Genetically engineered bacteria for efficient synthesis of lacto-n-difucohexaose and production method thereof

Abstract

This invention discloses a genetically engineered bacterium and its production method for efficiently synthesizing lactose-N-difucotetrasaccharide, belonging to the field of genetic engineering technology. Utilizing synthetic biology techniques, this invention combines and regulates manB, manC, gmd, and wcaG in the GDP-L-fucose synthesis pathway within a host bacterium. Based on this, mutants of the rate-limiting enzymes α-1,2-fucosyltransferase and α-1,3-fucosyltransferase are heterologously expressed. By optimizing pathway genes using plasmid copy number modules and upregulating the expression of the rate-limiting enzymes (enzyme source screening, truncation / extension mutations, site-directed mutagenesis, insertion site pattern optimization, and chromosome integration), a genetically engineered strain capable of efficiently producing lactose-N-difucotetrasaccharide de novo was obtained. The strain exhibits low production costs and high product yield, demonstrating significant potential for industrial-scale production.

Description

Technical Field

[0001] This invention relates to a genetically engineered bacterium and its production method for efficiently synthesizing lactose-N-difucotetrasaccharide, belonging to the field of genetic engineering technology. Background Technology

[0002] Human milk oligosaccharides (HMOs) are a family of structurally diverse non-conjugated polysaccharides naturally found in breast milk. They are not easily digested and absorbed by infants and can act as effective anti-adhesion and antibacterial agents, prebiotics, and regulators of inflammatory immune cell response cascades to improve neonatal health. Several simple HMOs, including 2'-fucosylated lactose (2'-FL), 3-fucosylated lactose (3-FL), lactose-N-tetrasaccharide (LNT), and lactose-N-neotetrasaccharide (LNnT), have been widely used in engineered microorganisms. Lactose-N-difucotetrasaccharide (LDFT) is an important component of fucosylated HMOs, its structure consisting of a core lactose unit with fucosylation at the C2' and C3 positions. Currently, LDFT has been approved by the U.S. Food and Drug Administration (FDA) as a Generally Recognized As Safe (GRAS) food and can be added to food as a novel food ingredient. Related studies have shown that LDFT can effectively prevent diarrhea caused by Campylobacter jejuni in newborns and inhibit platelet-induced inflammation. As a gastrointestinal and immunomodulator, the potential therapeutic applications of LDFT require further investigation. To further elucidate its mechanism of action, a large quantity of structurally homogeneous compounds of this type are needed. However, the yield from natural products is extremely low, and the high cost and limited market supply of LDFT (US$1400 / 10mg, Biosynth Carbosynth) hinder biological research. Therefore, finding a more efficient, environmentally friendly, and economical production method to obtain LDFT is a problem that needs to be solved.

[0003] There are three main methods for synthesizing LDFT: chemical synthesis, enzymatic synthesis, and fermentation synthesis. Chemical synthesis of LDFT requires numerous cumbersome protection and deprotection steps. Enzymatic synthesis is costly and unsuitable for industrial production due to the high price and low yield of the donor substrate, nucleotide sugars. Biosynthesis of LDFT using engineered bacteria allows for de novo preparation from inexpensive carbon sources (glucose, glycerol, lactose), a process characterized by its greenness, cleanliness, and sustainability. To date, only Zhang et al. and Lee et al. have synthesized LDFT in *E. coli* using a salvage synthesis pathway (Zhang et al., 2021; Lee et al., 2022). This method uses L-fucose as a substrate, involves ATP and GTP, and has high production costs and low yields, making industrial-scale production of LDFT still challenging. This invention aims to utilize synthetic biology techniques to achieve low-cost construction of antibiotic-free and antibiotic-resistant strains and efficient synthesis of LDFT through strategies such as reconstructing the de novo LDFT synthesis pathway, weakening metabolic branching pathways, optimizing pathway genes, and upregulating the expression of rate-limiting enzymes (enzyme source screening, truncation / extension mutations, site-directed mutagenesis, insertion site pattern optimization, and chromosome integration). This research method will enrich and develop the technical research on microbial metabolic regulation, providing new methods and cases for reconstructing microbial metabolic networks and improving carbon economy, and offering new ideas for the rational design and construction of next-generation microbial cell factories. It has significant value in both theoretical and practical applications. Summary of the Invention

[0004] [Technical Issues]

[0005] Existing technologies for producing lactose-N-difucotetrasaccharide are costly and have low yields, making them insufficient for industrial-scale production. They also fail to provide strains for efficient production of lactose-N-difucotetrasaccharide, nor do they offer cost-effective, green, and efficient methods for its production.

[0006] [Technical Solution]

[0007] This invention utilizes synthetic biology techniques, using inexpensive glycerol as a substrate, to de novo synthesize the key intermediate GDP-L-fucose through several consecutive biocatalytic processes. Subsequently, GDP-L-fucose and lactose undergo a two-step fucosylation process catalyzed by dual-enzyme catalysis of α-1,2-fucosyltransferase (α-1,2-FucT) and α-1,3-fucosyltransferase (α-1,3-FucT), ultimately synthesizing the high-value-added product LDFT. This method offers valuable insights into overcoming the technological bottlenecks of LDFT, providing an effective pathway for its mass production. It also possesses significant theoretical research value and socio-economic benefits, with broad market development prospects. Furthermore, to address the low yield of lactose-N-difucotetrasaccharide synthesized by existing biological methods, this invention provides a genetically engineered bacterium for producing lactose-N-difucotetrasaccharide and its construction method.

[0008] The first objective of this invention is to provide a chassis cell I, which uses Escherichia coli BZWNDPAL as a host and knocks out the glutamine-fructose-6-phosphate aminotransferase gene glmS.

[0009] The second objective of this invention is to provide a chassis cell II, which uses chassis cell I as a host, knocks out the ubiquinone-dependent pyruvate dehydrogenase gene poxB and the phosphoacetyltransferase-acetylkinase gene cluster pta-ackA, and integrates the expression of two copies of α-1,2-fucosyltransferase HpfutC and two copies of α-1,3-fucosyltransferase HpM32.

[0010] In one embodiment, the α-1,2-fucosyltransferase HpfutC is (a) or (b):

[0011] (a) The amino acid sequence of HpfutC is as shown in SEQ ID NO.15;

[0012] (b) Mutants based on (a): K102T, R105C, Y251F, K282E, K102T / R105C, Y251F / K282E, R105C / K282E, K102T / R105C / K282E or K102T / R105C / Y251F / K282E.

[0013] In one embodiment, (b) the amino acid sequence of the mutant is shown in SEQ ID NO.16 to SEQ ID NO.24.

[0014] In one embodiment, the α-1,3-fucosyltransferase HpM32 is any one of (a) to (c):

[0015] (a) The amino acid sequence of HpM32 as shown in SEQ ID NO.1;

[0016] (b) The truncated version obtained by truncating (a): the C-terminal 7-peptide repeat sequence of HpM32 was truncated by 49, 35, 14 and 7 amino acids respectively.

[0017] (c) The extended form obtained by extending (a): the C-terminal 7-peptide repeat sequence of HpM32 was extended by 7, 14, 21 and 28 amino acids, respectively.

[0018] In one embodiment, (b) the amino acid sequences of the mutant are shown as SEQ ID NO.7 to SEQ ID NO.10, respectively.

[0019] In one embodiment, in (c), the amino acid sequences of the mutants are shown as SEQ ID NO.11 to SEQ ID NO.14, respectively.

