Direct Evolution To Improve Xylose Tolerance Of Klebsiella Pneumoniae And It’s Regulate Mechanism

As a important chemical material,2,3-butanediol is widely used in many fields,such as chemical engineering,food industry,fucl 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.This study is aimed at acquiring strains that can utilize xylose efficiently in the process of 2,3-butancdiol producing.Through altering the expression quantity and genotype of the rpoD gene which encodes the sigma factor,a kind of global transcriptional regulatory factor,as well as direct selecting with stress-induced mutation theory.After that,we elaborate the mechanism of these changes at the molecular level,providing a reference for the biological transformation of 2,3-butanediol.1)We confirmed the threshold value of KPG’s tolerance of xylose is 75 g/L by comparing the growth conditions of KPG strain and contents of 2,3-butanediol and other by-products in different concentration of xylose medium.2)We get gene expression strains of rpoD using plasmid pUC18k and pDK7,and demonstrate that,to some extent,the changing of gene expression of’ rpoD can affects strains’ xylose tolerance through fermentation validation.Improving rpoD gene expression can accelerate the xylose consumption,raise the xylose consume rate.Compared to the original strain,the xylose consumption of KP-18-rpoD and KP-7-rpoD strain are increase by 167.2%and 127.3%respectively,and the production of 2,3-butanediol and other by-product also have a certain degree of improvement.3)We determined to use recA gene as reference gene in RT-PCR after expression analysis with recA,rpoB and ldh.On this basis,we measured the rpoD gene expression quantity in KPG,KP-18-rpoD and KP-7-rpoD respectively,the result indicated that the changing in rpoD gene expression quantity induced by plasmid pUC18k-rpoD and pDK7-rpoD have no significant difference.4)We established a gene library of rpoD through error-prone PCR,then get a mutant strain library.We also get some directed evolution strains based on stress-induced mutation theory.After screening by fermentation with high concentration of xylose,we acquired two mutant strains from above strains,named as KP2 and KPC,respectively.5)Through fermentation with high concentration of xylose,we tested the strain KP2 and KPC.We find that these two strains’ tolerance to xylose are enhanced.When ferment in a 125 g/L xylose medium with KP2 and KPC,respectively,the fermentation time are both doubled,and the consumption of xylose are increased by 117%and 168%separately,the yields of 2,3-butanediol are 35.78 g/L and 33.23 g/L respectively,increased by 132.4%and 115.8%,respectively,compared to the strain KPG.6)Through de novo transcriptome analysis,we get transcriptions(5693357 nt long totally)of KPG,KP2 and KPC that are cultured in high concentration of xylose medium after 48 hours.And 3990 of 4539 Unigenes are annotated after blast in NR,NT,Swiss-Prot,KEGG,COG and GO.Through differentially expressed gene analysis,505 and 674 differentially expressed genes(DEGs)are found in KP2 and KPC,compared with KPG separately.Among these DEGs,242 and 500 DEGs are up-regulated,respectively.The rest of 263 DEGs in KP2 and 174 DEGs in KPC are down-regulated.Via Pathway analysis of DEGs,we find that these DEGs regulate multiple processes,inculding signal transduction,membrane transport,carbohydrate metabolism,energy metabolism and other material metabolism,such as nucleotide and amino acid.These changed processes in transcriptional level resulted in the phenotypes defferences of KP2 and KPC.