Construction Of 2'-fucosyllactose-producing Strain And Fermentation Process Research

2'-fucosyllactose(2'-FL)is a kind of human milk oligosaccharides with probiotic function that is widely used in the fields of food and pharmaceutical,especially as an additive component of infant formula milk powder,which has attracted much attention in recent years.Traditionally,the methods for producing 2'-FL include natural product extraction and chemical synthesis,but both have disadvantages such as high cost and serious pollution.Therefore,the mildly reactive,green and environmentally friendly biological fermentation method has become one of the current research hotspots in 2'-FL production.In this study,Escherichia coli BL21(DE3)was used as the starting strain,and the 2'-FL production metabolic pathway was constructed in this strain.Then,the synthesis of metabolite was strengthened through modular metabolic engineering technology and gene editing technology.At last,the conditions of 2'-FL fermentation were optimized to further improve the growth of genetically modified strains.The yield of 2'-FL in the strain would provide a theoretical basis and industrial application basis for the production.Methods and experiment results were as follows:(1)De novo synthetic pathway to produce 2'-FL plasmid combinatorial construction.The clone was derived from the gene man B encoding phosphomannomutase,the gene man C encoding?-D-mannose-phosphate guanylyltransferase,the gene gmd encoding GDP-D-mannose-4,6-dehydratase,and the gene wca G encoding GDP-L-fucose synthase in Escherichia coli K12,and the gene fut C from Helicobacter pylori 26695 that encodes?-1,2-fucosyltransferase.At the same time,a modular metabolic engineering transformation theory was introduced.The 2'-FL synthesis pathway was divided into two modules,and five plasmids with different copy numbers were used to change the metabolic flux of the modules to adjust the expression level of key genes,so as to screen the best plasmid combination.(2)Metabolic transformation enhanced product synthesis.The 2'-FL production strain E.coli BL21(DE3)was modified using strategies such as blocking the metabolism of lactose branching and blocking the by-product colanic acid synthesis pathway at the genetic level.Using the CRISPR/Cas9 system,the lac Z and wac J genes were successfully knocked out,and three genetically modified strains of E.coli BL21(?lac Z),E.coli BL21(?wca J)and E.coli BL21(?wca J?lac Z)were obtained.(3)Optimization of fed-batch fermentation by de novo synthesis pathway.The fed-batch fermentation optimization was carried out through single factor experiments at the 3 L fermentor.The optimized fermentation conditions were as follows:DM medium was the fermentation medium,glycerol was the carbon source,the induction temperature was 30°C,the induction was performed when the cell mass(OD₆₀₀)reached 20,the concentration of the inducer(IPTG)was 0.5 mmol/L,and the p H feedback feeding method was used for feeding.The feeding concentration was 750 g/L glycerol and 20 g/L Mg SO₄·7H₂O.After optimization,the yield of 2'-FL could reach 18.22 g/L,which was 80.22%higher than the output before optimization.(4)Construction of a co-expression plasmid producing 2'-FL through the salvage synthetic pathway.The gene fkp encoding L-fucokinase/GDP-L-fucose pyrophosphorylase from Bacteroides fragilis was cloned,and the co-expression plasmid p ET-fkp-fut C was successfully constructed by heterologous expression of the genes fkp and fut C.(5)Optimization of shake flask culture conditions for salvage synthesis pathway.The culture conditions of strains E.coli BL21(DE3),E.coli BL21(?lac Z),E.coli BL21(?wca J)and E.coli BL21(?wca J?lac Z)in shaking flask fermentation to produce 2'-FL were optimized through single factor experiments.The optimized fermentation conditions were as follows:DM medium was the fermentation medium,the initial p H of the medium was 6.80,the inoculation volume was 5%,the induction temperature was 25°C and the concentration of inducer was 0.2 mmol/L.Cultured under this condition for 100 h,the 2'-FL yields of the four recombinant strains of E.coli BL21(DE3),E.coli BL21(?lac Z),E.coli BL21(?wca J)and E.coli BL21(?wca J?lac Z)increased by 1.87,1.69,1.23 and 1.56 times,respectively,and the highest accumulation of 2'-FL could reach 1.44 g/L.(6)The combined fermentation of the de novo synthesis pathway and the salvage synthesis pathway.The combined fermentation was carried out at the shake flask and fermentor levels respectively,and the results showed that the volume of bacteria in the combined pathway at the two levels was relatively reduced.At the same time,comparing the induction temperature of 25°C and 30°C at the fermenter level,the results showed that the yield of 2'-FL was relatively higher when the induction temperature was 30°C,which was2.47 g/L.However,the yield of combined fermentation did not reach the yield of single fermentation,but inhibited the volume of bacteria and the yield of 2'-FL.