Plasmid-free engineered escherichia coli for synthesizing lactoyl-N-fucosyl pentasaccharide I as well as construction method and application of plasmid-free engineered escherichia coli

Abstract

The invention discloses plasmid-free engineered Escherichia coli for synthesizing lactoyl-N-fucosyl pentasaccharide I as well as a construction method and application of the plasmid-free engineered Escherichia coli, and belongs to the field of genetic engineering. Starting from a genome integration strain for efficiently synthesizing lactoyl-N-tetraose, alpha-1, 2-fucosyltransferase TeSA is introduced, the integration copy number of the alpha-1, 2-fucosyltransferase TeSA is optimized, a plasmid-free strain for efficiently synthesizing the LNFPI is constructed through multi-step reinforcement of a GDP-fucose synthesis route and metabolic flux optimization, finally, the extracellular titer of the LNFPI is 3.98 g / L, and the extracellular titer of the LNFPI is 3.98 g / L; and the method has a good industrialization prospect.

Description

Technical Field

[0001] The present invention relates to a plasmid-free engineered Escherichia coli for synthesizing lactoyl-N-fucosylpentasaccharide I and a construction method and application thereof, belonging to the field of microbial genetic engineering. Background Art

[0002] Human milk oligosaccharides (HMOs) are key active components in breast milk, with unique structures and functions. They not only promote the growth of probiotics in the infant gut and enhance immunity, but also inhibit harmful pathogens and support brain development. As the main differentiating component between breast milk and regular formula, HMOs are being widely used in high-end infant formula and have shown great potential in nutritional supplementation, medical health and other fields. Lacto-N-fucosylpentaose I (LNFP I) is a representative fucosylated HMO and the second most abundant fucosylated oligosaccharide in breast milk after 2′-fucosyllactose. Structurally, LNFP I is a fucosylated derivative of a neutral core lacto-N-tetraose, composed of five monosaccharide units. Multiple studies have confirmed that LNFP I has significant antiviral activity, immunomodulatory functions, and the ability to improve intestinal flora. This ingredient has obtained safety certification from the U.S. Food and Drug Administration (FDA) and is approved as a functional ingredient for addition to infant formula and various dietary supplements to enhance the nutritional value and health benefits of the products.

[0003] Due to the scarcity of sources, obtaining LNFPⅠ on a large scale and efficiently is the focus of current research. Existing synthesis methods are mainly divided into three categories: chemical synthesis, enzymatic catalysis and microbial fermentation. Chemical synthesis requires complex protection-deprotection steps, harsh reaction conditions and limited yields; although enzymatic synthesis has good regioselectivity, it relies on expensive glycoside donors and cofactors, and there are still technical bottlenecks in obtaining efficient glycosyltransferases. In contrast, the construction of genetically engineered strains based on synthetic biology strategies and the optimization of metabolic pathways to achieve efficient synthesis of LNFPⅠ using cheap lactose as a substrate have shown significant economic and industrial potential and are considered the most promising large-scale production solution. The biosynthesis of LNFP I involves three sequential glycosylation steps: first, lactose is catalyzed by β-N-acetylglucosamine transferase (β-GlcNAcT) to generate LNTri II (GlcNAcβ1-3Galβ1-4Glc), which is then converted to LNT by β1,3-galactosyltransferase (β1,3-GalT), and finally to LNFP I by α1,2-fucosyltransferase (α1,2-FucT). These three key enzymatic reactions use UDP-GlcNAc, UDP-galactose, and GDP-L-fucose as specific glycosyl donors, respectively. To achieve efficient LNFP I synthesis, it is necessary not only to introduce these three glycosyltransferases into the engineered strain but also to strengthen the biosynthetic pathways of the relevant sugar nucleotide precursors to ensure flux balance and efficient metabolic flow throughout the entire synthetic pathway.

