Efficient asymmetric synthesis of ethyl (R)-3-hydroxybutyrate by recombinant Escherichia coli cells under high substrate loading using eco-friendly ionic liquids as cosolvent

Ionic liquids (ILs) which synthesized from bio-renewable materials have recently attracted much attention for their applications in biocatalysis. Ethyl ( R )-3-hydroxybutyrate (( R )-EHB) as a versatile chiral intermediate is of great interest in pharmaceutical synthesis. This study focuses on evaluating the performances of choline chloride (ChCl)-based and tetramethylammonium (TMA)-based neoteric ILs in the efficient synthesis of ( R )-EHB via the bioreduction of ethyl acetoacetate (EAA) at high substrate loading by recombinant Escherichia coli cells. It was found that choline chloride/glutathione (ChCl/GSH, molar ratio 1:1) and tetramethylammonium/cysteine ([TMA][Cys], molar ratio 1:1) as eco-friendly ILs not only enhanced the solubility of water-insoluble EAA in the aqueous buffer system, but also appropriately improved the membrane permeability of recombinant E. coli cells, thus boosting catalytic reduction efficiency of EAA to ( R )-EHB. In the developed ChCl/GSH- or [TMA][Cys]-buffer systems, the space-time yields of ( R )-EHB achieved 754.9 g/L/d and 726.3 g/L/d, respectively, which are much higher than neat aqueous buffer system (537.2 g/L/d space-time yield). Meanwhile, positive results have also been demonstrated in the bioreduction of other prochiral ketones in the established IL-buffer systems. This work exhibits an efficient bioprocess for ( R )-EHB synthesis under 325 g/L (2.5 M) substrate loading, and provides promising ChCl/GSH- and [TMA][Cys]-buffer systems employed in the biocatalysis for hydrophobic substrate.