Plasmid-free recombinant escherichia coli for synthesizing lactoyl-N-neotetraose as well as construction method and application of plasmid-free recombinant escherichia coli

Abstract

The invention discloses plasmid-free recombinant escherichia coli for synthesizing lactoyl-N-neotetraose as well as a construction method and application of the plasmid-free recombinant escherichia coli, and belongs to the field of gene engineering. An engineering strain MG-LNT-07 is used as a starting strain, beta-1, 4-galactosyl transferase Hplex2B is introduced, the copy number of the engineering strain is optimized, a multidrug efflux transporter gene mdfA is integrated, the expression of a glycoside donor pathway gene is fine-tuned, and a rapZ gene is knocked out, so that a plasmid-free strain for efficiently synthesizing the lactoyl-N-neotetraose is constructed, finally, the extracellular titer of LNnT is 7.76 g / L, and the expression of the lactoyl-N-neotetraose can be efficiently synthesized. And only 0.58 g / L of precursor LNTriII is remained, so that the method has a good industrial prospect.

Description

Technical Field

[0001] The invention relates to a plasmid-free recombinant Escherichia coli for synthesizing lactoyl-N-neotetraose and a construction method and application thereof, and belongs to the field of genetic engineering. Background Art

[0002] Breastfeeding has a crucial impact on infants' long-term health and basic development, offering significant advantages over formula. Breast milk composition has been extensively studied, gradually revealing its complex properties. Human milk oligosaccharides (HMOs) rank third in solids content, after lactose and lipids, and are considered the primary component distinguishing breast milk from animal milk. HMOs are composed of a variety of unconjugated oligosaccharides, typically consisting of five monosaccharides linked by different glycosidic bonds: glucose, galactose, N-acetylglucosamine (GlcNAc), L-fucose, and sialic acid. Over 200 different HMO structures have been identified. HMOs offer a variety of unique health benefits to infants, including prebiotic activity, protection against pathogen infection, and support for developmental and cognitive function. By adding multiple HMOs in scientifically defined proportions, formula can be endowed with a variety of functional nutritional properties. Furthermore, as research into the physiological functions of HMOs deepens, their application is expected to expand into industries such as dietary supplements, cosmetics, and pharmaceuticals.

[0003] Lactyl-N-neotetraose (LNnT, Galβ1,4GlcNAcβ1,3Galβ1,4Glc) is one of the most abundant neutral nuclear structures in breast milk and has been granted GRAS (Generally Recognized as Safe) certification by the U.S. Food and Drug Administration (FDA). Numerous studies have shown that LNnT has multiple beneficial physiological effects, such as promoting intestinal health, supporting immune function, and providing resistance to pathogen infection. Clinical studies have further confirmed its safety, making it a reliable ingredient in infant nutrition products. As the second HMO approved for inclusion in infant formula after 2'-fucosyllactose (2'-FL), LNnT is often used in combination with 2'-FL to optimize its functional and nutritional properties, thereby more closely resembling the benefits of breast milk.

[0004] Obtaining sufficient LNnT samples is a prerequisite for its commercial application and scientific research. However, its unique nature makes large-scale extraction of LNnT from natural sources a significant challenge. In recent years, microbial metabolic engineering has attracted widespread attention due to its cost-effectiveness and ease of scale-up. LNnT synthesis is typically achieved using lactose as a substrate by introducing two glycosyltransferases—β1,3-N-acetylglucosaminyltransferase (β1,3-GlcNAcT) and β1,4-galactosyltransferase (β1,4-GalT)—and utilizing naturally occurring UDP-GlcNAc and UDP-galactose in the host cell. Dong et al. successfully constructed a LNnT synthesis pathway in Bacillus subtilis by co-expressing lactose permease (LacY), β1,3-GlcNAcT (LgtA), and β1,4-GalT (LgtB). By optimizing LgtB expression levels, balancing the supply of key precursors, and downregulating the expression of branched pathway genes, LNnT production reached 5.41 g / L in a 3L bioreactor. In a recent study, Pichia pastoris was used as a host for LNnT production, but the ultimate yield was limited to 1.24 g / L. Besides the aforementioned studies, Escherichia coli is the primary host used in the vast majority of studies. In 2002, Priem et al. pioneered the production of LNnT in engineered E. coli JM109 through high-density culture, achieving a combined yield of LNnT and lactoyl-N-neohexose exceeding 5 g / L. In recent years, research on LNnT synthesis in E. coli has continued to emerge, with key strategies focusing on the identification of efficient glycosyltransferases, optimizing the glycosyl donor balance, and knocking out or enhancing key pathway genes. For example, by integrating LgtA and LgtB into the genome, connecting them using an enzyme assembly scaffold, and introducing the CRISPRi system to inhibit the expression of the competing pathway genes zwf and pfkA, researchers developed a plasmid-free engineered Escherichia coli K-12MG1655 strain. This strain produced 23.73 g / L of LNnT in a 3L bioreactor. In addition, Liao et al. developed a high-LNnT-producing Escherichia coli BL21 Star (DE3) strain. The main engineering strategies included identifying an efficient LNnT transporter and regulating carbon flux distribution through co-fermentation of glucose and glycerol, ultimately achieving the highest LNnT yield reported to date (112.47 g / L).