[0020] The third objective of this invention is to provide an engineered bacterium that produces lactose-N-difucotetrasaccharide, using chassis cell I or chassis cell II as a host, and freely expressing the phosphogannatase gene manB, mannose-1-phosphogguanine syltransferase gene manC, GDP-mannose-6-dehydrogenase gene gmd, GDP-fucose synthase gene wcaG, α-1,2-fucosyltransferase HpfutC, and α-1,3-fucosyltransferase HpM32.

[0021] In one embodiment, the α-1,2-fucosyltransferase HpfutC is (a) or (b):

[0022] (a) The amino acid sequence of HpfutC is as shown in SEQ ID NO.15;

[0023] (b) Mutants based on (a): K102T, R105C, Y251F, K282E, K102T / R105C, Y251F / K282E, R105C / K282E, K102T / R105C / K282E or K102T / R105C / Y251F / K282E.

[0024] In one embodiment, (b) the amino acid sequence of the mutant is shown in SEQ ID NO.16 to SEQ ID NO.24.

[0025] In one embodiment, the α-1,3-fucosyltransferase HpM32 is any one of (a) to (c):

[0026] (a) The amino acid sequence of HpM32 as shown in SEQ ID NO.1;

[0027] (b) The truncated version obtained by truncating (a): the C-terminal 7-peptide repeat sequence of HpM32 was truncated by 49, 35, 14 and 7 amino acids respectively.

[0028] (c) The extended form obtained by extending (a): the C-terminal 7-peptide repeat sequence of HpM32 was extended by 7, 14, 21 and 28 amino acids, respectively.

[0029] In one embodiment, (b) the amino acid sequences of the mutant are shown as SEQ ID NO.7 to SEQ ID NO.10, respectively.

[0030] In one embodiment, in (c), the amino acid sequences of the mutants are shown as SEQ ID NO.11 to SEQ ID NO.14, respectively.

[0031] In one implementation, manB, manC, gmd, and wcaG are expressed in free form using plasmids pRSFDuet-1, pETDuet-1, or pCDFDuet-1.

[0032] In one embodiment, HpM32 and dual-tandem HpfutC are expressed in free form using plasmids pRSFDuet-1, pETDuet-1, or pCDFDuet-1.

[0033] In one implementation, the original RBS of plasmids pRSFDuet-1, pETDuet-1, or pCDFDuet-1 is replaced with BBa_B0034.

[0034] In one embodiment, the tandemly linked HpfutC and HpM32 are inserted into the MCS1 and MCS2 regions of the plasmid, respectively.

[0035] In one embodiment, two copies of HpfutC are tandemly linked in the MCS1 region of the plasmid, and HpM32 is linked in the MCS2 region.

[0036] In one implementation, after knocking out the poxB site, the dual-tandem α-1,2-fucosyltransferase HpfutC is integrated and expressed at the poxB site.

[0037] In one implementation, after knocking out the pta-ackA site, the dual-tandem α-1,3-fucosyltransferase HpM32 is integrated and expressed at the pta-ackA site.

[0038] In one implementation, strong promoters are used to replace the promoters of the genes encoding manA, manC, manB, and gmd-wcaG in the E. coli genome.

[0039] In one implementation, a strong promoter is used to initiate the expression of HpfutC and HpM32.

[0040] In one implementation, the strong promoter includes the T7 promoter.

[0041] In one embodiment, the Escherichia coli is BL21(DE3)ΔlacZΔwcaJΔnudDΔpfkAΔlonΔglmS.

[0042] In one embodiment, the gene ID of the glutamine-fructose-6-phosphate aminotransferase gene glmS is 948241, the gene ID of the ubiquinone-dependent pyruvate dehydrogenase gene poxB is 946132, the gene ID of the phosphoacetyltransferase gene pta is 946778, the gene ID of the acetate kinase gene ackA is 946775, the gene ID of the mannose-6-phosphate isomerase gene manA is 944840, the gene ID of the phosphomannose mutase gene manB is 946574, the gene ID of the mannose-1-phosphate guanine transferase gene manC is 946580, the gene ID of the GDP-mannose-6-dehydrogenase gene gmd is 946562, and the gene ID of the GDP-fucose synthase gene wcaG is 946563.

[0043] The fourth objective of this invention is to provide a genetically engineered bacterium that produces 2'-fucosylated lactose or 3-fucosylated lactose, using chassis cell I or chassis cell II as the host, and freely expressing manB, manC, gmd, wcaG, and HpfutC or HpM32.

[0044] In one embodiment, genetically engineered bacteria expressing HpfutC in free form produce 2'-fucosylated lactose, and genetically engineered bacteria expressing HpM32 in free form produce 3-fucosylated lactose.

[0045] In one embodiment, the α-1,2-fucosyltransferase HpfutC is (a) or (b):

[0046] (a) The amino acid sequence of HpfutC is as shown in SEQ ID NO.15;

[0047] (b) Mutants based on (a): K102T, R105C, Y251F, K282E, K102T / R105C, Y251F / K282E, R105C / K282E, K102T / R105C / K282E or K102T / R105C / Y251F / K282E.

[0048] In one embodiment, (b) the amino acid sequence of the mutant is shown in SEQ ID NO.16 to SEQ ID NO.24.

[0049] In one embodiment, the α-1,3-fucosyltransferase HpM32 is any one of (a) to (c):

[0050] (a) The amino acid sequence of HpM32 as shown in SEQ ID NO.1;

[0051] (b) The truncated version obtained by truncating (a): the C-terminal 7-peptide repeat sequence of HpM32 was truncated by 49, 35, 14 and 7 amino acids respectively.

[0052] (c) The extended form obtained by extending (a): the C-terminal 7-peptide repeat sequence of HpM32 was extended by 7, 14, 21 and 28 amino acids, respectively.

[0053] In one embodiment, (b) the amino acid sequences of the mutant are shown as SEQ ID NO.7 to SEQ ID NO.10, respectively.

[0054] In one embodiment, in (c), the amino acid sequences of the mutants are shown as SEQ ID NO.11 to SEQ ID NO.14, respectively.

[0055] The fifth object of the present invention is to provide a method for producing lactose-N-difucotetrasaccharide, wherein the method involves fermenting lactose-N-difucotetrasaccharide using the engineered bacteria that produce lactose-N-difucotetrasaccharide as the fermentation strain.

[0056] In one embodiment, the method involves inoculating the engineered bacteria into a fermentation system using glycerol and glucose as carbon sources to produce lactose-N-difucotetrasaccharide.

[0057] In one embodiment, the strain is cultured in a shake-flask fermentation system to OD. 600 =0.6±0.1; add IPTG to a final concentration of 0.2-0.5mM, and simultaneously add lactose to a final concentration of 5-12g / L, and induce culture at 20-30℃ and 150-220rpm for no less than 60h.

[0058] In one embodiment, the strain is cultured in a fermenter system to OD. 600 =20±3; add IPTG to a final concentration of 0.2~0.5mM, and simultaneously add lactose to a final concentration of 5~12g / L, at 20~30℃, control the pH of the fermentation system to 6.5~7.0; control dissolved oxygen at 30%±5%, and ferment for no less than 70h.

