A fucosyltransferase polypeptide and its application

Abstract

The present invention provides a fucosyltransferase polypeptide, which can be applied to the synthesis of human milk oligosaccharides (HMOs) and belongs to the field of enzyme engineering technology. The fucosyltransferase polypeptide described in the present application comprises a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, and 100% identical amino acid sequence to the polypeptide shown in SEQ ID NO:1-25. The transferase polypeptide described in the present application improves the ability of the polypeptide shown in SEQ ID NO:1 to catalyze the synthesis of 2'-FL while also improving the specificity of the polypeptide catalyzing the substrate.

Description

Technical Field

[0001] The present invention relates to a series of fucosyltransferase polypeptides, which can be applied to the synthesis of human milk oligosaccharides (HMOs) and belong to the field of enzyme engineering technology. Background Art

[0002] Human milk oligosaccharides (HMOs) are a unique and diverse mixture of oligosaccharides found in human milk. They are the third largest solid component of breast milk, after fat and lactose. Among them, 2'-fucosyllactose (2'-FL) is one of the most abundant oligosaccharides in human milk, with a concentration of 2-5 g / L in human milk. 2'-FL has been shown to play an important role in the brain, intestines, and growth and development of infants. 2'-FL has been approved as an ingredient in infant formula in the United States, Australia, New Zealand, Canada, and other countries, and can be added to infant formula, regular foods, dietary supplements, and / or medical foods.

[0003] The synthesis of fucosyllactose is mostly carried out by chemical, enzymatic or microbial cell factory methods. Although the chemical synthesis method has made great progress, the synthesis process requires repeated and multiple protection and deprotection of the fucosyllactose molecules, resulting in low product yield and high production costs.

[0004] Chinese Patent No. 202110588386.2 discloses an in vitro multi-enzyme cascade catalytic method for synthesizing fucosylated lactose. This method utilizes a combination of an enzyme involved in GDP-L-fucose synthesis and an L-fucosyltransferase to catalyze the reaction between the substrate GDP-L-fucose and lactose to synthesize fucosylated lactose. The L-fucosyltransferase is selected from either α-1,2-fucosyltransferase or α-1,3-fucosyltransferase. The GDP-L-fucose synthesis enzyme and the fucosyltransferase dual enzyme coupled reaction can produce 200-500 mg / L of fucosyllactose in 2-12 hours.

[0005] The enzymatic synthesis of 2'-FL has mild and controllable reaction conditions, short reaction time, and easy product purification. However, since the glycosyl donor (GDP-L-fucose) for producing 2'-FL is expensive, improving its conversion rate will help reduce the production cost of 2'-FL.

[0006] 2'-FL synthesis requires a number of key enzymes, including GDP-mannose-4,6-dehydratase (Gmd), GDP-L-fucose synthase (WcaG), and α-1,2-fucosyltransferase (1,2-FT). In recent years, researchers have conducted extensive research on the source discovery and molecular engineering of these key enzymes. Numerous publications have explored and screened the sources of these key enzymes, with 1,2-FT (α-1,2-fucosyltransferase) being the most thoroughly studied. Huang et al. from Nankai University (Huang D, Yang KX, Liu J, et al. Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement. Metab Eng, 2017, 41: 23-38.) compared the α-1,2-fucosyltransferase genes futC, futL, futF, wbsJ, wbwK, wbgL, wbiQ, futG, futN, wcfB, and wcfW. The results showed that futC from Helicobacter pylori had the best effect.

[0007] Albermann et al. and Yu et al. (Albermann C, Piepersberg W, Wehmeier UF. Synthesis of the milk oligosaccharide 2'-fucosyllactose using recombinant bacterial enzymes. Carbohyd Res, 2001, 334(2): 97-103.; Yu S, Liu JJ, Yun EJ, et al. Production of a human milk oligosaccharide 2'-fucosyllactose by metabolically engineered Saccharomyces cerevisiae. Microb Cell Fact, 2018, 17: 101.) found that the substrate specificity of the FucT2 enzyme was not strong, which would cause the accumulation of by-products. For example, the undesired lactose difucosyllactose (DFL) was detected during the synthesis of 2'-FL in recombinant yeast and Escherichia coli. Therefore, it is necessary to study the substrate specificity of α-1,2-fucosyltransferase in order to improve the synthesis efficiency and conversion rate of 2'FL and reduce or eliminate the production of impurity sugars. The modification of key enzymes is crucial to improving the catalytic efficiency of enzymes. Summary of the Invention

[0008] The present invention aims to provide a fucosyltransferase polypeptide and its application in the synthesis of fucosyllactose to improve the production efficiency of fucosyllactose. The fucosyltransferase polypeptide is an α-1,2-fucosyltransferase polypeptide, and the fucosyllactose includes 2'-fucosyllactose (2'-FL).

[0009] The α-1,2-fucosyltransferase polypeptide described herein refers to a polypeptide that can catalyze the transfer of a fucose residue from a donor substrate to an acceptor molecule. The donor substrate for transferring the fucose residue to the acceptor molecule is typically guanosine diphosphate L-fucose (GDP-L-fucose). Suitable acceptor molecules for fucose residues include oligosaccharides, glycopeptides, glycoproteins, and glycolipids. Typically, the fucose residue is transferred to, for example, an N-acetylglucosamine residue, an N-acetylgalactosamine residue, a galactose residue, a fucose residue, a sialic acid residue, or a glucose residue of an oligosaccharide, or a sugar portion of a glycoprotein or glycolipid.

[0010] The technical solution of the present invention:

[0011] The applicant discovered an α-1,2-fucosyltransferase (α-1,2-fucosyltransferase) from Bacillus smithii in nature, named BSFut-wt, whose amino acid sequence is shown in SEQ ID NO: 1 and nucleotide sequence is shown in SEQ ID NO: 26. It has low catalytic activity in synthesizing 2'-FL, but is accompanied by the undesirable side activity of producing DFL (Difucosyllactose) synthesis.

