Fucosyltransferase polypeptide and application thereof

Abstract

The invention provides a fucosyl transferase polypeptide, which can be applied to the synthesis of human milk oligosaccharides (HMOs), and belongs to the technical field of enzyme engineering. The fucosyltransferase polypeptide provided by the invention comprises polypeptides with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% same amino acid sequences as polypeptides shown in amino acid sequences such as SEQ ID NO: 1-25. According to the transferase polypeptide disclosed by the invention, the specificity of a polypeptide catalytic substrate is also improved while the ability of the polypeptide as shown in SEQ ID NO: 1 to catalyze and synthesize 2 '-FL is improved.

Description

Technical Field

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

[0002] Human milk oligosaccharides (HMOs) are a unique and rich mixture of oligosaccharides in human milk. They are the third largest solid component in breast milk after fat and lactose. Among them, 2'-fucosyllactose (2'-FL) is one of the most abundant oligosaccharides in human milk oligosaccharides. The content of 2'-FL in human milk is 2-5g / L. It has been proven that 2'-FL plays an important role in the brain, intestines, growth and development of infants. 2'-FL has been approved as an ingredient for infant formula milk powder in the United States, Australia, New Zealand, Canada and other countries, and can be added to infant milk powder, ordinary food, dietary supplements and / or medical food.

[0003] The synthesis of fucosyllactose is mostly carried out by chemical method, enzymatic method or microbial cell factory synthesis method. Although the chemical synthesis method has made great progress, the fucosyllactose molecules need to be repeatedly and multiple times protected and deprotected during the synthesis process, resulting in low product yield and high production costs.

[0004] Chinese patent 202110588386.2 discloses a method for synthesizing fucosylated lactose by multi-enzyme cascade catalysis in vitro, which combines GDP-L-fucose synthesis-related enzymes and L-fucosyltransferase to catalyze the reaction of substrate GDP-L-fucose and lactose to synthesize fucosylated lactose. The L-fucosyltransferase is selected from one of α-1,2-fucosyltransferase and α-1,3-fucosyltransferase. The GDP-L-fucose synthesis-related enzyme and fucosyltransferase double enzyme coupling reaction can obtain 200-500 mg / L fucosylated lactose for 2-12 hours.

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

[0006] The synthesis of 2'-FL requires many 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 done a lot of work on the source mining and molecular modification of the above key enzymes. Many papers have conducted mining and screening analysis on the source of key enzymes, among which the most detailed research is 1,2-FT enzyme (α-1,2-fucosyltransferase). 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 in the synthesis of 2'-FL by 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 improve the catalytic efficiency of enzymes. Summary of the invention

[0008] Purpose of the invention: 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'-fucosyllactose, 2'-FL).

[0009] The α-1,2-fucosyltransferase polypeptide described in the present application refers to a polypeptide that can catalyze the transfer of a fucose residue from a donor substrate to an acceptor molecule. The donor substrate used to transfer the fucose residue to the acceptor molecule is usually 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 whose nucleotide sequence is shown in SEQ ID NO: 26. It has a low catalytic synthesis of 2'-FL activity, accompanied by the production of an undesirable side activity of synthesizing DFL (Difucosyllactose).

[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 similarity of 69.31% with the amino acid sequence of Bacillus cereus107 (as of October 20, 2023), which is the one with 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 the present application have carried out a series of modifications on the polypeptide shown in SEQ ID NO: 1, screened and obtained a series of derivative peptides that catalyze the synthesis of fucosyllactose, and the derivative peptides have improved ability to catalyze the synthesis of 2'-FL and specificity of catalytic substrates. The derivative peptides are transferase polypeptides formed by replacing one or more amino acid residues / or fragments in the amino acid sequence of the polypeptide shown in SEQ ID NO: 1.

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

[0016] The polypeptides of the present invention have higher lactose binding and / or transferase properties. In addition, compared with the currently known α-1,2-fucosyltransferase polypeptides, these polypeptides have no enzyme side activity, and when synthesizing 2'-fucosyllactose, no undesirable byproduct difucosyllactose (DFL) is formed.