[0059] In one embodiment, the fermentation medium comprises 20-40 g / L glycerol, 1-5 g / L casein amino acids, 4-10 g / L yeast extract, 1-5 g / L potassium dihydrogen phosphate, 5-10 g / L disodium hydrogen phosphate, 0.1-1.0 g / L ammonium chloride, 0.1-1.0 g / L sodium chloride, 1-1.5 g / L magnesium sulfate heptahydrate, 5-20 mg / L thiamine hydrochloride, and 0.1-1.0 mL / L trace metal solution.

[0060] In one embodiment, the trace metal solution comprises 20-30 g / L ferric chloride hexahydrate, 1-5 g / L calcium chloride dihydrate, 1-5 g / L zinc chloride, 1-5 g / L sodium molybdate dihydrate, 1-5 g / L copper sulfate pentahydrate, 1-5 g / L manganese sulfate monohydrate, and 0.1-1.0 g / L boric acid.

[0061] A sixth object of the present invention is to provide the application of said chassis cell I or said chassis cell II or said engineered bacteria producing lactose-N-difucotetrasaccharide or said method in the preparation of lactose-N-difucotetrasaccharide.

[0062] The present invention also provides the use of said chassis cell I or said chassis cell II in the preparation of human milk oligosaccharides.

[0063] In one embodiment, the human milk oligosaccharide comprises fucose-based lactose.

[0064] In one embodiment, the fucoidyl lactose is 2'-fucosyl lactose, 3-fucosyl lactose, or lactose-N-difucotetrasaccharide.

[0065] The beneficial effects of this invention are:

[0066] This invention involves the combined regulation of the phosphogannatase gene manB, mannose-1-phosphate guanine transferase gene manC, GDP-mannose-6-dehydrogenase gene gmd, and GDP-fucose synthase gene wcaG in the GDP-L-fucose synthesis pathway within the host bacterium *Escherichia coli* BL21(DE3)ΔlacZΔwcaJΔnudDΔpfkAΔlonΔglmS. Based on this, α-1,2-fucosyltransferase and α-1,3-fucosyltransferase mutants were heterologously expressed. By optimizing pathway genes using plasmid copy number modules and upregulating the expression of rate-limiting enzymes (enzyme source screening, truncation / extension mutagenesis, site-directed mutagenesis, insertion site pattern optimization, and chromosome integration), a genetically engineered strain capable of de novo, high-efficiency production of lactose-N-difucotetraose was obtained.

[0067] Through shake-flask fermentation, the ability of the genetically engineered bacteria constructed in this application to produce lactose-N-difucotetrasaccharide in shake flasks was increased from the initial 2.18 g / L to 4.68 g / L; in a 3L fermenter, the yield of lactose-N-difucotetrasaccharide reached 41.02 g / L, which is the highest yield reported to date. This invention lays the foundation for the industrial production of lactose-N-difucotetrasaccharide. Attached Figure Description

[0068] Figure 1 This is a metabolic pathway diagram of lactose-N-difucotetrasaccharide.

[0069] Figure 2 This is a comparative HPLC chromatogram of lactose-N-difucotetrasaccharide standard and fermentation sample.

[0070] Figure 3 Schematic diagram of truncation or extension of α-1,3-fucosyltransferase and the effect of mutants on LDFT generation.

[0071] Figure 4 Feed batch fermentation was carried out in a 3L fermenter for strain BDFL53. Detailed Implementation

[0072] The specific implementation of the present invention will be further described below with reference to examples and accompanying drawings.

[0073] 1. The plasmids, PCR reagents, restriction endonucleases, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercial products, and the specific operations are performed according to the kit instructions; the implementation of the present invention is not limited thereto, and other experimental operations and process parameters not specified are performed according to conventional techniques.

[0074] 2. Vectors pRSFDuet-1, pETDuet-1, and pCDFDuet-1 were purchased from Addgene.

[0075] 3. The sequencing of DNA products and plasmids was outsourced to Tianlin Biotechnology (Wuxi) Co., Ltd.

[0076] 4. Preparation of competent Escherichia coli cells: Shanghai Sangon Biotech Co., Ltd. reagent kit.

[0077] 5. LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0078] 6. LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar powder.

[0079] 7. Fermentation medium: Glycerol 30.0 g / L, casein amino acids 2.0 g / L, yeast extract 5.0 g / L, potassium dihydrogen phosphate 3.0 g / L, disodium hydrogen phosphate 6.8 g / L, ammonium chloride 1.0 g / L, sodium chloride 0.5 g / L, magnesium sulfate heptahydrate 1.4 g / L, thiamine hydrochloride 10.0 mg / L, trace metal solution 1.0 mL / L (ferric chloride hexahydrate 25.0 g / L, calcium chloride dihydrate 2.3 g / L, zinc chloride 2.6 g / L, sodium molybdate dihydrate 2.6 g / L, copper sulfate pentahydrate 2.0 g / L, manganese sulfate monohydrate 2.5 g / L, boric acid 0.7 g / L), pH 6.8.

[0080] 8. Shake-flask fermentation conditions for the strains in the following examples: A single colony of the engineered strain was inoculated into LB liquid medium and cultured in a shake flask at 37°C and 200 rpm for 12 h to obtain a seed culture; the seed culture was then inoculated into 50 mL of fermentation medium at an inoculation rate of 3% (v / v) and cultured in a shake flask at 37°C and 200 rpm until OD reached. 600 The concentration was 0.6; isopropyl-β-D-thiopyranogalactopyranoside (IPTG) was added to a final concentration of 0.3 mM, and lactose was added simultaneously to a final concentration of 8 g / L. The mixture was induced and cultured at 25 °C and 200 rpm for 72 h to obtain the fermentation broth. The content of lactose-N-difucotetraose was determined by HPLC. The fermentation conditions for 2'-fucosyllactose and 3-fucosyllactose were the same as those for the shake-flask fermentation of lactose-N-difucotetraose.

[0081] 9. Determination of Lactose-N-difucotetraose, 2'-fucosyllactose, and 3-fucosyllactose: HPLC was used. 1 mL of fermentation broth was boiled at 100℃ for 10 min, centrifuged at 12000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm membrane. HPLC was used to detect the production of lactose-N-difucotetraose, 2'-fucosyllactose, and 3-fucosyllactose, as well as the consumption of lactose and glycerol. HPLC detection conditions: Differential refractive index detector; Rezex ROA-organic acid column (Phenomenex, USA); column temperature 50℃; mobile phase 0.005 mol / L H₂SO₄ aqueous solution; flow rate 0.6 mL / min; injection volume 10 μL.

[0082] Example 1: CRISPR-Cas9 gene editing technology to knock out the glmS gene on the E. coli chromosome

[0083] Analysis of the de novo synthesis pathway of lactose-N-difucotetraose suggests that the branching pathway involving the conversion of the key intermediate fructose-6-phosphate to UDP-acetylglucosamine (a key intermediate in the synthesis of LNT, LNnT, 3'-SL, and 6'-SL) may hinder the biosynthesis and accumulation of LDFT. Figure 1 (As shown). Therefore, this embodiment uses Escherichia coli BL21(DE3)ΔlacZΔwcaJΔnudDΔpfkAΔlon(BZWNDPAL) as the starting strain (the construction method of strain BZWNDPAL has been disclosed in patent document with publication number CN114480240A). The glutamine-fructose-6-phosphate aminotransferase gene glmS in the genome of Escherichia coli BZWNDPAL was knocked out using the CRISPR-Cas9 gene knockout system. The specific steps are as follows (the primer sequences involved are shown in Table 1):

[0084] (1) The specific target gRNA (20 bp) of the glmS gene was found through http: / / www.regenome.net / cas-offinder. PCR amplification was performed using glmS-gRNA_F / glmS-gRNA_R primers with pTargetF plasmid (Addgene: #62226) as a template. The amplified product was digested with restriction endonuclease Dpn I to remove excess circular plasmid pTargetF. The amplified product was then transformed into E. coli DH5α competent cells, and the plasmid was extracted and identified by sequencing. The successfully constructed knockout plasmid was named pTargetF-glmS.

