Study On The Production Of Frucosylated Chondroitin By Escherichia Coli K4with Genetic Engineeirng

Chondroitin sulfate is an acid polysaccharide of glycosaminoglycans possessingmedicinal value. The capsule polysaccharide of Escherichia coli K4(K4CPS) can bedescribed as a chondroitin backbone decorated with fructose side branches on the C3positionof the GlcA residues named frucosylated chondroitin. So, E. coli K4is the main producingstrain in fermentation research. In this paper, in order to enhance metabolic flux of K4CPSsynthesis pathway and ultimately improve the production performance of K4CPS, the strain E.coli K4was modificated by three genetic engineering strategies, including overexpressing thekey enzyme, overexpressing the global transcriptional regulator and modifying the capsulargene cluster promoter. The main results are as follows:1. The polymerization of chondroitin backbone chain and the supply of monosaccharideprecursor are the key steps in the pathway of K4CPS synthesis, so we overexpressed geneskfoC and kfoA in E. coli K4, coding chondroitin polymerase gene andUDP-glucose-4-epimerase (catalyzing UDP-GlcNAc to UDP-GalNAc) separately. Theincrease of chondroitin polymerase and UDP-glucose-4-epimerase expression could promotethe utilization rate of glycerol. But the K4CPS production of strain E. coli APkfoAoverexpressing of gene kfoA decreased by19%compared to E. coli K4. Strain E. coli APkfoCand E. coli APkfoA-kfoC overexpressed a single gene of kfoC and coexpression of genes kfoAand kfoC separately, the yield of K4CPS increased by29%and39%than the wild type strain.It indicated that cooverexpression of genes kfoA and kfoC could promote the K4CPS synthesisability mostly. In addition, kfoA and kfoC gene can influence the synthesis of the highmolecular weight K4CPS polysaccharide.2. The transcription of the K4capsular gene cluster can be regulated by globaltranscription factor SlyA, which was overexpressed in E. coli K4by the constitutive andinducible method respectively. Constitutive overexpression of SlyA protein had a greaterinhibitory effect on the growth of cells, leading to K4CPS production and glycerolconsumption rate reducing by31%and24%respectively. While the strain grown to the end oflag phase, the overexpression of protein SlyA induced by IPTG can effectively improve theexpression level of the K4capsular gene cluster. The K4CPS yields of recombinant strain E.coli THslyA in5liter fermentor reached to2.64g/L, with an increase of25%compared withthe wild type. So, it is an effective way to enhance K4CPS production by transcriptionalregulation protein induced the expression of capsular gene cluster.3. The modification of3’ untranslated region (UTR) of the capsule gene cluster region3promoter (PR3) were investigated to further improve the productivity of K4CPS by E.coli K4.The fermentation and quantitative real time RCR results suggested that the changes of UTRsequence as ops sequence remain did not affect the production of K4CPS and the transcriptionof PR3. Compared to the wild type, PR3transcription level and the K4CPS yield of theinsertion mutant with ops sequence deletion were decreased. Mutant strain was constructed bydeleting both of UTR and ops sequence, however, its PR3transcription level was increased2 fold and the K4CPS yield reached0.75g/L, with an increase of46%.