Fucosyltransferase mutant and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a fucosyltransferase mutant and application thereof. According to the invention, a plurality of mutation sites of alpha-1, 2-fucosyltransferase (wcfB) of Bacteroides fragilis are obtained through bioinformatics analysis and mining, mutants are constructed through point mutation and combinatorial mutation, and the ability of the mutants to catalytically produce 2 '-FL is verified in Escherichia coli Nissle 1917. According to the alpha-1, 2-fucosyltransferase mutant obtained through screening, the yield of 2 '-FL is increased by 20% or above compared with the yield of a wild original recombinant strain, and the yield of the highest M51 is increased by 93.1%. The transformation strategies provided by the invention provide important theoretical and technical support for the alpha-1, 2-fucosyltransferase in the industrial production of breast milk oligosaccharides such as 2 '-FL and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular, the present application relates to a fucosyltransferase mutant and its application. BACKGROUND

[0002] Human Milk Oligosaccharides (HMOs) are also known as human milk oligosaccharides, which are the third largest solid component in breast milk, second only to lactose and fat. They are mainly divided into three categories: neutral fucosylated HMOs (including 2'-fucosyllactose 2'-FL, 3-fucosyllactose 3-FL) account for more than 80% of the total, neutral non-fucosylated HMOs (such as lactose-N-tetrasaccharide LNT, lactose-N-neotetrasaccharide LNnT), and acidic sialylated HMOs (such as 3'-sialyllactose 3'-SL, 6'-sialyllactose 6'-SL). Human milk oligosaccharides play an important role in the growth and development of infants, including promoting the growth of probiotics, inhibiting pathogen infection, regulating the immune system, promoting neurocognitive development, etc. The diversity of HMOs is partly due to modification reactions such as fucosylation, sialylation and acetylation. The α-1,2-fucosyltransferase in cells increases the structural complexity and functional diversity of HMOs by introducing fucosyl groups. In October 2023, two HMOs components 2'-FL (2'-fucosyllactose) and LNnT (lactose-N-neotetrasaccharide) were officially approved as food nutrient fortifiers in China, which can be applied to infant formula milk powder, formula milk powder (for children), and special medical infant food. With the advancement of synthetic biology and enzyme engineering technology, the production of HMOs using α-1,2-fucosyltransferase has broad prospects and is expected to be further applied in infant formula milk powder and functional food.

[0003] α-1,2-fucosyltransferase is an enzyme that catalyzes the attachment of fucose to acceptor molecules (such as lactose) through α-1,2-glycosidic bonds. It participates in various glycosylation reactions in living organisms, particularly in the synthesis of oligosaccharides and glycoconjugates. It is a key enzyme for the synthesis of certain HMOs, especially 2'-fucosyllactose (2'-FL) and lactose-N-fucopentaose (LNFP). α-1,2-fucosyltransferase is widely present in nature, including bacteria, fungi, plants and animals. In humans, the α-1,2-fucosyltransferase encoded by the FUT2 gene is closely related to the synthesis of blood group antigens and human milk oligosaccharides. α-1,2-fucosyltransferase plays a crucial role in the synthesis of human milk oligosaccharides, especially 2'-FL. Through catalyzing fucosylation reactions, this enzyme not only increases the structural diversity of HMOs, but also endows HMOs with various biological functions.

[0004] Although the current yield of biosynthesis of 2'-FL seems to have reached a high level, the synthesis efficiency (or conversion rate) of the product is still a thorny problem. Because the currently known α-1,2-fucosyltransferase has low activity, poor substrate recognition specificity and other problems, it is not suitable for large-scale industrial production of HMO. In the entire synthesis pathway, only the last step of the synthesis of 2'-FL by combining UDP-fucose and lactose catalyzed by α-1,2-fucosyltransferase is a heterologous synthesis reaction, and is unmodified. The glycosyl acceptor lactose is a simple β-1,4-type disaccharide, but it is not the natural acceptor of α-1,2-fucosyltransferase. α-1,2-fucosyltransferase has strong substrate specificity and is difficult to recognize β-1,4 substrates, which is consistent with the objective fact that α-1,2-fucosyltransferase has insufficient catalytic performance. In addition, studies have shown that the low activity of α-1,2-fucosyltransferase is mainly related to the three-dimensional structure and functional characteristics of the key amino acid sites. So far, little progress has been made in improving the catalytic efficiency of α-1,2-fucosyltransferase. On the one hand, it is due to the limited understanding of lactose, and on the other hand, the crystal structure and catalytic mechanism of α-1,2-fucosyltransferase have not been revealed, which also limits the functional enhancement and performance remodeling of α-1,2-fucosyltransferase through rational design and site-directed mutagenesis. Therefore, the modification of α-1,2-fucosyltransferase still needs further research and exploration. SUMMARY

