Enantioselective, continuous (R)- and (S)-2-butanol synthesis: achieving high space-time yields with recombinant E. coli cells in a micro-aqueous, solvent-free reaction system

The stereoselective production of (R)- or (S)-2-butanol is highly challenging. A potent synthesis strategy is the biocatalytic asymmetric reduction of 2-butanone applying alcohol dehydrogenases. However, due to a time-dependent racemisation process, high stereoselectivity is only obtained at incomplete conversion after short reaction times. Here, we present a solution to this problem: by using a continuous process, high biocatalytic selectivity can be achieved while racemisation is suppressed successfully. Furthermore, high conversion was achieved by applying recombinant, lyophilised E. coli cells hosting Lactobacillus brevis alcohol dehydrogenase in a micro-aqueous solvent-free continuous reaction system. The optimisation of residence time (τ) and 2-butanone concentration boosted both conversion (>99%) and enantiomeric excess (ee) of (R)-2-butanol (>96%). When a residence time of only τ=3.1 min was applied, productivity was extraordinary with a space-time yield of 2278±29g/(L×d), thus exceeding the highest values reported to date by a factor of more than eight. The use of E. coli cells overexpressing an ADH of complementary stereoselectivity yielded a synthesis strategy for (S)-2-butanol with an excellent ee (>98%). Although conversion was only moderate (up to 46%), excellent space-time yields of up to 461g/(L×d) were achieved. The investigated concept represents a synthesis strategy that can also be applied to other biocatalytic processes where racemisation poses a challenge.