[0004] Currently, research on the in vivo synthesis of LNFP I is very limited. Baumgartner et al. introduced α1,2-FucT (FutC) from Helicobacter pylori into an LNT-producing strain and enhanced the supply of GDP-L-fucose through a salvage pathway, resulting in the production of 271.6 mg / L of LNFP I in shake flasks. Hu et al. utilized a de novo GDP-L-fucose synthesis pathway based on three glycosyltransferases: LgtA, WbgO, and FutC. They produced 2.11 g / L of LNFP I in fed-batch culture. Li et al., based on a strain harboring multiple copies of LgtA, introduced WbgO and WbsJ using a three-plasmid system and overexpressed genes for the GDP-L-fucose synthesis pathway. Ultimately, they achieved an LNFP I yield of 30.47 g / L in a 5-L fermentor, while the byproducts LNTri II and LNT reached 5.36 g / L and 5.91 g / L, respectively. Currently, research on the biosynthesis of LNFP I primarily utilizes multi-plasmid expression systems. This approach not only requires the continuous addition of antibiotics to maintain plasmid stability but also relies on exogenous inducers to regulate gene expression. Plasmid genetic instability can easily lead to fluctuations in target enzyme expression. The use of antibiotics and inducers increases production costs and poses potential biosafety risks, making it difficult to meet the long-term stability requirements of industrial production. Therefore, the development of plasmid-free engineered strains constructed through genomic integration technology to achieve stable and efficient synthesis of LNFP I is crucial, providing a more reliable technical solution for the industrial production of LNFP I. Summary of the Invention

[0005] Purpose of the Invention

[0006] Current microbial production of LNFP I suffers from low yields, reliance on unstable plasmid expression systems, and the need for the addition of antibiotics and inducers. These factors not only lead to poor production stability, but also pose safety risks and significantly increase production costs. To address these technical issues, the present invention provides a recombinant strain capable of efficiently synthesizing LNFP I without the need for antibiotics and IPTG.

[0007] Technical Solution

[0008] A plasmid-free engineered Escherichia coli for synthesizing lactoyl-N-fucosylpentaose I is characterized by being prepared according to the following steps:

[0009] (1) Escherichia coli MG-LNT-11 was the starting strain, and its genotype was MG1655ΔwcaJΔlacZΔP lacY ::P J23119 ΔugdΔP g alE ::P tac ΔarsB / nagB / poxB::P J23119-lgtAΔldhA / wecB / manY / manZ / xylB::P J23119 -wbgO, three copies of the α1,2-fucosyltransferase encoding gene teSA were integrated at the yghWX, caiB, and hlyE sites;

[0010] (2) Replace the natural promoters of the gene clusters cpsBG and gmd-fcl with the strong constitutive promoter P J23119 , and gmd-fcl was additionally integrated at the adhE locus, where the cpsG gene encodes phosphomannose mutase, the gmd gene encodes GDP-mannose-4,6-dehydratase, and the fcl gene encodes GDP-L-fucose synthase;

[0011] (3) Integrate the wbgO gene at the ilvG site.

[0012] As an optimization method, the construction method of the recombinant bacteria using Escherichia coli MG-LNT-11 can be found 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 chromosomal integration of multicopy key gly cosyltransferase genes. Int. J. Biol. Macromol. 2025, 284 (Pt1), 137987). Specifically, the E. coli MG-LNT-11 was constructed from E. coli MG1655. First, the lacZ (β-galactosidase), wcaJ (UDP-glucose lipid transferase), and ugd (UDP-glucose-6-dehydrogenase) genes were deleted. Second, the natural promoters of lacY (β-galactosidase permease) and galE (UDP-galactose-4-epimerase) were replaced with constitutive promoters P, P, and P, respectively. J23119 and P tac ; Finally, lgtA (β-1,3-N-acetylglucosaminyltransferase) gene copies were integrated into multiple sites in the genome (arsB, nagB, poxB), and wbgO (β-1,3-galactosyltransferase) gene copies were integrated into the ldhA, wecB, manY, manZ, and xylB sites.

[0013] In one embodiment, the Escherichia coli is Escherichia coli MG1655.

[0014] In one embodiment, the teSA gene is derived from Thermoanaerobacterium sp. RBIITD, and its corresponding GenBank number is SNX52916.1.

[0015] In one embodiment, the Gene IDs of the genes cpsG, cpsB, gmd and fcl are 946574, 946580, 946562 and 946563, respectively.

[0016] In one embodiment, the gene wbgO is derived from Escherichia coli O55:H7, and its corresponding GenBank number is AAL67559.1.

[0017] In one embodiment, the Gene ID of the gene manA is 944840.

[0018] In one embodiment, the promoter P J23119 The nucleotide sequence of P is shown in SEQ ID NO.1. tac The nucleotide sequence is shown in SEQ ID NO.2.

[0019] The second object of the present invention is to provide a method for producing LNFP I, wherein the method uses the above-mentioned recombinant bacteria to produce LNFP I by fermentation.

