Breding Effciently Using Xylose Yield Of 2,3-butanediol Klebsiella Pneumoniae

As an important chemical material,2,3-butanediol is widely used in many fields,such as chemical engineering,food industry,fuel production and aerospace.Biological production of 2,3-butanediol with xylose as a raw material which is a widespread organic carbon source is competitive in the future.Therefore,making full use of xylose in cellulosic raw materials and improving the conversion of xylose 2,3-butanediol is one of the key issues for efficient production of 2,3-butanediol from cellulosic raw materials.In this study,through the validation of the gene function of Klebsiella pneumoniae with high xylose tolerance,the possible metabolic mechanism was preliminarily analyzed and the fermentation conditions of the strain were optimized.Finally,the strain with efficient use of xylose was genetically modified,the results provide theoretical and practical value for the efficient utilization of xylose by Klebsiella pneumoniae,which laid a solid foundation for the industrial production of 2,3-butanediol as well as provided some meaningful references for further strain transformation and fermentation process optimization.1)Overexpression of up-regulated genes,knockout of down-regulated genes,construction of 11 overexpression strains and 2 knockout strains,as well as selection of 9 genes for RT-PCR verification.The results showed that through overexpress the up-regulated genes involved in transmembrane transport,the key genes involved in the pentose phosphate pathway,the key genes of glycolysis pathway and the genes involved in signal transduction and electron transport.Under high xylose concentration,the xylose-consuming ability and the productivity of 2,3-butanediol were all improved to some extent compared with the parent strain;When knockout of the down-regulated gene that was connected with by-product pathway,the yield of 2,3-butanediol also increased to a certain extent.However,knockout of genes related to pyruvate metabolism the xylose consumption and 2,3-butanediol had little effect on the result.The RT-PCR verification results showed that the RT-PCR expression level is consistent with the up-down trend of RNA-seq.2)In 1L bioreactor,the Klebsiella pneumoniae strain with high xylose tolerance selected by adaptive evolution was optimized by response surface methodology.The optimal fermentation conditions were as follows:pH 5.9,temperature 35 ℃,under the optimal conditions,the yield of 2,3-butanediol was as high as 43.75 g/L with a speed of 230 r/min and aeration rate of 0.7 L/min,which was 32.7%higher than that before optimization.3)Complete knockout of phosphoenolpyruvate carboxylase gene ppc,succinate concentration and yield have greatly reduced the growth rate of strains slowed.But the yield of 2,3-butanediol was 27%higher than that of the parent strain,and the theoretical conversion rate(50%)of the mutant strain 2,3-butanediol was 21%higher than that of the parent strain.Both the xylose utilization and the 2,3-butanediol production rate decreased of knock-out ppc promoter,but the yield of 2,3-butanediol slightly increased.Both the concentration and yield of succinate decreased,but the yield of 2,3-butanediol didn’t improve significantly.Knockout of the fumarate reductase gene frdA and frdBC,which catalyzed the formation of succinate,slightly decreased the concentration of succinate,which was 16.27%and 25%lower than that of the parent strain respectively.The yield of 2,3-butanediol increased slightly,only increased by 7.8%and 10.2%respectively.The concentration of metabolites by-product ethanol increased by 13.14%and 17.26%respectively.