[0017] Specifically, in the first aspect, the present application provides a series of α-1,2-fucosyltransferase polypeptides for synthesizing fucosylated lactose, wherein the α-1,2-fucosyltransferase polypeptides are selected from the polypeptides shown in the amino acid sequences of 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, selected from any one of the following amino acid sequences.

[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 an 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 an 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 an amino acid sequence shown in SEQ ID NO:8 / or SEQ ID NO:9, respectively;

[0023] 5) In the amino acid sequence as shown in 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 an amino acid sequence as shown in 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 an amino acid sequence as shown in SEQ ID NO: 12 / or SEQ ID NO: 13, respectively;

[0025] 7) In the amino acid sequence shown in 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 shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively;

[0026] 8) In the amino acid sequence as shown in 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 an amino acid sequence as shown in 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 an amino acid sequence shown in SEQ ID NO: 18;

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

[0029] 11) In the amino acid sequence shown in 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 an amino acid sequence shown in SEQ ID NO: 20 / or SEQ ID NO: 21, respectively;

[0030] 12) In the amino acid sequence shown in SEQ ID NO:21, the amino acid residue at position 282 is replaced by E with D / or Y, thereby obtaining a polypeptide with an amino acid sequence shown in 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 replaced by 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 improved catalytic synthesis of 2'-FL activity comprises a polypeptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical to the polypeptide shown in SEQ ID NO: 1-25, and comprises an amino acid substitution mutation that is functionally equivalent to the schemes described in 1)-14) above. 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 schemes described in 1)-14) above.

[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 described in any one of SEQ ID NOs: 1 to 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%, 100% identical to the amino acid sequence of the polypeptides shown in SEQ ID NOs: 1 to 25, and comprises a polypeptide functionally equivalent to the amino acid sequence shown in SEQ ID NOs: 1 to 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 polypeptides presented in the present invention. Methods for preparing recombinant nucleotides, 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 a suitable 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 of 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 specific target nucleic acids, and the vector optionally contains a universal 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, and 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 above, or the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above.

[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] Further 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] Host cells can be cultivated in a nutrient medium suitable for producing polypeptides using methods known in the art. For example, cells can be cultured by shaking bottles, or in a suitable medium and under conditions that allow polypeptide expression and / or separation, small-scale or large-scale fermentations (including continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) can be carried out in a laboratory or industrial fermentor tank to cultivate cells. Cultivation is to use procedures known in the art, which occur in a suitable nutrient medium, and the medium comprises carbon and nitrogen sources and inorganic salts. Suitable medium can be purchased through commercial channels, or prepared according to disclosed compositions.

[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'-fucosyllactose, 2'-FL).

[0062] The application, suitable reaction conditions include: the presence of a suitable reaction substrate, the presence of a suitable polypeptide, or the presence of basic auxiliary factors 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 the present application, but the reaction conditions must enable the α-1,2-fucosyltransferase polypeptide described in the present 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 into 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 α-1,2-fucosyltransferase polypeptide listed above 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 above or the enzyme or enzyme composition described in the sixth aspect above. 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. Incubate 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 will be converted to 2'-FL by the enzyme using GDP-fucose, respectively. 2'-FL is then separated and purified by methods known in the art. At the end of the reaction or after separation and / or purification, HPLC chromatography is used to determine the yield of 2'-FL.

[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, but does not produce 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, etc., 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, colors 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 or 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 the identity of an amino acid sequence, and refers to the percentage of two or more sequences or subsequences that are identical or 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 analysis 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 connected to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to short chains (commonly referred to as peptides, oligopeptides and oligomers) and long chains (commonly referred to as proteins). "Polypeptides" include those modified by natural processes (such as, processing and other post-translational modifications), as well as by chemical modification techniques. These modifications are fully documented in basic textbooks, and in more detail in monographs, as well as in a large amount of research literature, and they are well known to technicians.

[0076] The term "nucleic acid sequence encoding an enzyme for 2'-FL synthesis" relates to a nucleic acid sequence encoding an enzyme essential in 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 and thereby producing 2'-FL.

[0077] Beneficial effects:

[0078] The present application provides α-1,2-fucosyltransferase polypeptides shown in SEQ ID NO:1-25, which improve the ability of the polypeptide shown in SEQ ID NO:1 to catalyze the synthesis of 2'-FL; and improve the specificity of its catalytic substrate, wherein the catalytic reaction of the polypeptides of SEQ ID NO:2-25 does not produce a byproduct DFL. In addition, the present application also provides polynucleotides capable of 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, etc.

