Beta 1,4-galactosyltransferase Habetagalt-s233p and its use in the synthesis of lnnt

Abstract

The application discloses a beta 1,4-galactosyltransferase Ha beta 4GalT-S233P and application thereof in synthesis of LNnT, and belongs to the technical field of functional enzymes. The amino acid sequence of the beta 1,4-galactosyltransferase Ha beta 4GalT-S233P is shown as SEQ ID NO. 3. The application discloses the application of the beta 1,4-galactosyltransferase Ha beta 4GalT-S233P in synthesis of LNnT. The beta 1,4-galactosyltransferase Ha beta 4GalT-S233P is obtained by modification of the beta 1,4-galactosyltransferase Ha beta 4GalT, has higher glycosyltransfer activity, and the conversion rate of synthesis of LNnT can reach 99.80%. The application lays a foundation for metabolic engineering of Escherichia coli for production of LNnT.

Description

Technical Field

[0001] This invention relates to a β1,4-galactosyltransferase Haβ4GalT-S233P and its application in the synthesis of LNnT, belonging to the field of functional enzyme technology. Background Technology

[0002] Breast milk is the optimal source of nutrition for infant growth and development, meeting almost all of their nutritional needs in early childhood. Human milk oligosaccharides (HMIs) are complex, non-conjugated carbohydrates found in breast milk, and are the third largest solid component after lactose and lipids. Lacto-N-neotetraose (LNnT) is one of the core structures of HMIs. It is a tetrasaccharide composed of lactose (Lac) as the reducing end, with N-acetylglucosamine and galactose alternately linked by β-1,3 and β-1,4 glycosidic bonds. LNnT possesses physiological activities such as regulating the intestinal epithelial barrier, promoting the proliferation of beneficial bacteria, and preventing necrotizing enterocolitis, making it a promising food fortifier. Therefore, the large-scale synthesis of LNnT is of great significance.

[0003] Currently, enzymatic and whole-cell synthesis are the mainstream methods for synthesizing LNnT. Both methods are based on β1,4-galactosyltransferases, but most of the β1,4-galactosyltransferases reported in existing technologies suffer from drawbacks such as poor soluble expression and low transglycosylation activity. Therefore, it is of great significance to explore β1,4-galactosyltransferases suitable for industrial applications. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a β1,4-galactosyltransferase Haβ4GalT-S233P and its application in the synthesis of LNnT, which belongs to the field of functional enzyme technology.

[0005] This invention is achieved through the following technical solution:

[0006] A β1,4-galactosyltransferase Haβ4GalT-S233P, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] The nucleotide sequence of the gene encoding the β1,4-galactosyltransferase Haβ4GalT-S233P is shown in SEQ ID NO.4.

[0008] Application of the β1,4-galactosyltransferase Haβ4GalT-S233P in the synthesis of LNnT.

[0009] The specific application method is as follows: using lacto-N-triose II (LNT II) as the donor and uridine diphosphate galactose (UDP-galactose, UDP-Gal) as the acceptor, LNnT is generated under the action of β1,4-galactosyltransferase Haβ4GalT-S233P.

[0010] A specific application method can also be as follows: using Escherichia coli as the host bacterium, construct a recombinant engineered bacterium expressing β1,4-galactosyltransferase Haβ4GalT-S233P; culture the recombinant engineered bacterium and add lactose as a substrate. Under the action of β1,4-galactosyltransferase Haβ4GalT-S233P expressed by the recombinant engineered bacterium, UDP-GlcNAc and UDP-Gal synthesized by the recombinant engineered bacterium itself are used as precursors to react with lactose to synthesize LNnT.

