Fucosyltransferase polypeptide and application thereof

Abstract

The invention relates to a series of fucosyl transferase polypeptides which can be applied to synthesis of human milk oligosaccharides (HMOs), and belongs to the technical field of enzyme engineering and genetic engineering. The invention provides an alpha-1, 3-fucosyltransferase polypeptide, which comprises a polypeptide with an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% identical to that of a polypeptide shown in SEQ ID NO: 1. Or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% of the same amino acid sequence as the polypeptide M1-21. The invention provides a polypeptide as shown in SEQ ID NO: 1 and M1-M21 polypeptides. The 3-FL catalytic synthesis capability of the polypeptide as shown in SEQ ID NO: 1 is improved; and the specificity of a catalytic substrate is improved.

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 technical fields of enzyme engineering and genetic engineering. Background Art

[0002] Human milk oligosaccharides (HMOs) are a unique and rich mixture of oligosaccharides present in human milk and are the third largest solid component in breast milk after fat and lactose.

[0003] 3-Fucosyllactose (3-FL) is a representative oligosaccharide of fucosylated HMOs and one of the most abundant human milk oligosaccharides. Among HMO monomers with known variations in concentration, 3-FL is the only one whose concentration increases during lactation. Compared to other HMOs, this increasing concentration of 3-FL during lactation suggests a broader range of applications in areas such as follow-on formula.

[0004] Research into the synthesis of 3-FL is crucial for the commercial application of HMOs, particularly identifying efficient, low-cost, and environmentally friendly synthetic routes. In recent years, research into the bioproduction of 3-FL has rapidly advanced, demonstrating numerous advantages over chemical synthesis routes. Therefore, the development of efficient methods for the production of 3-FL is urgently needed to lay a crucial foundation for the generation of more valuable human milk oligosaccharides.

[0005] Current research on 3-FL synthesis focuses on chemical, enzymatic, and microbial methods. Chemical synthesis often requires the introduction of protecting groups, resulting in tedious steps and potential problems such as inadequate protection, incomplete subsequent removal, and the occurrence of side reactions. In contrast, biological synthesis is more suitable for large-scale industrial production due to its high enzyme-substrate specificity, inexpensive substrates, simplified synthesis steps, minimal byproducts, and significantly improved yields. Currently, microbial fermentation is the primary method for synthesizing 3-fucosyllactose. In this microbial fermentation method for 3-FL synthesis, α-1,3-fucosyltransferase is a key enzyme, catalyzing the conversion of GDP-L-fucose and lactose. Currently, α-1,3-fucosyltransferases are mostly derived from Helicobacter pylori and Escherichia coli. Microbial methods for synthesizing 3-fucosyllactose often yield low yields, failing to meet the requirements for large-scale industrial production. Furthermore, the undesirable lactose difucosyllactose (DFL) is produced, making its isolation more difficult. Therefore, the selection of α-1,3-fucosyltransferase is crucial in the process of microbial synthesis of 3-FL. Therefore, for the needs of industrial development, α-1,3-fucosyltransferases that are more suitable for the production of 3-FL should be screened.

[0006] Patent No. 202111404385.4 discloses a recombinant Escherichia coli that expresses α-1,3-fucosyltransferase from Bacteroides gallinaceum, as well as genes for phosphomannose mutase, mannose-6-phosphate isomerase, GDP-L-fucose synthase, and GDP-D-mannose dehydrogenase from Escherichia coli; and knocks out UDP-glucose lipid carrier transferase and β-galactosidase. In a shake flask experiment, the recombinant E. coli had an ability to produce 3-FL of 2.01 g / L, and in a 3L fermenter, the yield of 3-FL reached 20.3 g / L. Patent No. 202011223864.1 discloses a recombinant Bacillus subtilis for producing 3-FL, its construction method, and application. The recombinant Bacillus subtilis 1643FL was obtained by integrating fusion gene fragments encoding fructose 6-phosphate isomerase, phosphomannose mutase, and mannose-1-phosphate guanylyltransferase at the manA locus of Bacillus subtilis, and fusion gene fragments encoding GDP-mannose dehydratase, GDP-L-fucose synthase, and α-1,3-fucosyltransferase at the xylA-xylB locus. The resulting recombinant Bacillus subtilis fermentation broth has a 3-FL concentration of 6.2 g / L. Patent 202310292697.3 discloses a mutant of Helicobacter pylori α-1,3-fucosyltransferase, which has mutations at amino acids 108 and / or 139 of the wild-type Helicobacter pylori α-1,3-fucosyltransferase. The mutant D139E has a Km of approximately 1 μM, and its enzyme activity is three times higher than the wild-type Km (approximately 3 μM).