[0012] The amino acid sequence of the α-1,2-fucosyltransferase polypeptide is shown in SEQ ID NO: 1. After comparison with the NCBI database, it has a 69.31% identity with the amino acid sequence of Bacillus cereus 107 (as of October 20, 2023), which is the highest similarity; it has a similarity of 33.57% with the α-1,2-fucosyltransferase (α-1,2-fucosyltransferase,) derived from Helicobacter pylori, named HPFut-wt, the amino acid sequence is shown in SEQ ID NO: 27, and the nucleotide sequence is shown in SEQ ID NO: 28. Figure 8 ).

[0013] The fucosyltransferase polypeptide is a lactose-accepting fucosyltransferase polypeptide.

[0014] The inventors of this application have conducted a series of modifications to the polypeptide represented by SEQ ID NO:1, screening and obtaining a series of derivative peptides that catalyze the synthesis of fucosyllactose. These derivative peptides exhibit improved 2'-FL synthesis and substrate specificity. These derivative peptides are transferase polypeptides derived from the polypeptide represented by SEQ ID NO:1 by replacing one or more amino acid residues or fragments with the amino acid sequence.

[0015] This application describes exemplary substitutions or combinations of substitutions for the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1.

[0016] The polypeptides of the present invention have enhanced lactose binding and / or transferase properties. Furthermore, compared to currently known α-1,2-fucosyltransferase polypeptides, these polypeptides lack enzymatic side activities and, when synthesizing 2'-fucosyllactose, do not form the undesirable byproduct difucosyllactose (DFL).

[0017] Specifically, in the first aspect, the present application provides a series of α-1,2-fucosyltransferase polypeptides that synthesize fucosylated lactose, wherein the α-1,2-fucosyltransferase polypeptides are selected from the polypeptides whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25. Among them, SEQ ID NO: 2-25 is obtained by replacing some amino acids in the amino acid sequence shown in SEQ ID NO: 1, specifically, the amino acid sequence is selected from any one of the following.

[0018] 1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid fragment at positions 5-7 is replaced by QVY to GIW, and the amino acid fragment at positions 28-41 is replaced by NDVYLDSSTSYEKY to DDVYLDIETYFEKN / or

[0019] KDVYLDTVTWYEKY, thereby obtaining a polypeptide with an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3;

[0020] 2) In the amino acid sequence shown in SEQ ID NO: 2, the amino acid fragment at positions 55-62 is replaced by HIKPKHAS with HTKPKLAK or PIKPKEAK, thereby obtaining a polypeptide with the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5, respectively;

[0021] 3) In the amino acid sequence shown in SEQ ID NO: 5, the amino acid fragment at positions 68-74 is replaced by NLSDLDE with KLSDIDE or ELGSFDD, thereby obtaining a polypeptide with the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7, respectively;

[0022] 4) In the amino acid sequence shown in SEQ ID NO: 6, the amino acid fragment at positions 87-93 is replaced by KKSTYVE with KKDTVIV / or EKKTMIK, thereby obtaining a polypeptide with the amino acid sequence shown in SEQ ID NO: 8 / or SEQ ID NO: 9, respectively;

[0023] 5) In the amino acid sequence of SEQ ID NO: 9, the amino acid fragment at positions 110-117 is replaced by TYFKGYY with SFLYGYW or AYLEGYW, thereby obtaining a polypeptide with the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, respectively;

[0024] 6) In the amino acid sequence shown in SEQ ID NO: 10, the amino acid residue at position 122 is replaced by W to Y, and the amino acid fragment at positions 126-136 is replaced by EEDLLKDYQFT to KEDLLKDYQFL or LEDLKKAFQFK, thereby obtaining a polypeptide with the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13, respectively;

[0025] 7) In the amino acid sequence of SEQ ID NO: 12, the amino acid fragment at positions 166-173 is replaced by WLNKEYRD with YLNKEYEE or YLNKEYEE, thereby obtaining polypeptides with amino acid sequences as shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively;

[0026] 8) In the amino acid sequence of SEQ ID NO: 14, the amino acid fragment at positions 181-194 is replaced by LEWYLKAIAYVEKR with EEYYLKAIAYVEER or VDYYLKAINYVLEK, thereby obtaining a polypeptide with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17, respectively;

[0027] 9) In the amino acid sequence shown in SEQ ID NO: 16, the amino acid fragment at positions 201-205 is replaced by EIFSY with YIFSD, thereby obtaining a polypeptide with the amino acid sequence shown in SEQ ID NO: 18;

[0028] 10) In the amino acid sequence of SEQ ID NO: 18, the amino acid residue at position 211 is substituted from Q to K, and the amino acid residue at position 224 is substituted from D to S, thereby obtaining a polypeptide with the amino acid sequence of SEQ ID NO: 19;

[0029] 11) In the amino acid sequence of SEQ ID NO: 19, the amino acid fragment at positions 259-264 is replaced by NKWKDK with NKYKDK or CEDEDA, thereby obtaining a polypeptide with the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, respectively;

[0030] 12) In the amino acid sequence of SEQ ID NO: 21, the amino acid residue at position 282 is replaced by E with D or Y, thereby obtaining a polypeptide with the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23;

[0031] 13) In the amino acid sequence shown in SEQ ID NO: 22, the amino acid residue at position 286 is substituted from K to D, thereby obtaining a polypeptide with the amino acid sequence shown in SEQ ID NO: 24;

[0032] 14) In the amino acid sequence shown in SEQ ID NO: 24, the amino acid fragment at positions 289-291 is replaced by IQL to LLLEKEEIEE, thereby obtaining the amino acid sequence shown in SEQ ID NO: 25.

[0033] The α-1,2-fucosyltransferase polypeptide with an amino acid sequence as shown in SEQ ID NO: 2-25 completely eliminates the production of the byproduct DFL (Difucosyllactose).