[0020] In one embodiment, the method comprises: inoculating the recombinant strain conventionally cultured on a plate into a seed culture medium, performing seed culture at a temperature range of 36-39°C for 8-14 hours to obtain a seed culture solution, then transferring the seed solution to a fermentation medium at an inoculation ratio of 0.5%-2%, and when the absorbance value of the culture at a wavelength of 600 nm (OD 600 ) reached the range of 0.6–0.8, lactose substrate was added and the culture temperature was adjusted to the range of 28–32°C, under which conditions the fermentation production of LNFP I was carried out.

[0021] 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.

[0022] 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: 30 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.

[0023] In one embodiment, the trace metal element components include: 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 components include: 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.

[0024] In one embodiment, the fermentation time is 72-96 hours.

[0025] 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.

[0026] In one embodiment, the application includes producing LNFP I or a product containing LNFP I using the above-mentioned recombinant bacteria or the method.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention further improves upon the prior art by integrating the relevant enzymes into the genome. The results of the copy number optimization of the TeSA gene in Example 2 show that when the copy number of the TeSA gene is greater than 5, the conversion from LNTriⅡ to LNT is inhibited, which is detrimental to the accumulation of the final product, LNFP I. When the copy number is increased to 4, it may impose an unnecessary burden on the host, thereby reducing the residual amounts of both the precursors LNT and LNTriⅡ. Therefore, three copies of the TeSA gene are most effective.

[0029] Further enhancement of the key GDP-fucose synthesis pathway revealed that LNFPI production significantly increased with the continuous enhancement of the GDP-fucose synthesis pathway genes cpsBG and gmd-fcl, reaching LNFPI levels of 0.94 g / L, 3.02 g / L, 3.50 g / L, and 0.14 g / L in MF531, MF532, MF533, and MF534, respectively. However, further enhancement of the cpsBG gene caused LNFP I production to plummet to 0.14 g / L, while residual LNT increased to 6.24 g / L. This may have disrupted the steady-state supply of GDP-fucose, leading to the strain's loss of the ability to convert LNT to LNFP I. The gmd-fcl gene promoter enhancement had the most significant effect, increasing LNFP I production by 221.3%. The cpsBG gene promoter enhancement and the additional integration of the gmd-fcl gene also had some effects. The MF533 strain performed best, with an LNFP I content of 3.50 g / L and residual amounts of the precursors LNT and LNTriⅡ of 1.43 g / L and 2.56 g / L, respectively.

[0030] Synthesis route optimization results showed that strain MF535, formed by further stacking the wbgO gene on MF533, and strain MF536, formed by replacing the native promoter of the manA gene with PJ23119, exhibited a decrease in LNFP I production to 2.73 g / L after manA was enhanced, with residual LNT and LNTriⅡ levels of 1.57 g / L and 2.78 g / L, respectively. However, MF535 performed best, with LNFP I production further increased to 3.98 g / L and LNT reduced to 0.85 g / L.

[0031] (2) The recombinant strain developed by the present invention does not require the exogenous addition of antibiotics or IPTG inducers, which not only significantly reduces the economic cost and biosafety risks of industrial production, but also greatly improves the feasibility and promotion value of this technology in actual industrial applications.

[0032] (3) The strain of the present invention can produce 3.98 g / L of LNFP I after 96 hours of culture under shake flask fermentation conditions, showing excellent prospects for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the metabolic pathway for engineered Escherichia coli to produce LNFP I.

[0034] Figure 2 A graph comparing the yields of engineered E. coli after integrating different copies of the teSA gene.

[0035] Figure 3This is a comparison chart of the yield after strengthening the GDP-fucose synthesis pathway. DETAILED DESCRIPTION

[0036] The present invention will be further described in more detail through specific implementation examples.

[0037] The experimental materials used in the following examples include, but are not limited to, various commercial plasmids, restriction endonucleases, PCR polymerases, column-based DNA extraction kits, and DNA gel purification kits. Their use strictly follows the standard operating procedures provided by the manufacturer. All routine molecular biology experiments, such as colony PCR, agarose gel electrophoresis, heat shock transformation, electroporation transformation, competent cell preparation, and bacterial genome extraction and preservation, were performed in accordance with the standard methods described in Molecular Cloning: A Laboratory Manual (Fourth Edition). DNA sequencing analysis was provided by Suzhou Jinweizhi Biotechnology Co., Ltd. with technical service support.

[0038] The culture media involved in the fermentation synthesis of LNFP I are as follows:

[0039] (1) LB solid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 15 g / L agar powder.

[0040] (2) LB liquid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride.

