| Welan gum is an extracellular polysaccharide.Due to its unique physical and chemical properties,it has been used in petroleum,food,concrete and other industries in recent years.Based on the genome sequencing of Sphingomonas sp.WG and the predicted synthesis pathway of welan gum,the function of glycosyltransferase WelK and hybrid two-component protein WelA in the synthesis of welan gum was studied by gene knockout and overexpression.This study will lay a theoretical foundation for the subsequent transformation of strains through genetic engineering methods and regulation of the synthesis of welan gum.The research content of this thesis is as follows:First,a glycosyltransferase WelK knockout strain,a complementary strain,and an overexpression strain were constructed.The welK knockout cassette was constructed by fusion PCR and ligated to the vector p JQ200 SK to obtain the knockout plasmid p JQ200SK-ΔwelK,and the welK knockout strain ΔwelK was constructed by homologous recombination.The welK gene was amplified and ligated to the vector p BBR1MCS-3 to construct the recombinant plasmid p BBR1MCS-3-welK,which was transformed into ΔwelK and Sphingomonas sp.WG,respectively,to construct ΔwelK/p BBR1MCS-3-welK and overexpression strain Sphingomonas sp.WG/p BBR1MCS-3-welK.Second,in order to analyze the effect of the glycosyltransferase WelK on the synthesis of welan gum,wild strain,welK knockout strains,and welK complementary strains were cultured in a fermentation medium.At 48 h of fermentation,the viscosity of the fermentation broth of the knockout strain was reduced by 99.2% compared with the wild strain,but the viscosity of the complementary strain was 2.5 times that of the knockout strain,but only 1.5% of the wild strain.The yield of welK knockout strain was 1.7 g/L,which was about 71.5% lower than that of wild strain.After complement,the yield recovered to 3.4 times of the knockout strain and97.4% of the wild strain.It is indicated that the glycosyltransferase WelK plays a key role in the synthesis of welan gum.At the same time,a preliminary structural analysis of the purified polysaccharide samples obtained from the fermentation of wild strain and welK complementary strain were performed.The polysaccharides produced by wild strain and welK complementary strain have similar functional groups.The total sugar content and uronic acid content of the polysaccharides produced by welK complementary strain were lower than that of wild strain,and the content of acetyl groups is about 80.7% higher than that of the wild strain.In the composition of neutral monosaccharides,the content of mannose and galactose increased,and the content of glucose and rhamnose decreased.Next,wild strain and welK overexpressing strain were fermented in medium at low glucose concentration(3%)and high glucose concentration(6.7%).At 84 h of fermentation,the welan gum production of overexpressing strain increased by 12.5% compared with wild strain and the viscosity decreased by 27.1% when the strain was cultured at low glucose concentration.At high sugar concentration,the viscosity of overexpressing strain decreased by 57.9% compared with wild strain,and the yield increased by 34.1% compared with wild strain.The highest yield reached 43.13 g/L.The results indicated that the overexpression of welK resulted in an increase in the production of welan gum and a decrease of the viscosity of the fermentation broth.In addition,the structural analysis of welan gum obtained from the fermentation of wild strain and welK overexpressing strain showed that the polysaccharides produced by wild strain,and welK overexpressing strain have similar functional groups.The total sugar content and uronic acid content of the polysaccharides produced by welK overexpressing strain were higher than that of wild strain,and the acetyl content was decreased by about 26.5% compared to the wild strain.It is indicated that the overexpression of welK has an effect on the structure of welan gum.Finally,the knockout strain ΔwelA of the hybrid two-component protein WelA was also constructed by homologous recombination.The effects of WelA on the synthesis of welan gum were analyzed by fermentation results.Compared with the wild strain,the viscosity of the fermentation broth of welA knockout strain decreased by 99.5%,and the yield of welan gum decreased by 29.3%,indicating that WelA plays an important role in the synthesis of welan gum. |
