Construction Of Lactoyl-N-neotetracose-producing Strain And Fermentation Process Research

Lacto-N-neotetraose(LNn T)is a kind of neutral,non-rocky glycosylated human milk oligosaccharides(HMOs),accounting for about 6% of human milk oligosaccharide content.It is widely used in infant formula,geriatric food,functional beverages and health products because of its prebiotic,immunomodulatory,antibacterial and antiviral physiological functions.Currently,LNn T has been approved by the U.S.FDA(U.S.Food and Drug Administration)and the European Union as a Generally Recognized as Safe(GRAS)and Novel Food(NF)to be added to infant formula.LNn T has not yet been officially approved in mainland China,however,the importance of LNn T in China is increasing day by day,and its market space is vast and its application potential is huge.Traditional chemical and enzymatic synthesis of LNn T has problems such as cumbersome reaction process and expensive key precursors.Microbial cell factory preparation of LNn T can better meet the needs of low cost,simple process,low carbon and environmental protection for industrial production.The production of LNn T using integrated E.coli BL21(DE3)is still not reported.In this project,we have systematically modified the chassis microorganism E.coli BL21(DE3)through gene screening,metabolic pathway reconstruction,module optimization,weakening substrate and key precursor branching pathway,RBS optimization of rate-limiting enzyme,and construction of antibacterial-free strain and other genetic manipulation to achieve the construction of plasmid-based and integrated LNn T production strains.The research provides new ideas for solving the key problems of efficient biosynthesis of LNn T and the development of other HMOs and other heterosaccharides.The main research contents and results are as follows:(1)A microbial cell factory for ab initio synthesis of LNn T was constructed based on the p CDFDuet-1 and p ETDuet-1 dual plasmid expression system using strain E.coli BL21(DE3)as the host strain.Gene screening and plasmid construction were performed for key enzyme genes(glm S,glm M and glm U)of the UDP-Glc NAc module and key enzyme genes(pgi,pgm,gal U and gal E)of the UDP-galactose module.strains B-R1(containing glm M),B-R2(containing glm U),B-R3(containing glm S)LNn T titers were 0.354 g/L,0.214 g/L,and 0.456g/L,respectively,with the glm S-containing strain B-R3 showing better performance in LNn T production;the UDP-galactose modules B-C1(containing pgi),B-C2(containing pgm),B-C3(containing gal U),B-C4(containing gal E),and B-C5(containing gal U-gal E)LNn T titers were0.2836 g/L,0.2534 g/L,0.6198 g/L,0.742 g/L,and 0.3736 g/L,respectively,with the better performance of LNn T production by gal E-containing strain B-C4.(2)To analyze the competitive pathway,the competing target genes lac Z and wec B in E.coli BL21(DE3)were deleted using the CRISPR-Cas9 gene editing system to block substrate lactose degradation and promote intracellular supply of intermediate UDP-Glc NAc to maximize the flow of carbon flux to the biosynthetic pathway of LNn T,and to study the production of recombinant bacteria at the shake flask level The fermentation performance of LNn T production by recombinant bacteria at the shake flask level was investigated.Recombinant strains BL21(DE3)Δlac Z,BL21(DE3)Δwec B and BL21(DE3)Δlac ZΔwec B were obtained,respectively.the yields of LNn T obtained by the above three defective bacteria were0.350,0.315 and 0.452 g/L,respectively,which were increased by 26%,14% and 64%compared to the control strain(0.276 g/L),14% and 64%,respectively,compared to the control strain(0.276 g/L).(3)The pathway genes were assembled by plasmid expression system and expression vectors with different copy numbers were constructed to increase the expression levels of upstream key genes and downstream glycosyltransferases to compare the LNn T production performance of different combinations of strains.The media(M9,M9 CA,MOPS and DM)and induction conditions(temperature,concentration and time)were optimized for the best combination of strains and fermentation was performed in 3L fermenters with supplemental fractions.The fermentation results showed that the yields of LNn T after shake flask fermentation of a total of 12 strains from SA1 to 12 were 1.168 g/L,0.423 g/L,0.693 g/L,0.187g/L,0.645 g/L and 0.452 g/L,0.365 g/L,0.420 g/L,0.463 g/L,0.105 g/L,0.243 g/L,0.243 g/L The highest yield of 1.168 g/L was obtained for the engineered bacterium SA1 containing recombinant plasmids p ET-glm S-gal E and p RSF-lgt A-Nmlgt B.The optimal culture conditions for the combined strains were the use of DM medium,induction temperature of 25°C,induction concentration of 0.2 m M,induction time of 8 h,and the highest yield of 14.25 g/L of fermentation in a 3L fermenter with supplemental fractionation.(4)The expression intensity and translation initiation rate of the rate-limiting enzyme β-1,4-galactosyltransferase were screened by RBS and fine-tuned to study the production level of LNn T containing different RBS engineered bacteria at the shake flask level,and the best screened strains were subjected to supplemental fermentation in 3 L fermenters to make the strains have the potential for industrial application.The strain containing RBS(BBa＿B0064)gave the best results,producing 3.34 g/L of LNn T.The supplemental batch fermentation in 3 L fermenters eventually produced 15.68 g/L of LNn T,which was 4.715 times higher than that of the shake flask culture.(5)The metabolic network of E.coli BL21(DE3)Δlac ZΔwec B was remodeled and an LNn T biosynthetic pathway was constructed on the genome.To prevent degradation of the key intermediate UDP-galactose,the ugd gene was deleted and the gal E gene was integrated at this locus;to inhibit the effect of the by-product acetic acid on product synthesis,the pox B and ack A-pta genes were deleted and the lgt A and glm S genes were integrated at this locus;in addition,the key rate-limiting enzyme gene Nmlgt B was integrated into the glk locus.The LNn T production capacity of each strain was tested at shake flask and fermenter levels without the addition of antibiotics and inducers.the yields of BZW3 and BZW4 were 0.23 g/L and 0.73g/L,respectively.the transmission stability test of strains BZW3 and BZW4 showed that the titers of LNn T of strains BZW3 and BZW4 at generation 6 were 0.21 and 0.76 g/L,which were91.3% and 95.9% of the yields of generation 0 strains.For the high-density supplemental fermentation of the engineered strain BZW4 in 3 L fermenter in p H-stat mode,the resistant gene-free engineered bacterium BZW4 produced 2.42 g/L of LNn T at the end of the fermentation,which was 1.40 times higher than that at the shake flask level.