[0011] Furthermore, the recombinant engineered bacteria expressing β1,4-galactosyltransferase Haβ4GalT-S233P are constructed as follows:

[0012] (1) Construction of the basic strain for LNnT production

[0013] Using Escherichia coli BL21(DE3) as the host bacterium, the UDP-N-acetyl-2-epimerase gene was knocked out using the CRISPR-Cas9 gene editing system. wecB Purine synthesis repressor protein gene purR and β-galactosidase gene lacZ and in wecB Integrating sugar transporter gene at the gene locus nagE ,exist purR PRPP synthase gene integrated at the gene locus prs The basic strain for LNnT production was constructed;

[0014] UDP-N-acetyl-2-epimerase gene wecB The nucleotide sequence is shown in SEQ ID NO.5; purine synthesis repressor protein gene. purR The nucleotide sequence is shown in SEQ ID NO.6; β-galactosidase gene lacZ The nucleotide sequence is shown in SEQ ID NO.7; Glycotransporter gene nagE The nucleotide sequence is shown in SEQ ID NO.9; PRPP synthase gene prs The nucleotide sequence is shown in SEQ ID NO.11;

[0015] (2) Construction of LNnT producing strain

[0016] The β1,3-N-acetylglucosyltransferase gene was inserted at MCS1 of plasmid pETDueT-1. NmlgtA and lactose permease gene lacY The gene encoding β1,4-galactosyltransferase Haβ4GalT-S233P was inserted into the MCS2 region of plasmid pETDueT-1 to construct a recombinant plasmid; this recombinant plasmid was then introduced into the LNnT-producing strain to construct the LNnT-producing strain.

[0017] β1,3-N-acetylglucosyltransferase gene NmlgtA The nucleotide sequence is shown in SEQ ID NO.13; lactose permease gene lacY The nucleotide sequence is shown in SEQ ID NO.15; the gene encoding β1,4-galactosyltransferase Haβ4GalT-S233P is shown in SEQ ID NO.4.

[0018] The β1,4-galactosyltransferase Haβ4GalT-S233P of this invention is obtained by modifying the β1,4-galactosyltransferase Haβ4GalT, exhibiting higher transglycosylation activity and achieving a conversion rate of 99.80% for LNnT synthesis. This invention also constructs a recombinant engineered bacterium expressing β1,4-galactosyltransferase Haβ4GalT-S233P. Cultivating this recombinant engineered bacterium and adding exogenous lactose allows for the synthesis of LNnT. This invention lays the foundation for metabolic engineering of Escherichia coli to produce LNnT.

[0019] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0020] Figure 1 SDS-PAGE electrophoresis results: Lane 1: protein marker; Lane 2: pure enzyme solution; Lane 3: elution buffer; Lane 4: breakthrough buffer; Lane 5: crude enzyme solution.

[0021] Figure 2 Results of relative enzyme activity determination under different temperature conditions.

[0022] Figure 3 Results of relative enzyme activity determination under different pH conditions.

[0023] Figure 4 Results of relative enzyme activity determination under different metal ions and chemical reagent conditions.

[0024] Figure 5 : Results of relative yield determination under different donor-acceptor ratios.

[0025] Figure 6 The results of relative yield determination under different enzyme dosages and reaction times.

[0026] Figure 7 Results of the determination of the relative transglycosylation activity of each mutant enzyme.

[0027] Figure 8 Schematic diagram showing the changes in cell density, LNnT concentration, LNT II concentration, and lactose concentration during fermentation. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0029] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0030] The plasmids pMal-C5X and pETDuet-1 used in this invention were preserved in the inventor's laboratory; these plasmids are commonly used gene expression vectors in the field and can be purchased from the market.

[0031] The Escherichia coli BL21(DE3) competent cells used in this invention were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0032] The LB medium used in this invention consists of the following components: 10 g / L sodium chloride; 10 g / L tryptone; 5 g / L yeast extract; 15 g / L agar powder (added when preparing solid medium); the remainder is water; sterilization conditions: 115℃, 30 min.

[0033] The M9 culture medium used in this invention was purchased from Aili Biotechnology Co., Ltd.

[0034] The UDP-Gal used in this invention was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0035] The LNT II used in this invention was purchased from Shandong Henglu Biotechnology Co., Ltd.