[0005] However, the existing microbial synthesis method for producing LNnT has low yields or relies on large-scale tank verification, which brings experimental burdens. The use of IPTG also brings safety risks and cost issues. Summary of the Invention

[0006] Technical Solution

[0007] In response to the above technical problems, the present invention provides a recombinant bacterium that can efficiently synthesize LNnT without relying on antibiotics and IPTG.

[0008] The first object of the present invention is to provide a recombinant bacterium, wherein the recombinant bacterium is based on Escherichia coli MG-LNT-07 (E. coli MG1655ΔlacZΔP lacY ::P J23119 ΔarsB::P J23119 -lgtAΔnagB::P J23119 -lgtAΔpoxB::P J23119 -lgtAΔldhA::P J23119 -wbgOΔwcaJΔugdΔP galE ::P tac ) is the starting strain, and the related strain is disclosed in the literature Qian, Q.; Yang, L.; Zhao, C.; Tao, M.; Zhang, W.; Mu, W. Highly efficient production of lacto-N-tetraose in plasmid-free Escherichia coli through chromosomalin integration of multicopy key glycosyltransferase genes. Int. J. Biol. Macromol. 2025, 284 (Pt1), 137987. The literature review is as follows: The chassis strain MG-LNT-07 is derived from Escherichia coli MG1655, and the β-galactosidase encoding gene lacZ, the UDP-glucose lipid carrier transferase encoding gene wcaJ and the UDP-glucose-6-dehydrogenase encoding gene ugd on the genome are knocked out, and the promoter P J23119 Replace the promoter of the β-galactosidase permease gene lacY on the genome with the promoter P tac The promoter of the UDP-galactose-4-epimerase gene, galE, was replaced on the genome. Additionally, a copy of the β-1,3-N-acetylglucosaminyltransferase gene, lgtA, was integrated into each of the genomic loci, arsB, nagB, and poxB. A copy of the β-1,3-galactosyltransferase gene, wbgO, was integrated into the ldhA locus. By overexpressing key pathway genes and blocking branching pathways, this strain is able to efficiently synthesize lactoyl-N-tetraose (LNT).

[0009] The following genetic engineering operations were performed on the starting strain MG-LNT-07:

[0010] (1) One to five copies of the β-1,4-galactosyltransferase encoding gene hplex2B were integrated into the wbgO, manX, manZ, wecB, and xylB loci;

[0011] (2) integration of the multidrug efflux pump encoding gene mdfA at the hlyE locus;

[0012] (3) Integrate three promoters P with different strengths at the yghwX, intQ, caiB, ybeQ, and rph sites. J23119 、P tac 、P trc The five different pathway genes controlled by the gene include glucosamine-6-phosphate synthase encoding gene glmS, glucosamine phosphoisomerase encoding gene glmM, N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase encoding gene glmU, glucose-1-phosphate uridyltransferase encoding gene galU, and UDP-glucose-4-epimerase encoding gene galE;

[0013] (4) Knock out the RNA-binding protein encoding gene rapZ in the genome.

[0014] In one embodiment, the Escherichia coli is Escherichia coli MG1655.