[0034] In certain embodiments, the polypeptide having enhanced catalytic activity for the synthesis of 2'-FL comprises a polypeptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a polypeptide of SEQ ID NOs: 1-25, and comprises an amino acid substitution mutation that is functionally equivalent to the above schemes 1)-14). In certain embodiments, the substitution mutation comprises a mutation to a charged residue; in certain embodiments, the substitution mutation comprises a mutation to a basic residue. In certain embodiments, the substitution mutation comprises a mutation homologous to the amino acid sequence of the above schemes 1)-14).

[0035] In certain embodiments, the polypeptide having increased activity in catalyzing the synthesis of fucosylated lactose comprises a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-25. In certain embodiments, the polypeptide comprises a polypeptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the polypeptides set forth in SEQ ID NOs: 1-25, and comprises a polypeptide functionally equivalent to the amino acid sequence set forth in SEQ ID NOs: 1-25.

[0036] In a second aspect, the present application provides a polynucleotide encoding the polypeptide as described in the first aspect above.

[0037] Polynucleotides encoding α-1,2-fucosyltransferase polypeptides can be prepared using recombinant DNA techniques known in the art. These methods include, for example, cloning, recombination, in vitro synthesis, in vitro amplification, and / or other available methods. A variety of methods can be used to express expression vectors encoding the polypeptides presented herein. Methods for preparing recombinant nucleic acids, expressing, and isolating expression products are known and described in the Examples.

[0038] In a third aspect, the present application provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above.

[0039] The nucleic acid construct preferably further comprises one or more regulatory sequences operably linked thereto, and the regulatory sequences can direct the production of the polypeptide in an appropriate expression host.

[0040] In a fourth aspect, the present application provides an expression vector comprising the polynucleotide as described in the second aspect above, or comprising the nucleic acid construct as described in the third aspect above.

[0041] The vector has a nucleotide or nucleic acid construct according to an embodiment of the present invention operably linked to a regulatory sequence capable of achieving expression of the DNA fragment, such as a promoter region.

[0042] A variety of kits are commercially available for purifying plasmids and other related nucleic acids from cells. Any isolated and / or purified nucleic acid can be further manipulated to produce other nucleic acids for transfection into cells, integration into related vectors to infect organisms for expression, etc. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters that can be used to regulate expression of a specific target nucleic acid. The vector optionally includes a general expression cassette.

[0043] In a specific embodiment, the expression cassette comprises all elements for expressing the α-1,2-fucosyltransferase polypeptide, including elements necessary for transcription and translation in the host cell. For example, the expression cassette comprises a promoter and a terminator. The promoter and terminator are not particularly limited and may be promoters and terminators known in the art that can achieve expression of the variant.

[0044] In a fifth aspect, the present application provides a transformed host cell, which is transformed with the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above.

[0045] In some embodiments, the transformed host cell is transformed with the polynucleotide described in the second aspect or the nucleic acid construct described in the third aspect.

[0046] In some embodiments, the preferred host cell is Escherichia coli, or other host cells capable of transforming the polynucleotide as described in the second aspect, or the nucleic acid construct as described in the third aspect, or the expression vector as described in the fourth aspect.

[0047] The host cell is preferably selected from Escherichia coli, Bacillus, and yeast; the yeast is selected from Saccharomyces cerevisiae, Kluyveromyces, and Yarrowia lipolytica;

[0048] More preferably, the host cell is the genetically engineered Escherichia coli BL21 (DE3).

[0049] The term "host cell" is defined as a cell that has been transformed or transfected, or is capable of being transformed or transfected, with an exogenous polynucleotide sequence, thereby comprising at least one non-naturally occurring sequence in the host cell.

[0050] In a sixth aspect, the present application provides an enzyme agent or enzyme composition, which comprises the polypeptide as described in the first aspect above.

[0051] The enzyme or enzyme composition preferably contains one or more of the polypeptides with amino acid sequences as shown in SEQ ID NOs: 1-25 of the present invention.

[0052] In a seventh aspect, the present application provides a method for producing the polypeptide as described in the first aspect above, comprising:

[0053] (1) culturing a transformed host cell under conditions suitable for expressing the polypeptide; the transformed host cell is as described in the fifth aspect above; and

[0054] (2) Recovering the polypeptide.

[0055] In a specific embodiment, the step (1) comprises: firstly introducing a nucleic acid construct or a recombinant expression vector encoding the polypeptide as described in the first aspect above into a host cell to construct an engineered host cell expressing the polypeptide; then, culturing the engineered host cell and inducing it to express the polypeptide.

[0056] In a specific embodiment, the step (2) includes the steps of isolating and purifying the polypeptide from the culture.

[0057] Can use methods known in the art, in the nutrient medium that is suitable for producing polypeptide, cultivate host cell.For example, can pass through shake flask culture, or in applicable substratum and under the condition of allowing polypeptide expression and / or separation, carry out small-scale or large-scale fermentation (comprising continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) in laboratory or industrial fermentor tank and cultivate cell.Cultivation is to use program known in the art, occurs in applicable nutrient medium, and described substratum comprises carbon and nitrogen source and inorganic salt.Suitable substratum can be purchased through commercial channels, or according to disclosed composition preparation.

[0058] The polypeptide may be recovered from the culture using methods known in the art. For example, the variant may be recovered from the nutrient medium by a variety of conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0059] In addition, the polypeptide can be purified by various procedures known in the art to obtain substantially pure polypeptides, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography), electrophoresis procedures (e.g., preparative isoelectric focusing), differential solubility methods (e.g., ammonium sulfate precipitation), SDS-PAGE, salting out, etc. or a combination thereof; further preferably, purification can be performed by Ni column affinity chromatography.

[0060] In an eighth aspect, the present application provides the use of the α-1,2-fucosylfucotransferase polypeptide described in the first aspect, the transformed host cell described in the fifth aspect, or the enzyme or enzyme composition described in the sixth aspect in synthesizing fucosyllactose;

[0061] Preferably, the fucosyllactose includes 2'-fucosyllactose (2'-FL).