[0005] In order to further solve the problems of activity, specificity and stability of α-1,2-fucosyltransferase, the present application optimizes the substrate binding site, enhances the efficiency of catalytic residues, improves the structural stability and reduces the product inhibition, and obtains a high-activity and reliable α-1,2-fucosyltransferase mutant, which lays a foundation for further improving the yield of 2'-FL. Thus, the present application is completed.

[0006] In one aspect of the present application, the present application provides an α-1,2-fucosyltransferase mutant, which is mutated at positions 138, 140, 148, 149, 236, 247 and 248 in the amino acid sequence of wild-type α-1,2-fucosyltransferase. The amino acid sequence of the wild-type α-1,2-fucosyltransferase is shown in SEQ ID NO. 7.

[0007] In one embodiment, the α-1,2-fucosyltransferase mutant of the present application is one or more of K138R, P140A, K148Y, S149D, S236A, V247E and S248E.

[0008] In one embodiment, the mutant of the alpha-1, 2-fucosyltransferase of the present application is K148Y / S149D or V247E / S248E.

[0009] In one embodiment, the mutant of the alpha-1, 2-fucosyltransferase of the present application is a combination of two or more of P140A, K148Y, K148Y / S149D, S236A, V247E / S248E, K138R.

[0010] In one embodiment, the mutant of the alpha-1, 2-fucosyltransferase of the present application is K138R / S236A; S236A / V247E / S248E, K148Y / S149D / V247E / S248E, K138R / V247E / S248E, K138R / K148Y / S149D, S236A / K148Y / S149D. Preferably, the mutant is K138R / V247E / S248E, K138R / K148Y / S149D.

[0011] In the second aspect of the present application, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the mutant of the alpha-1, 2-fucosyltransferase of the first aspect of the present application.

[0012] In the third aspect of the present application, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect of the present application. In one embodiment, the expression vector is a plasmid vector.

[0013] In the fourth aspect of the present application, the present application provides a genetically engineered bacterium comprising the expression vector of the third aspect of the present application. The chassis bacterium of the genetically engineered bacterium is one of Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis.

[0014] In one embodiment, the chassis bacterium is Escherichia coli, and the Escherichia coli strain comprises BL21, C41, OrigamB, Rosetta, Turner, JM109, HMS174, NovaBlue, AD494, Origami, Nissle 1917.

[0015] Preferably, in the present application, the chassis bacterium used is Escherichia coli Nissle 1917, which does not contain pathogenic factors such as enterotoxin, hemolytic toxin, and cytotoxin, and has no safety risk to the host.

[0016] In one embodiment, the E. coli Nissle 1917 in the present application is a genetically modified E. coli Nissle 1917, and the modification includes integrating a T7 RNA polymerase expression frame of a specific sequence at a specific site of the Nissle 1917 strain, deleting cryptic plasmids, knocking out two other interfering genes, an endA gene and an ompT gene on the genome; see CN202310516515.6 for specific modification methods.

[0017] In a fifth aspect of the present application, a method for producing an alpha-1,2-fucosyltransferase is provided, wherein the method uses the aforementioned alpha-1,2-fucosyltransferase mutant as a catalyst to catalyze the formation of alpha-1,2-fucosyl lactose from fucose and lactose.

[0018] Beneficial effects

[0019] The present application obtains multiple mutation sites of alpha-1,2-fucosyltransferase (wcfB) of Bacteroides Fragilis through bioinformatics analysis and mining, and verifies the ability of the mutants to catalyze the production of 2'-FL in E. coli Nissle 1917 through point mutation and combination mutation. The alpha-1,2-fucosyltransferase mutants obtained by screening in the present application significantly improve the yield of 2'-FL, which is more than 20% higher than that of the wild original recombinant strain, and the highest M51 is increased by 93.1%. The screening and modification strategies of the present application provide important theoretical and technical support for the industrial production of alpha-1,2-fucosyltransferase in 2'-FL and other human milk oligosaccharides. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 , R96-Y103 alpha-helix domain in alpha-1,2-fucosyltransferase wcfB.