[0015] In one embodiment, the hplex2B gene is derived from Helicobacter pylori 26995, and its nucleotide sequence is shown in SEQ ID NO.1.

[0016] In one embodiment, the Gene ID of the gene mdfA is 945448.

[0017] In one embodiment, the Gene IDs of the genes glmS, glmM, glmU, galU and galE are 948241, 947692, 948246, 945730 and 945354, respectively.

[0018] In one embodiment, the promoter P J23119 、P tac and P trc The nucleotide sequences are shown in SEQ ID No. 2, No. 3 and No. 4.

[0019] In one embodiment, the Gene ID of the gene rapZ is 947727.

[0020] The second object of the present invention is to provide a method for producing lactoyl-N-neotetraose, wherein the method uses the above-mentioned recombinant bacteria to ferment and produce lactoyl-N-neotetraose.

[0021] In one embodiment, the method comprises: inoculating the recombinant bacteria growing normally on the plate into a seed culture medium, culturing at 36-39°C for 8-14 hours to obtain a seed solution, transferring the seed solution to a fermentation medium at an inoculum volume of 0.5%-2%, and culturing until the OD 600 When the pH value is 0.6–0.8, lactose is added and the culture temperature is lowered to 28–32°C to ferment and produce lactoyl-N-neotetraose.

[0022] In one embodiment, the seed culture medium comprises: 1-10 g / L yeast extract, 5-20 g / L peptone, and 5-20 g / L sodium chloride. Alternatively, the seed culture medium comprises: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride.

[0023] In one embodiment, the fermentation medium comprises: 10–30 g / L glycerol, 1–10 g / L yeast extract, 5–20 g / L potassium dihydrogen phosphate, 1–10 g / L diammonium hydrogen phosphate, 0.5–3 g / L citric acid, 0.5–3 g / L magnesium sulfate heptahydrate, 1–20 ml / L trace metal elements, and 1–10 mg / L thiamine. Alternatively, the fermentation medium comprises: 20 g / L glycerol, 5 g / L yeast extract, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate, 10 ml / L trace metal elements, and 4.5 mg / L thiamine.

[0024] In one embodiment, the trace metal element formula includes: 5-20g / L ferrous sulfate, 1-5g / L zinc sulfate heptahydrate, 0.5-2g / L anhydrous copper sulfate, 0.1-1g / L manganese sulfate monohydrate, 0.1-1g / L sodium borate decahydrate, 0.1-1g / L ammonium molybdate, and 0.5-5g / L calcium chloride dihydrate. Alternatively, the trace metal element formula includes: 10g / L ferrous sulfate, 2.25g / L zinc sulfate heptahydrate, 1.0g / L anhydrous copper sulfate, 0.35g / L manganese sulfate monohydrate, 0.23g / L sodium borate decahydrate, 0.11g / L ammonium molybdate, and 2.0g / L calcium chloride dihydrate.

[0025] In one embodiment, the fermentation time is 72-96 hours.

[0026] The third object of the present invention is to provide applications of the above-mentioned recombinant bacteria or the above-mentioned method in the fields of medicine, food or chemical industry.

[0027] In one embodiment, the application includes producing lacto-N-neotetraose or a product containing lacto-N-neotetraose using the above-mentioned recombinant bacteria or the method.

[0028] Innovations of the present invention:

[0029] 1. The present invention sequentially integrates the hplex2B gene at five different sites (wbgO, manX, manZ, wecB, and xylB) to form strains (Mn01-Mn05) to investigate their effects on lactoyl-N-neotetraose;

[0030] Results showed that strain Mn01 produced 1.09 g / L of LNnT in the culture supernatant after shake flask fermentation, while the byproduct LNTriⅡ was only 0.09 g / L, less than one-tenth of LNnT. This result demonstrates that the Hplex2B enzyme from Helicobacter pylori 26995 efficiently synthesizes LNnT with high purity, meeting application requirements without the need for additional purification. Furthermore, LNnT yields of Mn01-Mn05 were 1.09, 1.35, 1.67, 2.83, and 2.17 g / L, respectively. Among them, strain Mn04, which carries four copies of the hplex2B gene, achieved the highest LNnT yield of 2.83 g / L. This also suggests that more copies of the hplex2B gene are not necessarily better: underexpression can limit product accumulation, while overexpression can increase the metabolic burden on the host.

