Optically Pure Acetoin Production Through Cell-free Metabolic Engineering

Acetoin,3-hydroxy-2-butanone,is a kind of food additive As a important 4 carbon chemical,it has been defined as one of the 30 platform chemicals with priority for the development by the U.S.Department of Energy and widely used in cosmetic,agricultural and chemical industries.In our work,acetoin was efficient produced from meso-2,3-butanediol with NAD~+regeneration through cell-free metabolic engineering in vitro.The optimal enzyme for NAD~+regeneration and meso-2,3-butanediol dehydrogenase were chosen through screening two enzymes,detecting kinetic parameter and comparing the performance of acetoin production through the two enzyme coupling.Lastly,acetoin was efficiently produced by optimization the system conditions.Firstly,meso-2,3-butanediol was chosen for acetoin production and the other enzyme was coupled with meso-2,3-butanediol dehydrogenase for NAD~+regeneration.Enzymes involed in acetoin production were selected.Two enzymes for NAD~+regeneration were selected,which were NADH oxidase from Bacillus subtilis(BS-NOX)and xylose reductase from Candida tenuis(CT-XR).Four butanediol dehydrogenase from Bacillus subtilis,Corynebacterium glutamicum,Pyrococcus furiosus and Parageobacillus thermoglucosidans(BS-BDH、CG-BDH、PF-BDH and PT-BDH)were screened.To select a better enzyme for NAD~+regeneration,acetoin was produced using BS-BDH coupling with BS-NOX and CT-XR,respectively.CT-XR was chosen due to the higher acetoin titer.And to select a better enzyme for pure acetoin production,acetoin was produced using CT-XR coupling with BS-BDH、CG-BDH、PF-BDH and PT-BDH,respectively.According to the production and optical purity of acetoin,the couple,CT-XR and CG-BDH,was chosen to produce D-(-)-acetoin.Then,the biocatalysis conditions of D-(-)-acetoin production from meso-2,3-butanediol through the two-enzyme cascade were optimized.The best reaction conditions were in PBS buffer at 37°C and pH 7.5.The optimal ratio of meso-2,3-butanediol to xylose was 1:1.5,and 1:1 was the the optimal ratio of CG-BDH to CT-XR.Under the optimized conditions,the substrate conversion was successfully improved to50.1%.Finally,to obtain a higher production of acetoin,the concentation of meso-2.3-butanediol,xylose were increased to 584.9 mM and 847.7 mM.As a result,323.9 mM(28.5 g/L)D-(-)-acetoin with an optical purity of 95.2%and 268.6 mM(40.29 g/L)xylitol were obtained.The meso-2,3-butanediol convertion was 55.4%.The acetoin productivity was 4.75 g/(L?h),and the yield was 99%of the theoretical yield.This work therefore offers a novelly efficient strategy for NAD~+regeneration.The two-enzyme cascade in this work is a promising,economical and green alternative strategy for the optically pure acetoin biosynthesis.