[0036] The lactose used in this invention was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0037] Example 1: Discovery of β1,4-galactosyltransferase Haβ4GalT

[0038] To discover β1,4-galactosyltransferases with high transglycosylation activity, this invention screened a strain derived from *Helicobacter pectinus* from the NCBI website. Helicobacter acinonychis The gene fragment (GenBank: WP_011577652.1) encodes a protein whose amino acid sequence is shown in SEQ ID NO.1. This protein may possess β1,4-galactosyltransferase activity and is named β1,4-galactosyltransferase Haβ4GalT. The nucleotide sequence of the gene encoding β1,4-galactosyltransferase Haβ4GalT (codon optimized) is shown in SEQ ID NO.2. This invention involves heterologous expression of this enzyme and investigation of its enzymatic activity to assess its suitability for industrial production of LNnT.

[0039] Example 2: Heterologous expression and purification of β1,4-galactosyltransferase Haβ4GalT

[0040] The steps are as follows:

[0041] (1) Construction of recombinant plasmid: The gene fragment (nucleotide sequence as shown in SEQ ID NO.2) was artificially synthesized and ligated into the pMal-C5X expression vector to obtain the recombinant plasmid.

[0042] (2) Construction of recombinant engineered bacteria: The above recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and positive transformants were screened using LB plates containing ampicillin resistance to construct a recombinant strain expressing β1,4-galactosyltransferase Haβ4GalT.

[0043] (3) Expression of β1,4-galactosyltransferase Haβ4GalT: The recombinant strain was inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 12 h; then, 1% (volume percentage) of the inoculum was added to 50 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm until OD. 600 The concentration was 0.6; isopropyl-β-D-thiogalactoside (IPTG) was added to a concentration of 0.1 mM, and expression was induced at 20℃ for 20 h.

[0044] (4) Extraction: Take the culture medium, centrifuge at 4℃ and 8000 rpm for 10 min, collect the bacterial cells, resuspend them in ultrapure water, sonicate for 30 min, centrifuge at 4℃ and 8000 rpm for 15 min, obtain the supernatant, filter with a 0.45 μm filter membrane, which is the crude enzyme solution.

[0045] (5) Purification: The purification was performed using linear starch resin affinity chromatography. The purification column was rinsed with 30 mL of ultrapure water and equilibrated with 30 mL of CB buffer. The crude enzyme solution was added to the purification column and allowed to drip naturally. This process was repeated 3 times. Unbound contaminants were then rinsed with CB buffer until the permeate could no longer turn Coomassie Brilliant Blue blue. The target protein was eluted with maltose elution buffer to obtain the eluent.

[0046] The eluent was concentrated using an ultrafiltration tube with a cutoff of 30 kDa, centrifuged and replaced at 4℃ and 4000 r / min to obtain a pure enzyme solution containing β1,4-galactosyltransferase Haβ4GalT. The concentration of the pure enzyme solution was determined to be 467.09 μg / mL.

[0047] The CB buffer solution was prepared by dissolving 2.4 g Tris, 11.7 g NaCl and 0.3 g EDTA in 1 L of ultrapure water and adjusting the pH to 7.5 with hydrochloric acid.

[0048] The maltose elution buffer is prepared by dissolving 3.4 g of maltose in 1 L of CB buffer.

[0049] (6) Electrophoresis detection: SDS-PAGE detection was performed on the crude enzyme solution and the pure enzyme solution. The SDS-PAGE electrophoresis detection results are as follows: Figure 1 As shown, the purified protein band is single, with a molecular weight slightly less than 75.0 kDa, consistent with the prediction. The purified protein is a Haβ4GalT protein fused with the fusion-aiding tag MBP. There are no obvious contaminating proteins near the target band, indicating high purity.

[0050] Example 3 Determination of transglycosylation activity

[0051] The method for determining transglycosylation activity is as follows: A 150 μL reaction system contains: 1.5 mM LNT II, ​​1.5 mM UDP-Gal, and 10 mM Mg. 2+ Add 10 μL of pure enzyme solution (in the form of MgCl2) and Tris-HCl buffer (50 mM, pH 7.0). Mix all components and react in a 37°C water bath for 2 h. After the reaction, inactivate the enzyme by boiling in a water bath for 10 min. Centrifuge at 12000 rpm for 10 min, collect the supernatant, filter through a 0.22 μm filter membrane, and perform liquid chromatography analysis.