[0031] 2. Existing evidence shows that MdfA protein promotes the transport and biosynthesis of various HMOs, but it has not been shown to promote the synthesis of lactoyl-N-neotetraose. The present invention discovered that mdfA can promote the synthesis of LNnT, as follows:

[0032] Specifically, the Mn06 strain (Mn04 with an additional mdfA gene integrated into its genome) achieved an LNnT production of 3.61 g / L, a significant 27.5% increase compared to the parent strain Mn04. At the same time, the accumulation of the precursor byproduct LNTriⅡ was 0.37 g / L, showing a slightly upward trend. This not only confirms the positive regulatory effect of the mdfA gene on LNnT biosynthesis, but also provides a new modification target for further improving the production performance of the engineered strain.

[0033] 3. Promoters of different strengths have different effects on LNnT synthesis efficiency, specifically: glmS, glmM, glmU, galU and galE and three constitutive promoters of different strengths (P J23119 、P tac and P trc ) constructed 15 expression units and integrated them into the Mn06 genome to obtain engineered strains Mn07–Mn21. tacThe expression of galU driven by the promoter showed the best effect, increasing the LNnT production to 5.31g / L, which was 47.09% higher than the control strain Mn06 (3.61g / L). It is worth noting that the expression of galU regulated by different promoters showed significant differences: P J23119 -galU and P trc -galU reduced the yield to 3.11 g / L and 3.16 g / L, respectively. tac -glmS,P trc -glmM and P trc Integration of -galE also showed a positive effect, with the LNnT production of the corresponding strains reaching 3.93 g / L, 4.12 g / L, and 4.04 g / L, respectively.

[0034] To further explore the gene synergistic effect, the optimal strain Mn17 (P tac -galU) were integrated on the basis of trc -galE, P tac -glmS and P trc -glmM, and constructed the Mn22–Mn24 strains. Among them, Mn24 (integrated P trc -glmM) achieved LNnT production of 5.97 g / L, while other combinations showed no significant synergistic effect. These results indicate that a precise balance must be maintained in the supply of UDP-GlcNAc and UDP-Gal precursors during LNnT biosynthesis. This not only confirms the importance of screening for key genes but also highlights the necessity of fine-tuning gene expression through promoter engineering for optimizing biosynthetic systems.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The recombinant strain constructed by the present invention does not require the addition of antibiotics or IPTG, which reduces the cost and safety risks of industrial production and is more suitable for industrial promotion and application.

[0037] (2) The strain of the present invention can produce 7.76 g / L of LNnT after 96 h of fermentation in a shake flask, with only 0.58 g / L of the precursor LNTriⅡ remaining, which has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the metabolic pathway for engineered Escherichia coli to produce lactoyl-N-neotetraose.

[0039] Figure 2 A graph comparing the yields of engineered E. coli after integrating different copies of the hplex2B gene.

[0040] Figure 3Schematic diagram of the LNTriⅡ synthesis pathway (A) and a comparison of yields after knocking out the rapZ gene (B). DETAILED DESCRIPTION

[0041] The present invention will be further described in more detail through specific implementation examples.

[0042] The commercial products used in the following examples, including plasmids, restriction enzymes, PCR polymerases, column-based DNA extraction kits, and DNA gel purification kits, were used according to the manufacturer's instructions. Routine experimental procedures, including colony PCR, agarose gel electrophoresis, heat shock transformation, electroporation, competent cell preparation, and bacterial genome extraction and storage, were performed according to the standard procedures in Molecular Cloning: A Laboratory Manual (Fourth Edition). DNA sequencing analysis was performed by Suzhou GeneWeiZ Biotechnology Co., Ltd.

[0043] The culture media involved in the fermentation synthesis of LNnT are as follows:

[0044] (1) LB solid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 15 g / L agar powder.

[0045] (2) LB liquid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride.

[0046] (3) Fermentation medium: 20 g / L glycerol, 5 g / L yeast extract, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate, and 10 ml / L trace metal elements (10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, 2.0 g / L calcium chloride dihydrate). The pH of the medium was adjusted to approximately 6.8 with solid sodium hydroxide and sterilized at 115°C for 30 min. In addition, 4.5 mg / L thiamine was added before use.

