Metabolic Engineering To Synthesize 2'-fucosyllactose In Escherichia Coli

2'-Fucosyllactose(2'-FL)was the most abundant HMO of the total HMOs.It plays an important role in promoting infants'health.For newborns,it has the effect of modulation of the immune system and promotion of brain development.2'-FL has received considerable attention because of its pontential application values in pharmaceutical and food industries.2'-FL can be synthesized by chemical method,but the strict requirements for protection and deprotection procedures make the large-scale 2'-FL production challenging.Enzymatic synthesis involves expensive and complex procedures for the preparation of fucosylated donor,which renders this approach inapplicable for the mass production of 2'-FL.Currently,the cell factory method uses microorganisms such as Escherichia coli as fermentation strains and uses cheaper raw materials as fermentation substrates,which provides more potentials for large-scale 2'-FL production.In this study,the de novo and salvage pathways for GDP-fucose synthesis were engineered and optimized in Escherichia coli BL21(DE3)to improve the production of 2'-FL.Then,the genes in bypass pathways,were inactivated to improve 2'-FL production.In addition,the shake fermentation conditions were optimized.Finally,the best 2'-FL producing strain BWLF3 was cultivated in 3-L fermenter to further enhance the titer of 2'-FL.The main results of this study are as follow:(1)E.coli BL21(DE3)was used as a host strain to construct three metabolically engineering strains harboring the de novo pathway,salvage pathway,and combinational pathway,respectively.These three strains,labeled as 21-bcgw(harboring plamid p AC-BCGW),21-fkp(harboring plamid p ET-F),and 21-bcgw F(harboring plamid p AC-BCGW and p ET-F),respectively.By comparison,strain 21-bcgw F produced the highest titer of GDP-fucose,and was 2-fold and 1.48-fold of that produced by 21-bcgw and 21-fkp.(2)To construct the biosynthetic pathway of 2'-FL in E.coli BL21(DE3),the fuc T2 gene from Helicobacter pylori,encoding?-1,2-fucosyltransferase,was cloned into p ET-F to achieve high metabolic flux toward 2'-FL.Two plasmids,p AC-BCGW and p ET-FF,were cotransformed into E.coli BL21(DE3)to create Strain1.The SDS-PAGE result revealed that the genes from the de novo and salvage pathways for GDP-fucose production(man C,man B,gmd,wca G,and fkp),and fuc T2,were successfully expressed in E.coli.After 70 h of microbial fermentation,Strain1 produced 0.22 g/L 2'-FL.(3)To enhance higher metabolic flux toward 2'-FL biosynthesis,the genes in bypass pathways,including lac Z,fuc I,fuc K,and wca J,were inactivated by using CRISPR-Cas9system.The p AC-BCGW and p ET-FF plasmids were introduced to BW(?wca J),BWL(?wca J/?lac Z),and BWLF(?wca J/?lac Z/?fuc IK)strains to construct three engineered strains,BW1,BWL1,and BWLF1.The titer of 2'-FL produced by BWLF1 was the highest,and it was10.36-fold of that produced by Strain1.With the deletion of gene lac Z,the yield of 2'-FL from lactose enhanced to 36%in BWL1 strain.Compared to BWL1,the yield of 2'-FL produced by BWLF1 increased 19%.(4)A combinatorial optimization of gene expression levels was also employed toward the key genes from 2'-FL biosyhthetic pathways to enhance 2'-FL prduction.Two clusters man C-man B and gmd-wca G from the de novo pathway on GDP-fucose synthesis were placed into a single plasmid,and the genes fuc T2 and fkp were placed into another one.The expression levels of genes were regulated by varying plasmid copy numbers.The results revealed that BWLF3strain(harboring plamids p CD-BCGW and p ET-FF)produced the highest titer of 2'-FL,and the titer was 10%higher than that produced by BWLF1(harboring plamids p AC-BCGW and p ET-FF).(5)Finally,shaking flask fementation conditions,including induction OD_600,IPTG concentration,induction temperature and carbon resources were optimized.The fementation results revealed that the optimum induction temperature was 37°C,and the optimum IPTG concentration was 0.2 m M,and the optimum induction time was OD₆₀₀ 1.2.Then,the best 2'-FL producing strain BWLF3 was cultivated in optimized shake fementation conditions with 20g/L glycerol.The titer of 2'-FL reached 2.62 g/L in optimized shake-flask fermentation condition.After cultivated in 3 L fermenter by fed-batch,the final production of 2'-FL increased to 14.1 g/L.