[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 .HPLC spectrum of lactose.

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

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

[0083] Figure 4 .HPLC spectrum of the polypeptide reaction liquid 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 the LC-MS analysis spectrum of the substance with rt of about 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 implementation methods 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 concept and scope of use of the present invention all fall within the protection scope of the present invention.

[0089] It should be noted that the terms used are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates 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 embodiments of the present invention are to describe specific embodiments, rather than to limit the scope of protection of the present invention. If the experimental methods of specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology within the art are generally followed, 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 according to the conditions recommended by the manufacturer.

[0090] In the following specific embodiments, if the experimental methods for specific conditions are not specified, they are usually based on conventional methods and conditions of molecular biology within the technology of the field, 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 an amino acid sequence as shown in SEQ ID NO: 1 (nucleotide sequence as shown in SEQ ID NO: 26) as a template, synthesize the polypeptide shown in SEQ ID NO: 1, and finally construct it 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 Molecular Cloning Experiment Guide, 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. The above recombinant plasmids were transformed 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 melt, 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 heat shock for 45 seconds, then immediately cool them in an ice bath for 2 minutes, add 1 mL of fresh LB medium (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, 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 out positive transformants.

[0097] 4. Cultivate 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, place 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) inoculation amount and shake and culture at 37°C until OD 600 The expression of the expression vector was 0.8, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG), the final concentration of IPTG was 0.1mmol / L, and the induction was carried out at 16℃ for 12h, the rotation speed was 200rpm; after induction of expression, the fermentation broth was centrifuged at 5000r / min for 30min at 4℃ to collect the bacteria; the bacteria were resuspended in 20mM pH7.4 PBS buffer, and ultrasonicated at a frequency of plus on 5s / off 5s for 30min to break the bacteria; the liquid after breaking was centrifuged at 13000×g and 4℃ for 30min to remove cell debris and collect the supernatant.

[0099] 6. The soluble polypeptide sequence was purified by nickel column affinity chromatography. The process was as follows: deionized water was added to the top of the nickel column, and 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, and the sample was fully combined with 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, and finally the target protein was eluted with 5 times the 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. It can be seen that the polypeptides shown in SEQ ID NO: 1-25 were obtained. For detailed information, see Table 1.

[0101] Table 1. Correspondence 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 shown in SEQ ID NOs: 1-25, and α-1,2-fucosyltransferase derived from Helicobacter pylori shown in SEQ ID NO: 27) to serve as catalysts for catalytic synthesis of 2'-FL.

[0105] Using the purified polypeptides shown in SEQ ID NOs: 1-25 obtained in Example 1 and the α-1,2-fucosyltransferase from Helicobacter pylori shown in 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] 15mM lactose, 15mM GDP-L-fucose, 10mM MnCl 2 , 25 mM Tris-HCl, pH 7.5, add the purified polypeptides shown in SEQ ID NO: 1-25 obtained in Example 1 and α-1,2-fucosyltransferase from Helicobacter pylori shown in SEQ ID NO: 27 as catalysts 0.2 mg / mL, mix well, react at 37° C. for 6 hours, terminate the reaction, and purify it 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 aqueous solution, flow rate: 0.5 ml / min, column temperature: 35°C, injection volume: 10 μL, evaporative light detector, evaporation temperature 75°C, nebulization temperature 45°C.

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

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

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

[0112] The standard and reaction liquid were tested according to the above analysis method. The HPLC analysis results showed that the peak time rt of the 2'-FL standard was 15.06 min. The reaction liquids of the polypeptides described in Example 1 all showed a strong absorption peak near 15.06 min, which was consistent with the peak time of the 2'-FL standard, indicating that the catalytic reaction of the polypeptide described 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 shown in 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 HPLC chromatogram peak of the reaction solution 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 spectrum results of the 2'-FL standard are consistent with the theoretical molecular weight of 2'-FL 488.44, which is within the allowable error range, thus proving that the polypeptides shown in SEQ ID NO: 1-25 successfully synthesized 2'-FL. This indicates that SEQ ID NO: 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 detected by the above HPLC analysis method, and the results are recorded in Table 2.