[0047] Antibiotic concentration: kanamycin 50 mg / L, spectinomycin 50 mg / L.

[0048] LNnT production by shake flask fermentation: The engineered strain was inoculated into 4 mL of LB liquid medium and cultured at 37°C and 200 rpm for 10–12 h. The seed liquid was inoculated into 20 mL of fermentation medium at a 2% inoculum volume and cultured at 37°C and 200 rpm. When the OD 600When the cell density increased to 0.6–0.8, lactose was added to a final concentration of 5 g / L, and the culture temperature was lowered to 30°C and cultured at 200 rpm for 96 h.

[0049] The content of each component (LNnT, LNTri II, lactose, and glycerol) in the fermentation broth was determined using high-performance liquid chromatography (HPLC). The procedure was as follows: 1 mL of fermentation broth sample was centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected and filtered through a 0.22 μm aqueous filter. The resulting filtrate was then analyzed by HPLC to quantify the content of each target substance.

[0050] HPLC detection conditions: high performance liquid chromatography (HPLC) system (Waters e2695); chromatographic column: Rezex ROA-organic acid H+ (8%); detector: Waters 2414RIDetector differential detector; mobile phase: 5 mM H2SO4; flow rate: 0.6 mL / min; column temperature: 60°C; injection volume: 10 μL.

[0051] Example 1: Construction of the lactoyl-N-neotetraose synthesis pathway and optimization of multi-copy integration of β1,4-GalT

[0052] Using MG-LNT-07 as the base chassis strain, the host strain was modified using the pEcCpf1 / pcrEG dual-plasmid gene editing system. The detailed description of the relevant method is published in the literature Zhu, X.; Wu, Y.; Lv, X.; Liu, Y.; Du, G.; Li, J.; Liu, L. Combining CRISPR-Cpf1 and recombineering facilitates fast and efficient genome editing in Escherichia coli. ACS Synth. Biol. 2022, 11(5), 1897–1907.

[0053] First, the hplex2B gene was integrated at the wbgO site to construct a synthetic pathway for lactoyl-N-neotetraose. The resulting strain was named Mn01. Subsequently, the hplex2B gene was sequentially integrated at four different sites, manX, manZ, wecB, and xylB, to obtain hosts Mn02, Mn03, Mn04, and Mn05 with 2–5 copies of the gene integrated. The lactoyl-N-neotetraose production of the strain was verified by fermentation.

[0054] like Figure 2As shown, strain Mn01 produced 1.09 g / L of LNnT in the culture supernatant after shake flask fermentation, while the byproduct LNTriⅡ was only 0.09 g / L, less than one-tenth of LNnT. This result demonstrates that the hplex2B enzyme is capable of efficiently synthesizing LNnT, and the product is highly pure, meeting application requirements without the need for additional purification steps. Further analysis of the fermentation performance of engineered strains Mn02–Mn05 revealed LNnT yields of 1.35, 1.67, 2.83, and 2.17 g / L, respectively, with corresponding LNTriⅡ yields of 0.11, 0.14, 0.24, and 0.18 g / L. Notably, this study successfully constructed an LNnT-producing strain by integrating the hplex2B gene into the MG-LNT-07 chassis. Among them, strain Mn04, carrying four copies of the hplex2B gene, achieved the highest LNnT yield of 2.83 g / L, demonstrating that more copies of the hplex2B gene are not necessarily better. Experimental data indicate that the expression level of pathway enzymes has a decisive influence on product synthesis: insufficient expression leads to limited product accumulation, while overexpression may increase the metabolic burden on the host. These results confirm that optimizing the integrated copy number of key enzyme genes is a simple and efficient strain improvement strategy during the construction of plasmid-free engineered strains.