[0021] Figure 2 , Comparison of 2'-FL production by shake flask fermentation of combination mutants of alpha-1,2-fucosyltransferase.

[0022] Figure 3 , HPLC detection of M51 fermentation broth and standard product. (A) Liquid chromatogram of M51 fermentation 69h sample, and the peak times of 2'-FL, lactose and glucose are 8.111min, 8.774min and 10.271min respectively. (B) Liquid chromatogram of 2'-FL, lactose and glucose standard sample, and the peak times are 8.094min, 8.759min and 10.257min respectively. DETAILED DESCRIPTION

[0023] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used, and the molecular biology methods, bioinformatics methods involved in the examples can refer to the relevant textbooks, reference books, teaching websites, including but not limited to "Molecular Biology", "Bioinformatics", "Gene Engineering", "Molecular Cloning Experiment Guide", "Molecular Biology Experiment Guide". If the manufacturer of the reagent or instrument used is not specified, it is a conventional product that can be obtained by market purchase.

[0024] Example 1: Construction of 2'-FL synthesis strain

[0025] (1) According to the method described in patent No. CN202310516515.6, in the original strain Escherichia coli Nissle 1917 (E. coli Nissle 1917), based on gene editing technology, the optimized T7RNAP expression frame is inserted into the Nissle 1917 bacterial genome, and the insertion site of the expression frame is the attB site on the genome, and then the endA and ompT genes are knocked out to prepare EcNcΔattB(lacUV5-T7)ΔendAΔompT.

[0026] (2) According to the method described in patent No. CN202311431659.8, in EcNcΔattB(lacUV5-T7)ΔendAΔompT, based on gene editing technology, the lacZ, wcaJ, lon, pfkA and nudD genes are knocked out to prepare EcNcΔattB(lacUV5-T7)ΔendAΔompTΔlacZΔwcaJΔlonΔpfkAΔnudD.

[0027] (3) According to the method described in patent number CN202311431659.8, the genomic of Escherichia coli K-12 MG1655 was used as a template, and the CB-F / CB-R, GW-F / GW-R, and LacY-F / LacY-R primers were used to clone the gene fragments of ManB-ManC (shown in SEQ ID NO. 1 and SEQ ID NO. 2), Gmd-WcaG (SEQ ID NO. 3 and SEQ ID NO. 4), and LacY (SEQ ID NO. 5), respectively. The recovered gene fragments of ManB-ManC and Gmd-WcaG were connected to the vector pCOLADuet through a homologous recombination seamless cloning kit (ProLigation-Free Cloning Kit, abm company, item number E086) to obtain the recombinant plasmid pCOLADuet-manB-manC-gmd-wcaG. Using the same connection method, the recovered LacY gene fragment was connected to the pCDFDuet plasmid to construct the plasmid pCDFDuet-lacY.

[0028] The codon-optimized alpha-1,2-fucosyltransferase gene wcfB (derived from Bacteroides Fragilis) was synthesized by Wuhan Tianyi Huayu Technology Co., Ltd. The optimized wcfB sequence is shown as SEQ ID NO. 6. The synthesized gene fragment was connected to the vector pCDFDuet-lacY through a homologous recombination seamless cloning kit to construct the plasmid pCDFDuet-lacY-wcfB.

[0029] The plasmids pCOLADuet-manB-manC-gmd-wcaG and pCDFDuet-lacY-wcfB were co-transformed into the strain EcNcΔattB(lacUV5-T7)ΔendAΔompTΔlacZΔwcaJΔlonΔpfkAΔnudD to obtain the strain CK.

