimibe production. The regeneration efficiency of the cofactor, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is one of the main restricted factor. Recombinant Escherichia coli strain (smCR125) coexpressing carbonyl reductase (CR125) and glucose dehydrogenase were successfully constructed and applied for the production of (S)-ET-5 for the first time. Without extra addition of the coenzyme NADPH, the yield of 99.8% and the enantiomeric excess (e.e.) of 99.9% were achieved under ET-4 concentration of 200 g/L. Using a substrate fed-batch strategy, under the optimal conditions, the substrate ET-4 concentration was increased to 250 g/L with the yield of 98.9% and the e.e. of 99.9% after 12 hr reaction. The space-time yield of 494.5 g L −1 d −1 and the space-time yield per gram biocatalyst of 24.7 g L −1 d −1 g −1 DCW were achieved, which were higher than ever reported for the biosynthesis of the ezetimibe intermediate.
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