Metabolic Engineering Of Escherichia Coli For Production Of 2'-Fucosyllactose

2'-fucosyllactose as a new type of rare Human milk oligosaccharide(HMO)have caused widespread research interest,because of its stimulating the growth of intestinal probiotics,protecting the intestinal health of infants,and improving the immune system.At present,2'-fucosyllactose has been approved by FDA as an infant milk powder additive with broad application prospects.A strain(Kosakonia sp.CCTCCM2018092)with high yield of fucoidan was isolated by our laboratory,whose genomic information was obtained by whole-genome sequencing.Then,we preliminary analyzed the reasons of its high-yield fucoidan and identified its GDP-L-fucose biosynthesis pathway.Meanwhile,genes of GDP-L-fucose synthesis of Kosakonia sp CCTCCM 2018092 strain were heterologously expressed to the E.coli host to construct an engineered strain for producing GDP-L-fucose.The method of gene knockout,overexpression,promoter engineering were used to further increase the production of GDP-L-fucose downstream product 2'-fucosyllactose.The main contents of this article are as follows:1.The reasons of high-yield fucoidan of Kosakonia sp CCTCCM 2018092 was analyzed by bioinformatics,and the results shown that the synthetic genes of fucoidan has multiple copies.We proposed that multiple copies of synthetic genes of fucoidan are responsible for the high-yield fucoidan of Kosakonia sp CCTCCM 2018092,which also provides a theoretical basis for the high fucoidan mechanism of this strain.2.Using different genotypes of E.coli hosts(BL21(DE3),JM109(DE3),Turner(DE3),HMS174(DE3),DH10B(DE3))as the starting strain.We expressed heterologously GDP-L-fucose biosynthesis pathway derived from Kosakonia sp CCTCCM 2018092 to construct an engineered strain,which can produce GDP-L-fucose.In order to screen the ideal E.coli host for production of GDP-L-fucose,we performed shake flask fermentation.The shake flask results shown that the strain JM109(DE3)was the ideal host for production GDP-L-fucose with 7.96 mg/L,whether at 22 °C or25 °C.3.Based on the above strain capale of producing GDP-L-fucose,we expressed heterologously ?-1,2-fucosyltransferase(Fut C)from Helicobacter pylori to construct de novo pathway engineered strain of 2'-fucosyllactose.Fusion tag-maltose binding protein(MBP)was used for fusion expression of ?-1,2-fucosyltransferase(Fut C).Surprisingly,we found that the expression of the fusion protein in vivo did not affect the function of Fut C,but instead can promote the soluble expression of Fut C.And the shake flask results shown that 30.1mg/L of 2'-fucosyllactose was obtained by engineered strain.However,?-1,2-fucosyltransferase(Fut C)without fusion tag expressed in engineered strain cannot detect the target product 2'-fucosyllactose.To further increase the production of 2'-fucosyllactose(2'-FL),gene(wca J,ack A,edd,eda)were deleted.Compared with the control strain,the production of 2'-fucosyllactose increased 10 times,in the engineered strains ?ack A?wca J.4.In order to further increase the production of 2'-fucosyllactose,a heterologous bifunctional enzyme(FKP)of L-fucose kinase/GDP-L-fucose pyrophosphatase derived from B.fragile 9343 and Fut C were co-expressed in JM109(DE3)strain to construct salvage pathway engineered strain of 2'-fucosyllactose.Like the de novo pathway strain,the engineered strain expressed ?-1,2-fucosyltransferase(Fut C)without fusion tag cannot detect the metabolite 2'-fucosyllactose(2'-FL).But ?-1,2-fucosyltransferase(Fut C)with fusion tag can detect the metabolite 2'-fucosyllactose with 197 mg/L in engineered strain.To further the production of 2'-fucosyllactose(2'-FL),the two genes(arabinose isomerase(Ara A)and rhamnose isomerase(Rha A)),whcih can decompose the substrate L-fucose,were deleted at the same time.The shake flask fermentation shown that the engineered strain can produc 974 mg/L of 2'-fucosyllactose,which was4.7-fold that of the control strain.5.Through bioinformatics,we found a new ?-1,2-fucosyltransferase(Fut C)derived from Kosakonia sp.CCTCCM2018092,and Coexpressed it with L-fucose kinase / GDP-L-fucose pyrophosphorylase bifunctional enzyme(FKP)to to construct engineered strain of p ET-Fut Cks-FKP.The shake flask fermentation shown that the titer of 2'-fucosyllactose(2'-FL)can be detected in culture.6.In order to realize directed evolution of ?-1,2-fucosyltransferase,we constructed a biosensor which can detect concentration of L-fucose to reflect the production of 2'-fucosyllactose.