[0030] Example 2 Mutation site analysis of alpha-1,2-fucosyltransferase mutants

[0031] 2.1. Consensus Finder multiple sequence alignment (MSA) restores conserved amino acids

[0032] B. fragilis (NCBI ACCESSION: WP_005813010) contains 864 bp and encodes 287 amino acids (SEQ ID NO. 7). So far, no crystal structure of a-1,2-FucT with high homology to wcfB has been reported. The poor enzymatic activity of a-1,2-fucosyltransferase and the poor soluble expression in E. coli are the key to limit the further improvement of 2’-FL production, while codon optimization can only improve the transcription efficiency of wcfB in E. coli, but cannot improve the solubility of protein expression. Therefore, to further promote the correct folding of wcfB in E. coli, the inventors first tried to restore the non-conserved amino acids in wcfB to conservative amino acids to promote protein solubility. The wcfB amino acid sequence was submitted to ConsensusFinder http: / / cbs-kazlab.oit.umn.edu / ), and non-conserved residues were automatically selected from the queried sequence by BLASTp (NCBI), CD-HIT, and Clustal Omega in three steps, and the originally conserved amino acids were calculated for replacement. After screening, Q198S was found to be the recommended replacement site by Consensus Finder.

[0033] When Consensus Finder performs multiple sequence alignment, it not only refers to the sequences of B. fragilis family glycosyltransferases, but also includes glycosyltransferase sequences from other microorganisms in the NCBI database. Since the CD-HIT step of Consensus Finder only selects one representative sequence for MSA from each group of highly similar sequences (> 95%), it is impossible to compare highly similar sequences in the same microbial species to find non-conserved residues. Therefore, in order to study a-1,2-fucosyltransferases from B. fragilis species, 250 a-1,2-fucosyltransferase sequences from different B. fragilis were found using the BLAST function of the NCBI website and multiple sequence alignment was performed, and single residues W114H and N115R with a conservation rate of more than 70% were selected. The results of multiple sequence alignment also show that W114H and N115R are conserved at the same time in most cases, so W114H / N115R double mutant sequences are also designed for testing.

[0034] 2.2. EVcouplings multiple sequence alignment to restore conservative amino acids

[0035] Evolutionary studies have shown that conserved residues are critical for protein structure and enzyme stability. Based on multiple sequence alignment, site-directed saturation mutation was applied to design and improve the catalytic activity of wcfB. In addition, 13583 homologous sequences were identified using the EVcouplings (https: / / evcouplings.org / ) server, combined with computer recommendations and empirical screening results, and finally 8 mutants including K80T; K80P; K80I; K80N; K80V; E91C; E91C / K92R; V250F were determined.

[0036] 2.3. Improve the hydrophilicity of the alpha-helix domain

[0037] Using the alpha-helix rule, the amino acid helix wheel of the 9 alpha-helix in wcfB was first analyzed, and based on previous studies, it was identified that the aggregation hotspot in the amino acid sequence of wcfB is located in the alpha-helix domain of R96-Y103 (as shown in Figure 1 The alpha-helix domain was subjected to site-directed saturation mutagenesis and combinatorial mutation simulation, and 4 mutant combinations with the highest potential to improve the hydrophilicity of the alpha-helix domain were screened, i.e. R96S / V99A / L100M; R96D / S97I / V98S / V99A / L100R; V99G / L100V; V99T / L100P.

[0038] 2.4. Remodel the substrate binding region

[0039] Firstly, the wcfB protein structure was predicted from scratch using the trRosetta software. The obtained model was evaluated to have the correct topology, with more than 90% of residues located within the allowed and additional allowed regions. Therefore, this model is reliable and can be used for subsequent work. The amino acids in the enzyme active center intervene in the substrate recruitment and binding process, which in turn affects the catalytic performance of the enzyme. Therefore, the targeted design of key amino acids to drive changes in the microenvironment of the active center is the key to functional customization. Determining the scope of the active center and remodeling the substrate binding region can be a feasible method to improve its catalytic ability. By using computer-aided screening and site-directed mutagenesis to narrow down the range of residue candidates, a small and smart mutant library was constructed. According to the calculation, 15 amino acid residues were selected as candidates: 90S, 137L, 138K, 139L, 140P, 145F, 147G, 148K, 149S, 231M, 235I, 236S, 237V, 238K, 239L. From the spatial distribution of these amino acids, they are clustered in the pocket area in the center of the model, covering almost the entire area. Generally, an active center that is too hydrophilic or too hydrophobic is not conducive to the binding or release of substrates. Site-directed mutagenesis was performed on the above 15 candidate amino acids, resulting in 19 mutants, S90F; L137V / K138R; K138R; L137V / K138R / L139I; L139I / P140A; P140A; F145L; F145V; G147H; K148Y; K148Y / S149D; G147H / K148Y / S149D; M231W; I235A; S236A; V237A; K238A; L239G; L239A.