[0085] (2) Using the genome of Escherichia coli BZWNDPAL as a template, homologous arm sequence fragments were amplified by primers glmS-US_F / glmS-US_R for the upstream homologous arm and glmS-DS_F / glmS-DS_R for the downstream homologous arm. After purification and recovery of the products, the upstream and downstream homologous arm fragments were ligated by primers glmS-US_F / glmS-DS_R using SOE-PCR to obtain a gene homologous repair template.

[0086] (3) Electroporate pCas9 plasmid (Addgene:#62225) into E. coli BZWNDPAL electroporation competent cells. After placing on ice for 5 min, thaw the competent cells. Add 10 μL of plasmid to 100 μL of competent cells and mix gently. Transfer the plasmid and electroporated competent cells into a pre-chilled electroporation cuvette and electroporate at 2.5 kV for 5 ms. Immediately after electroporation, add pre-chilled LB liquid and mix gently by pipetting. Transfer the culture medium containing plasmid and competent cells to a new centrifuge tube for expansion culture for 1.5 h. Centrifuge at 6000 r / min for 2 min, discard the supernatant, spread the bacterial cells on LB agar plates containing kanamycin, and incubate overnight at 30℃.

[0087] (4) Single colonies of *E. coli* BZWNDPAL / pCas9 were picked and cultured in LB medium at 30°C for 1.0 h. L-arabinose was then added to a final concentration of 30 mM to induce λ-red system expression. When OD... 600 When the concentration reaches 0.6-0.8, prepare Escherichia coli BZWNDPAL / pCas9 competent cells.

[0088] (5) Electroporate 500 ng of the plasmid pTargetF-glmS constructed in step (1) and 1000 ng of the gene homology repair template constructed in step (2) to the E. coli BZWNDPAL / pCas9 competent cells prepared in step (4), spread them on LB plates (kanamycin and spectinomycin), and incubate at 30℃ for 16-24 h. Perform colony PCR verification on the single colonies that grow on the plates, screen for positive transformants and perform gene sequencing.

[0089] (6) For verified single colonies, the pTargetF-glmS and pCas9 plasmids were eliminated. Single colonies were inoculated into LB liquid medium (kanamycin resistant) and cultured at 30°C and 200 rpm until the logarithmic growth phase. IPTG was added to a final concentration of 0.5 mmol / L and cultured overnight to induce pTargetF-glmS plasmid inactivation. The bacterial suspension was streaked onto LB plates containing kanamycin and cultured at 30°C and 200 rpm for 12 h. Single colonies were spotted onto plates resistant to both kanamycin and spectinomycin. No colony growth indicated successful pTargetF-glmS plasmid elimination.

[0090] (7) The pCas9 plasmid is a temperature-sensitive plasmid. Single colonies of the successfully eliminated pTargetF-glmS plasmid were transferred to LB broth without antibiotics and passaged at 42°C to eliminate the pCas9 plasmid. After streaking the bacterial solution onto antibiotic-free LB plates, the culture was kept at 37°C. Single colonies were spotted onto LB broth containing kanamycin. If no single colony grew, it indicated that the pCas9 plasmid was successfully eliminated. The constructed pTargetF-glmS-free and pCas9 plasmid-deleted strains were stored at -80°C for later use.

[0091] (8) The successfully constructed Escherichia coli chassis host was named BZWNDPALS.

[0092] Table 1. GlmS gene knockout primers

[0093]

[0094] Example 2: De novo synthesis of lactose-N-difucotetrasaccharide and module-optimized gene expression

[0095] Using BZWNDPAL and BZWNDPALS from Example 1 as chassis hosts, pathway genes were co-expressed using a dual plasmid system. The specific steps for constructing the recombinant plasmids and strains are as follows (primer sequences involved are shown in Table 2):

[0096] (1) Obtaining manB, manC, gmd and wcaG fragments: Using the genome of Escherichia coli K12 as a template, PCR amplification was performed using primers manCB_F / R and GW_F / R, and DNA was recovered by gel to obtain manC-manB and gmd-wcaG gene fragments.

[0097] (2) Using pRSFDuet-1, pETDuet-1, and pCDFDuet-1 as templates, the vector backbone sequence was amplified using primers V1_F / R and V2_F / R. According to In-Fusion cloning technology, fragments manC-manB were inserted between the Nco I / BamHI restriction sites of the above vectors, and fragment gmd-wcaG was inserted between the Nde I / Xho I restriction sites of the above vectors. Positive clones were screened and sequenced, ultimately yielding recombinant plasmids pRSF-CBGW, pET-CBGW, and pCDF-CBGW.

[0098] (3) Obtaining HpfutC and HpM32 gene fragments: Tianlin Biotechnology (Shanghai) Co., Ltd. was commissioned to synthesize the HpfutC gene sequence (SEQ ID NO.15) from Helicobacter pylori ATCC 26695 and the mutant HpM32 gene sequence (SEQ ID NO.1) (S45F / D127N / R128E / H131I / Y199N / E340D / V368A) from Helicobacter pylori NCTC11639. The synthesized HpfutC fragment was ligated between the Nco I / BamHI restriction sites of the vector pETDuet-1 using a seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.) to obtain the plasmid pET-HpfutC. Then, the HpM32 gene fragment was ligated between the Nde I / Xho I restriction sites of the vector plasmid pET-HpfutC to obtain the plasmid pET-HpfutC-HpM32. Furthermore, the -35 and -10 regions of the original RBS(T7) in plasmid pET-HpfutC-HpM32 were replaced with RBS(BBa_B0034). The RBS(BBa_B0034) sequence and primer sequences are shown in Table 2. Using pET-HpfutC, pET-HpM32, and pET-HpfutC-HpM32 plasmids as templates, the vector backbone sequence was amplified using primers BBa_B0034_CF / R and BBa_B0034_MF / R. The DNA was recovered by gel extraction, and the product was digested with the restriction endonuclease Dpn I to remove excess circular template. The linear fragment was recombined using a seamless cloning kit, then transformed into E. coli DH5α and cultured overnight. Single clones were picked from the plate and transferred to 4 mL of LB medium for expansion culture. Plasmids were extracted and sequenced. The successfully constructed plasmids were named pET-R34-HpfutC, pET-R34-HpM32 and pET-R34-HpfutC-R34-HpM32, respectively.

[0099] (4) Using pRSFDuet-1 and pCDFDuet-1 as templates, the vector backbone fragment was amplified using primers V3_F / R; using pET-R34-HpfutC-R34-HpM32 as a template, the R34-HpfutC-R34-HpM32 gene fragment was amplified using primers CM_F / R, and the R34-HpfutC-R34-HpM32 and the vector backbone fragment were seamlessly ligated to obtain the recombinant plasmids pRSF-R34-HpfutC-R34-HpM32 and pCDF-R34-HpfutC-R34-HpM32.