[0052] The conditions for liquid chromatography detection were as follows: the detector was a differential refractive index detector, the chromatographic column was an Aminex HPX-87H column (4.6 mm × 150 mm), the analysis temperature was 50℃, the mobile phase was 5 mM sulfuric acid solution, the flow rate was 0.4 mL / min, and the injection volume was 20 μL.

[0053] Glycosyl activity definition (U): The amount of enzyme required to catalyze the synthesis of 1 nmol LNnT per minute under specific conditions.

[0054] Transglycosylation specific activity (U / mg): The transglycosylation activity unit per milligram of enzyme.

[0055] Liquid chromatography results showed that the peak elution time of the synthesized product was consistent with that of the LNnT standard, confirming that the enzyme could catalyze the synthesis of LNnT. The transglycosylation activity of Haβ4GalT in synthesizing LNnT was determined to be 6.82 U / mg.

[0056] Example 4 Determination of Enzymatic Properties

[0057] (1) Determination of optimal temperature: Enzyme activity was measured at different temperatures (25℃, 30℃, 35℃, 37℃, 40℃, 45℃, 50℃) within the range of 25-50℃, following the method described in Example 3. The highest enzyme activity was taken as 100%, and the relative enzyme activity under different temperature conditions was calculated. The results of the relative enzyme activity determination under different temperature conditions are as follows: Figure 2 As shown, the optimal reaction temperature for β1,4-galactosyltransferase Haβ4GalT is 37℃.

[0058] (2) Determination of optimal pH: Enzyme activity was measured at different pH values ​​within the range of pH 5.0–9.5 (the buffers used were: MES-NaOH buffer at pH 5.0–6.5, HEPES-NaOH buffer at pH 6.5–7.5, and Tris-HCl buffer at pH 7.0–9.5), following the method described in Example 3. The relative enzyme activity under different pH conditions was calculated with the highest enzyme activity defined as 100%. The results of the relative enzyme activity measurements under different pH conditions are shown below. Figure 3 As shown, the optimal pH for β1,4-galactosyltransferase Haβ4GalT is 6.5.

[0059] (3) Effects of metal ions and chemical reagents on transglycosylation activity: Following the determination method in Example 3, a 50 mM HEPES-NaOH solution at pH 6.5 was used as a buffer solution, and metal ions (Mg2+, ... 2+ Mn 2+ Ni 2+ Ca 2+All were added in the form of chloride salts) and chemical reagents (EDTA and DTT) were used to replace 10 mM Mg. 2+ Enzyme activity was determined under different metal ion and chemical reagent conditions. The enzyme activity of the blank group (without any added metal ions) was taken as 100%, and the relative enzyme activity under different metal ion and chemical reagent conditions was calculated. The results of the relative enzyme activity determination under different metal ion and chemical reagent conditions are shown below. Figure 4 As shown. It can be seen that Ni 2+ EDTA at 1 mM and 10 mM significantly inhibited transglycosylation activity, while Mg 2+ Mn 2+ Ca 2+ Both 1 mM DTT and 1 mM DTT significantly promoted enzyme activity, among which 1 mM Mn 2+ The transglycosylation activity was increased to 299.85%, indicating that β1,4-galactosyltransferase Haβ4GalT is a metal ion-dependent enzyme.

[0060] Example 5: Optimization of the reaction system for synthesizing LNnT

[0061] (1) LNnT was synthesized using LNT II as the donor and UDP-Gal as the acceptor under the action of β1,4-galactosyltransferase Haβ4GalT. The 150 μL reaction system contained: 1.5 mM LNT II, ​​3.75 mM UDP-Gal, and 1 mM Mn 2+ Added in the form of MnCl2, an appropriate amount of pure enzyme solution (the enzyme concentration in the reaction system is 190 μg / mL, which is used for acceptor ratio screening and has been preliminarily verified to effectively catalyze the reaction), and HEPES-NaOH buffer (concentration 50 mM, pH 6.5). Mix all components and react in a water bath at 37℃ for 2 h; after the reaction, inactivate by boiling in a water bath for 10 min; centrifuge at 12000 rpm for 10 min, collect the supernatant, filter it through a 0.22 μm filter membrane, and perform liquid chromatography analysis under the same conditions as in Example 3.

