Glycosyltransferase mutant and application thereof

Abstract

The invention belongs to the field of enzyme engineering and microbial fermentation, and particularly relates to a glycosyl transferase mutant and application thereof, and the amino acid sequence of the glycosyl transferase mutant is shown as SEQ ID NO.2. According to the invention, the structure of glycosyl transferase (FutRb) is studied, and a directed evolution technology is combined to mutate three sites of a FutRb protein, specifically, alanine at the 98th site of an amino acid sequence is mutated into arginine, valine at the 106th site of the amino acid sequence is mutated into glutamic acid, and alanine at the 232 site of the amino acid sequence is mutated into serine, so that the glycosyl transferase mutant (FutRbM3) is constructed. The invention also constructs a recombinant strain, and the recombinant strain is used for synthesis of 3-fucosyllactose. Experimental results show that the FutRbM3 greatly increases the synthesis yield of the 3-fucosyllactose.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and microbial fermentation, and specifically relates to a glycosyltransferase mutant and its application. Background Technology

[0002] Human milk oligosaccharides are important immunologically active components in breast milk, playing a crucial role in infant nutrition and immune function. Traditional human milk oligosaccharide production is limited by factors such as breast milk source and extraction efficiency, making it difficult to meet market demand. Therefore, finding new production methods and improving existing methods has become a current research hotspot. 3-Fucosyllactose is one of the simplest human milk oligosaccharides, accounting for 5% of total human milk oligosaccharides. α-1,3-Fucosyltransferase, as a key enzyme in the metabolic pathway of 3-fucosyllactose synthesis in organisms, plays a vital role in determining whether 3-fucosyllactose can be industrially applied.

[0003] The synthesis of 3-fucosylated lactose in organisms uses GDP-L-fucose and lactose as substrates. In existing technologies, a de novo synthetic pathway is typically employed to produce GDP-L-fucose. Specifically, in *E. coli*, the intermediate GDP-L-fucose is generated by enhancing the expression of phosphogmannose mutase (manB), mannose-1-guanylate transferase (manC), GDP-D-mannose-4,6-dehydratase (gmd), and GDP-L-fucose synthase (wcaG). Simultaneously, the degradation of lactose in *E. coli* is prevented by knocking out the LacZ gene. The resulting GDP-L-fucose is then linked to lactose via α-1,3-fucosyltransferase to synthesize 3-fucosylated lactose. However, the wild-type α-1,3-fucosyltransferases reported for the synthesis of 3-fucosylated lactose generally exhibit poor activity. Therefore, by studying α-1,3-fucosyltransferase, we explored and constructed a mutant of α-1,3-fucosyltransferase with high catalytic activity, which is of great significance for improving the production efficiency of 3-fucosyl lactose. Summary of the Invention

[0004] The primary objective of this invention is to discover a glycosyltransferase capable of efficiently synthesizing 3-fucosylated lactose, named FutRb. Through structural studies of FutRb and combined with directed evolution technology, a glycosyltransferase mutant is provided. This mutant exhibits better enzymatic activity for the synthesis of 3-fucosylated lactose using GDP-L-fucose and lactose as substrates, thereby enhancing the industrial application value of glycosyltransferases.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0006] Through big data mining, a wild-type glycosyltransferase (FutRb) capable of synthesizing 3-fucosylated lactose was identified, with its amino acid sequence shown in SEQ ID NO.1. Structural studies of the glycosyltransferase were conducted, and directed evolution of its related amino acids was performed. Specifically, alanine at position 98 was mutated to arginine, valine at position 106 to glutamic acid, and alanine at position 232 to serine. Glycosyltransferase mutants were obtained by mutating at least one of these positions.

[0007] SEQ ID NO.1

[0008] MIDPRTSDFLAEFLASANRDPALLDRFLLHGPDRGGRGARPRLKIAFFDFWPDFDFSANFFVEILSSRFEVSVVENDCDLAIVSVFGTRHREARTARALFFTGENVRPPLDGVDMSVSFDRIDDPRHYRLPLYVMHAWEHLREGATPHFCHPVLPPAPPTREE AAKRKFCAFLYKNPNCARRNDFFQMLCARRHVESVGWLLNNTGSVVKMGWLPKIRVFSRYRFAFAFENASYPGYLTEKILDAFQAGAVPLYWGDPGVLRDVAAGSFIDMSRYSSDEEAIDAILAADDDYDTYRRYRGTAPFLGTEDFYFDAYRLAEWIESRL.

