Constructing Cell Factories For High-Yield Of Optically Pure(R,R)-2,3-Butanediol And Optimizing Their's Fermentation

(R,R)-2,3-butanediol is an important platform compound with great value and bright future.Its synthesis through biological fermentation has attracted the attentions of a large number of researchers due to those renewable raw materials and environmental friendly reactions.Many works previous had been done mainly on screening high-yielding strains of(R,R)-2,3-butanediol from the nature environment.With the development of synthetic biology,constructing microbial cell factories for high-yielding(R,R)-2,3-butanediol becomes an effective way,by technical supports of metabolic analysis and genetic engineering.In this study,E.coli strain MG1655 was used as a base cell after systematically analysis of its main metabolism and by-product synthesis pathways.Metabolic engineering strategies were applied to modify those node genes on key pathways.As the result,an artificial route for(R,R)-2,3-butanediol was built and introduced into base cells,and obtained many microbial cell factories.Those cells which could produce high level of optically pure(R,R)-2,3-butanediol were selected as the objects to optimize fermentation parameters,including those feeding processes.The main content and results were shown as follows:(1)Some key enzyme genes related to the by-products of synthesis(butadione reductase gene dar,fumarate reductase gene frd ABCD,pyruvate formate lyase gene pfl B,alcohol dehydrogenase gene adh E,lactic acid dehydrogenation Enzyme gene ldh A,phosphoenolpyruvate synthase gene pps and phosphoacetyl transferase gene pta)were knocked out and modified.Three key enzyme genes(?-acetolactate synthase gene bud B,?-acetolactate decarboxylase gene bud A,and(R,R)-2,3-butanediol dehydrogenase gene bdh)that carry(R,R)-2,3-butanediol were introduced plasmid p Trc99A-bud B-bud A-bdh to build a series of cell factories.In shake flask(50 m L/250 m L)fermentation and fermentation tank level(0.5 L/1 L)fermentation,screening for synthesis(R,R)-2,3-butanediol production higher cell factory GXASY8 bdh,its on the fermentation tank at the initial 24 h,100 g/L glucose fermentation(R,R)-2,3-butanediol production is 24.60 g/L,the yield of 0.25 g/g substrates,optical purity 97.68%,(R,R)-2,3-butanediol production increased by 34.21% before modification.The concentrations of acetic acid,formic acid and lactic acid were reduced by 83.77%,69.13% and 66.30%,respectively.No succinic acid and ethanol were detected.(2)At the level of the 1 L fermentation tank(0.5 L/1 L),fermentation with cassava powder hydrolysate as a carbon source to obtain 22.02 g/L(R,R)-2,3-butanediol,which was only slightly lower than that of glucose as a carbon source.(R,R)-2,3-butanediol concentration increases with substrate concentration.The fermentation conditions were the temperature was 37°C,the aeration was 0.5 L/min and the p H was controlled in two stages.The initial value was 7.0.When it droped to 6.5,the p H was kept at 6.5 constantly until the end of fermentation.The concentration of(R,R)-2,3-butanediol was 28.26 g/L,the production intensity was 1.4 g/(L·h)and the yield is 0.29 g/g substrate.In optimization,the production of(R,R)-2,3-butanediol was 28.34% higher than before.(3)At the level of the 3 L fermentation tank(1.5 L/3 L),fermentation conditions: temperature 37°C,ventilation 1.5 L/min,p H 6.5,when the fermentation progressed to a substrate concentration between 30-40 g/L,the cassava flour-cotton seed powder hydrolysate was supplemented to a substrate concentration of 130 g/L,and the p H was adjusted by ammonia water to ferment 52 h.The concentration of(R,R)-2,3-butanediol was 108.80 g/L,which was 342.28% higher than shake flask fermentation(24.6 g/L)and 285% higher than batch fermentation(28.26 g/L).The fed-batch fermentation(94.93 g/L),which was only supplemented with the cassava flour enzymatic hydrolysis solution,increased by 14.61%,the production intensity was 2.09 g/(L·h),and the yield was 0.53 g/g substrate.In this study,we constructed cell factories,and optimized the fermentation process using cassava powder as raw material,and then achieved a higher(R,R)-2,3-butanediol output.The work provides new knowledge for developping biobased chemicals from cassava.