[0007] Chinese Patent No. 202110588386.2 discloses an in vitro multi-enzyme cascade catalytic method for synthesizing fucosylated lactose. This method utilizes a combination of GDP-L-fucose synthesis-related enzymes and L-fucosyltransferase to catalyze the reaction between the substrate GDP-L-fucose and lactose to synthesize fucosylated lactose. The document discloses that the production of 3-FL by a dual-enzyme coupling of L-fucokinase / GDP-L-fucose pyrophosphorylase and α-1,3-fucosyltransferase was 302.2 mg / L; and that by a triple-enzyme coupling of L-fucokinase / GDP-L-fucose pyrophosphorylase, α-1,3-fucosyltransferase, and polyphosphate kinase, the production of 3-FL was 506.7 mg / L.

[0008] In summary, it is necessary to study the substrate specificity of α-1,3-fucosyltransferase in order to improve the synthesis efficiency and conversion rate of 3-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

[0009] 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-1,3-fucosyltransferase polypeptide, and the fucosyllactose includes 3-fucosyllactose (3-FL).

[0010] The α-1,3-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.

[0011] The technical solution of the present invention:

[0012] The applicant discovered an α-1,3-fucosyltransferase polypeptide (α-1,3-fucosyltransferase) from Bacillus smithii in nature, named BSFut3t-wt, whose amino acid sequence is shown in SEQ ID NO: 1 and nucleotide sequence is shown in SEQ ID NO: 3. It has low catalytic activity in synthesizing 3-FL, accompanied by an undesirable side activity of synthesizing DFL (Difucosyllactose). It has an amino acid sequence similarity of 33.96% with the α-1,3-fucosyltransferase (α-1,3-fucosyltransferase) from Helicobacter pylori, named HPFutA-wt, whose amino acid sequence is shown in SEQ ID NO: 2. Figure 4 ).

[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 3-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 derivative polypeptides of the present invention have enhanced lactose binding and / or transferase properties. Furthermore, compared to currently known α-1,3-fucosyltransferase polypeptides, these polypeptides have reduced enzymatic side activities and reduced / absent the formation of the undesirable byproduct lacto-N-difucotetraose (DFL) when synthesizing 3-fucosyllactose.

[0017] Specifically, in a first aspect, the present application provides a series of α-1,3-fucosyltransferase polypeptides that synthesize fucosylated lactose, wherein the α-1,3-fucosyltransferase polypeptides are selected from the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 and its derivative peptides M1-M21, and the derivative peptides M1-M21 are as follows:

[0018] 1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid at position 2 is substituted from K to T, thereby obtaining polypeptide M1;

[0019] 2) In the amino acid sequence of M1, the amino acid at position 17 is replaced by K to R, thereby obtaining polypeptide M2;

[0020] 3) In the amino acid sequence of M2, the amino acid segment at positions 21-22 is replaced by KN to NS, thereby obtaining polypeptide M3;

[0021] 4) In the amino acid sequence of M3, the amino acid at position 27 is replaced by K to N, thereby obtaining polypeptide M4;

[0022] 5) In the amino acid sequence of M4, the amino acid fragment at positions 45-48 is replaced by FFSD to IYSC, thereby obtaining polypeptide M5;

[0023] 6) In the amino acid sequence of M5, the amino acid fragment at positions 69-72 is replaced by EPDF with QPNL, thereby obtaining polypeptide M6;

[0024] 7) In the amino acid sequence of M6, the amino acid segment at positions 97-103 is replaced by YKYKEAL to FLYTQDY, thereby obtaining polypeptide M7;

