Regular articleAsymmetric whole-cell bioreduction of (R)-carvone by recombinant Escherichia coli with in situ substrate supply and product removal

•(R)-carvone was reduced to (2R,5R)-dihydrocarvone by recombinant Escherichia coli.•No alcohols were formed as side-products in biotransformations up to the L-scale.•In situ substrate feeding and product removal (SFPR) improved the bioreduction.•Using ionic liquids or adsorbent resins for SFPR led to high conversions (>85%).•Concomitantly, the product was gained in high diastereomeric excess (up to 99.2%).

The chiral building block (2R,5R)-dihydrocarvone can be synthesized by asymmetric reduction of the CC bond of (R)-carvone using ene-reductases. However, whole-cell biotransformations are challenging due to the toxicity of the substrate, its low solubility in aqueous media and the formation of alcohols as by-products. Here, (2R,5R)-dihydrocarvone was produced using Escherichia coli overexpressing an ene-reductase from Nostoc sp. PCC 7120. A NADP+-accepting mutant of the formate dehydrogenase from Mycobacterium vaccae regenerated cofactors. Whereas alcohols as by-products were not detected during biotransformation of 50 mM (R)-carvone, a host cell-mediated product isomerization was observed leading to a low diastereomeric excess (de) of 81.7% after 5 h. Moreover, the toxicity of the substrate resulted in a low conversion of 27.2%. Both effects were successfully prevented by in situ substrate feeding and product removal using water-immiscible ionic liquids or hydrophobic adsorbent resins. The biphasic bioreductions were optimized and the reaction system with the highest space-time yield was transferred to the liter scale. Under the optimized conditions (300 mM (R)-carvone, 400 mM formate, 36 g L−1 biocatalyst, XAD4 at a resin to substrate mass ratio of 5, 300 mM phosphate buffer, pH 6.3), (2R,5R)-dihydrocarvone was obtained with 96.5% de and 96.8% conversion within 9 h.

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