| Human milk oligosaccharides(HMOs)play an important physiological role in the growth of infants and children,three of them,lacto-N-neotetraose(LNn T),lacto-N-tetraose(LNT),and lacto-N-fucopentaose Ⅰ(LNFP Ⅰ)have received widespread attention because of their high content in human milk,and studies have continuously confirmed their physiological value as bioactive components.The above three HMOs have been approved to be added to some food as novel food ingredients,the market demand of domestic and international is still expanding,therefore,how to meet the market demand for HMOs has become the focus.Currently,the main way to obtain the above three HMOs is to produce them by microbial synthesis.E.coli BL21(DE3)is well characterized at the phenotypic and genotypic level,which is routinely used in the manufacture of food and pharmaceutical ingredients.Derivatives from the host organism E.coli BL21(DE3)have proven their safety as LNn T and LNT production strains in metabolism,toxicology and genotoxicity studies.In this paper,a genetically stable LNTri II-producing strain was generated by genetically engineering,based on this,microbial synthetic strains that can efficiently produce LNn T,LNT,and LNFP Ⅰ were generated respectively,the main research results are as follows:(1)Genetically stable LNTri II-producing strains were constructed for the biosynthesis of LNn T,LNT,and LNFP I.By knocking out the gene lac Z to delete the lactose intracellular degradation pathway.Bacterial multicopy chromosomal integration using CRISPR-associated transposases insert the recombinant Ptac-lgt A cassette into the the IS186 locus in the E.coli BL21(DE3)chromosome to allow Lgt A to be expressed in E.coli BL21(DE3).The endogenous-generated UDP-Glc NAc and exogenous-added lactose constructed the de novo biosynthesis pathway of LNTri II,then six engineered E.coli BL21(DE3)were obtained,and the highest LNTri II titer was 6.83 g/L compared to the biosynthetic capacity of LNTri II.(2)Integration of the gene gal E into the E.coli BL21(DE3)genome to generate the precursor UDP-Gal,six host strains with different copies of lgt A inserted into the IS186 locus of E.coli BL21(DE3),paired with overexpression of the heterologous gene Aa-β1,4-Gal T using Duet-1 series plasmid vectors with different copy numbers.The strain EA21,which inserted three copies of lgt A into the chromosomal locus and contained plasmid p AC-Aa,was the best strain for LNn T biosynthesis in the optimised combination,with an LNn T titer of 0.88 g/L.The synthesis of LNn T in engineered E.coli BL21(DE3)was achieved through the expression of two key enzymes Lgt A and Aa-β1,4-Gal T.By knocking out the gene nag B encoding glucosamine-6-phosphate deaminase and the gene wec B encoding UDP-N-acetylglucosamine-2-epimerase in the UDP-Glc NAc synthesis pathway,the LNn T titer of strain EA37 was 0.98g/L,an increase of 11.6% compared to the control strain EA21.(3)On the basis of stable synthesis of LNTri II strain,gal E gene was integrated into the genome and introduced the β1,3-galactotransferase Wbg O derived from E.coli O55:H7 by the plasmid p CD-wbg O constructed the de novo biosynthesis pathway for LNT,and the titer of LNT in shake flask of strain EB05 reached 4.97 g/L,by blocked the competitive pathway between UDP-Glc NAc and UDP-Gal,the titer of LNT was successfully increased to 6.07 g/L.(4)The biosynthetic pathway of LNFP I was successfully constructed by introduced the heterologous GDP-Fuc pathway and α1,2-fucosyltransferase based on the efficient synthesis of LNT strains.The de novo and salvage pathways of GDP-Fuc were introduced into highly efficient LNT-producing chassis strains,the comparison revealed that the de novo pathway of GDP-Fuc was more favourable for LNFP I synthesis.Seven reported α1,2-fucosyltransferases were screened by comparing their characteristics for LNFP I biosynthesis and found that Wbs J derived from E.coli O128 increased the LNFP I titer to 2.53 g/L and without by-product 2’-FL produced in LNFP I biosynthesis. |