[0025] 8) In the amino acid sequence of M7, the amino acid at position 118 is replaced by E to K, thereby obtaining polypeptide M8;

[0026] 9) In the amino acid sequence of M8, the amino acid fragment at positions 125-127 is replaced by CFN to NFI, thereby obtaining polypeptide M9;

[0027] 10) In the amino acid sequence of M9, the amino acid fragment at positions 133-135 is replaced by YDE to AHR, thereby obtaining polypeptide M10;

[0028] 11) In the amino acid sequence of M10, the amino acid fragment at positions 138-139 is replaced by IK to FE, thereby obtaining polypeptide M11;

[0029] 12) In the amino acid sequence of M11, the amino acid at position 141 is substituted from Y to 1, thereby obtaining polypeptide M12;

[0030] 13) In the amino acid sequence of M12, the amino acid at position 156 is replaced by T to I, thereby obtaining polypeptide M13.

[0031] 14) In the amino acid sequence of M13, the amino acid fragment at positions 162-165 is replaced by FLIE with YRVD, thereby obtaining polypeptide M14;

[0032] 15) In the amino acid sequence of M14, the amino acid at position 223 is replaced by T to C, thereby obtaining polypeptide M15;

[0033] 16) In the amino acid sequence of M15, the amino acid segment at positions 227-229 is replaced by SSY to KSK, thereby obtaining polypeptide M16;

[0034] 17) In the amino acid sequence of M16, the amino acid at position 243 is substituted from K to D, thereby obtaining polypeptide M17;

[0035] 18) In the amino acid sequence of M17, the amino acid segment at positions 247-248 is replaced by TE to SQ, thereby obtaining polypeptide M18;

[0036] 19) In the amino acid sequence of M18, the amino acid at position 255 is substituted from F to L, thereby obtaining polypeptide M19;

[0037] 20) In the amino acid sequence of M19, the amino acid fragment at positions 260-263 is replaced by NPEE with DPEK, thereby obtaining polypeptide M20;

[0038] 21) In the amino acid sequence of M20, the amino acid at position 268 is substituted from A to E, thereby obtaining polypeptide M21.

[0039] Polypeptides M1-M21 reduce or eliminate the production of the byproduct DFL (Difucosyllactose).

[0040] In certain embodiments, the polypeptide having enhanced catalytic activity for 3-FL synthesis 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 the polypeptide set forth in SEQ ID NO: 1 and polypeptides M1-M21, and comprises an amino acid substitution mutation that is functionally equivalent to the amino acid substitutions described in 1)-21) 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 described in 1)-21) above.

[0041] In a second aspect, the present application provides a polynucleotide encoding the polypeptide as described in the first aspect above.

[0042] Polynucleotides encoding the polypeptide of SEQ ID NO: 1 and polypeptides M1-M21 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.

[0043] In a third aspect, the present application provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] In a specific embodiment, the expression cassette comprises all elements for expressing the α-1,3-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.

[0049] 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.

[0050] In some embodiments, the host cell includes but is not limited to natural strains or genetically modified strains of bacteria, yeast, mold, etc.;

[0051] Further preferably, the host cell includes but is not limited to natural strains or genetically modified strains of Escherichia sp., Bacillus sp., Kluyveromyces sp., etc.;

[0052] Further preferably, the host cell includes but is not limited to natural strains or genetically modified strains such as Saccharomyces cerevisiae, Kluyveromyces marxianus, Kluyveromyces lactis, and Yarrowia lipolytica.

[0053] More preferably, the host cell is the genetically engineered Escherichia coli BL21 (DE3).

[0054] 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.

[0055] The enzyme or enzyme composition preferably contains one or more of the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 and polypeptides M1-21 of the present invention.

[0056] Depending on the reaction substrate and product, the enzyme or composition may also contain bifunctional enzymes such as L-fucose pyrophosphorylase (L fucokinase / GDP-L-fucosepyrophosphorylase, FKP), glucokinase (glucokinase, Glk), mannose-phosphate mutase (mannose-phosphate mutase, ManB), mannose-1-phosphate guanylate transferase (ManC), GDP-mannose dehydratase (GDP-mannose-4,6-dehydratase, GMD), etc., which are beneficial for the synthesis of fucosyllactose.

