Study On Reducing The Capsular Polysaccharide Synthesis Of 1,3-Propanediol-Producing Klebsiella Pneumoniae

1,3-propanediol(1,3-PDO)is an important platform compound and has broad application in many fileds.Klebsiella pneumoniae(K.pneumoniae)is an excellent 1,3-PDO biosynthetic cell factory.However,the accumulation of the capsular polysaccharide in K.pneumoniae increase the viscosity of fermentation broth,and impact the mass transfering efficiency and the efficient transport of substrates and products,resulting in an obstacle in downstream extraction and industrialization of 1,3-PDO.In this study,according to the capsular polysaccharide synthesis pathway of K.pneumoniae,overexpression of the transcriptional inhibitory factor lon of the capsular polysaccharide synthesis pathway and knocking out the related genes on the capsular polysaccharide gene cluster by the Red recombination technique were performed,to reduce the content of the capsular polysaccharide in K.pneumoniae.To investigate the effect of the transcription level of the capsular polysaccharide synthesis-related,gnd and rcsA,on the production of 1,3-PDO,the transcriptional inhibitory factor lon gene was overexpressed in the wild strain.The transcriptional levels of gnd and rcsA were reduced by about 66% and 63% of the K.pneumoniae(pRSFDuet-tac-lon),respectively.In addtion,the content of capsular polysaccharide was decreased by 10.5%,the viscosity of the fermentation broth was decreased significantly,and the yield of 1,3-PDO was increased by 16.4%.Meanwhile,the molar conversion of 1,3-PDO was increased by 21.1% and up to 0.69 mol·mol-1,indicating that inhibition of the expression of capsular polysaccharide synthesis genes may improve the molar conversion of 1,3-PDO.To weaken the expression of capsular polysaccharide gene cluster and enhance the synthesis of the 1,3-PDO,gene-deficient strains were constructed.The content of capsular polysaccharide of the K.pneumoniae ΔwecA,K.pneumoniae Δgnd and K.pneumoniae Δwzi were reduced by 6.8%,9.5% and 20.6%,respectively,and the viscosity of fermentation broth were decreased,then the total yields of 1,3-PDO of the K.pneumoniae ΔwecA and K.pneumoniae Δwzi were increased by 4.0% and 17.4%,respectively.1,3-PDO titer per cell of the K.pneumoniae Δgnd was increased by 10.3%.The 1,3-PDO molar conversion of the K.pneumoniae Δgnd,K.pneumoniae Δwzi and K.pneumoniae ΔwecA were increased by 21.5%,15.8% and 3.7%,respectively.Besides,the amount of capsular polysaccharide of the K.pneumoniae ΔgndΔwecA,K.pneumoniae ΔwziΔwecA and K.pneumoniae ΔgndΔwzi were reduced by 16.8%,38.5% and 71.9%,respectively.The K.pneumoniae ΔwziΔwecA harvested a highest 1,3-PDO production by 23.0% and up to 20.01 g·L-1,the 1,3-PDO production per cell and molar conversion were increased by 5.8% and 21.2%,respectively.The titer per cell of 1,3-PDO was significantly improved by about 35.9% of the K.pneumoniae ΔgndΔwzi.In addition,according to the results of the transmission electron microscopy,the content of capsular polysaccharide was significantly decreased of the double-deficient strains.Meanwhile,the cells fimbriae synthesis ability and the adhesion were reduced.To further reduce the content of the capsular polysaccharide,the K.pneumoniae Δwzi(pRSFDuet-tac-lon)and K.pneumoniae ΔwziΔwecA(pRSFDuet-tac-lon)were constructed.In comparison,the contents of capsular polysaccharide were reduced by 4.9% and 3.5%,respectively.Besides,the viscosity of the fermentation broth were reduced.At the same time,the titers per cell of 1,3-PDO were increased by 19.5% and 32.3%,respectively.The results showed that the decline of the capsular polysaccharide content reduced the viscosity of the fermentation broth,then enhanced the permeability of the membrane,and finally strengthened the synthesis of 1,3-PDO.This study provided a novel prospect on development of K.pneumoniae on the production of 1,3-PDO.