[0117] Table 2. Research 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 showed 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 yield of the byproduct DFL (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 as shown in SEQ ID NO:1, and the yield was 0.04 mg / mL; while DFL was not detected in the catalytic reaction liquid of the α-1,2-fucosyltransferase polypeptide as 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 NO: 8-25 improve the activity of the polypeptide of SEQ ID NO: 1 in synthesizing 2'-FL to varying degrees. Among them, the α-1,2-fucosyltransferase polypeptides shown in SEQ ID NO: 23-25 ​​catalyze the synthesis of 2'-FL with an output of 5 times or more than that of the polypeptide shown in SEQ ID NO: 1.

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

[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-constructed PET32a-M1 and PET32a-M24 were transferred 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 single colonies were inoculated into 20 mL of liquid LB and cultured in 250 mL shake flasks 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 the culture was inoculated into 100 mL of LB medium containing 36 g / L glucose (or glycerol) as a carbon source for growth in 500 mL shake flasks. When OD 600 When the pH reached about 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 the production of 2′-FL. At the same time, the culture medium needed to be supplemented with ampicillin at a final concentration of 100 μg / ml. After the end, the culture solution was boiled for 10 minutes, and the supernatant was centrifuged to detect the production of 2′-FL and DFL. The results are recorded in Table 3.

[0128] Table 3

[0129]

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

[0131] Construct Bacillus subtilis chassis cells, refer 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 Bacillus subtilis chassis cells BSP43-manB-P43-manC-P43-gmd-P43-wcaG. At the same time, the 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 cultured on LB solid medium at 37°C for 10-12 hours, and single colonies were inoculated into 20 mL of liquid LB in a 250 mL shake flask at 37°C and 220 rpm for 10-12 hours. Te seed culture was further inoculated into 30 mL of fermentation medium at a rate of 10% and stirred at 37°C and 220 rpm for 72 hours in a 250 mL shake flask. A medium with the following composition was used during shake flask fermentation: 6 g / L trypsin, 12 g / L yeast extract, 12.5 g / L K 2 HPO 4 ·3H 2 O, 2.5 g / L KH 2 PO 4 and 10 mL / L of trace metal solution (composition: 4 g / L FeSO 4 7H 2 O, 4 g / L CaCl 2 , 1g / L MnSO4·H 2 O, 0.2g / L NaMoO 4 ·2H 2 O, 0.2g / LZnSO 4 7H 2 O, 0.1 g / L AlCl 3 6H2O, 0.1g / LCuCl 2 ·2H 2 O, and 0.05 g / L H 3 BO 4Sucrose and lactose were sterilized and added to the sterilized shake flask to a final concentration of 20 and 10 g / L, respectively. After the culture was boiled for 10 min, the supernatant was centrifuged and the 2'-FL and DFL yields were measured. 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 simultaneously scaled up to a 1L fermenter, and continuous feeding fermentation was performed. The sucrose content in the fermentation broth was controlled within the range of 17g / L to 22g / L; the lactose concentration was within the range of 10g / L to 20g / L, and the fermentation was terminated after continuous feeding for 30 hours. After the fermentation was completed, the culture broth was boiled for 10 minutes, and the supernatant was centrifuged to detect the 2'-FL content, which was 20g / L in the BS-M1 fermentation broth and 30g / L in the BS-M24 fermentation broth.

[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 stable phase. Streak the strains in solid culture medium such as YDP, culture at 30℃ for 2-3 days, pick a single colony and inoculate it in 1.5mL YPD liquid culture medium, and culture it at 30℃ and 200rpm overnight. Then inoculate it in a 50mL liquid culture medium shake flask at a 2% inoculum, and culture it at 30℃ and 200rpm until OD 600=1, and inoculated into 1.5L YPD medium (yeast extract powder 10g / L, peptone 20g / L, glucose 20g / L) (3L volume fermenter) at an inoculum of 2%, and after culturing for 5-6h, began to flow sucrose (mother liquor concentration 50%), and the sucrose addition flow rate was controlled at 8mL / h. At the same time, lactose (mother liquor concentration was 40%) was added to maintain the final lactose concentration of 15g / L. The total fermentation time was 72h. After 72h of fermentation, the fermentation liquid was centrifuged, and the supernatant and precipitate were collected respectively. The precipitate was crushed by a high-pressure homogenizer, boiled and centrifuged to remove protein, and the supernatant was combined to obtain the final fermentation product of yeast. The 2'-FL and DFL yields in the supernatant were detected, and the results were recorded 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 chassis.