[0057] In a seventh aspect, the present application provides a method for producing the polypeptide as described in the first aspect above, comprising:

[0058] (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

[0059] (2) Recovering the polypeptide.

[0060] 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.

[0061] In a specific embodiment, the step (2) includes the steps of isolating and purifying the polypeptide from the culture.

[0062] 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) to cultivate cell in laboratory or industrial fermentor tank.Cultivation is to use program known in the art, occurs in applicable substratum, and described substratum comprises carbon and nitrogen source and inorganic salt.Applicable substratum can be purchased through commercial channels, or according to disclosed composition preparation.

[0063] 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.

[0064] 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.

[0065] In an eighth aspect, the present application provides the use of the α-1,3-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;

[0066] Preferably, the fucosyllactose includes but is not limited to 3-fucosyllactose (3-FL).

[0067] 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-1,3-fucosyltransferase polypeptide described in this application to exert its enzymatic activity.

[0068] In one embodiment, the method comprises: adding the α-1,3-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.

[0069] Preferably, the application uses GDP-L-fucose and lactose as substrates and the α-1,3-fucosyltransferase polypeptide described in the present application as a catalyst to catalyze the synthesis and obtain 3-FL.

[0070] Preferably, the application is to prepare the α-1,3-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-1,3-fucosyltransferase polypeptide listed above can be isolated and optionally further purified. Specifically, it includes:

[0071] a) catalyzing the synthesis of 3-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:

[0072] b) Optionally, a step of purifying and / or recovering 3-FL.

[0073] In one embodiment, at least one of the α-1,3-fucosyltransferase polypeptides described herein is mixed with GDP-L-fucose, a buffer solution (such as Tris-HCl or HEPES), and lactose, and 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 this period, lactose reacts with GDP-L-fucose to convert it into 3-FL. 3-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 3-FL yield is determined by HPLC chromatography.

[0074] The application of the α-1,3-fucosyltransferase polypeptide of the present invention uses acceptor lactose and donor GDP-L-fucose as substrates to produce 3-FL through a catalytic reaction. The reaction solution contains no or a trace amount of undesirable DFL, which is beneficial for the separation and purification of the reaction product.

[0075] In this step, the mixture containing 3-FL can be separated and purified in a conventional manner. Preferably, the mixture containing 3-FL is clarified by centrifugation, flocculation, decantation and / or filtration.

[0076] Preferably, substantially all proteins, as well as amino acids, RNA and DNA, etc., are removed from the mixture containing 3-FL (preferably after clarification). In this step, proteins and related impurities can be removed from the mixture containing 3-FL in a conventional manner. Optionally, purification can be performed 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.

[0077] 3-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.

[0078] 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.

[0079] 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.

[0080] The term "a nucleic acid sequence encoding an enzyme for 3-FL synthesis" relates to a nucleic acid sequence encoding an enzyme essential for the synthesis pathway of 3-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, thereby producing 3-FL.

[0081] Beneficial effects:

[0082] This application provides polypeptides represented by SEQ ID NO: 1 and polypeptides M1-M21, which enhance the ability of the polypeptide represented by SEQ ID NO: 1 to catalyze the synthesis of 3-FL and improve its substrate specificity. The catalytic reaction of polypeptides M1-M21 reduces the DFL-producing side effect of the polypeptide represented by SEQ ID NO: 1. Furthermore, this application provides polynucleotides encoding the polypeptides and related nucleic acid products, transformed host cells, enzymes containing the polypeptides, and methods for catalytically synthesizing 3-FL using the polypeptides as catalysts.

[0083] 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

[0084] Figure 1 HPLC chromatograms of the reaction solutions of lactose, 3-FL standard, DFL standard, the polypeptide represented by SEQ ID NO: 1, and polypeptide M18 are shown. The RT of lactose is 9.98 min, the RT of the 3-FL standard is 11.43 min, and the RT of the DFL standard is 13.00 min.