[0041] (3) Fermentation medium: 30 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, and 2.0 g / L calcium chloride dihydrate). The pH of the culture medium was adjusted to 6.8 ± 0.1 with solid sodium hydroxide. After autoclaving at 115°C for 30 minutes, 4.5 mg / L thiamine solution was aseptically added before use.

[0042] Antibiotic concentration: kanamycin 50 mg / L, spectinomycin 50 mg / L.

[0043] LNFP I shake flask fermentation production: First, the engineered strain was inoculated into 4 mL of LB liquid medium and cultured at 37°C and 200 rpm for 10–12 hours to prepare the seed solution. The seed solution was then transferred to 20 mL of fermentation medium at a 2% (v / v) inoculum and cultured at the same temperature and rotation speed. 600 When the value reached 0.6–0.8, lactose was added to a final concentration of 5 g / L, and the culture temperature was adjusted to 30 °C (maintaining a rotation speed of 200 rpm), and the culture was continued for 96 h to complete the fermentation process.

[0044] Fermentation broth composition analysis utilizes high-performance liquid chromatography (HPLC) to quantitatively determine the content of target substances, including LNFP I, LNT, LNTri II, lactose, and glycerol. The specific testing procedure involves taking a 1 mL sample of fermentation broth and centrifuging it at 10,000 × g for 10 minutes to remove bacterial cells. The supernatant is collected and filtered through a 0.22 μm aqueous microporous filter. The resulting clear filtrate is then directly sampled for HPLC analysis.

[0045] 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.

[0046] Example 1: Construction of LNFP I Synthesis Pathway

[0047] MG-LNT-08 and MG-LNT-11 were used as the base strains. Related strains are reported 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 chromosomal integration of multicopy key glycosyltransferase genes. Int. J. Biol. Macromol. 2025, 284(Pt1), 137987). Strains MG-LNT-08 and MG-LNT-11 integrate two and five copies of the β-1,3-galactosyltransferase encoding gene, wbgO, respectively. By strengthening the target biosynthesis pathway and deleting competing bypass metabolic genes, LNT biosynthesis efficiency was significantly improved, making them ideal bases for constructing LNFP I production strains. Their host genotypes and fermentation yields are shown in Table 1.

[0048] Table 1 Information on chassis engineering strains for efficient LNT synthesis

[0049]

[0050] The host strain was modified by the pEcCpf1 / pcrEG dual-plasmid gene editing system to introduce the necessary α1,2-FucT encoding gene teSA. The detailed operation process of this method is disclosed 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. The primers involved in the gene manipulation process are summarized in Table 2. First, the teSA gene was integrated into the yghWX site of the two chassis strains to construct the LNFP I synthesis pathway. The resulting strains were named MF21 and MF51, respectively. To systematically compare the advantages and disadvantages of these two chassis, the teSA gene was then sequentially superimposed and integrated at two other different sites (caiB and hlyE) to obtain hosts MF22, MF23, MF52, and MF53 with 2–3 copies integrated (see Table 3 ). Shake flask fermentation was then used to verify the production of various products of the strains.

[0051] Table 2 Primers involved in gene manipulation

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] Table 3 Host genotypes of MF22, MF23, MF52 and MF53

[0058]

[0059] Table 4 Shake flask fermentation information of engineered strains for LNFP I production

[0060]

[0061] Preliminary shake flask fermentation results are shown in Table 4. In MG-LNT-08 and MG-LNT-11, strains with 1–3 copies of the teSA gene were integrated, respectively, to synthesize LNFP I. The final LNFP I yield ranged from 0.44 to 0.74 g / L. Strain MF53 performed best, reaching a yield of 0.74 g / L. LNFP I yield increased with increasing teSA gene copy number. Regarding the residual levels of the precursors LNT and LNTriII, strains harboring a higher copy number of the wbgO gene exhibited higher levels of LNT residuals and lower levels of LNTriII residuals, consistent with the levels observed prior to teSA gene integration. Engineered strains harboring a higher copy number of the wbgO gene exhibited enhanced glycosyltransferase activity, effectively promoting the conversion of lactose to LNT. Since LNT is a key precursor for LNFP I synthesis, this efficient conversion capability lays a crucial foundation for the large-scale production of LNFP I. In contrast, host strains with low-copy wbgO genes performed poorly in terms of LNFP I synthesis efficiency. Based on the above metabolic advantages, MF53 strain was selected as the starting strain for further genetic modification.

