| Microbial extracellular polysaccharides have attracted more and more attention due to their good physical and chemical properties,and they have a broad prospect.This article takes Sphingomonas sp.WG as the main research object to study the effect of fusion expression of glycosyltransferases Wel B,Wel K,Wel L,and Wel Q,responsible for assembly of tetrasaccharide repeat unit on polysaccharide synthesis in Sphingomonas sp.WG.Firstly,a LB clone vector was constructed,and several glycosyltransferase genes were fused by using the vector in different fusion sequences and expressed in the Sphingomonas sp.WG.By constructing a multi-enzyme cascade system to simulate the substrate channel transfer effect,to construct a high-producing sphingans strain,which not only provides a more efficient method for further research on the relationship between the structure and function of the fusion enzyme,laid a theoretical foundation for the metabolic pathway of sphingans.The research contents of this paper are as follows:Firstly,to eliminate the restriction of restriction enzyme sites and construct an expression vector that can flexibly change the direction of multiple genes and multiple fusions,this research designed and developed an LB cloning system.The cloning system is constructed by using the broad host plasmid p BBR1MCS-3 as the initial vector and inserting the LB fragment(GCTCTTCCTCAGC)into its multiple cloning site by PCR.The LB fragment contains Lgu Ⅰ and Bbv C Ⅰ partially overlapping recognition sites.Both of the two restriction endonucleases can generate the same non-palindromic sequence.This property can be used to quickly and flexibly insert multiple genes into the expression vector step by step.To verify the effectiveness of the cloning system,the four glycosyltransferases genes wel B,wel K,wel L,and wel Q from Sphingomonas sp.WG were fused to the LB cloning vector gradually.Then,the recombinant plasmids p BBR1MCS-3-LB-wel BK,p BBR1MCS-3-LB-wel KB,p BBR1MCS-3-LB-wel BKLQ,p BBR1MCS-3-LB-wel KBLQ,p BBR1MCS-3-LB-wel KLBQ,p BBR1MCS-3-LB-wel KLQB and p BBR1MCS-3-LB-wel QLKB were transformed into Sphingomonas sp.WG,which by the method of three-parent hybridization.The results of fermentation data show that the expression of double-gene fusion has little effect on the yield of sphingans,but it has a great influence on the viscosity.When the recombinant strain Sphingomonas sp.WG/ p BBR1MCS-3-LB-wel KB was fermented for 84 h,the broth viscosity increased by nearly 24.7% compared with the wild strain;the four-gene fusion expression had a greater yield of sphingans,the yield of sphingomonas sp.WG/ p BBR1MCS-3-LB-wel BKLQ was 29.7% higher than that of the wild strain in the fermentation medium containing 30 g/L glucose,and the glucose utilization rate of some strains was also significantly improved.Finally,through such methods as ethanol precipitation to extract polysaccharide from the fermentation,and different samples of sphingans were obtained.And through the infrared spectrum analysis,determination of uronic acid content and acetyl content and means of sphingans gas chromatography gel structure parsing,glycosyltransferases to research complex effects on sphingans structure.The results showed that the acetyl group content of the recombinant strain was higher than that of the wild strain,and the composition of monosaccharides varies greatly. |