[0100] Table 2. Primers for plasmid construction

[0101]

[0102]

[0103] (5) The above-mentioned pathway genes were assembled using a plasmid expression system, and expression vectors with different copy numbers were constructed to increase the expression levels of upstream key genes and downstream glycosyltransferases. Module optimization was performed using three expression vectors with different strengths: pCDFDuet-1 (copy number 20–40), pETDuet-1 (copy number > 40), and pRSFDuet-1 (copy number > 100). The key genes gmd, wcaG, manC, and manB were assigned to module one, and HpfutC and HpM32, which contain fucosyltransferases, were assigned to module two. Seven LDFT engineered strains were constructed under different plasmid and strain combinations (see Table 3), and the production performance of LDFT strains under different combinations was compared.

[0104] Table 3. Detailed information on engineered bacteria using modular plasmid combinations.

[0105]

[0106] (6) The shake-flask fermentation experiment showed that the optimal plasmid combination for LDFT production using E. coli BL21(DE3)ΔlacZΔwcaJΔnudDΔpfkAΔlonΔglmS(BZWNDPALS) as the host strain was pRSF-CBGW and pET-R34-HpfutC-R34-HpM32, i.e., strain BDFL2. Under fixed expression in module two, product accumulation increased with the increase of expression intensity in module one. After fixing the expression of module one, more product accumulated in module two when the expression intensity was slightly lower than that of module one. The optimal strain BDFL2 achieved the highest LDFT yield of 2.18 g / L after 60 h of fermentation.

[0107] Example 3: Screening of α-1,3-fucosyltransferases from different sources

[0108] (1) Obtaining HpM32, Hp3 / 4FT, HpfutA, Hc3FT, Hh3FT and Bf3FT gene fragments: Tianlin Biotechnology (Shanghai) Co., Ltd. was commissioned to synthesize the following gene sequences: HpM32 (S45F / D127N / R128E / H131I / Y199N / E340D / V368A) mutant from Helicobacter pylori NCTC 11639, Hp3 / 4FT gene sequence from Helicobacter pylori UA948, HpfutA gene sequence from Helicobacter pylori ATCC 26695, Hc3FT gene sequence from Helicobacter pylori gastricis, Hh3FT gene sequence from Helicobacter hepatis and Bf3FT gene sequence from Bacteroides fragilis. The synthesized target gene fragment was ligated between the Nde I / Xho I restriction sites of the vector pET-R34-HpfutC using a seamless cloning kit (Nanjing Novizan Biotechnology Co., Ltd.), resulting in plasmids pET-R34-HpfutC-R34-HpM32, pET-R34-HpfutC-R34-Hp3 / 4FT, pET-R34-HpfutC-R34-HpfutA, pET-R34-HpfutC-R34-Hc3FT, pET-R34-HpfutC-R34-Hh3FT, and pET-R34-HpfutC-R34-Bf3FT.

[0109] (2) The expression vector pRSF-CBGW from Example 2 and several prokaryotic α-1,3-fucosyltransferase expression vectors from (1) above were co-transformed into E. coli BZWNDPALS, and the resulting strains are shown in Table 4. The shake-flask fermentation results showed that, by measuring the yield of LDFT, the mutant HpM32 (S45F / D127N / R128E / H131I / Y199N / E340D / V368A) derived from Helicobacter pylori NCTC 11639 had a higher LDFT production capacity.

[0110] Table 4. Engineered bacteria containing α-1,3-fucosyltransferase from different sources

[0111]

[0112] Example 4: Effects of truncated or extended mutants of α-1,3-fucosyltransferase on lactose-N-difucotetrasaccharide yield Impact

[0113] α-1,2-fucosyltransferases and α-1,3-fucosyltransferases participate in the final two steps of LDFT biosynthesis, catalyzing the transfer of the fucose group from GDP-L-fucose and linking the galactosyl and glucose groups of lactose via α-1,2 and α-1,3 bonds. According to previous reports, poor solubility and low catalytic activity of fucosyltransferases have become common problems in the microbial preparation of fucosyllactose or lactose-N-difucotetrasaccharide. The C-terminus of α-1,3-fucosyltransferases consists of a heptapeptide repeat sequence and an amphiphilic helix, responsible for enzyme dimerization and peripheral membrane anchoring, respectively. In most α-1,3-fucosyltransferases, the C-terminus of the catalytic domain has a conserved heptapeptide repeat sequence of 2-10 sequences. Truncation or extension of the heptapeptide repeat sequence and / or amphiphilic helical region may lead to increased enzyme activity and soluble expression levels.

[0114] This embodiment optimizes the C-terminal heptapeptide repeat sequence by truncating or extending the HpM32 mutant (with 7 heptapeptide repeats at the C-terminus) derived from Helicobacter pylori NCTC 11639. By regulating the solubility and expression intensity of α-1,3-fucosyltransferase, an engineered strain capable of efficiently producing LDFT was constructed and screened. Eight heptapeptide repeat sequences of different lengths were selected. The constructed plasmid mutants are shown in Table 4, and the engineered strains are shown in Table 5, containing 4 truncated mutants and 4 extended mutants. The specific construction process is as follows (primer sequences involved are shown in Table 5).

[0115] (1) Taking the truncated mutant H6 and the extended mutant H8 as examples, primers H6_F / R and H8_F / R were used to amplify the vector backbone sequence using the pET-R34-HpfutC-R34-HpM32 plasmid constructed in Example 2 as a template. The DNA was recovered by gel extraction, and the product was digested with restriction endonuclease Dpn I to remove excess circular template. The linear fragment was recombined using the One Step Cloning Kit (Vazyme), and then transformed into E. coli DH5α and cultured overnight. Single clones on the plate were picked and transferred to 4 mL of LB medium for expansion culture. The plasmid was extracted and sequenced. The successfully constructed knockout plasmids were named pET-R34-HpfutC-R34-H6 and pET-R34-HpfutC-R34-H8. The same method was used to construct mutants H0, H2, H5, H9, H10, and H11, respectively. The plasmid templates for the truncated mutants H0, H2, and H5 were pET-R34-HpfutC-R34-HpM32, and the plasmid templates for the extended mutants H9, H10, and H11 were pET-R34-HpfutC-R34-H8, pET-R34-HpfutC-R34-H9, and pET-R34-HpfutC-R34-H10, respectively.

[0116] Table 5. Primer sequences for truncated or extended mutants

[0117]

[0118]

[0119] (2) The plasmid successfully constructed in the above steps was transformed into the host Escherichia coli BZWNDPALS containing pRSF-CBGW, and the resulting engineered strains are shown in Table 6.

[0120] (3) The obtained engineered bacteria were fermented in shake flasks to produce LDFT, and the results were simultaneously presented in [the table / document / etc.]. Figure 3 Extending the length of the heptapeptide repeat sequence can increase the yield of LDFT and the soluble expression of the enzyme. The optimal strain BDFL19 achieved an LDFT yield of 3.03 g / L.