[0009] A second object of the present invention is to provide a gene encoding the above-mentioned glycosyltransferase mutant, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] SEQ ID NO.2

[0011] MIDPRTSDFLAEFLASANRDPALLDRFLLHGPDRGGRGARPRLKIAFFDFWPDFDFSANFFVEILSSRFEVSVVENDCDLAIVSVFGTRHREARTARRLFFTGENERPPLDGVDMSVSFDRIDDPRHYRLPLYVMHAWEHLREGATPHFCHPVLPPAPPTREE AAKRKFCAFLYKNPNCARRNDFFQMLCARRHVESVGWLLNNTGSVVKMGWLPKIRVFSRYRFAFAFENSSYPGYLTEKILDAFQAGAVPLYWGDPGVLRDVAAGSFIDMSRYSSDEEAIDAILAADDDYDTYRRYRGTAPFLGTEDFYFDAYRLAEWIESRL.

[0012] A third object of the present invention is to provide a gene containing a glycosyltransferase mutant, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0013] SEQ ID NO.3

[0014] ATGATCGATCCGCGCACCAGTGATTTTCTGGCAGAATTTCTGGCAAGTGCAAATCGCGATCCGGCACTGCTGGATCGTTTTCTGCTGCATGGCCCGGATCGTGGTGGTCGCGGCGCAAGACCGCGCTTAAAAATTGCCTTTTTCGATTTTTGGCCGGATTTTGATTTTAGTGCAAATTTCTTTGTGGAGATTCTGAGCAGTCGTTTTGAAGTGAGCGTTGTTGAAAATGATTGTGATCTGGCCATTGTGAGTGTTTTTGGTACACGCCATCGTGAAGCCCGTACCGCACGTCGCCTGTTTTTCACCGGCGAAAATGAACGTCCGCCGCTGGATGGCGTGGATATGAGCGTGAGCTTTGATCGCATTGATGATCCGCGTCATTATCGTCTGCCGCTGTATGTGATGCATGCATGGGAACATCTGCGCGAAGGCGCAACCCCGCATTTTTGTCATCCGGTTCTGCCGCCGGCCCCGCCTACAAGAGAAGAAGCCGCAAAACGTAAATTTTGTGCCTTTCTGTATAAGAATCCGAATTGTGCCCGCCGCAATGATTTCTTTCAGATGCTGTGTGCACGCCGTCATGTTGAAAGCGTTGGCTGGCTGCTGAATAATACCGGCAGTGTGGTGAAAATGGGTTGGCTGCCGAAAATTCGTGTTTTTAGCCGTTATCGTTTTGCCTTTGCATTTGAAAATAGTAGCTATCCGGGCTATCTGACCGAAAAAATTCTGGATGCATTTCAGGCCGGTGCAGTTCCGCTGTATTGGGGCGATCCGGGTGTGCTGCGTGATGTTGCCGCCGGTAGCTTTATTGATATGAGTCGTTATAGTAGTGACGAAGAAGCAATTGATGCCATTCTGGCCGCAGATGATGATTATGATACCTATCGTCGTTATCGTGGCACCGCACCGTTTCTGGGCACCGAAGATTTTTATTTTGATGCATATCGTCTGGCAGAATGGATTGAAAGCCGTCTGTAA。

[0015] A fourth objective of this invention is to provide a recombinant expression plasmid containing the aforementioned genes.

[0016] A fifth objective of this invention is to provide an engineered bacterial strain comprising the aforementioned recombinant expression plasmid. Preferably, *Escherichia coli*, which produces high levels of GDP-L-fucose, is selected as the host bacterium for constructing the engineered strain. The method for constructing this strain is detailed in Chinese Invention Patent Publication No. CN116426452A.

[0017] The sixth objective of this invention is to provide the application of the above-mentioned glycosyltransferase mutant in the synthesis of 3-fucosylated lactose.

[0018] In one preferred embodiment, the application includes: inducing the generation of a glycosyltransferase mutant in gene-edited Escherichia coli to synthesize 3-fucosylated lactose.

[0019] In a preferred embodiment, the gene-edited Escherichia coli is a recombinant genetically engineered bacterium with LacZ knocked out.

[0020] In a preferred embodiment, the recombinant genetically engineered bacteria synthesize 3-fucosylated lactose at 25°C.