[0055] Example 2: Effect of additional integration of the mdfA gene on lactoyl-N-neotetraose synthesis

[0056] Research has shown that the mdfA gene from Escherichia coli encodes a membrane-localized multidrug transporter (MMT) that utilizes the proton electrochemical gradient as a driving force to mediate the efflux of a variety of drugs, including chloramphenicol, erythromycin, and some aminoglycoside antibiotics, thereby conferring drug resistance to the host. Notably, evidence suggests that MdfA promotes the transport and biosynthesis of various HMOs. Based on this finding, an engineered strain, Mn06, was constructed by integrating an additional copy of the mdfA gene into the genome of strain Mn04 (LNnT production of 2.83 g / L) to investigate its regulatory role in LNnT biosynthesis. Fermentation experiments revealed that strain Mn06 produced 3.61 g / L of LNnT, a significant 27.5% increase compared to the parent strain Mn04. Furthermore, the accumulation of the precursor byproduct, LNTriⅡ, reached 0.37 g / L, showing a slightly increasing trend (Table 1). This result not only confirms the positive regulatory effect of the mdfA gene on LNnT biosynthesis, but also provides a new modification target for further improving the production performance of the engineered strain.

[0057] Table 1 Shake flask fermentation information of engineered strains with additional integration of mdfA gene for LNnT production

[0058]

[0059] Example 3: Enhancement and Optimization of Glycoside Donor Synthesis

[0060] The expression of five key genes (glmS, glmM, glmU, galU and galE) in the UDP-GlcNAc and UDP-Gal biosynthesis pathways was systematically regulated. J23119 、P tac and P trc ) constructed 15 expression units and integrated them into the Mn06 genome to obtain engineered strains Mn07–Mn21. The experimental results showed (Table 2) that overexpression of the glmU gene led to a decrease in LNnT production, while fine-tuning the expression of the glmS, glmM, galU, and galE genes through promoter optimization significantly improved LNnT synthesis efficiency. tac The expression of galU driven by the promoter showed the best effect, increasing the LNnT production to 5.31g / L, which was 47.09% higher than the control strain Mn06 (3.61g / L). It is worth noting that the expression of galU regulated by different promoters showed significant differences: P J23119 -galU and P trc -galU reduced the yield to 3.11 g / L and 3.16 g / L, respectively. tac -glmS,P trc -glmM and P trc Integration of -galE also showed a positive effect, with the LNnT production of the corresponding strains reaching 3.93 g / L, 4.12 g / L, and 4.04 g / L, respectively.

[0061] To further explore the gene synergistic effect, the optimal strain Mn17 (P tac -galU) were integrated on the basis of trc -galE, P tac -glmS and P trc -glmM, and constructed the Mn22–Mn24 strains. Among them, Mn24 (integrated P trc -glmM) achieved LNnT production of 5.97 g / L, while other combinations showed no significant synergistic effect. These results indicate that a precise balance must be maintained in the supply of UDP-GlcNAc and UDP-Gal precursors during LNnT biosynthesis. This not only confirms the importance of screening for key genes but also highlights the necessity of fine-tuning gene expression through promoter engineering for optimizing biosynthetic systems.

[0062] Table 2 Enhancement and optimization of glycoside donor synthesis for LNnT production by shake flask fermentation of engineered strains

[0063]

[0064] Example 4: Effect of knocking out the rapZ gene on product synthesis

[0065] Since the residual amount of the precursor LNTriⅡ in the engineered strains involved is low, this indicates that insufficient LNTriⅡ synthesis may be the key bottleneck limiting further increase in LNnT production. Studies have shown that in Escherichia coli, the RNA binding protein RapZ regulates the expression of GlmS mRNA through the synergistic effect with the small RNA GlmY and GlmZ: when the concentration of intracellular glucosamine-6-phosphate (GlcN6P) is high, RapZ binds to GlmZ as a feedback inhibitor and degrades it by recruiting RNase E, resulting in downregulation of glmS expression. Based on this regulatory mechanism, the rapZ gene was knocked out in the optimal strain Mn24 (LNnT production 5.97g / L), and the Mn25 engineered strain was successfully constructed. The transformation achieved remarkable results, increasing the LNnT production to 7.76g / L, an increase of 29.98%, while the LNTriⅡ concentration was maintained at 0.58g / L ( Figure 3 This result not only confirms that insufficient UDP-GlcNAc supply is the key rate-limiting factor in this study system, but also effectively relieves metabolic feedback inhibition by precisely targeting the rapZ regulatory node, providing a new strategy for metabolic engineering of microbial LNnT synthesis.