[0062] For such applications, suitable reaction conditions include: the presence of a suitable reaction substrate, a suitable polypeptide, or the presence of essential cofactors such as monovalent or divalent ions, a pH value within an appropriate range, a suitable temperature, etc. It is not necessary to meet the optimal value of each factor affecting the polypeptide described in this application, but the reaction conditions must enable the α-1,2-fucosyltransferase polypeptide described in this application to exert its enzymatic activity.

[0063] In one embodiment, the method comprises: adding the α-1,2-fucosyltransferase polypeptide described in the present application to a reaction system, contacting the polypeptide with an acceptor and a fucosyl donor in the reaction system, and performing a catalytic reaction to synthesize the desired fucosylated lactose.

[0064] Preferably, the application uses GDP-L-fucose and lactose as substrates and the α-1,2-fucosyltransferase polypeptide described in the present application as a catalyst to catalyze the synthesis and obtain 2'-FL.

[0065] Preferably, the application is to prepare the α-1,2-fucosyltransferase polypeptide in a cell-free expression system (such as but not limited to the PURExpress system (NEB)) or in a host organism (such as but not limited to Escherichia coli or Saccharomyces cerevisiae), after which the above-listed α-1,2-fucosyltransferase polypeptide can be isolated and optionally further purified. Specifically, it includes:

[0066] a) catalyzing the synthesis of 2'-FL from substrates GDP-L-fucose and lactose using the polypeptide described in the first aspect or the enzyme or enzyme composition described in the sixth aspect. Further preferably, the method further comprises:

[0067] b) Optionally, a step of purifying and / or recovering 2'-FL.

[0068] In one embodiment, at least one of the α-1,2-fucosyltransferase polypeptides described herein is mixed with GDP-L-fucose, a buffer solution (such as Tris-HCl or HEPES), and lactose. The mixture is incubated at a specific temperature (e.g., 35°C, 37°C, or 40°C) for a specific period of time (e.g., 24 hours), during which lactose or 2'FL is converted to 2'-FL by the enzyme using GDP-fucose. 2'-FL is then isolated and purified by methods known in the art. At the end of the reaction or after isolation and / or purification, the yield of 2'-FL is determined by HPLC chromatography.

[0069] The application of the α-1,2-fucosyltransferase polypeptide of the present invention uses acceptor lactose and donor GDP-L-fucose as substrates to produce 2'-FL through catalytic reaction without producing undesirable DFL, which is beneficial to the separation and purification of the reaction products.

[0070] In this step, the mixture containing 2'-FL can be clarified in a conventional manner. Preferably, the mixture containing 2'-FL is clarified by centrifugation, flocculation, decantation and / or filtration.

[0071] Preferably, substantially all proteins, as well as amino acids, RNA, and DNA, are removed from the mixture containing 2'-FL (preferably after clarification). In this step, proteins and related impurities can be removed from the mixture containing 2'-FL in a conventional manner. Preferably, proteins, salts, by-products, color, and other related impurities are removed from the mixture containing 2'-FL by ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated carbon or carbon treatment, chromatography, ion exchange chromatography (such as, but not limited to, cation exchange, anion exchange, mixed bed ion exchange), hydrophobic interaction chromatography, and / or gel filtration (i.e., size exclusion chromatography), in particular by chromatography, more particularly by ion exchange chromatography, hydrophobic interaction chromatography, or ligand exchange chromatography.

[0072] 2'-FL is further isolated from the reaction mixture and subjected to further purification steps using techniques well known in the art such as evaporation, lyophilization, crystallization, precipitation and / or drying, spray drying.

[0073] The term "identity" refers to amino acid sequence identity and refers to the percentage of two or more sequences or subsequences that are identical, or that have identical amino acid residues or nucleotides, when compared and aligned for maximum correspondence. An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0074] Proteins and / or protein sequences are "homologous" when they are derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and / or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence.

[0075] The term "polypeptide" refers to any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to short chains (commonly referred to as peptides, oligopeptides, and oligomers) as well as long chains (commonly referred to as proteins). "Polypeptides" include those modified by natural processes (such as processing and other post-translational modifications) and by chemical modification techniques. These modifications are fully documented in basic textbooks and in more detail in monographs, as well as in a large body of research literature, and are well known to those skilled in the art.

[0076] The term "a nucleic acid sequence encoding an enzyme for 2'-FL synthesis" relates to a nucleic acid sequence encoding an enzyme essential for the synthesis pathway of 2'-FL, for example, an enzyme capable of transferring the fucose moiety of a GDP-fucose donor substrate to the 2'-hydroxyl group of the galactose moiety of lactose to thereby produce 2'-FL.

[0077] Beneficial effects:

[0078] This application provides α-1,2-fucosyltransferase polypeptides represented by SEQ ID NOs: 1-25, which enhance the ability of the polypeptide represented by SEQ ID NO: 1 to catalyze the synthesis of 2'-FL and improve its substrate specificity. The catalytic reactions of the polypeptides represented by SEQ ID NOs: 2-25 do not produce DFL as a byproduct. Furthermore, this application provides polynucleotides encoding the polypeptides and related nucleic acid products, transformed host cells, enzymes containing the polypeptides, and methods for catalyzing the synthesis of 2'-FL using the polypeptides as catalysts.

[0079] The technical solution of the present application has positive significance for the industrial production of human milk oligosaccharides. The method is green, efficient, and sustainable, is conducive to the application of industrial large-scale production, and has important practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 .Lactose HPLC spectrum.

[0081] Figure 2 .HPLC spectrum of 2'-FL standard.

[0082] Figure 3 .HPLC spectrum of DFL standard.

[0083] Figure 4 .HPLC spectrum of the reaction solution of the polypeptide shown in SEQ ID NO:1.

[0084] Figure 5 .HPLC spectrum of the reaction solution of the polypeptide shown in SEQ ID NO:20.