[0021] Beneficial effects:

[0022] Based on the structural study of the existing α-1,3-fucosyltransferase FutRb, this invention, through rational design, performed site-directed mutations on amino acids 106 and 232, located on the outer side of the enzyme's catalytic pocket, to enhance hydrogen bonding near the active pocket and improve its thermal stability. Simultaneously, a mutation was performed on amino acid 98, located inside the catalytic pocket, to facilitate the correct orientation of the substrate GDP-L-fucose. The resulting mutant enzyme was named FutRb. M3 Compared to wild-type FutRb, FutRb M3 The ability to catalyze the synthesis of 3-fucosylated lactose in the Escherichia coli expression system was significantly improved, and the yield was significantly increased, demonstrating good potential for industrial application. Attached Figure Description

[0023] Figure 1 This is a diagram of the 3-fucosylation pathway in the recombinant strain.

[0024] Figure 2 For recombinant expression plasmid pETDuet-FutRb M3 Structural diagram.

[0025] Figure 3 For containing FutRb M3 Figure showing the accumulation of 3-fucosylated lactose by recombinant strains expressing plasmids in a 5L fermenter.

[0026] Figure 4 Figure showing the accumulation of 3-fucosylated lactose by recombinant strains containing the FutRb expression plasmid in a 5L fermenter.

[0027] Figure 5 High performance liquid chromatogram of 3-fucosyllactose standard.

[0028] Figure 6 The image shows the high-performance liquid chromatogram of the fermentation broth sample from a 5L fermenter. Detailed Implementation

[0029] The present invention will be further described below through specific embodiments. It should be noted that these descriptions of embodiments are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0031] In this invention, the amino acid sequence of the wild-type glycosyltransferase from the strain *Azospirillum lipoferum* is shown in SEQ ID NO.1, and the glycosyltransferase mutant (FutRb) is also described. M3 The amino acid sequence of the gene is shown in SEQ ID NO.2. We commissioned General Biotech (Anhui) Co., Ltd. to synthesize the gene.

[0032] All gene sequencing services involved in this invention are outsourced to General Biotechnology (Anhui) Co., Ltd.

[0033] Example 1 Screening of glycosyltransferases

[0034] This embodiment mainly describes the screening process of glycosyltransferases capable of synthesizing 3-fucosylated lactose. Specifically, the amino acid sequences of reported Helicobacter pylori-derived glycosyltransferases were compared against the entire NCBI database using these sequences as templates. Each amino acid sequence was analyzed, including but not limited to MBO5664686.1, RGY65243.1, WP_007483358.1, and WP_235339153.1. These sequences were then expressed in plasmids in *E. coli* strains that efficiently synthesize GDP-L-fucose, confirming their ability to synthesize 3-fucosylated lactose. Furthermore, fermentation analysis revealed that the wild-type glycosyltransferase from *Azospirillum lipoferum* strain exhibited the best 3-fucosylated lactose synthesis ability; therefore, this enzyme was selected as the template for subsequent targeted modification. The metabolic pathway of this enzyme in the recombinant strain catalyzing the synthesis of 3-fucosylated lactose is as follows: Figure 1 As shown.

[0035] Example 2: Plasmid construction and directed mutagenesis of glycosyltransferase

[0036] This embodiment mainly describes the plasmid construction of pETDuet-FutRb and the directed mutagenesis of FutRb. Specifically, alanine at position 98 of the amino acid sequence was mutated to arginine, valine at position 106 to glutamic acid, and alanine at position 232 to serine. The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO.1. After codon optimization of this amino acid sequence in E. coli, the gene encoding this amino acid sequence was synthesized by General Biotechnology (Anhui) Co., Ltd.

[0037] The pETDuet-FutRb plasmid was constructed using a one-step cloning method. The specific steps were as follows: The pETDuet plasmid (purchased from Shanghai Zeye Biotechnology Co., Ltd.) was double-digested with Nde I and Xho I restriction endonucleases, and the 5364 bp linearized plasmid fragment was recovered. After FutRb gene sequence synthesis, PCR amplification was performed using FutRb-F / FutRb-R primers. After amplification, the fragment was recovered via gel extraction. The enzyme digestion and gel extraction reagents were used according to the reagent instructions. The linearized pETDuet plasmid and the FutRb fragment with homologous arms were then used to construct the pETDuet-FutRb plasmid according to the one-step cloning kit instructions. After the pETDuet-FutRb plasmid was constructed, sequencing was performed to ensure the accuracy of the inserted sequence.