[0085] Figure 2 .Mass spectrometry analysis of 3-FL standard. The results show that the sodium addition mode of 3-FL standard is 511.17 and the molecular weight of 3-FL is 488.2, which is within the allowable error range with the theoretical molecular weight of 488.4.

[0086] Figure 3 . Figure 1 The LC-MS analysis of the substance with rt of about 11.43min in the HPLC spectrum of the peptide M18 reaction liquid showed that it was consistent with the mass spectrum of the 3-FL standard, thus proving the successful synthesis of 3-FL.

[0087] Figure 4 .Comparison of amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2. 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 indicated, 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] In the following examples, the alpha-1,3-fucosyltransferase (FutC) involved is from Bacillus smithii, with an amino acid sequence as shown in SEQ ID NO: 1 and a nucleotide sequence as shown in SEQ ID NO: 3; or is from Helicobacter pylori, with an amino acid sequence as shown in SEQ ID NO: 2.

[0092] 3-FL and DFL standards are produced by ELICITYL, France.

[0093] Example 1. Expression of the polypeptide shown in SEQ ID NO: 1 and polypeptides M1-M21 in Escherichia coli.

[0094] Using the amino acid sequences of the polypeptides shown in SEQ ID NO: 1 and SEQ ID NO: 2 as templates, their nucleotide sequences were synthesized respectively and finally constructed into the PET32a vector to obtain PET32a-wt and PET32a-HPFutA plasmids.

[0095] 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: 3 of PET32a-wt was used to identify the mutation sites using Primer-BLAST ( Primer designing tool(nih.gov) ) mutant primers were designed, and a recombinant plasmid series (pET32a-M1 to pET32a-M21) were constructed using a point mutagenesis kit (Fast Mutagenesis Kit).

[0096] 3. Transform the above recombinant plasmids into Escherichia coli BL21 (DE3) according to the following steps:

[0097] 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 to pET32a-M21 recombinant plasmids (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 positive transformants.

[0098] 4. Culture positive transformants, extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the recombinant plasmids pET32a-M1 to pET32a-M21 are successfully introduced into E. coli.

[0099] 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.

[0100] 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.

[0101] 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 and SEQ ID NO: 2, as well as M1-M21 polypeptides were obtained. The relevant information is shown in Table 1.

[0102] Table 1. Corresponding numbers of α-1,3-fucosyltransferase polypeptides and their amino acid sequences

[0103]

[0104]

[0105] Example 2. Determination of the ability of the polypeptides obtained in Example 1 (polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 2, and polypeptides M1-M21) to serve as catalysts for the synthesis of 3-FL.

[0106] 3-FL was synthesized using the purified polypeptide represented by SEQ ID NO: 1 or SEQ ID NO: 2 or one of the M1-M21 polypeptides obtained in Example 1 as a catalyst, and lactose and GDP-L-fucose as reaction substrates, respectively. The specific procedures are as follows:

[0107] 0.2 mg / mL of the purified polypeptide represented by SEQ ID NO: 1 or SEQ ID NO: 2 or one of the M1-M21 polypeptides obtained in Example 1 was added to 40 mM lactose, 15 mM GDP-L-fucose, 10 mM MnCl2, and 25 mM Tris-HCl, pH 7.5, respectively. The mixture was mixed and reacted at 37° C. for 6 hours. The reaction was terminated and the product was purified by gel column method.

[0108] Identification of catalytic synthesis products:

[0109] 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.

[0110] LC-MS analysis conditions are as follows:

[0111] 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

[0112] =65:35; ESI-MS mode, molecular weight scanning range 100~800.

[0113] The standard and reaction solution were tested using the aforementioned analytical method. HPLC analysis revealed that the peak elution time (RT) of the 3-FL standard was 11.43 min. The reaction solutions of the polypeptide described in Example 1 all exhibited a strong absorption peak around 11.43 min, consistent with the peak elution time of the 3-FL standard, indicating that the polypeptide-catalyzed reaction in Example 1 produced 3-FL.

[0114] In addition: HPLC analysis of DFL standard (attached Figure 1 )rt is 13.00min, the reaction solutions of the polypeptide shown in SEQ ID NO: 1 and polypeptides M1 and M3 have a weak peak at rt of 13.00min; the reaction solutions of polypeptide M2 ​​and polypeptides M4-M21 have no peak at this position, among which the HPLC spectrum of the reaction solution of polypeptide M18 (attached) Figure 1 ).