[0085] Figure 6 .Mass spectrometry analysis of 2'-FL standard.

[0086] Figure 7 . Figure 5 The HPLC spectrum shown is an LC-MS analysis spectrum of a substance having an rt of approximately 15.06 min.

[0087] Figure 8 .Comparison of amino acid sequences of SEQ ID NO:1 and SEQ ID NO:27. DETAILED DESCRIPTION

[0088] The present invention is further described in detail below through examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Modifications or replacements to the details and forms of the technical solution without departing from the structural ideas and scope of use of the present invention fall within the scope of protection of the present invention.

[0089] It should be noted that the terms used are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. In the following specific embodiments, if the experimental methods for specific conditions are not specified, they are generally in accordance with the conventional methods and conditions of molecular biology within the technology of the art, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: A Laboratory Manual", or in accordance with the conditions recommended by the manufacturer.

[0090] In the following specific embodiments, if the experimental methods for specific conditions are not specified, they are generally based on conventional methods and conditions of molecular biology within the art, and such techniques and conditions are fully explained in the literature; all materials, reagents, etc., unless otherwise specified, can be obtained from commercial channels.

[0091] 2'-FL and DFL standards were produced by ELICITYL, France.

[0092] Example 1. Expression of polypeptides shown in SEQ ID NOs: 1-25 in Escherichia coli.

[0093] 1. Using the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 (nucleotide sequence shown in SEQ ID NO: 26) as a template, the polypeptide shown in SEQ ID NO: 1 was synthesized and finally constructed into the PET32a vector to obtain the PET32a-wt plasmid.

[0094] 2. Different mutation sites were obtained by computer-assisted rational design. According to the method of the Molecular Cloning Experiment Guide, the sequence of SEQ ID NO: 26 of PET32a-wt was used to perform Primer-BLAST ( Primer designing tool(nih.gov)) mutant primers were designed and a recombinant plasmid series (pET32a-M1-M24) was constructed using a point mutagenesis kit (Fast Mutagenesis Kit).

[0095] 3. Transform the above recombinant plasmids into Escherichia coli BL21 (DE3) according to the following steps:

[0096] Take the prepared Escherichia coli BL21 (DE3) competent cells, place them on ice for 30 minutes to thaw, take 100 μL of competent cells and 10 μL of pET32a--M1-M24 recombinant plasmid (concentration 50 ng / μL), respectively, mix them, place them in a 42°C water bath for 45 seconds, then immediately cool them in an ice bath for 2 minutes, add 1 mL of fresh LB medium (LB medium: 1.0% peptone, 0.5% yeast extract, 1.0% NaCl, and 1.5% agar powder on the plate), and resuscitate and culture at 37°C and 100 rpm for 1 hour. Then, take 100 μL of the bacterial solution and spread it on an LB plate containing ampicillin (100 μg / mL). After culturing in a 37°C constant temperature incubator for 12 hours, pick a single colony for colony PCR to screen for positive transformants.

[0097] 4. Culture the positive transformants, extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the pET32a-M1-M24 recombinant plasmid has been successfully introduced into E. coli.

[0098] 5. Inoculate the correct transformant into LB liquid medium and culture it on a shaker at 37°C and 200 rpm for 12 hours to obtain seed solution. Then, inoculate the seed solution into fresh LB medium at a 1% (v / v) inoculum and culture it at 37°C with shaking until the OD 600 The expression of the culture medium was 0.8, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG) with a final IPTG concentration of 0.1 mmol / L, and induced at 16°C for 12 h at a speed of 200 rpm; after induction of expression, the fermentation broth was centrifuged at 5000 r / min for 30 min at 4°C to collect the bacteria; the bacteria were resuspended in 20 mM pH 7.4 PBS buffer and ultrasonicated at a frequency of plus on 5s / off 5s for 30 min to break the bacteria; the broken liquid was centrifuged at 13000×g and 4°C for 30 min to remove cell debris, and the supernatant was collected.

[0099] 6. The soluble polypeptide sequence was purified using nickel column affinity chromatography. The process was as follows: deionized water was added to the top of the nickel column. After natural elution, it was eluted with 5 volumes of Binding buffer. The crude enzyme solution filtered through a 0.45 μm filter membrane was then loaded onto the column. The sample was fully bound to the nickel column at a flow rate of 1.5 mL / min. After the sample was dried, it was continuously eluted with 5 column volumes of Washing buffer to remove impurities. Finally, the target protein was eluted with 5 times the volume of Elution buffer and the eluate was collected. Then, the expression of the target protein was analyzed by SDS-PAGE.

[0100] The SDS-PAGE results showed that the genetically engineered bacteria had obvious specific expression bands after induction, and the molecular weight of the bands was basically consistent with the expected molecular weight of 35.3 kDa. Therefore, it can be seen that the polypeptides shown in SEQ ID NO: 1-25 were obtained. For detailed information, please see Table 1.

[0101] Table 1. Corresponding relationship between α-1,2-fucosyltransferase polypeptides and their amino acid sequence numbers

[0102]

[0103]

[0104] Example 2. Determination of the ability of the polypeptides obtained in Example 1 (polypeptides represented by SEQ ID NOs: 1-25, and the α-1,2-fucosyltransferase derived from Helicobacter pylori represented by SEQ ID NO: 27) to serve as catalysts for the synthesis of 2'-FL.

[0105] Using the purified polypeptides represented by SEQ ID NOs: 1-25 obtained in Example 1 and the Helicobacter pylori-derived α-1,2-fucosyltransferase represented by SEQ ID NO: 27 as catalysts, and lactose and GDP-L-fucose as reaction substrates, 2'-FL was synthesized respectively. The specific procedures are as follows:

[0106] 15 mM lactose, 15 mM GDP-L-fucose, 10 mM MnCl2, 25 mM Tris-HCl, pH 7.5, were added to each of the purified polypeptides shown in SEQ ID NOs: 1-25 obtained in Example 1, and 0.2 mg / mL of α-1,2-fucosyltransferase from Helicobacter pylori shown in SEQ ID NO: 27 as a catalyst, mixed well, reacted at 37°C for 6 hours, terminated the reaction, and purified by gel column method.