[0038] Site-directed mutagenesis was performed on amino acid 98 of FutRb, changing alanine to arginine. Specifically, the FutRb gene fragment was used as a template, and FutRb-F / A98R-R and A98R-F / FutRb-R primers were used for the first round of PCR amplification. The two amplified fragments were recovered from the gel. Using the two recovered fragments as templates, FutRb-F / FutRb-R primers were used for the second round of PCR amplification. The gene fragment obtained in the second round of amplification was the nucleotide sequence corresponding to the mutation of alanine to arginine at amino acid 98 of FutRb.

[0039] Site-directed mutagenesis was performed on amino acid 106 of FutRb to mutate valine to glutamic acid. Specifically, the FutRb gene fragment was used as a template, and FutRb-F / V106E-R and V106E-F / FutRb-R were used as primers for the first round of PCR amplification. The two amplified fragments were recovered from the gel. Using the two recovered fragments as templates, FutRb-F / FutRb-R were used as primers for the second round of PCR amplification. The gene fragment obtained in the second round of amplification was the nucleotide sequence corresponding to the mutation of valine to glutamic acid at amino acid 106 of FutRb.

[0040] Site-directed mutagenesis was performed on amino acid 232 of FutRb, changing alanine to serine. Specifically, using the FutRb gene fragment as a template, a first round of PCR amplification was performed using FutRb-F / A232S-R and A232S-F / FutRb-R primers. The two amplified fragments were recovered from the gel. Using the two recovered fragments as templates, a second round of PCR amplification was performed using FutRb-F / FutRb-R primers. The gene fragment obtained in the second round of amplification was the nucleotide sequence corresponding to the mutation of alanine to serine at amino acid 232 of FutRb.

[0041] The glycosyltransferase resulting from targeted mutations at positions 98, 106, and 232 was named FutRb. M3 According to the construction method of the pETDuet-FutRb plasmid described in this embodiment, FutRb is obtained after directed mutation. M3 The recombinant expression plasmid was named pETDuet-FutRb. M3 .

[0042] pETDuet-FutRb M3 A schematic diagram of the structure of the recombinant expression plasmid is shown below. Figure 2 As shown.

[0043] Directed mutation of glycosyltransferase FutRb M3The amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence after codon optimization is shown in SEQ ID NO.3.

[0044] The primer sequences involved in this embodiment are shown in Table 1.

[0045] Table 1

[0046] Primer sequence number Primer name Primer sequence (5'→3') SEQ ID NO.4 FutRb-F AGTTAAGTATAAGAAGGAGATATACATATGATCGATCCGCGCAC SEQ ID NO.5 FutRb-R CGGTTTCTTTACCAGACTCGAGTTACAGACGGCTTTTCAATC SEQ ID NO.6 A98R-F CGTACCGCACGTCGCCTGTTTTTCACCGGCGAAAATG SEQ ID NO.7 A98R-R CGGTGAAAAACAGGCGACGTGCGGTACGGGCTTCACCGAT SEQ ID NO.8 V106E-F CCGGCGAAAATGAACGTCCGCCGCTGGATGGCGT SEQ ID NO.9 V106E-R AGCGGCGGACGTTCATTTTCGCCGGTGAAAAACAGG SEQ ID NO.10 A232S-F GCATTTGAAAATAGTAGCTATCCGGGCTATCTGACCG SEQ ID NO.11 A232S-R TAGCCCGGATAGCTACTATTTTCAAATGCAAAGGCAAAACG

[0047] Example 3 Construction of 3-fucosylated lactose-producing strains

[0048] The chassis strain used in this embodiment is Escherichia coli BL21(DE3) with double knockout of the β-galactosidase encoding gene lacZ and the UDP-glucose lipotransferase encoding gene wcaJ, which was previously constructed in our laboratory. The construction method of this strain is detailed in Chinese invention patent with publication number CN116426452A.