[0062] Example 2: Copy number optimization of TeSA gene

[0063] Studies have shown that optimizing the copy number of the target gene is a key regulatory strategy to improve the efficiency of product synthesis. Specifically, when the gene copy number is insufficient, its expression level is difficult to meet the metabolic needs of the product synthesis pathway; and excessive integration will cause the host metabolic load to be too heavy, thereby affecting the normal physiological function of the bacteria. In Example 1, as the gradient of the TeSA gene copy number increased, the LNFP I production also gradually increased. Therefore, by systematically optimizing the integrated copy number of the TeSA gene, it is expected to establish the optimal balance between expression level and metabolic load. Based on this, the TeSA gene was further integrated on the basis of MF53 to determine its optimal expression copy number range. The TeSA gene was sequentially integrated at the fucK, wcaC and ybeQ sites to obtain 4-6 copies of hosts MF54, MF55 and MF56, and the shake flask fermentation results are shown in the figure. Figure 2 As shown, no significant increase in LNFP I production was observed with further increases in copy number. LNFP I yields in MF54, MF55, and MF56 were 0.61 g / L, 0.79 g / L, and 0.76 g / L, respectively. Compared to MF53, the accumulation of both the precursors LNT and LNTriⅡ decreased in MF54, while the product distribution patterns of MF55 and MF56 changed significantly, with LNT accumulation decreasing to 0.64 g / L and 0.59 g / L, respectively, while LNTriⅡ accumulation increased significantly to 6.39 g / L and 6.82 g / L. This suggests that when the TeSA gene copy number exceeds 5, the conversion of LNTriⅡ to LNT is inhibited, hindering the accumulation of the final product LNFP I. Increasing the copy number to 4 may impose an unnecessary burden on the host, resulting in a decrease in the residual amounts of both the precursors LNT and LNTriⅡ. Therefore, a three-copy TeSA gene is most effective.

[0064] Example 3: Enhancement of the key GDP-fucose synthesis pathway

[0065] Based on the highly efficient LNT-biosynthesizing chassis strain MG-LNT-11, the α1,2-FucT-encoding gene teSA was integrated to construct the LNFP I biosynthesis pathway. However, as the teSA gene copy number increased, LNFP I production remained low, reaching a maximum of 0.79 g / L, far below the requirements for industrial production. The conversion of LNT to LNFP I requires two key factors: the efficient and specific α1,2-FucT and an adequate supply of GDP-fucose. Therefore, following the integration of α1,2-FucT, it is necessary to strengthen the GDP-fucose biosynthesis pathway genes.

[0066] Using MF53 as the base strain, the following operations were performed in sequence: using the strong promoter P J23119 Replace the natural promoter of the gene cluster cpsBG with a strong promoter PJ23119 The natural promoter of the gene cluster gmd-fcl was replaced, gmd-fcl was additionally integrated at the adhE locus, and cpsBG was integrated at the ycgH locus to obtain strains MF531, MF532, MF533, and MF534. Detailed information on the strains is shown in Table 5 .

[0067] Table 5 Information on engineering strains that enhance the GDP-fucose synthesis pathway

[0068]

[0069] The synthesis of LNFP I and the residues of precursors LNT and LNTriⅡ of each strain were verified by shake flask fermentation. Figure 3 As shown in Figure 2, LNFP I production increased significantly with the continuous enhancement of the GDP-fucose biosynthesis pathway genes cpsBG and gmd-fcl. LNFP I levels in MF531, MF532, MF533, and MF534 were 0.94 g / L, 3.02 g / L, 3.50 g / L, and 0.14 g / L, respectively. Enhancement of the gmd-fcl gene promoter had the most significant effect, increasing LNFP I production by 221.3%. Enhancement of the cpsBG gene promoter and the additional integration of the gmd-fcl gene also had some effects. MF533 performed best, with an LNFP I content of 3.50 g / L and residual levels of its precursors LNT and LNTriⅡ of 1.43 g / L and 2.56 g / L, respectively. However, further stacking of the cpsBG gene caused the LNFP I production to drop sharply to 0.14 g / L, while the LNT residual content increased to 6.24 g / L. This may have destroyed the supply homeostasis of GDP-fucose, resulting in the strain losing the ability to convert from LNT to LNFP I.