[0107] Identification of catalytic synthesis products:

[0108] HPLC detection method: Detection conditions: chromatographic column model: Shodex Asahipak NH2P-50 4E, mobile phase: 65% acetonitrile in water, flow rate: 0.5 ml / min, column temperature: 35°C, injection volume: 10 μL, evaporative light detector, evaporation temperature 75°C, and nebulization temperature 45°C.

[0109] LC-MS analysis conditions are as follows:

[0110] Chromatographic column model: Shodex Asahipak NH2P-50 4E, detector: UV detector (Hitachi Chromaster), detection wavelength: 210 nm, injection volume: 10 μL, flow rate: 0.5 mL / min, column temperature: 35 °C, mobile phase: acetonitrile: water

[0111] =65:35; ESI-MS mode, molecular weight scanning range 100~800.

[0112] The standard and reaction solution were tested using the aforementioned analytical method. HPLC analysis revealed that the peak elution time of the 2'-FL standard was 15.06 min. The reaction solutions of the polypeptide described in Example 1 all exhibited a strong absorption peak around 15.06 min, consistent with the peak elution time of the 2'-FL standard, indicating that the polypeptide-catalyzed reaction in Example 1 produced 2'-FL.

[0113] In addition: HPLC analysis of DFL standard ( Figure 3 ) rt is 17.7min, and a weak peak appears in the reaction solution of the polypeptide represented by SEQ ID NO: 1 at rt of 17.7min (Appendix Figure 4 ); The reaction solutions of the polypeptides described in SEQ ID NO: 2-25 did not produce any peak at this position, and the HPLC spectrum of the reaction solution of the polypeptide described in SEQ ID NO: 20 (attached Figure 5 ).

[0114] The LC-MS analysis conditions are shown above, and the analysis results show:

[0115] The product of the reaction solution HPLC chromatogram peak near rt = 15.06min was analyzed by LC-MS, and its spectrum is shown in the attached Figure 7 The MH value of the fermentation broth was 487.17, which was consistent with the Figure 6The mass spectrometry results of the 2'-FL standard were consistent with the theoretical molecular weight of 2'-FL, 488.44, within the allowable error range. This demonstrates that the polypeptides represented by SEQ ID NOs: 1-25 were successfully synthesized from 2'-FL. This suggests that SEQ ID NOs: 1-25 derived from Bacillus smithii can replace the previously reported SEQ ID NO: 27 derived from Helicobacter pylori for the synthesis of 2'-FL.

[0116] The yields of 2'-FL and DFL in the fermentation broth were determined by the HPLC analysis method described above. The results are recorded in Table 2.

[0117] Table 2. Studies on the synthesis of 2'-FL catalyzed by various peptides

[0118] Peptide amino acid sequence number 2'-FL yield, mg / mL DFL yield, mg / mL HPFutc 0.5 0.2 SEQ ID NO: 1 1.0 0.04 SEQ ID NO:2 0.6 Not detected SEQ ID NO:3 0.8 Not detected SEQ ID NO:4 0.7 Not detected SEQ ID NO:5 0.9 Not detected SEQ ID NO:6 0.96 Not detected SEQ ID NO:7 0.85 Not detected SEQ ID NO:8 1.2 Not detected SEQ ID NO:9 1.8 Not detected SEQ ID NO: 10 1.5 Not detected SEQ ID NO:11 1.86 Not detected SEQ ID NO:12 1.79 Not detected SEQ ID NO:13 2.3 Not detected SEQ ID NO:14 2.1 Not detected SEQ ID NO:15 2.3 Not detected SEQ ID NO:16 2.2 Not detected SEQ ID NO: 17 2.8 Not detected SEQ ID NO: 18 3.6 Not detected SEQ ID NO: 19 3.8 Not detected SEQ ID NO:20 3.6 Not detected SEQ ID NO:21 4.6 Not detected SEQ ID NO:22 4.5 Not detected SEQ ID NO:23 5.6 Not detected SEQ ID NO:24 5.8 Not detected SEQ ID NO:25 5.1 Not detected

[0119] Table 2 Data Description:

[0120] (1) The catalytic reaction liquid of α-1,2-fucosyltransferase from Helicobacter pylori had a 2'-FL yield of 0.5 mg / mL and a DFL yield of 0.2 mg / mL. This indicates that compared with the α-1,2-fucosyltransferase from Helicobacter pylori, the α-1,2-fucosyltransferase from Bacillus smithii not only had a higher 2'-FL yield (1.0 mg / mL) but also a lower byproduct DFL yield (0.04 mg / mL).

[0121] (2) In addition to 2'FL, a byproduct DFL was detected in the catalytic reaction liquid of the α-1,2-fucosyltransferase polypeptide with the amino acid sequence shown in SEQ ID NO:1, with a yield of 0.04 mg / mL; while DFL was not detected in the catalytic reaction liquid of the α-1,2-fucosyltransferase polypeptide shown in SEQ ID NO:2-25. This result proves that after the α-1,2-fucosyltransferase from Bacillus smithii was modified, the catalytic specificity of all mutants was improved and the byproduct DFL was not synthesized.

[0122] (3) The α-1,2-fucosyltransferase polypeptides shown in SEQ ID NOs: 8-25 increased the activity of the polypeptide shown in SEQ ID NO: 1 in synthesizing 2'-FL to varying degrees. Among them, the α-1,2-fucosyltransferase polypeptides shown in SEQ ID NOs: 23-25 ​​catalyzed the synthesis of 2'-FL with a yield that was 5 times or more that of the polypeptide shown in SEQ ID NO: 1.