[0049] To enhance the ability of the chassis strain to supply lactose and GDP-L-fucose, in this embodiment, phosphogannatase (manB), mannose-1-guanylate transferase (manC), GDP-D-mannose-4,6-dehydratase (gmd), GDP-L-fucose synthase (wcaG), and lactose permease (lacY) were overexpressed on the pCDFDouet plasmid, and the overexpressed plasmid was named pCDF-CBGWY. The gene fragments corresponding to the above amino acid sequences were all obtained by PCR amplification from Escherichia coli BL21(DE3) strain. The construction method of pCDF-CBGWY plasmid can be referred to Chinese invention patent with publication number CN116426452A.

[0050] 3-Fucosyllactose-producing strains were developed by introducing pCDF-CBGWY plasmid and pETDuet-FutRb into Escherichia coli BL21(DE3) with lacZ and wcaJ knocked out. M3 In this embodiment, plasmid introduction is performed using electroporation. First, competent *E. coli* BL21(DE3) cells need to be prepared. The specific steps are as follows:

[0051] (1) Inoculate a single clone of Escherichia coli BL21(DE3) with double knockout of lacZ and wcaJ into a test tube containing 5 mL of LB medium and incubate overnight at 37°C and 200 rpm.

[0052] (2) Inoculate 1% of the solution into 50 mL of LB medium and incubate at 37°C until OD. 600 It reaches approximately 0.5.

[0053] (3) Transfer the bacterial culture to a 50 mL pre-cooled centrifuge tube, place it on ice for 20 min, and centrifuge at 4℃ and 4000 rpm for 5 min.

[0054] (4) Discard the supernatant, add 15 mL of pre-cooled 10% glycerol solution to suspend the cells, and centrifuge at 4℃ and 4000 rpm for 5 min.

[0055] (5) Repeat step (4) twice.

[0056] (6) Discard the supernatant, add 500 μL of pre-cooled 10% glycerol solution, gently suspend the bacterial cells, and then dispense 100 μL / tube into sterilized 1.5 mL centrifuge tubes and store them in a -80℃ freezer for later use.

[0057] Plasmids containing pCDF-CBGWY and pETDuet-FutRb were prepared by electroconversion. M3 Recombinant Escherichia coli strains:

[0058] (1) Add 2 μL of pCDF-CBGWY plasmid and pETDuet-FutRb plasmid to 100 μL of E. coli BL21(DE3) competent cells with double knockout of lacZ and wcaJ. M3 Mix the plasmid gently and incubate on ice for 20 minutes.

[0059] (2) Wipe the moisture off the side of the pre-cooled 0.2cm electroporation cup, add all the competent cells into the electroporation cup, and perform electroconversion at 2.5 KV.

[0060] (3) Immediately after the electric shock, add 700 μL of antibiotic-free LB culture medium and incubate at 30°C for 1 h to allow the bacteria to recover.

[0061] (4) Spread the bacterial cells evenly on LB plates containing spectinomycin and ampicillin resistance, and incubate them in a 30°C incubator for 16 hours.

[0062] The single clones that grow on the plate are recombinant strains with the ability to produce 3-fucosylated lactose.

[0063] Example 4: FutRb containing directed mutations M3 Application of recombinant strains

[0064] In this embodiment, the recombinant strain constructed in Example 3 was used as the fermentation strain, and a 5-L fermenter was used as the fermentation vessel. At the same time, in order to verify the production capacity of the fermentation strain, a recombinant strain expressing wild-type FutRb plasmid was used as a control strain for fermentation verification and comparison experiments. The only difference between the control strain and the recombinant strain constructed in Example 3 was whether FutRb was modified by point mutation. The fermentation medium and fermentation process control were the same. The fermentation medium was an optimized M9 medium, namely: 12.8 g / L disodium hydrogen phosphate heptahydrate, 3 g / L potassium dihydrogen phosphate, 2 g / L ammonium chloride, 0.5 g / L sodium chloride, 0.25 g / L magnesium sulfate heptahydrate, 15 mg / L calcium chloride dihydrate, 2 g / L yeast extract, and 10 mL / L trace element mixture. The trace element mixture contains: 5 g / L LEDTA, 0.83 g / L ferric chloride hexahydrate, 84 mg / L zinc chloride, 0.13 mg / L copper chloride dihydrate, 10 mg / L cobalt chloride dihydrate, 10 mg / L boric acid, 1.6 mg / L manganese chloride tetrahydrate, and an initial glucose concentration of 20 g / L.