[0040] In addition, the present application also found that the three amino acids 41L, 42L and 169D, although not directly combined with the substrate, are close to the pocket area of the enzyme active center after folding, and there is a hydrogen bond effect, which may affect the enzyme-substrate binding and catalytic efficiency, so the mutants L41A; L41D; L42A; L42D; L41A / L42A; L41D / L42D; D169A are designed, which is beneficial to eliminate the hydrogen bond effect, enhance the affinity of the enzyme and the substrate, and improve the flexibility of GDP-fucose in the active center of the glycosyltransferase.

[0041] 2.5. Site-directed mutagenesis

[0042] The VESPAl (Variant Effect Score Prediction without Alignments) tool and the GEMME (Global Epistatic Model Predicting Mutational Effects) tool were used to predict the site-directed mutation sites of wcfB, and finally four mutation sites with the highest scores and the best predicted mutation effects were selected: V53A; V53Q; V247E; V247E / S248E.

[0043] Example 3 Construction of a-1, 2-fucosyltransferase mutants and synthesis of 2'-fucosyllactose by shake flask fermentation

[0044] 3.1 Obtaining of recombinant plasmids containing a-1, 2-fucosyltransferase mutant genes

[0045] The recombinant plasmids containing a-1, 2-fucosyltransferase mutant genes were constructed and obtained by whole plasmid amplification: see Table 1, for the mutation sites involved in sections 2.1-2.5 in Example 2, the upstream and downstream primers were designed according to the desired mutant site, and the whole plasmid was amplified using PrimeSTAR HS DNA Polymerase (Takara) with plasmid pCDFDuet-lacY-wcfB as the template. The plasmid containing the wcfB gene mutation sequence was amplified by PCR, transformed into E. coli, and then picked and plated into LB liquid medium for overnight culture, after which the plasmid was extracted and sequenced for verification. All the above recombinant plasmids containing mutations that were correctly verified by sequencing were co-transformed with plasmid pCOLADuet-manB-manC-gmd-wcaG into strain EcNcAattB(lacUV5-T7)AendAAlacZAwcaJAlonApfkAAnudD, to obtain recombinant strains containing the designed 2'-fucosyllactose transferase mutants.

[0046] 3.2 Screening test of the level of 2'-FL produced by mutant recombinant strains in fermentation

[0047] All the recombinant strains were inoculated into 5 mL fresh LB medium (containing 50 μg / mL streptomycin, 50 μg / mL kanamycin) respectively, and cultured at 37 °C in a shaker (220 rpm) for 12 h. The seed liquid was inoculated into 25 mL DM medium (containing 50 μg / mL streptomycin, 50 μg / mL kanamycin) at a 2% inoculation amount, and cultured at 37 °C in a shaker (220 rpm) until the OD600 value reached 0.6-0.8. Then, 0.3 mM IPTG and 12 g / L lactose were added to induce, and then the culture was placed in a shaker (220 rpm) at 25 °C for 72 h. 1 mL of the fermentation broth was taken, centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane into a liquid sample bottle for detection of product yield.

[0048] All the samples were detected by using an Agilent HPLC 1260 high-performance liquid chromatograph equipped with a parallax refractive detector (RID). The separation chromatographic column was a BIO-RAD Aminex HPX-87H column, the column temperature was 40 °C, and the mobile phase was 5 mM sulfuric acid aqueous solution, and the flow rate was 0.5 mL / min. This method can be used for quantitative detection of glucose, lactose and 2'-FL, and the external standard method is used for quantification.

[0049] Table 1: Mutation sites of mutants and test results of 2'-FL fermentation yield

[0050]

[0051] As shown in Table 1, in this embodiment, compared with CK, the 40 mutant enzymes have reduced or lost activities, and the corresponding recombinant strains have significantly reduced 2'-FL synthesis ability. The recombinant strains M19, M21, M22, M29, M36 and M46 containing the mutation sites P140A, K148Y, K148Y / S149D, S236A, V247E / S248E and K138R, respectively, have 2'-FL yields of 8.45, 8.34, 8.54, 8.92, 6.91 and 7.74 g / L in shake flask fermentation, respectively, which are increased by 50.1%, 48.1%, 51.7%, 58.4% and 37.5%, respectively, compared with the original fermentation strain.