[0123] Example 3. Synthesis of 2'-fucosyllactose using Escherichia coli as a substrate.

[0124] References Huang et al. (Huang, D., Yang, K., Liu, J., Xu, Y., Wang, Y., Wang, R., Liu, B., &

[0125] Feng, L. (2017). Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement. Metabolic engineering, 41, 23–38.) The strain BL21ΔlacZΔlonΔwcaJ was constructed, and the above-mentioned constructs PET32a-M1 and PET32a-M24 were transformed into the Escherichia coli chassis cells BL21ΔlacZΔlonΔwcaJ by electroporation to construct EC-M1 and EC-M4.

[0126] Method for culturing Escherichia coli to produce 2'-FL:

[0127] E. coli strains EC-M1 and EC-M4 were cultured on LB solid medium at 37°C for 10-12 hours, and a single colony was inoculated into 20 mL of liquid LB and cultured in a 250 mL shake flask at 37°C and 220 rpm for 10-12 hours. Each strain was inoculated into 5 mL of LB, and when the strain reached stationary phase, 1 mL of culture was inoculated into 100 mL of LB medium containing 36 g / L glucose (or glycerol) as a carbon source for growth in a 500 mL shake flask. When the OD 600 When the pH reached approximately 0.6, 0.1 mM IPTG was added at 25°C for induction. After 2 hours and 10 hours of additional culture, 5 g / L lactose was added to supplement 2′-FL production. At the same time, the culture medium was supplemented with ampicillin to a final concentration of 100 μg / mL. After 10 minutes of induction, the culture was boiled and centrifuged for 10 minutes. The supernatant was then collected and analyzed for 2′-FL and DFL production. The results are recorded in Table 3.

[0128] Table 3

[0129]

[0130] Example 4. Synthesis of 2'-fucosyllactose using Bacillus as the chassis.

[0131] The Bacillus subtilis chassis cells were constructed according to Zhang et al. (Zhang, Q., Liu, Z., Xia, H., Huang, Z., Zhu, Y., Xu, L., Liu, Y., Li, J., Du, G., Lv, X., & Liu, L. (2022). Engineered Bacillus subtilis for the de novo production of 2'-fucosyllactose. Microbial cellfactories, 21(1), 110.) to construct the Bacillus subtilis chassis cells BSP43-manB-P43-manC-P43-gmd-P43-wcaG. At the same time, P43-M1 and P43-M24 expression cassettes were constructed, and the expression cassettes were transferred into BSP43-manB-P43-manC-P43-gmd-P43-wcaG and inserted into the manP gene, thereby constructing BS-M1 and BS-M24.

[0132] Bacillus subtilis strains BS-M1 and BS-M24 were grown on LB solid medium at 37°C for 10-12 hours. A single colony was inoculated into 20 mL of liquid LB in a 250 mL shake flask and incubated at 37°C and 220 rpm for 10-12 hours. The Te seed culture was further inoculated into 30 mL of fermentation medium at a rate of 10% and incubated in a 250 mL shake flask at 37°C and 220 rpm for 72 hours. The following culture medium was used during shake flask fermentation: 6 g / L trypsin, 12 g / L yeast extract, 12.5 g / L KHPO 3H O, 2.5 g / L KH PO , and 10 mL / L trace metal solution (composition: 4 g / L FeSO 7H O, 4 g / L CaCl , 1 g / L MnSO 7H O, 0.2 g / L NaMoO 2H O, 0.2 g / L ZnSO 7H O, 0.1 g / L AlCl 6H O, 0.1 g / L CuCl 2 2H O, and 0.05 g / L H BO ). Sucrose and lactose were sterilized and added to the sterilized shake flasks to final concentrations of 20 and 10 g / L, respectively. After the culture was completed, the culture medium was boiled for 10 minutes, and then centrifuged to collect the supernatant to detect the production of 2'-FL and DFL. The results are recorded in Table 4.

[0133] Table 4

[0134] Genetically modified cells 2'-FL yield, mg / mL DFL yield, mg / mL BS-M1 1.6 Not detected BS-M24 3.2 Not detected

[0135] The above process was scaled up to a 1L fermenter using continuous fed-batch fermentation. Sucrose concentrations in the fermentation broth were maintained between 17g / L and 22g / L, and lactose concentrations were maintained between 10g / L and 20g / L. Fermentation was terminated after 30 hours of continuous feeding. The culture broth was boiled for 10 minutes, and the supernatant was centrifuged and analyzed for 2'-FL content. The 2'-FL content in the fermentation broth for BS-M1 was 20g / L, and that for BS-M24 was 30g / L.

[0136] Example 5. Synthesis of 2'-fucosyllactose using Saccharomyces cerevisiae as a substrate.

[0137] The Saccharomyces cerevisiae chassis cells were constructed according to Xu et al. (Xu, M., Meng, X., Zhang, W., Shen, Y., & Liu, W. (2021). Improved production of 2'-fucosyllactose in engineered Saccharomyces cerevisiae expressing a putative α-1,2-fucosyltransferase from Bacillus cereus. Microbial cell factories, 20(1), 165.), and SC-Δgal80-Pgal1-lac12-Pgal1-gmd-wcaG was constructed. The pRS305-Pgal-M1 and pRS305-Pgal-M24 plasmids were constructed and transformed into SC-Δgal80-Pgal1-lac12-Pgal1-gmd-wcaG to obtain strains SC-M1 and SC-M24.