[0121] Table 6. Detailed information on truncated or extended mutant engineered bacteria

[0122]

[0123] Example 5: Effect of α-1,2-fucosyltransferase mutants on lactose-N-difucotetrasaccharide yield

[0124] It has been reported that most α-1,2-fucosyltransferases exhibit low catalytic activity and poor soluble expression in microbial expression systems, and no crystal structures of microbial-derived α-1,2-fucosyltransferases have been reported. This embodiment is based on previous research on mutants (disclosed in patent document CN116064455A), selecting several single-point (K102T, R105C, Y251F, K282E) and combined mutants (K102T / R105C, Y251F / K282E, R105C / K282E, K102T / R105C / K282E, K102T / R105C / Y251F / K282E) of α-1,2-fucosyltransferases with enhanced catalytic activity, and applying them to the fermentation production of lactose-N-difucotetraose. The mutant was constructed based on plasmid pET-R34-HpfutC-R34-H10. The specific procedure and primers used are as follows (Table 7):

[0125] (1) Taking the mutant K102T as an example, plasmid was constructed using pET-R34-HpfutC-R34-H10 as a template and K102T_F / R as upstream and downstream primers. The expression plasmid was linearized and amplified by reverse PCR. Subsequently, the template plasmid was digested with Dpn I enzyme, and the linearized vector fragment was purified and recovered. The linear fragment was recombinantly ligated using the One Step Cloning Kit (Vazyme). The recombination reaction system was reacted at 37℃ for 30 min, and then transformed into E. coli DH5α and cultured overnight. Single clones on the plate were picked and transferred to 4 mL of LB medium for expansion culture. The plasmid was then extracted and sequenced. The successfully constructed plasmid was named pET-R34-K102T-R34-H10. The plasmid templates for the other single-point mutants and V1 are the same as K102T. The plasmid templates for the combined mutants V7, V8, V9 and V10 are based on the single-point mutants and are superimposed mutations. The corresponding templates are Y251F, R105C, V1 and V9, respectively. Finally, the single-point mutant plasmids obtained were pET-R34-K102T-R34-H10, pET-R34-R105C-R34-H10, pET-R34-Y251F-R34-H10, and pET-R34-K282E-R34-H10, and the combined mutant plasmids obtained were pET-R34-V1-R34-H10, pET-R34-V7-R34-H10, pET-R34-V8-R34-H10, pET-R34-V9-R34-H10, and pET-R34-V10-R34-H10.

[0126] Table 7. Primer sequences for α-1,2-fucosyltransferase mutants

[0127]

[0128] (2) The plasmid successfully constructed in the above steps was transformed into host Escherichia coli BZWNDPALS containing pRSF-CBGW, and the resulting engineered strains are shown in Table 8.

[0129] (3) The engineered bacteria were fermented in shake flasks to produce LDFT. The yield results are shown in Table 7.

[0130] Table 8. Detailed information on engineered bacteria with different α-1,2-fucosyltransferase mutants

[0131]

[0132]

[0133] Example 6: Effects of the insertion site and pattern of the rate-limiting enzyme on the yield of lactose-N-difucotetrasaccharide

[0134] Studies have shown that the insertion location and pattern of pathway genes at the multiple cloning site (MCS) can affect the synthesis of target compounds. To increase the synthesis efficiency of lactose-N-difucotetrasaccharide, this example compares the insertion order, location, and tandem copy number of two rate-limiting enzymes.

[0135] To verify the effect of the insertion site on the expression of the rate-limiting enzyme gene and LDFT production, the α-1,2 / 3-fucosyltransferase mutant V10 / H10 was inserted into the MCS1 and MCS2 regions of the vector pETDuet-1, which replaced the original RBS(T7) with BBa_B0034, respectively, resulting in four plasmids: pET-R34-1H10, pET-R34-2H10, pET-R34-1V10, and pET-R34-2V10. Subsequently, to verify the effects of different pathway configurations and sequences on product synthesis, the rate-limiting enzyme was optimized using operons and pseudooperons, resulting in four plasmids: pET-R34-1V10-R34-1H10, pET-R34-2V10-R34-2H10, pET-R34-1V10-R34-2H10, and pET-R34-1H10-R34-2V10. Finally, to enhance the expression of the rate-limiting enzyme, the α-1,2 / 3-fucosylation transfer gene was tandemly inserted into the NotI restriction site after the MCS1 region of plasmid pET-R34-1V10-R34-2H10, resulting in plasmids pET-R34-1V10-R34-2V10-R34-3H10 and pET-R34-1V10-R34-2H10-R34-3H10. Based on plasmid pET-R34-1V10-R34-2V10-R34-3H10, the α-1,3-fucosylation transfer gene was inserted into the BamHI restriction site, resulting in plasmid pET-R34-1V10-R34-2V10-R34-3H10-R34-4H10. The plasmid construction method was the same as in Example 2, and the primers used are listed below (Table 9). Among them, plasmids pET-R34-1H10 and pET-R34-1V10, whose target gene is located in the MCS1 region, use 1H_F / R and 1V_F / R as primers, respectively, while the vector backbone uses the common primer V_1H / V_F / R; plasmids pET-R34-2H10 and pET-R34-2V10, whose target gene is located in the MCS2 region, use 2H_F / R and 2V_F / R as primers, respectively, while the vector backbone uses the common primer V_2H / V_F / R. Based on this, plasmids pET-R34-1V10-R34-1H10 and pET-R34-2V10-R34-2H10 amplified the target gene using 1V1H_F / R and 2V2H_F / R, and amplified the vector backbone using V_1V1H-F / R and V_2V2H_F / R. Plasmid pET-R34-1H10-R34-2V10 used pET-R34-1H10 as a template to amplify the gene and vector fragment using 2V_F / R and V_2H / V_F / R, respectively.In addition, the target gene amplification primers used for the gene tandem plasmids pET-R34-1V10-R34-2V10-R34-3H10 and pET-R34-1V10-R34-2H10-R34-3H10 were 1V2V3H_F / R and 1V2H3H_F / R, respectively, and the vector amplification primers were V_1V2V / H3H_F / R; the target gene was amplified using 1V2V2H3H_F / R for plasmid pET-R34-1V10-R34-2V10-R34-3H10-R34-4H10, and the vector backbone was amplified using V_1V2V2H3H_F / R.

[0136] The constructed expression vector was transformed into *Escherichia coli* BZWNDPALS containing pRSF-CBGW, resulting in 10 engineered strains, as shown in Table 10. The engineered strains were then used to produce LDFT via shake-flask fermentation. The engineered strain containing the recombinant plasmids pRSF-CBGW and pET-R34-1V10-R34-2V10-R34-3H10 (i.e., strain BDFL37) achieved the highest yield of 3.94 g / L.

[0137] Table 9. Insertion sites of rate-limiting enzymes and primers for plasmid construction with optimized patterns

[0138]

[0139]

[0140] Table 10. Detailed information on engineered bacteria

[0141]

[0142] Example 7: Production of LDFT by Chromosomal Integration Enhancement Strains of Pathway Enzymes

[0143] To enhance the synthesis of the precursor GDP-L-fucose, this embodiment employs a promoter engineering strategy to moderately regulate the expression levels of key enzymes in the GDP-L-fucose module. Specifically, the strong promoter T7 replaces the promoters of manA, manB, manC, and gmd on the *E. coli* genome. Furthermore, to mitigate the accumulation of acetic acid, a byproduct of glycolysis, the α-1,2-fucosyltransferase mutant V10-V10 (double tandem) from the downstream rate-limiting enzyme is integrated into the poxB site on the chromosome, and the α-1,3-fucosyltransferase mutant H10-H10 (double tandem) is integrated into the ackA-pta site on the chromosome. The gene editing steps involved in the lactose-N-difucotetrasaccharide synthesis pathway are as follows (primer sequences are shown in Table 11):

[0144] (1) Taking the integration of the V10-V10 gene at the poxB site as an example, the specific target gRNA (20bp) of the poxB gene was searched using http: / / www.regenome.net / cas-offinder. Using the poxB-gRNA_F / poxB-gRNA_R primers, PCR amplification was performed with the pTargetF plasmid (Addgene: #62226) as a template. The amplified product was digested with the restriction endonuclease Dpn I to remove the redundant circular plasmid pTargetF. The amplified product was then transformed into E. coli DH5α competent cells, and the plasmid was extracted and identified by sequencing. The successfully constructed knockout plasmid was named pTargetF-poxB.

