Research On Microbial Production Of (R,R)-2,3-Butanediol

2,3-butanediol, a potential bulk platform chemical, is widely applied in pharmaceutical, food, fuel and other fields. Due to the presence of two chiral carbon atoms in the molecular structure of 2,3-butanediol, so there are three kinds of isomers, namely (R,R)-, (S,S)-and meso-o-2,3-butanediol. (R,R)-2,3-butanediol not only shares the basic function with a mixture of stereoisomers of 2,3-butanediol, but also has special application in asymmetric synthesis of high-value liquid crystals, agricultural compounds, as well as Pharmaceuticals. In the study, According to the current problems that high cost of raw materials and low optical purity of products exist in (R,R)-2,3-butanediol production, the main work was carried out as follows:1.In this study, the mora meal instand of yeast extract was first used as nitrogen source for the (R,R)-2,3-butanediol production. And the mora meal simultaneous hydrolyzation and fermentation process was optimized to achieve a low-cost and efficient (R,R)-2,3-butanediol production technology. On the basis of single factor experiments, the optimized fermentation media components were obtained by using Plackett-Burman Design and Response Surface Methodology. On the basis of above conditions, up to 68.43 g/L (R,R)-2,3-butanediol(optical purity reaches 98.5%) could be achieved within 54 h with a productivity of 1.27 g/(L·h). In order to futher reduce the cost of production, less expensive cassava starch was used as carbon source to replace sucrose on the basis of previous optimized media. Cassava starch simultaneous saccharification and fermentation process were further optimized, and the final optimized results were as follows:Cassava starch 125 g/L, a-amylase 0.2 KNU/g Cassava starch, Saccharifying enzyme 3 AGU/g Cassava starch, Mora meal 60 g/L, (NH4)2SO4 1 g/L, MgSO4 0.3 g/L, Na2HPO4 1.05 g/L, KH2PO4 6 g/L, Tween-80 2 mL/L, CH3COONa 6.09 g/L, Neutral protease 0.52 g/L, Trace element solution 3 mL/L, initial pH 6.4. Using the optimized fermentation media,71.13 g/L (R,R)-2,3-butanediol(optical purity reaches 98.4%) was obtained within 50 h with a productivity of 1.42 g/(L·h), and this is the highest (R,R)-2,3-butanediol(e.e.>98%) titer so far reported from low-cost raw materials. In summary, this paper studied the production of (R,R)-2,3-butanediol by P. polymyxa through Cassava starch simultaneous saccharification and Mora meal simultaneous enzymatic hydrolysis, and achieved a low-cost and efficient (R,R)-2,3-butanediol production process.2.According to the gene prediction information, the key genes of (R,R)-2,3-butanediol pathway, namely gene alsS encoding the acetolactate synthetase, gen alsD encoding the acetolactate decarboxylase and gene bdh encoding the (R,R)-2,3-butanediol dehydrogenase were amplified and cloned. Recombinant plasmid pETDuet-alsS-alsD-bdh was constructed and was successfully transformed into E. coli BL21(DE3). when 50 g/L glycerol was used as the substrate, 12.04 g/L (R,R)-2,3-butanediol(optical purity reaches 99.2%) was produced within 36 h by the recombinant strain.