[0052] Example 4 Combination mutation of α-1,2-fucosyltransferase and shake flask fermentation

[0053] Based on the screening results of Example 3, it is shown that the mutations of P140A, K148Y, K148Y / S149D, S236A, V247E / S248E, K138R, etc. significantly improve the 2'-FL yield of the recombinant strain in the shake flask. Therefore, the six mutation sites are combined to design six combined mutants, which are K138R / S236A; S236A / V247E / S248E; K148Y / S149D / V247E / S248E; K138R / V247E / S248E; K138R / K148Y / S149D; S236A / K148Y / S149D. Among them, the mutant plasmid is obtained, the recombinant strain is constructed, and the test method steps of the 2'-FL production ability are referred to Example 3, and the strain number is shown in Table 2.

[0054] Results: See Table 2 and Figure 2 As shown in Table 2 and Figure 3 The fermentation yield of the recombinant strain M50 obtained by the K138R / V247E / S248E three-site combined mutation is 7.34 g / L. The fermentation yield of the recombinant strain M51 obtained by the K138R / K148Y / S149D three-site combined mutation is 10.62 g / L, which is 93.1% higher than that of the original strain, 37.2% higher than that of M46, and 24.4% higher than that of M22. The expression of M47, M48, M49 and M52 does not have a significant increase.

[0055] Table 2 Combination of α-1,2-fucosyltransferase mutations and test results of 2'-FL production in shake flask fermentation

[0056]

[0057] Example 5 Verification of Fermentor Yield of Recombinant Strain M51

[0058] Glycerol bacteria M51 was inoculated into fresh LB medium for overnight activation culture, 1% inoculation amount was transferred into 25 mL LB for further culture for 5 h as seed liquid, and all was inoculated into 500 mL DM medium (1.5 L fermentor). The fermentation conditions were controlled to carry out fermentation culture and induction to produce 2'-FL. After detection, M51 produced 53.1 g / L 2'-fucosyl lactose after 69 h of fermentation; while the yield of the unmutated CK strain was only 16.4 g / L, and the small test level of the mutant strain was increased by 223.8%.

[0059] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. An alpha- 1,2-fucosyltransferase mutant, characterized in that, The mutant is mutated at positions 138, 140, 148, 149, 236, 247, 248 in the amino acid sequence of wild-type α-1, 2-fucosyltransferase, and the amino acid sequence of the wild-type α-1, 2-fucosyltransferase is shown as SEQ ID NO.

7.

2. The a-1,2-fucosyltransferase mutant according to claim 1, characterized in that, The mutation is one or more of K138R, P140A, K148Y, S149D, S236A, V247E, S248E.

3. The a-1,2-fucosyltransferase mutant according to claim 2, characterized in that, The mutation is K148Y / S149D or V247E / S248E.

4. The alpha- 1,2-fucosyltransferase mutant of claim 2, wherein, The mutation is a combination of two or more of K138R, P140A, K148Y, K148Y / S149D, S236A, V247E / S248E.

5. The a-1,2-fucosyltransferase mutant according to claim 4, characterized in that, The mutation is K138R / S236A, S236A / V247E / S248E, K148Y / S149D / V247E / S248E, K138R / V247E / S248E, K138R / K148Y / S149D, or S236A / K148Y / S149D; preferably, the mutation is K138R / V247E / S248E, K138R / K148Y / S149D.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the α-1, 2-fucosyltransferase mutant of any one of claims 1-5.

7. An expression vector, characterized by, The expression vector contains the nucleic acid molecule of claim 6; preferably, the expression vector is a plasmid vector.

8. A genetically engineered bacterium, characterized by, The genetically engineered bacteria contain the expression vector of claim 7; preferably, the chassis bacteria of the genetically engineered bacteria is one of Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis. 9.The genetically engineered bacteria according to claim 8, characterized in that, The chassis bacteria is Escherichia coli, and the Escherichia coli strain includes BL21, C41, OrigamB, Rosetta, Turner, JM109, HMS174, NovaBlue, AD494, Origami, Nissle 1917.

10. A method for producing an α-1, 2-fucosyltransferase, characterized by, The method is to use the α-1, 2-fucosyltransferase mutant of any one of claims 1-5 as a catalyst to catalyze the formation of α-1, 2-fucosyl lactose from lactose and lactose.

Images