[0145] (2) Using the Escherichia coli BZWNDPAL genome as a template, three sequence fragments were amplified using the upstream homologous arm primer poxB-US_F / poxB-US_R, the midstream homologous arm primer V10-V10-MS_F / V10-V10-MS_R, and the downstream homologous arm primer poxB-DS_F / poxB-DS_R, respectively. After purification and recovery of the products, the three fragments were ligated using the SOE-PCR method with primers poxB-US_F / poxB-DS_R to obtain the gene homologous repair template.

[0146] (3) Electroporate pCas9 plasmid (Addgene:#62225) into E. coli BZWNDPALS electroporation competent cells. After placing on ice for 5 min, thaw the competent cells. Add 10 μL of plasmid to 100 μL of competent cells and mix gently. Transfer the plasmid and electroporated competent cells into a pre-chilled electroporation cuvette and electroporate at 2.5 kV for 5 ms. Immediately after electroporation, add pre-chilled LB liquid and mix gently by pipetting. Transfer the culture medium containing plasmid and competent cells to a new centrifuge tube for expansion culture for 1.5 h. Centrifuge at 6000 r / min for 2 min, discard the supernatant, spread the bacterial cells on LB agar plates containing kanamycin, and incubate overnight at 30℃.

[0147] (4) Single colonies of *E. coli* BZWNDPAL / pCas9 were picked and cultured in LB medium at 30°C for 1.0 h. L-arabinose was then added to a final concentration of 30 mM to induce λ-red system expression. When OD... 600 When the concentration reaches 0.6-0.8, prepare Escherichia coli BZWNDPAL / pCas9 competent cells.

[0148] (5) Electroporate 500 ng of pTargetF-poxB constructed in step (1) and 1000 ng of gene homology repair template constructed in step (2) to E. coli BZWNDPAL / pCas9 competent cells prepared in step (4), spread them on LB plates (kanamycin and spectinomycin), and incubate at 30℃ for 16-24 h. Perform colony PCR verification on single colonies growing on the plates, screen positive transformants and perform gene sequencing.

[0149] (6) For verified single colonies, the pTargetF-poxB and pCas9 plasmids were eliminated. Single colonies were inoculated into LB liquid medium (kanamycin resistant) and cultured at 30°C and 200 rpm until the logarithmic growth phase. IPTG was added to a final concentration of 0.5 mmol / L and cultured overnight to induce pTargetF-poxB plasmid inactivation. The bacterial suspension was streaked onto LB plates containing kanamycin and cultured at 30°C and 200 rpm for 12 h. Single colonies were spotted onto plates resistant to both kanamycin and spectinomycin. No colony growth indicated successful pTargetF-poxB plasmid elimination.

[0150] (7) The pCas9 plasmid is a temperature-sensitive plasmid. Single colonies of successfully eliminated pTargetF-poxB plasmid were transferred to LB broth without antibiotics and passaged at 42°C to eliminate the pCas9 plasmid. After streaking the bacterial solution onto antibiotic-free LB plates, the culture was kept at 37°C. Single colonies were spotted onto LB broth containing kanamycin. If no single colony grew, it indicated that the pCas9 plasmid was successfully eliminated. The constructed gene-deleted strains without pTargetF-poxB plasmid and pCas9 plasmid were stored at -80°C for later use.

[0151] (8) Refer to the above steps for promoter replacement of the "key enzyme" on the chromosome and integration of mutant H10.

[0152] Table 11. Primers for promoter substitution and rate-limiting enzyme integration

[0153]

[0154]

[0155] The engineered strains constructed in this embodiment are shown in Table 12. The LDFT synthesis capacity, cell biomass, and byproduct accumulation of the engineered strains were tested. After deleting the poxB and ackA-pta genes, no acetic acid accumulation was detected in the recombinant strains during fermentation. The optimal antibiotic-free strain BDFL46 achieved an LDFT synthesis yield of 1.68 g / L, while the optimal antibiotic-treated strain BDFL53 increased the LDFT yield to 4.68 g / L, a 114% increase compared to the initial BDFL1 strain.

[0156] Table 12. Detailed information on engineered bacteria with integrated lactose-N-difucotetrasaccharide metabolic pathway.

[0157]

[0158] Example 8: Application of novel chassis host strains in the production of 2'-fucosylated lactose and 3-fucosylated lactose

[0159] 2'-FL, 3-FL, and LDFT are the most abundant fucosylated human milk oligosaccharides secreted in breast milk. 2'-FL and 3-FL are precursors for LDFT synthesis, and their intracellular availability is crucial for efficient microbial LDFT synthesis. To verify the beneficial effects of the novel chassis strain constructed in this invention on precursor synthesis, the expression plasmid pRSF-CBGW required for 2'-FL / 3-FL production and a mutant plasmid containing α-1,2 / 3-fucosylation enzymes were co-transformed into the host strain. The novel chassis strains selected were *Escherichia coli* BZWNDPAL, BZWNDPALS, and BDFL46. The selected α-1,2-fucosyltransferase plasmids were pET-R34-HpfutC, pET-R34-V10, and pET-R34-1V10-R34-2V10, and the selected α-1,3-fucosyltransferase plasmids were pET-R34-HpM32, pET-R34-H10, and pET-R34-1H10-R34-2H10. Among these, plasmids pET-R34-HpfutC and pET-R34-HpM32 were derived from Example 2. The primers for constructing the other plasmids are shown in Table 13 below. Using plasmids pET-R34-HpfutC and pET-R34-HpM32 as vector templates, linear amplification of the target fragments V10 and H10 was performed using primers V10_F / R and H10_F / R. The target fragments were then replaced at the HpfutC and HpM32 positions of the vector, respectively, to finally obtain plasmids pET-R34-V10 and pET-R34-H10. Based on plasmids pET-R34-V10 and pET-R34-H10, linear fragments of R34-2V10 and R34-2H10 were amplified using primers R34-V10_F / R and R34-H10_F / R, and then inserted into the MCS2 region of vectors pET-R34-V10 and pET-R34-H10 to obtain dual tandem α-1,2 / 3-fucosyltransferase plasmids pET-R34-1V10-R34-2V10 and pET-R34-1H10-R34-2H10.