[0062] (2) Optimization of donor-acceptor ratio: In the reaction system described in (1) above, the concentrations of UDP-Gal were adjusted to 1.5 mM, 2.25 mM, 3 mM, 3.75 mM, 4.5 mM, 5.25 mM, and 6 mM, respectively, i.e., the donor-acceptor ratio (molar ratio of LNT II to UDP-Gal) was 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, and 1:4. The relative yields under different donor-acceptor ratios were calculated with the highest LNnT yield as 100%. The results of the relative yield measurements under different donor-acceptor ratios are as follows: Figure 5 As shown, the optimal donor-acceptor ratio is 1:2.5.

[0063] (3) Optimization of enzyme dosage and reaction time: From an economic and efficiency perspective, in the reaction system described in (1) above, a better low enzyme concentration range was optimized, and the enzyme dosage was adjusted so that the enzyme concentrations in the reaction system were 60 μg / mL, 80 μg / mL, and 100 μg / mL, respectively. The reaction was carried out in a water bath at 37℃ for 10 h, with samples taken at 2-h intervals. Taking the highest LNnT yield as 100%, the relative yields under different enzyme dosages and reaction times were calculated. The results of the relative yield determination under different enzyme dosages and reaction times are as follows: Figure 6 As shown, the optimal enzyme dosage was 100 μg / mL, and the optimal reaction time was 4 h. Under these optimal conditions, the enzyme activity of β1,4-galactosyltransferase Haβ4GalT in synthesizing LNnT was 0.37 U, the specific enzyme activity was 24.85 U / mg, and the conversion efficiency was 85.54%.

[0064] Example 6 Mutant modification of β1,4-galactosyltransferase Haβ4GalT

[0065] The results of Example 5 show that the β1,4-galactosyltransferase Haβ4GalT can synthesize LNnT via a transglycosylation reaction using LNT II and UDP-Gal as substrates, but its enzyme activity is relatively low. Therefore, this invention mutates and modifies it to obtain a β1,4-galactosyltransferase with higher transglycosylation activity and more suitable for industrial applications.

[0066] A three-dimensional structural model of the β1,4-galactosyltransferase Haβ4GalT was constructed using AlphaFold2. The Haβ4GalT 3D structure obtained through homology modeling was submitted to HotSpot Wizard 3.1, and mutation hotspots were predicted based on NST energy calculations. The following 11 sites were selected for mutation: A47T, A93G, N97S, E112Q, D116E, I195L, V202K, R208K, R209K, I211V, and S233P.

[0067] Mutant plasmids were constructed and corresponding primers were designed using SnapGene software. Using the recombinant plasmid synthesized in Example 2 as a template, PCR amplification was performed to obtain recombinant plasmids containing the encoding genes of each mutant enzyme. The recombinant engineered bacteria were transformed according to the method in Example 2, and the purified enzyme solutions of each mutant enzyme were collected for the following studies.

[0068] Example 7 Determination of transglycosylation activity of mutant enzyme

[0069] The 150 μL reaction system contained: 1.5 mM LNT II, ​​3.75 mM UDP-Gal, and 1 mM Mn. 2+Add an appropriate amount of pure enzyme solution (100 μg / mL concentration in the reaction system) (in the form of MnCl2), and use HEPES-NaOH buffer (50 mM, pH 6.5) as the buffer. Mix all components and react in a 37°C water bath for 4 h; after the reaction, inactivate the enzyme by boiling in a water bath for 10 min; centrifuge at 12000 rpm for 10 min, collect the supernatant, and filter it through a 0.22 μm filter membrane. Perform initial screening using TLC detection. Perform liquid chromatography detection under the same conditions as in Example 3.

