Effects Of Key Enzyme Overexpression On The Synthesis Of Welan Gum

Welan gum is a class of macromolecule hetero-polysaccharides secreted by Sphingomonas sp.WG.Because of its unique molecular structure and excellent rheological properties,welan gum has been used in the areas of oil drilling,food industry,concrete,and so on.To determine the effects of UgpG and WelB overexpression on the yield,viscosity and structure of the Welan gum,we constructed the key enzymes UDP-glucose pyrophosphorylase(UgpG)and glucose isoprene transferase(WelB)overexpressing strains in this paper.This work will provide a theoretical foundation for the subsequent construction of high welan gum-producing strains by genetic engineering.The main contents are as follows:First of all,key enzyme overexpressing strains of Sphingomonas sp.WG were constructed.The gene ugpG and welB were amplified by PCR using the genomic DNA of Sphingomonas sp.WG as template.The two fragments were ligated to the vector pBBR1MCS-3 to construct the recombinant plasmid pBBR1MCS-3-ugpG/welB,respectively.Two recombinant plasmids were transferred to Sphingomonas sp.WG to construct the recombinant strains Sphingomonas sp.WG/pBBR1MCS-3-ugpG(strain A)and Sphingomonas sp.WG/pBBR1MCS-3-welB(strain B).Subsequently,in order to confirm the effects of key enzymes on the synthesis of welan gum,wild strain,strain A and B were cultured in LB medium and fermentation medium respectively and the production of welan gum and viscosity of fermentation broth were determined.The results showed that,the welan gum production and viscosity of strain A increased by 8.58% and 42.71% compared with that of the wild strain when they were cultured in LB medium for 96 h.Its welan gum production and the viscosity of the fermentation broth increased by 27.75% and 6.86% when cultured in the fermentation medium for 72 h.The viscosity of the fermentation broth of strain B was 9.87-fold that of the wild strain when cultured in LB medium for 96 h.Compared with the wild strain,its production of welan gum increased by 21.02% when cultured in the fermentation medium for 48 h.These results indicate that the overexpression of UgpG and WelB has a significant effect on the yield of welan gum and the viscosity of fermentation broth.Besides,the structures of the polysaccharides produced by three strains were preliminarily elucidated.The results showed that the polysaccharide samples had very similar functional groups,however,compared with the wild strain,the total sugar content,uronic acid content and acetyl content of strain A and B were improved.This work confirms the important role of the key enzymes UgpG and WelB overexpression in the structure of welan gum.Finally,exogenous proteins contain extra ?-galactosidase short peptides when using the series of pBBR1 MCS vector.In order to overcome this shortcoming,we used pBBRMCS-3 plasmid as template for transformation to constructed a new and efficient expression vector pBBRtac3.Three specific DNA sequences including Ptac and Ter from pGEX4T-1 and MCS from pBBR1MCS-3 were obtained using PCR amplification,respectively.Then they were ligated to obtain the the expression element PMT by the fusion PCR.At the same time,pMCS3 fragment including the pBBR1MCS-3 sequence except for Ptac,LacZ? and MCS was amplified by PCR.Finally,the PMT and pMCS3 were digested by two enzymes and ligated by DNA ligase to obtain pBBRtac3.At the same time,in order to study the expression efficiency of this vector,we expressed the key enzyme UgpG in E.coli BL21(DE3).The results showed that,compared with pBBR1MCS-3,the expression of UgpG was significantly increased when pBBRtac3 was used as expression vector.This vector might lay foundation for further construction of high welan gum producing strains using genetic engineering method.