[0115] HPLC analysis results showed that the reaction solution of the polypeptide represented by SEQ ID NO: 2 also had a weak peak at rt of 13.00 min, indicating that the reaction products of the polypeptide represented by SEQ ID NO: 1 and SEQ ID NO: 2 both contained undesirable DFL.

[0116] The LC-MS analysis conditions are shown above, and the analysis results show:

[0117] The product of the HPLC chromatogram peak of the reaction solution at rt = 11.43 min was analyzed by LC-MS. Figure 2 The M+Na value in the fermentation broth was 511.17, which was consistent with the Figure 2The mass spectrometry results of the 3-FL standard are consistent with the theoretical molecular weight of 3-FL, 488.4, within the allowable error range. This demonstrates that the polypeptides represented by SEQ ID NO:1 and M1-M21 were successfully synthesized from 3-FL. This suggests that the polypeptides represented by SEQ ID NO:1 and M1-M21, derived from Bacillus smithii, can replace the previously reported polypeptide represented by SEQ ID NO:2, derived from Helicobacter pylori, for the synthesis of 3-FL.

[0118] The yields of 3-FL and DFL in the fermentation broth were detected by the above-mentioned HPLC analysis method, and the results are recorded in Table 2.

[0119] Table 2. Studies on the synthesis of 3-FL catalyzed by various peptides

[0120]

[0121]

[0122] Table 2 Data Description:

[0123] (1) The catalytic reaction liquid of α-1,3-fucosyltransferase from Helicobacter pylori showed a 3-FL yield of 0.2 mg / mL and a DFL yield of 0.1 mg / mL, which proved that the α-1,3-fucosyltransferase from Bacillus smithii had a higher yield of 3-FL (0.5 mg / mL) than that from Helicobacter pylori.

[0124] (2) In the catalytic reaction liquids of the α-1,3-fucosyltransferase polypeptide having the amino acid sequence shown in SEQ ID NO: 1, DFL was detected as a byproduct in addition to 3-FL. In the catalytic reaction liquids of M2, M4-M21, only 3-FL was detected, and no byproduct DFL was detected.

[0125] The above results demonstrate that the peptides M2, M4-M21 derived from α-1,3-fucosyltransferase of Bacillus smithii have improved specificity in catalyzing the synthesis of 3-FL and eliminated the side activity of synthesizing DFL.

[0126] (3) Peptides M2-M5 and M7-M21 increased the activity of the polypeptide shown in SEQ ID NO:1 in synthesizing 3-FL.

[0127] Example 3. Synthesis of 3-fucosyllactose using Escherichia coli as a substrate.

[0128] Referring to Huang et al. (Huang, D., Yang, K., Liu, J., Xu, Y., Wang, Y., Wang, R., Liu, B., & 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.), Escherichia coli BL21 was used as the starting strain to construct the strain BL21ΔlacZΔlonΔwcaJ, and the above-mentioned constructs PET32a-M1 and PET32a-M21 were transformed into the Escherichia coli chassis cells BL21ΔlacZΔlonΔwcaJ by electroporation to construct EC-M1 and EC-M21.

[0129] Method for culturing Escherichia coli to produce 3-FL:

[0130] E. coli strains EC-M1 and EC-M21 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 3-FL production. At the same time, the culture medium was supplemented with ampicillin to a final concentration of 100 μg / mL. After 10 minutes, the culture was boiled and centrifuged, and the supernatant was collected for analysis of 3-FL and DFL production. The results are reported in Table 3.

[0131] Table 3

[0132]

[0133]

[0134] Example 4. Synthesis of 3-fucosyllactose using Bacillus as the chassis.

[0135] To 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.), and use Bacillus subtilis with the deposit number of CCTCC N0: M 20231126 (BStgtP8) as the starting strain to construct Bacillus subtilis chassis cells BSP43-manB-P43-manC-P43-gmd-P43-wcaG. At the same time, P43-M1 and P43-M21 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-M21.