[0070] Initial screening using TLC removed the obviously inactive mutant enzymes A93G and R208K. Using the transglycosylation activity of β1,4-galactosyltransferase Haβ4GalT as 100%, the relative transglycosylation activity of each mutant enzyme was calculated. The results of the relative transglycosylation activity determination for each mutant enzyme are as follows: Figure 7 As shown in the figure, most mutant enzymes showed varying degrees of increased transglycosylation activity. Among them, the mutant enzyme S233P showed the greatest increase in transglycosylation activity, with an enzyme activity of 1.78 U for synthesizing LNnT, a specific enzyme activity of 118.60 U / mg, and a conversion efficiency of 99.80%.

[0071] The mutant enzyme S233P was named β1,4-galactosyltransferase Haβ4GalT-S233P, and its amino acid sequence is shown in SEQ ID NO.3. The nucleotide sequence of its encoding gene is shown in SEQ ID NO.4.

[0072] Example 8 Construction of the basic strain for LNnT production

[0073] LNnT synthesis requires the supply of Lac, UDP-GlcNAc, and UDP-Gal. Therefore, to enhance precursor accumulation, this embodiment weakens the byproduct pathway and strengthens precursor supply at the genomic level to construct a basic strain for LNnT synthesis. The construction method is as follows: using *Escherichia coli* BL21(DE3) as the host bacterium, the UDP-N-acetyl-2-epimerase gene was knocked out using the CRISPR-Cas9 gene editing system. wecB Purine synthesis repressor protein gene purR and β-galactosidase gene lacZ and in wecB Integrating sugar transporter gene at the gene locus nagE ,exist purR PRPP synthase gene integrated at the gene locus prs The recombinant strain obtained was named LNnT-1, which is Escherichia coli BL21(DE3)Δ wecB::nagE Δ purR::prs Δ lacZ .

[0074] UDP-N-acetyl-2-epimerase gene wecB The nucleotide sequence is shown in SEQ ID NO.5.

[0075] Purine synthesis repressor protein gene purR The nucleotide sequence is shown in SEQ ID NO.6.

[0076] β-galactosidase gene lacZ The nucleotide sequence is shown in SEQ ID NO.7.

[0077] Glycotransfer protein gene nagE The nucleotide sequence is shown in SEQ ID NO.9, and the amino acid sequence of the sugar transporter nagE is shown in SEQ ID NO.8.

[0078] PRPP synthase gene prs The nucleotide sequence of the PRPP synthase prs is shown in SEQ ID NO.11, and the amino acid sequence of the PRPP synthase prs is shown in SEQ ID NO.10.

[0079] Example 9 Construction of LNnT producing strain

[0080] The β1,3-N-acetylglucosyltransferase gene was inserted at MCS1 of the free plasmid pETDueT-1. NmlgtA and lactose permease gene lacY Insert the β1,4-galactosyltransferase gene at MCS2. MBP - Haβ4GalT- S233P The recombinant plasmid pETDueT-1-NmlgtA-lacY-MBP-Haβ4GalT-S233P was constructed. This recombinant plasmid was introduced into the strain LNnT-1 from Example 8, and after sequencing verification, the recombinant strain was named LNnT-1.1.

[0081] β1,3-N-acetylglucosyltransferase gene NmlgtA The nucleotide sequence is shown in SEQ ID NO.13, and the amino acid sequence of β1,3-N-acetylglucosyltransferase NmlgtA is shown in SEQ ID NO.12.

[0082] lactose permease gene lacY The nucleotide sequence of the enzyme is shown in SEQ ID NO.15, and the amino acid sequence of the lactose permease lacY is shown in SEQ ID NO.14.