[0065] The specific fermentation process was as follows: The recombinant strain was inoculated into LB liquid medium containing spectinomycin and ampicillin and cultured overnight at 37°C. A 1% (v / v) inoculum was transferred to 200 mL of fresh LB medium as a secondary seed culture. When the OD600 of the culture reached approximately 4, the secondary seed culture was inoculated into a fermenter at a 10% (v / v) inoculum for scale-up culture at 37°C. During fermentation, the dissolved oxygen concentration was maintained at 20%–40%, the pH was controlled at 6.8–7.2, ammonia was used as a pH adjuster, and the aeration ratio was 1 vvm. When the OD600 of the cells reached 25, the temperature was slowly lowered to 25°C, and 0.1 mM IPTG was added as an inducer and 10 g / L lactose. During fermentation, lactose was fed in as a substrate, maintaining a lactose concentration of 5–10 g / L.

[0066] Fermentation results as follows Figure 3 and Figure 4 As shown. By Figure 3 It was found that after IPTG induction at 6 h, the recombinant strain showed a simultaneous and significant increase in cell growth (OD600) and the concentration of the product 3-fucosyllactose (3-FL) during fermentation from 13 to 55 h, exhibiting a trend of simultaneous growth and synthesis. During this period, the accumulation rate of 3-FL was approximately 1.7 g / L / h. At 63 h of fermentation, the yield of 3-fucosyllactose reached 78.9 g / L. The fermentation curve of the control strain was as follows... Figure 4As shown, the control strain grew slowly in the early stage, and the product accumulation rate in the middle stage was much lower than that of the mutated recombinant strain. After 63 hours of fermentation, the final product yield was 43.6 g / L. The fermentation experiment in this example demonstrates that the recombinant strain of the present invention, under the described fermentation process, can efficiently convert the substrate into the target product and achieve high-yield accumulation.

[0067] Example 5: Determination of 3-fucosyllactose

[0068] 3-Fucose-based lactose was detected using high-performance liquid chromatography (HPLC). Before analysis, the fermentation broth sample required pretreatment. The specific pretreatment method was as follows: the fermentation broth was centrifuged at 12000 rpm for 2 min. The supernatant was diluted with water to a concentration range of 25 mg / L–500 mg / L, mixed thoroughly, and filtered through a 0.22 μm aqueous membrane. The filtered sample was then transferred to a sample vial for HPLC analysis.

[0069] The high-performance liquid chromatography (HPLC) instrument used was an Agilent 1200, and the chromatographic column was a Rezex ROA Organic Acid column (250*4.6 mm*2.7 μm). A differential detector was used, the column temperature was set to 50℃, the mobile phase was 100% 2.5 mM sulfuric acid aqueous solution, the flow rate was set to 0.4 mL / min, and the injection volume was 5 μL per injection.

[0070] from Figure 5 As can be seen, the peak elution time of the 3-fucosyllactose standard is 9.9 min. Under the same detection conditions, such as... Figure 6 As shown, a peak was also detected in the fermentation broth sample at 9.9 min, which can be confirmed as the product 3-fucosylated lactose.

[0071] The above embodiments are merely illustrative of the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solutions based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.

Claims

1. A glycosyltransferase capable of synthesizing 3-fucosylated lactose, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A glycosyltransferase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2. The glycosyltransferase mutant is obtained by using the glycosyltransferase described in claim 1 as a template through the following three site-directed mutations: alanine at position 98 of the amino acid sequence is mutated to arginine, valine at position 106 is mutated to glutamic acid, and alanine at position 232 is mutated to serine.

3. A gene encoding a glycosyltransferase mutant as described in claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

3.

4. A recombinant expression plasmid comprising the gene as described in claim 3.

5. An engineered strain comprising the recombinant expression plasmid as described in claim 4.

6. The use of the glycosyltransferase mutant as described in claim 2 in the synthesis of 3-fucosylated lactose.

7. The application according to claim 6, characterized in that, The plasmid of the glycosyltransferase mutant was introduced into a strain that produces high GDP-L-fucose to synthesize 3-fucosyl lactose.

8. The application according to claim 7, characterized in that, The strains include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris series bacteria and fungi.

9. The application according to claim 8, characterized in that, The strain was used as the fermentation strain to produce 3-fucosylated lactose.

10. The application according to claim 9, characterized in that, The strains include those that ferment 3-fucosylated lactose using one or more of glucose, glycerol, xylose, and methanol as carbon sources and lactose as a common substrate.

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