[0066] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A plasmid-free recombinant Escherichia coli for synthesizing lactoyl-N-neotetraose, characterized in that: The following genetic engineering operations were performed based on strain MG-LNT-07: (1) 1 to 5 copies of the β-1,4-galactosyltransferase encoding gene hplex2B were sequentially integrated at the wbgO, manX, manZ, wecB, and xylB sites; (2) integration of the multidrug efflux pump encoding gene mdfA at the hlyE locus; (3) Integrate three promoters P with different strengths at the yghwX, intQ, caiB, ybeQ, and rph sites. J23119 、P tac 、P trc The five different pathway genes controlled by the gene include glucosamine-6-phosphate synthase encoding gene glmS, glucosamine phosphoisomerase encoding gene glmM, N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase encoding gene glmU, glucose-1-phosphate uridyltransferase encoding gene galU, and UDP-glucose-4-epimerase encoding gene galE; (4) Knockout of the RNA-binding protein encoding gene rapZ on the genome; Protein:Protein MG-LNT-07 of E.coli MG1655ΔlacZΔP lacY ::P J23119 ΔarsB::P J23119 -lgtAΔnB::P J23119 -lgtAΔpoxB::P J23119 -lgtAΔldhA::P J23119 -wbgOΔwcaJΔugdΔP galE ::P tac 。 。 2. The plasmid-free recombinant Escherichia coli for synthesizing lactoyl-N-neotetraose according to claim 1, characterized in that The following genetic engineering operations were performed based on strain MG-LNT-07: (1) Four copies of the β-1,4-galactosyltransferase encoding gene hplex2B were sequentially integrated into the wecB locus; (2) integration of the multidrug efflux pump encoding gene mdfA at the hlyE locus; (3) Knock out ybeQ and integrate P at its site tac -galU, after knocking out intQ and integrating P at its site trc -glmM. (4) Knock out the RNA-binding protein encoding gene rapZ in the genome.

3. The plasmid-free recombinant Escherichia coli for synthesizing lactoyl-N-neotetraose according to claim 1 or 2, characterized in that: The nucleotide sequence of hplex2B is shown in SEQ ID NO.1; the Gene ID of the gene mdfA is 945448; the Gene IDs of the genes glmS, glmM, glmU, galU and galE are 948241, 947692, 948246, 945730 and 945354 respectively; and the Gene ID of the gene rapZ is 947727.

4. A method for producing lactoyl-N-neotetraose, characterized in that: Lactoyl-N-neotetraose is fermented and synthesized by using the recombinant Escherichia coli according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The recombinant Escherichia coli according to any one of claims 1 to 3 is inoculated into a seed culture medium and cultured at 36-39°C for 8-14 hours to obtain a seed solution, the seed solution is transferred to a fermentation medium at an inoculum volume of 0.5%-2%, and cultured until the OD 600 When the pH value is 0.6–0.8, lactose is added and the culture temperature is lowered to 28–32°C to ferment and produce lactoyl-N-neotetraose.

6. The method according to claim 5, characterized in that The seed culture medium comprises: yeast extract 1–10 g / L, peptone 5–20 g / L, and sodium chloride 5–20 g / L; The formula of the fermentation medium includes: 10-30 g / L glycerol, 1-10 g / L yeast extract, 5-20 g / L potassium dihydrogen phosphate, 1-10 g / L diammonium hydrogen phosphate, 0.5-3 g / L citric acid, 0.5-3 g / L magnesium sulfate heptahydrate, 1-20 ml / L trace metal elements, and 1-10 mg / L thiamine.

7. The method according to claim 6, characterized in that The trace metal element formula includes: 5–20 g / L ferrous sulfate, 1–5 g / L zinc sulfate heptahydrate, 0.5–2 g / L anhydrous copper sulfate, 0.1–1 g / L manganese sulfate monohydrate, 0.1–1 g / L sodium borate decahydrate, 0.1–1 g / L ammonium molybdate, and 0.5–5 g / L calcium chloride dihydrate. Alternatively, the trace metal element formula includes: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, and 2.0 g / L calcium chloride dihydrate.

8. The method according to claim 5, characterized in that The fermentation time is 72–96 h.

9. Use of the Escherichia coli according to any one of claims 1 to 3 in the preparation of lactoyl-N-neotetraose products.

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