Study On Production Of Chiral Acetoin And (2S,3S)-2, 3-butanediol By Biocatalysis

Acetoin is a valuable flavor naturally existing in wine,honey,cocoa,butter,coffee, strawberry,garnet berry,etc.It has an asymmetric carbon,and has two stereoisomers:(3R)-acetoin and(3S)-acetoin.Acetoin plays important role in pharmaceutical synthesis and food-additive synthesis as intermediates.Chiral AC has been used to synthesizeα-hydroxyketone derivatives that are novel optically active compounds and to synthesize liquid crystal compositions.2,3-Butanediol (2,3-BD) is the hydrogenation product of AC.2,3-BD is not only a kind of fuel,but also a valuable chemical feedstock.It has the potential applications in the manufacture of printing inks,perfumes,fumigants,moistening and plasticizers.As to(2S,3S)-2,3-BD,it can also be used as antifreeze agent due to its low freezing point (-60℃).For the production of AC,usually a mixture of both isomers was produced by wild strain fermentation.By biocatalysis process,the production of chiral AC had been achieved,but the concentration and productivity were not high enough. Therefore,it is essential to find an effective method to realize chiral AC production.An NAD-dependent 2R,3R-butanediol dehydrogenase(2R,3R-BDH) from Bacillus subtilis 168 ATCC 23857 was suggested to have the ability to catalyze 2R,3R-butanediol((2R,3R)-2,3-BD) and meso-2,3-butanediol(meso-2,3-BD) to R-acetoin((3R)-AC) and S-acetoin((3S)-AC),respectively,but no activity on 2S,3S-butanediol((2S,3S)-2,3-BD).The NADH oxidase(NOX) from Lactobacillus brevis CICC 6004 could be employed for the regeneration of the cofactor NAD⁺.A whole cell biocatalyst for the effective production of chiral AC from 2,3-BD was constructed by co-expression of the genes encoding 2R,3R-BDH and NOX in Escherichia coli BL21(DE3).Under optimal conditions,36.7 g 1^-1 of(3S)-AC and 41.8 g 1^-1 of(3R)-AC both with the enantiomeric excess(ee) of 96.0%were produced from 43.0 g 1^-1 of meso-2,3-BD and 45.0 g 1^-1(2R,3R)-2,3-BD by 3.0 g dry cell weight (DCW) 1^-1 of the biocatalyst in 12 h,respectively,which were the highest yield among those reported till now.The biocatalyst could also be used in the kinetic resolution of(2S,3S)-2,3-BD from the mixture of 2,3-BD,and from 20.0 g 1^-1 of 2,3-BD (meso-2,3-BD 75.0%,(2R,3R)-2,3-BD 12.6%,(2S,3S)-2,3-BD 12.4%),2.4 g 1^-1 of (2S,3S)-2,3-BD(ee 99.0%) was obtained in 10 h.The results showed that the recombinant cell harboring 2R,3R-BDH and NOX is a promising alternative in the future industrial production of chiral(3S)-AC,(3R)-AC,and(2S,3S)-2,3-BD.Due to the existing of a 2R,3R-BDH,a mixture of both isomers of AC was usually produced by B.subtilis 168 fermentation.(3R)-AC can be obtained after the knock out of the gene ydjL,which encodes 2R,3R-BDH.Two fragments of ydjL were obtained by PCR and had been successfully ligated to the integration vector. Different conditions of electrothansformation were optimized and the optimal combination was obtained.Unfortunately,the right transformate was not obtained finally.