[0160] The 2'-FL and 3-FL engineered strains constructed in this embodiment are shown in Tables 14 and 15. The shake-flask production performance of each engineered strain was compared. The shake-flask fermentation conditions were the same as those for the production of lactose-N-difucotetrasaccharide. The fermentation results showed that the modified strain BZWNDPALS had a higher capacity for producing 2'-FL and 3-FL than BZWNDPAL, and the α-1,2 / 3-fucosyltransferase mutant plasmid showed a significant advantage in product synthesis. Specifically, when two copies of the mutant gene were tandemly attached to the plasmid, the yields of 2'-FL and 3-FL in the host strain BZWNDPALS increased by 60% and 93% compared to the control group (B2FL1 and B3FL1), respectively. In the modified strain BDFL46, the yields of 2'-FL and 3-FL were further increased by 80% and 114% compared to the control group (B2FL1 and B3FL1). These results indicate that the deletion of the glmS gene, the positive mutation of the key rate-limiting enzyme, and the chromosomal integration of the pathway enzyme in the chassis strain can significantly enhance the carbon flux of the 2'-FL / 3-FL metabolic pathway, promote the correct folding and functional expression of the rate-limiting enzyme, and thus achieve efficient bioprocessing of 2'-FL / 3-FL.

[0161] Table 13. Primer sequences for α-1,2 / 3-fucosyltransferase mutants

[0162]

[0163] Table 14.2'-Detailed information on engineered FL bacteria

[0164]

[0165] Table 15.3 - Detailed information on engineered FL bacteria

[0166]

[0167]

[0168] Example 9: Feed-in batch fermentation of lactose-N-difucotetrasaccharide in a 3L fermenter

[0169] To prepare high-yield LDFT, recombinant strain BDFL53 was used for high-density fed-batch fermentation in 3L fermenters.

[0170] Fermentation conditions: Thaw the glycerol bacteria stored at -80℃ on ice. Use an inoculation loop to take a small amount of the bacterial culture and streak it in a "Z" pattern onto a solid LB agar plate (ampicillin 100 μg / mL, kanamycin sulfate 50 μg / mL). Incubate overnight at 37℃. Pick a single colony from the plate and inoculate it onto a 50 mL agar plate. mThe seed culture was prepared by culturing L of LB liquid medium at 37°C and 200 rpm for 12 hours. A 3-L fermenter containing 1L of fermentation medium (initial glycerol concentration 30 g / L) was used to inoculate the strain at a rate of 5% (v / v). The pre-induction culture temperature was set at 37°C until OD... 600 To achieve a concentration of 20, IPTG was added to induce protein expression, resulting in a concentration of 0.2 mmol / L in the fermentation system. The initial lactose concentration was 10 g / L, and the induction fermentation temperature was 25℃. Throughout the fermentation process, NH4OH was used to maintain a constant pH of 6.80 in the tank. To maintain cell growth and fucoidan synthesis, after the initial glycerol was consumed, 800 g / L of glycerol (containing 20 g / L MgSO4·7H2O) was added to replenish the carbon source. pH feedback was used to maintain the glycerol concentration in the fermentation system at a low level (glycerol is used for cell growth and metabolism, with a concentration of approximately 0 g / L, i.e., just consumed by the strain) until the end of fermentation. After the initial lactose was consumed, 300 g / L of lactose was added to maintain a final concentration of approximately 10 ± 0.5 g / L in the fermentation system. If the lactose concentration decreased during fermentation, lactose was continuously added until the end of fermentation. The system is cascaded and controlled during fermentation. By adjusting the rotation speed, aeration rate and oxygen, the dissolved oxygen in the tank is kept at 30% ± 5%.

[0171] Sampling was performed regularly throughout the fermentation process, and cell OD was measured. 600 1 mL of fermentation broth was boiled for 15 min to completely lyse the cells, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm membrane. The amount of LDFT generated during fermentation was detected by HPLC. Figure 2 The study also examined the consumption of lactose and glycerol. Results showed that after fermentation (a total of 80 hours), the concentration of LDFT in the product reached 41.02 g / L. Figure 4 ).

[0172] The concentrations of LDFT were 32.46, 38.88, and 41.02 g / L at 64, 72, and 80 hours of fermentation, respectively, and the conversion rates reached 0.49, 0.52, and 0.53 mol LDFT / mol lactose, respectively.

[0173] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An engineered bacterium producing lactose-N-difucotetrasaccharide, characterized in that, Using E. coli as the host, knockout β- galactosidase gene lacZ UDP-glucose lipid carrier transferase gene wcaJ GDP-mannose-mannosyl hydrolase gene nudD 6-phosphofructokinase-1 gene pfkA protease gene lon Glutamine-fructose-6-phosphate aminotransferase gene glmS ; Free expression of phosphomannose mutase gene manB mannose-1-phosphate guanine transferase gene manC GDP-mannose-6-dehydrogenase gene gmd GDP-fucose synthase gene wcaG、α -1,2-fucosyltransferase HpfutC and / or α -1,3-fucosyltransferase HpM32 ; The α -1,2-fucosyltransferase HpfutC For (a) or (b): (a) The amino acid sequence is shown in SEQ ID NO.

15. HpfutC ; (b) Mutants based on (a): K102T, R105C, Y251F, K282E, K102T / R105C, Y251F / K282E, R105C / K282E, K102T / R105C / K282E, or K102T / R105C / Y251F / K282E; The α -1,3-fucosyltransferase HpM32 For any one of (a) to (c): (a) The amino acid sequence is as shown in SEQ ID NO.

1. HpM32 ; (b) The truncated body obtained by truncating (a): truncated HpM32 The C-terminal 7-peptide repeat sequences were truncated by 49, 35, 14 and 7 amino acids, respectively; (c) An extension obtained by extending (a): extension HpM32 The C-terminal 7-peptide repeat sequence was extended by 7, 14, 21 and 28 amino acids, respectively.

2. The engineered bacteria according to claim 1, characterized in that, Using plasmids pRSFDuet-1, pETDuet-1, or pCDFDuet-1 for free expression manB , manC , gmd and wcaG Using plasmids pRSFDuet-1, pETDuet-1, or pCDFDuet-1 for free expression HpM32 and double series HpfutC .

3. The engineered bacteria according to claim 1, characterized in that, Replace the original RBS of plasmids pRSFDuet-1, pETDuet-1, or pCDFDuet-1 with BBa_B0034.

4. The engineered bacteria according to claim 2 or 3, characterized in that, Double series HpfutC and HpM32 Insert the plasmid into the MCS 1 and MCS 2 regions respectively.

5. The engineered bacteria according to claim 1, characterized in that, The ubiquinone-dependent pyruvate dehydrogenase gene was also knocked out. poxB and phosphorylated acetyltransferase-acetylkinase gene cluster pta-ackA .

6. The engineered bacteria according to claim 5, characterized in that, Knockout poxB After the site, poxB Site integration expression dual tandem α -1,2-fucosyltransferase HpfutC ; Knockout pta - ackA After the site, pta - ackA Site integration expression dual tandem α -1,3-fucosyltransferase HpM32 .

7. The engineered bacteria according to any one of claims 1 to 3, characterized in that, Replace the E. coli genome with a strong promoter manA, manC, manB and gmd - wcaG The promoter of the gene is started using a strong promoter. HpfutC and HpM32 The expression.

8. The engineered bacteria according to claim 4, characterized in that, Replace the E. coli genome with a strong promoter manA, manC, manB and gmd - wcaG The promoter of the gene is started using a strong promoter. HpfutC and HpM32 The expression.

9. The engineered bacteria according to claim 5 or 6, characterized in that, Replace the E. coli genome with a strong promoter manA, manC, manB and gmd - wcaG The promoter of the gene is started using a strong promoter. HpfutC and HpM32 The expression.

10. A method for producing lactose-N-difucotetrasaccharide, characterized in that, The method involves using any one of the engineered bacteria described in claims 1 to 9 as the fermentation strain to produce lactose-N-difucotetrasaccharide.

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