[0070] Example 4: Synthesis Pathway Optimization

[0071] After GDP-fucose supply enhancement, the engineered strain MF533 showed excellent LNFP I synthesis ability, with a yield of 3.50 g / L, and the residual amounts of the precursors LNT and LNTriⅡ were 1.43 g / L and 2.56 g / L, respectively. Judging from the accumulation concentrations of the three oligosaccharides, the LNT concentration was low, so the wbgO gene was further stacked on the basis of MF533 to form the strain MF535 to promote the conversion of LNTriⅡ to LNT. The shake flask fermentation results are shown in Table 6. The LNFP I yield of MF535 was further increased to 3.98 g / L, and LNT was reduced to 0.85 g / L. Based on MF535, the natural promoter of the manA gene was replaced with P J23119, resulting in strain MF536. manA encodes mannose-6-phosphate isomerase, the first step in the GDP-fucose biosynthesis pathway. After enrichment with manA, LNFP I production decreased to 2.73 g / L, while residual levels of LNT and LNTriⅡ were 1.57 g / L and 2.78 g / L, respectively.

[0072] Table 6 Synthesis route optimization for LNFP I production engineered strain shake flask fermentation information

[0073]

[0074] 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 engineered Escherichia coli for synthesizing lactoyl-N-fucosylpentaose I, characterized in that: Prepared as follows: (1) Escherichia coli MG-LNT-11 was the starting strain, and its genotype was MG1655ΔwcaJΔlacZΔP lacY ::P J23119 ΔugdΔP galE ::P tac ΔarsB / nagB / poxB::P J23119 -lgtAΔldhA / wecB / manY / manZ / xylB::P J23119 -wbgO, three copies of the α1,2-fucosyltransferase encoding gene teSA were integrated at the yghWX, caiB, and hlyE sites; (2) Replace the natural promoters of the gene clusters cpsBG and gmd-fcl with the strong constitutive promoter P J23119 , and gmd-fcl was additionally integrated at the adhE locus, where the cpsG gene encodes phosphomannose mutase, the gmd gene encodes GDP-mannose-4,6-dehydratase, and the fcl gene encodes GDP-L-fucose synthase; (3) Integrate the wbgO gene at the ilvG site.

2. The plasmid-free engineered Escherichia coli for synthesizing lactoyl-N-fucosylpentaose I according to claim 1, characterized in that: The E. coli MG-LNT-11 was constructed by the following steps: using E. coli MG1655 as the starting strain, firstly knocking out the lacZ, wcaJ and ugd genes; secondly replacing the natural promoters of lacY and galE with the constitutive promoter P J23119 and P tac ; Finally, the lgtA gene copy was integrated into the arsB, nagB, and poxB sites of the genome, and the wbgO gene copy was integrated into the ldhA, wecB, manY, manZ, and xylB sites.

3. The plasmid-free engineered Escherichia coli for synthesizing lactoyl-N-fucosylpentaose I according to claim 1, characterized in that: The GenBank accession number of the teSA gene is SNX52916.

1.

4. The plasmid-free engineered Escherichia coli for synthesizing lactoyl-N-fucosylpentaose I according to claim 1, characterized in that: The Gene IDs of the genes cpsG, cpsB, gmd and fcl are 946574, 946580, 946562 and 946563 respectively.

5. The plasmid-free engineered Escherichia coli for synthesizing lactoyl-N-fucosylpentaose I according to claim 1, characterized in that: The GenBank accession number of the gene wbgO is AAL67559.

1.

6. A plasmid-free engineered Escherichia coli for synthesizing lactoyl-N-fucosylpentaose I according to claim 1 or 2, characterized in that: The promoter P J23119 The nucleotide sequence of P is shown in SEQ ID NO.

1. tac The nucleotide sequence is shown in SEQ ID NO.

2.

7. A method for producing LNFP I, characterized in that: LNFP I is produced by fermentation using the engineered Escherichia coli according to any one of claims 1 to 6.

8. The method according to claim 7, characterized in that The engineered E. coli cultured on a plate was inoculated into the seed culture medium, and the seed culture was carried out at a temperature range of 36-39°C for 8-14 hours to obtain the seed culture solution. The seed solution was then transferred to the fermentation medium at an inoculation ratio of 0.5%-2%. When the absorbance value of the culture at a wavelength of 600 nm was OD 600 When the concentration reached 0.6–0.8, lactose substrate was added and the culture temperature was adjusted to 28–32°C for LNFP I fermentation production.

9. The method according to claim 8, characterized in that The components of the seed culture medium include: yeast extract 1-10 g / L, peptone 5-20 g / L, sodium chloride 5-20 g / L. Optionally, the components of the seed culture medium include: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L; 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: 30 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.

10. Use of the plasmid-free engineered recombinant Escherichia coli for synthesizing lactoyl-N-fucosylpentaose I according to claims 1-6 in producing LNFP I or products containing LNFP I.

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