[0136] Bacillus subtilis strains BS-M1 and BS-M21 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. During shake flask fermentation, the following culture medium was used: 6 g / L trypsin, 12 g / L yeast extract, 12.5 g / L K2HPO4·3H2O, 2.5 g / L KH2PO4, and 10 mL / L trace metal solution (composed of 4 g / L FeSO4·7H2O, 4 g / L CaCl2, 1 g / L MnSO4·H2O, 0.2 g / L NaMoO4·2H2O, 0.2 g / L ZnSO4·7H2O, 0.1 g / L AlCl3·6H2O, 0.1 g / L CuCl2·2H2O, and 0.05 g / L H3BO4). Sterilized sucrose and lactose were added to the sterilized shake flasks to final concentrations of 20 and 10 g / L, respectively. Following fermentation, the culture was boiled for 10 minutes, and the supernatant was centrifuged and analyzed for 3-FL and DFL production. The results are reported in Table 4.

[0137] Table 4

[0138] Genetically modified cells 3-FL yield, mg / mL DFL yield, mg / mL BS-M1 1.3 Not detected BS-M21 5.2 Not detected

[0139] 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 3-FL content. The results were: 10.8g / L in the BS-M1 fermentation broth, and 29.7g / L in the BS-M24 fermentation broth.

[0140] Example 5. Synthesis of 3-fucosyllactose using Saccharomyces cerevisiae as a substrate.

[0141] 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.), with the deposit number CCTCC The Saccharomyces cerevisiae strain NO:M20231127 (SctgtP8) was used as the starting strain, and SC-Δgal80-Pgal1-lac12-Pgal1-gmd-wcaG was constructed. The pRS305-Pgal-M1 and pRS305-Pgal-M21 plasmids were constructed and transformed with SC-Δgal80-Pgal1-lac12-Pgal1-gmd-wcaG to obtain strains SC-M1 and SC-M21.

[0142] Take Saccharomyces cerevisiae SC-M1 and SC-M21 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 YPD, 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 2% of the inoculum into 50mL liquid culture medium shake flasks and culture at 30℃ and 200rpm until the OD 600=1, and were 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 h 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 h. After 72 h 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 tested for 3-FL and DFL production, and the results are recorded in Table 5.

[0143] Table 5

[0144] Genetically modified cells 3-FL yield, g / L DFL yield, g / L SC-M1 15.7 Not detected SC-M21 32.1 Not detected

[0145] Example 6. Synthesis of 3-fucosyllactose using Kluyveromyces as a substrate.

[0146] 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 Novo Lacto-N-biose Synthesis. Journal of agricultural and food chemistry, 71(23), 9031–9039.

[0147] https: / / doi.org / 10.1021 / acs.jafc.3c01779) construction and transformation methods, The lactic acid Kluyveromyces strain with the deposit number of CCTCC N0: M2022118 (Kluyveromyces HLLWF3) was used as the starting strain. Obtained strain KL- ΔLAC4 ::GMD-ΔXK::GMER, and simultaneously constructed expression cassettes ΔGK::Ptef1-M1 and ΔGK::Ptef1-M21, and transferred the expression cassettes into Strain KL-ΔLAC4 ::GMD-ΔXK::GMER, and obtained KL-M1 and KL-M21 strains.

[0148] Take Kluyveromyces KL-M1 and KL-M21 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 YPD, 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 were inoculated at a 2% inoculum 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 h of culture, sucrose (mother liquor concentration of 50%) was added at a rate of 8 mL / h. Simultaneously, lactose (mother liquor concentration of 40%) was added to maintain a final lactose concentration of 15 g / L. The total fermentation time was 72 h. After 72 h 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 3-FL and DFL contents in the supernatants were measured, and the results are recorded in Table 6.

[0149] Table 6

[0150] Genetically modified cells 3-FL yield, g / L DFL yield, g / L KL-M1 16.7 Not detected KL-M21 33.8 Not detected

[0151] The present invention discloses a series of α-1,3-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.