[0142] The 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 NovoLacto-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 the 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 stable phase. Streak the strains in solid culture medium such as YDP, culture at 30℃ for 2-3 days, pick a single colony and inoculate it in 1.5mL YPD liquid culture medium, and culture it at 30℃ and 200rpm overnight. Then inoculate them in 50mL liquid culture medium shake flasks at a 2% inoculation rate, and culture them at 30℃ and 200rpm until OD 600 =1, and inoculated into 1L YPD medium (yeast extract powder 10g / L, peptone 20g / L, glucose 20g / L) (3L volume fermenter) at an inoculum of 2%, and after culturing for 6-7h, began to flow sucrose (mother liquor concentration 50%), and the sucrose addition flow rate was controlled at 8mL / h, and lactose (mother liquor concentration was 40%) was added at the same time to maintain the final lactose concentration of 15g / L, and the total fermentation time was 72h. After 72h of fermentation, the fermentation liquid was centrifuged, and the supernatant and precipitate were collected respectively. The precipitate was crushed by a high-pressure homogenizer, boiled and centrifuged to remove the protein, and the supernatant was combined to obtain the final fermentation product of the yeast, and the 2'-FL and DFL contents in the supernatant were detected. 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, preparation methods and applications thereof, 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 particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all 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 clear understanding, it will be apparent to one of ordinary skill 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 present invention.

Claims

1. An α-1,2-fucosyltransferase polypeptide comprising an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the polypeptides of 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. The transferase polypeptide of claim 1, wherein the amino acid sequence is 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, and SEQ ID NO:

25.

3. A polynucleotide encoding the transferase polypeptide according to any one of claim 1 or claim 2.

4. A nucleic acid construct comprising the polynucleotide according to claim 3; 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 a suitable expression host.

5. An expression vector comprising the polynucleotide according to claim 3, or comprising the nucleic acid construct according to claim 4.

6. A transformed host cell transformed with the polynucleotide according to claim 3, or the nucleic acid construct according to claim 4, or the expression vector according to claim 5; The host cell is preferably a host cell capable of transforming the polynucleotide according to claim 3, the nucleic acid construct according to claim 4, or the expression vector according to claim 5; The host cell is preferably selected from Escherichia coli, Bacillus, and yeast; the yeast is selected from Saccharomyces cerevisiae, Kluyveromyces, and Yarrowia lipolytica; More preferably, it is the genetically engineered Escherichia coli BL21 (DE3).

7. An enzyme agent or enzyme composition comprising one or more of the transferase polypeptides according to any one of claim 1 or claim 2.

8. The method for producing the transferase polypeptide according to any one of claims 1 or 2, characterized in that: The steps include: (1) culturing the transformed host cell under conditions suitable for expressing the transferase polypeptide; and (2) Recovering the polypeptide. Preferably, 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. Preferably, the step (2) includes the steps of separating and purifying the polypeptide from the culture.

9. Use of the transferase polypeptide according to any one of claims 1 or 2, or the transformed host cell according to claim 6, or the enzyme agent or enzyme composition according to claim 7 in synthesizing fucosyllactose; Preferably, the fucosyllactose includes 2'-fucosyllactose (2'-fucosyllactose, 2'-FL); The application, preferably, uses GDP-L-fucose and lactose as substrates; The use is preferably to prepare the transferase polypeptide in a cell-free expression system or in a host organism.

10. The use according to claim 9, characterized in that: The steps include: a) using the transferase polypeptide according to any one of claims 1 or 2, or the transformed host cell according to claim 6, the enzyme agent or enzyme composition according to claim 7 as a catalyst to catalyze the synthesis of 2'-FL from substrates GDP-L-fucose and lactose; More preferably, the method further comprises: b) optionally, a step of purifying and / or recovering 2'-FL.

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