[0138] Take Saccharomyces cerevisiae SC-M1 and SC-M24 respectively and let them grow rapidly until they enter the late logarithmic phase or the stationary phase. Streak the strains on solid culture medium such as YDP, culture at 30℃ for 2-3 days, pick a single colony and inoculate it into 1.5mL YPD liquid culture medium, and culture it at 30℃ and 200rpm overnight. Then, inoculate it into 50mL liquid culture medium shake flasks at a 2% inoculum volume and culture it at 30℃ and 200rpm until the OD 600= 1, and inoculated at a 2% inoculum into 1.5 L of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) (3 L fermentor). After 5-6 hours of culture, sucrose (50% mother liquor concentration) was added at a controlled rate of 8 mL / h. Simultaneously, lactose (40% mother liquor concentration) was added to maintain a final lactose concentration of 15 g / L. The total fermentation time was 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and the supernatant and precipitate were collected separately. The precipitate was disrupted using a high-pressure homogenizer, boiled, and centrifuged to remove protein. The supernatants were combined to obtain the final yeast fermentation product. The supernatants were assayed for 2'-FL and DFL production, and the results are reported in Table 5.

[0139] Table 5

[0140] Genetically modified cells 2'-FL yield, g / L DFL yield, g / L SC-M1 25 Not detected SC-M24 32 Not detected

[0141] Example 6. Synthesis of 2'-fucosyllactose using Kluyveromyces as a substrate.

[0142] Kluyveromyces lactis chassis cells were constructed according to the reference Li et al. (Li, F., Ma, W., Liu, L., Niu, K., Liu, D., Yin, W., Zhang, X., Han, L., & Fang, X. (2023). Reprogramming the Metabolic Network in Kluyveromyces lactis with a Transcriptional Switch for De Novo Lacto-N-biose Synthesis. Journal of agricultural and food chemistry, 71(23), 9031–9039.

[0143] https: / / doi.org / 10.1021 / acs.jafc.3c01779 ) construction and transformation method to obtain strain KL-Δ LAC4 ::GMD-ΔXK::GMER, and simultaneously constructed expression cassettes ΔGK::Ptef1-M1 and ΔGK::Ptef1-M24, and transferred the expression cassettes into Strain KL-ΔLAC4 ::GMD-ΔXK::GMER, and obtained KL-M1 and KL-M24 strains.

[0144] Take Kluyveromyces KL-M1 and KL-M24 respectively and let them grow rapidly until they enter the late logarithmic phase or the stationary phase. Streak the strains on solid culture medium such as YDP, culture at 30℃ for 2-3 days, pick a single colony and inoculate it into 1.5mL YPD liquid culture medium, and culture it at 30℃ and 200rpm overnight. Then inoculate it into 50mL liquid culture medium shake flasks at a 2% inoculum volume and culture it at 30℃ and 200rpm until the OD 600 =1, and 2% inoculum was inoculated into 1 L of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) (3 L fermentor). After 6-7 hours of culture, sucrose (50% mother liquor concentration) was added at a rate of 8 mL / h. Simultaneously, lactose (40% mother liquor concentration) was added to maintain a final lactose concentration of 15 g / L. The total fermentation time was 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and the supernatant and precipitate were collected separately. The precipitate was disrupted using a high-pressure homogenizer, boiled, and centrifuged to remove protein. The supernatants were combined to obtain the final yeast fermentation product. The 2'-FL and DFL contents in the supernatants were measured, and the results are recorded in Table 6.

[0145] Table 6

[0146] Genetically modified cells 2'-FL yield, g / L DFL yield, g / L KL-M1 28 Not detected KL-M24 35 Not detected

[0147] The present invention discloses a series of α-1,2-fucosyltransferase polypeptides, their preparation methods and applications, DNA molecules encoding the polypeptides, vectors, and host cells. Those skilled in the art can refer to the content of the present invention and appropriately improve the process parameters to achieve the desired results. It should be noted in particular that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0148] Although the present invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that any equivalent aspects or modifications may be implemented. Therefore, the present description and examples should not be construed as limiting the scope of the invention.

Claims

1. An α-1,2-fucosyltransferase polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO:

25. 2 . A polynucleotide encoding the α-1,2-fucosyltransferase polypeptide according to claim 1 . A nucleic acid construct comprising the polynucleotide according to claim 2 . 4 . An expression vector comprising the polynucleotide according to claim 2 or the nucleic acid construct according to claim 3 .

5. A transformed host cell transformed with the polynucleotide of claim 2, the nucleic acid construct of claim 3 or the expression vector of claim 4.

6. The host cell transformed as claimed in claim 5, selected from Escherichia coli, Bacillus or yeast strains.

7. The host cell transformed according to claim 6, wherein The yeast strain is selected from Yarrowia lipolytica.

8. The host cell transformed as claimed in claim 5, selected from Escherichia coli E. coli BL21(DE3). 9 . An enzyme agent comprising one or more of the α-1,2-fucosyltransferase polypeptides according to claim 1 .

10. The method for producing the α-1,2-fucosyltransferase polypeptide according to claim 1, characterized in that: The steps include: (1) culturing the transformed host cell according to claim 5 under conditions suitable for expressing the α-1,2-fucosyltransferase polypeptide; and (2) Recovering the α-1,2-fucosyltransferase polypeptide.

11. The production method according to claim 10, characterized in that The step (1) comprises: firstly introducing the nucleic acid construct according to claim 3 or the expression vector according to claim 4 into a host cell to construct an engineered host cell, and then culturing the engineered host cell and inducing it to express the α-1,2-fucosyltransferase polypeptide.

12. The production method according to claim 10, characterized in that: The step (2) includes the steps of isolating and purifying the α-1,2-fucosyltransferase polypeptide from the culture.

13. Use of the α-1,2-fucosyltransferase polypeptide of claim 1, the transformed host cell of claim 5, or the enzyme of claim 9 in synthesizing 2'-fucosyllactose.

14. The use according to claim 13, characterized in that GDP-L-fucose and lactose were used as substrates.

15. The use according to claim 13, characterized in that The steps include: a) using the α-1,2-fucosyltransferase polypeptide of claim 1, the transformed host cell of claim 5, or the enzyme agent of claim 9 as a catalyst to catalyze the synthesis of 2'-fucosyllactose from substrates GDP-L-fucose and lactose.

16. The use according to claim 15, characterized in that Also includes: b) a step of purifying and / or recovering 2'-fucosyllactose.

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