Construction Of Escherichia Coli Cell Factory For Production Of 2’-fucosyllactose

2’-Fucosyllactose(2’-FL)is one of the most important components in humanmilk,accounting for 31%of humanmilk oligosaccharides,which has important biological significance and physiological roles.2’-FL inhibits pathogen infection,regulates intestinal flora and improves immunity and plays an important role in the growth and development of infants.The use of a metabolic engineering strategy to construct 2’-FL synthesis recombinant strains to achieve efficient 2’-FL synthesis is bound to promote its commercial application.In this study,the 2’-FL synthesis pathway was constructed in E.coli BL21(DE3)Δwca JΔlonΔlac Z,a combinatorial modularization strategy was adopted to optimize the expression pathway,and CRISPR-Cas9 gene editing technology was employed to transform the host to enhance 2’-FL synthesis,which provided strong theoretical support for the industrial application of 2’-FL.The main research contents are as follows:(1)Construction of the heterologous synthesis pathway of 2’-FL by E.coli.The genes encoding key enzymes of the GDP-L-fucose biosynthesis pathway(manB,manC,gmd,wcaG,and fut C)encoding mannose phosphate mutase,guanine phosphate guanosine transferase,GDP-D-mannose 4,GDP-L-fucose synthase and α-1,2-pyrosyltransferase were overexpressed,and the plasmids pRSF-CB-GW and pET-F were obtained.Strain BWLZ01 was obtained by cotransformation of the plasmid E.coli BL21(DE3)Δwca JΔlonΔlac Z,which successfully synthesized 1.18 g/L 2’-FL.(2)Combining a modularization strategy to optimize 2’-FL synthesis.The de novo synthesis pathway of GDP-L-fucose was used as module I,and the fucosylation pathway of GDP-L-fucose and lactose was used as module II.The expression intensity of the module was changed by optimizing plasmid combinations with different replication abilities and different pathway configurations,and the recombinant strain BWLZ13(including plasmid pRSF-CBGW-F)was obtained.The 2’-FL yield of this strain was 2.56 g/L,2.17 times higher than that of the original strain.(3)Optimize the synthetic pathway of 2’-FL.The recombinant strain BWLZ16(including plasmid pRSF-GWCB-F)was constructed by optimizing the expression sequence of manC,manB,gmd and wcaG in the operon in module I.The yield of 2’-FL reached 2.94 g/L,which was 15% higher than that of strain BWLZ13.By optimizing the RBS intensity of the module I gene expression element,the recombinant strain BWLZ25(including plasmid pRSF-(RWT)GW(R34)C(R34)B-F)was obtained,and its 2’-FL yield was further increased to 3.73 g/L,which was 27% higher than that of strain BWLZ16.The expression of protein FutC in module Ⅱ was preferably enhanced by the promoter and protein solubilization tag,and the recombinant strain BWLZ30(including plasmid pRSF-(RWT)GW(R34)C(R34)B-(DDD)F)was obtained.Its 2’-FL yield was increased to 4.02 g/L,which was 8% higher than that of strain BWLZ25.(4)Chassis host modification.Fifteen bypass genes were screened by analyzing the 2’-FL metabolic network.By using the CRISPR-Cas9 gene editing technique,the yields of iclR(encoding a glyoxylate operon repressor)and ldhA(encoding D-lactate dehydrogenase)reached 5.25 g/L,which was 31% higher than that of strain BWLZ30,indicating that the optimization of bypass metabolism reduced substrate loss and byproduct production.The metabolic flux was directed to the target product 2’-FL.(5)Improving 2’-FL production by fed-batch fermentation.To verify the industrial production potential of the optimal strain 2’-FL,the recombinant strain E20 was fed in a 5 L fermenter.After 90 hours of fermentation,the yield of 2’-FL reached 40.05 g/L,indicating that the constructed strain has good application potential.