[0083] Example 10 Fermentation Production of LNnT

[0084] The strain LNnT-1.1 from Example 9 was inoculated into 5 mL of LB liquid medium and cultured at 37°C and 220 r / min for 10 h to obtain a seed culture. The seed culture was then transferred at a 2% (v / v) inoculation rate to a baffled Erlenmeyer flask containing M9 medium. 1‰ (v / v) of vitamins and 1‰ (v / v) of trace elements were added, along with glucose (as a carbon source) to a concentration of 20 g / L, and ampicillin to a concentration of 100 μg / mL. The mixture was cultured at 37°C and 130 r / min with shaking until OD reached. 600 The pH was 0.80 (approximately 5 h). IPTG was added to a concentration of 0.3 mM to induce expression, and lactose (as a substrate) was added to a concentration of 8 g / L. Fermentation was carried out at 20°C for 180 h. Phenol red was used to monitor pH changes during fermentation, and ammonia was added dropwise to maintain a neutral fermentation environment. Glucose solution (600 g / L) was used as the fed-batch medium, employing a gradient feeding strategy: 2-hour intervals in the early stage (0–48 h), 4-hour intervals in the middle stage (48–96 h), and 8-hour intervals in the late stage (96–180 h), with 8‰ (volume percentage) of the fed-batch medium added each time. The changes in cell density, LNnT concentration, LNT II concentration, and lactose concentration during fermentation are illustrated in the diagram below. Figure 8 As shown, the yield of LNnT reached 4.99 g / L after 180 h of fermentation.

[0085] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A β1,4-galactosyltransferase Haβ4GalT-S233P, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. The gene encoding the β1,4-galactosyltransferase Haβ4GalT-S233P as described in claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

4.

3. The application of the β1,4-galactosyltransferase Haβ4GalT-S233P described in claim 1 in the synthesis of LNnT.

4. The application of the β1,4-galactosyltransferase Haβ4GalT-S233P according to claim 3 in the synthesis of LNnT, characterized in that, The specific application method is as follows: using LNT II as the donor and UDP-Gal as the acceptor, LNnT is generated by the reaction under the action of β1,4-galactosyltransferase Haβ4GalT-S233P.

5. The application of the β1,4-galactosyltransferase Haβ4GalT-S233P according to claim 3 in the synthesis of LNnT, characterized in that, The specific application method is as follows: using Escherichia coli as the host bacterium, a recombinant engineered bacterium expressing β1,4-galactosyltransferase Haβ4GalT-S233P is constructed; the recombinant engineered bacterium is cultured and lactose is added as a substrate. Under the action of β1,4-galactosyltransferase Haβ4GalT-S233P expressed by the recombinant engineered bacterium, UDP-GlcNAc and UDP-Gal synthesized by the recombinant engineered bacterium itself are used as precursors to react with lactose to synthesize LNnT.

6. The application of the β1,4-galactosyltransferase Haβ4GalT-S233P according to claim 5 in the synthesis of LNnT, characterized in that, The recombinant engineered bacteria expressing β1,4-galactosyltransferase Haβ4GalT-S233P were constructed as follows: (1) Construction of the basic strain for LNnT production Using Escherichia coli BL21(DE3) as the host bacterium, the UDP-N-acetyl-2-epimerase gene was knocked out using the CRISPR-Cas9 gene editing system. wecB Purine synthesis repressor protein gene purR and β-galactosidase gene lacZ and in wecB Integrating sugar transporter gene at the gene locus nagE ,exist purR PRPP synthase gene integrated at the gene locus prs The basic strain for LNnT production was constructed; UDP-N-acetyl-2-epimerase gene wecB The nucleotide sequence is shown in SEQ ID NO.5; purine synthesis repressor protein gene. purR The nucleotide sequence is shown in SEQ ID NO.6; β-galactosidase gene lacZ The nucleotide sequence is shown in SEQ ID NO.7; Glycotransporter gene nagE The nucleotide sequence is shown in SEQ ID NO.9; PRPP synthase gene prs The nucleotide sequence is shown in SEQ ID NO.11; (2) Construction of LNnT producing strain The β1,3-N-acetylglucosyltransferase gene was inserted at MCS1 of plasmid pETDueT-1. NmlgtA and lactose permease gene lacY The gene encoding β1,4-galactosyltransferase Haβ4GalT-S233P was inserted into the MCS2 region of plasmid pETDueT-1 to construct a recombinant plasmid; this recombinant plasmid was then introduced into the LNnT-producing strain to construct the LNnT-producing strain. β1,3-N-acetylglucosyltransferase gene NmlgtA The nucleotide sequence is shown in SEQ ID NO.13; lactose permease gene lacY The nucleotide sequence is shown in SEQ ID NO.15; the gene encoding β1,4-galactosyltransferase Haβ4GalT-S233P is shown in SEQ ID NO.4.

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