[0152] 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-1,3-fucosyltransferase polypeptide comprising a polypeptide having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the polypeptide of SEQ ID NO: 1; or a polypeptide having an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to polypeptide M1-21; Preferably, the amino acid sequence of the α-1,3-fucosyltransferase polypeptide is as shown in SEQ ID NO:

1.

2. The α-1,3-fucosyltransferase polypeptide according to claim 1, which is obtained by modifying at least one of positions 2, 17, 21-22, 27, 45-48, 69-72, 97-103, 118, 125-127, 133-135, 138-139, 141, 156, 162-165, 223, 227-229, 243, 247-248, 255, 260-263, and 268 of the amino acid sequence shown in SEQ ID NO: 1; Preferably, the modification is a mutation, and the mutation is selected from any one of the following mutations: 1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid at position 2 is substituted from K to T, thereby obtaining polypeptide M1; 2) In the amino acid sequence of M1, the amino acid at position 17 is replaced by K to R, thereby obtaining polypeptide M2; 3) In the amino acid sequence of M2, the amino acid segment at positions 21-22 is replaced by KN to NS, thereby obtaining polypeptide M3; 4) In the amino acid sequence of M3, the amino acid at position 27 is replaced by K to N, thereby obtaining polypeptide M4; 5) In the amino acid sequence of M4, the amino acid fragment at positions 45-48 is replaced by FFSD to IYSC, thereby obtaining polypeptide M5; 6) In the amino acid sequence of M5, amino acid residues 69-72 are replaced by EPDF with QPNL, thereby obtaining polypeptide M6; 7) In the amino acid sequence of M6, the amino acid segment at positions 97-103 is replaced by YKYKEAL to FLYTQDY, thereby obtaining polypeptide M7; 8) In the amino acid sequence of M7, the amino acid at position 118 is replaced by E to K, thereby obtaining polypeptide M8; 9) In the amino acid sequence of M8, the amino acid fragment at positions 125-127 is replaced by CFN to NFI, thereby obtaining polypeptide M9; 10) In the amino acid sequence of M9, the amino acid fragment at positions 133-135 is replaced by YDE to AHR, thereby obtaining polypeptide M10; 11) In the amino acid sequence of M10, the amino acid fragment at positions 138-139 is replaced by IK to FE, thereby obtaining polypeptide M11; 12) In the amino acid sequence of M11, the amino acid at position 141 is substituted from Y to 1, thereby obtaining polypeptide M12; 13) In the amino acid sequence of M12, the amino acid at position 156 is replaced by T to I, thereby obtaining polypeptide M13. 14) In the amino acid sequence of M13, the amino acid fragment at positions 162-165 is replaced by FLIE with YRVD, thereby obtaining polypeptide M14; 15) In the amino acid sequence of M14, the amino acid at position 223 is replaced by T to C, thereby obtaining polypeptide M15; 16) In the amino acid sequence of M15, the amino acid segment at positions 227-229 is replaced by SSY to KSK, thereby obtaining polypeptide M16; 17) In the amino acid sequence of M16, the amino acid at position 243 is substituted from K to D, thereby obtaining polypeptide M17; 18) In the amino acid sequence of M17, the amino acid segment at positions 247-248 is replaced by TE to SQ, thereby obtaining polypeptide M18; 19) In the amino acid sequence of M18, the amino acid at position 255 is substituted from F to L, thereby obtaining polypeptide M19; 20) In the amino acid sequence of M19, the amino acid fragment at positions 260-263 is replaced by NPEE with DPEK, thereby obtaining polypeptide M20; 21) In the amino acid sequence of M20, the amino acid at position 268 is substituted from A to E, thereby obtaining polypeptide M21. 3 . A polynucleotide encoding the α-1,3-fucosyltransferase polypeptide according to claim 1 .

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, which can direct the production of the polypeptide in an appropriate expression host cell. 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, the nucleic acid construct according to claim 4, or the expression vector according to claim 5; Preferably, the nucleic acid construct according to claim 5 or the host cell according to claim 6 is characterized in that, The host cells include but are not limited to natural strains or genetically modified strains such as bacteria, yeast, and mold; Further preferably, the host cell includes but is not limited to natural strains or genetically modified strains of Escherichia sp., Bacillus sp., yeast sp., etc.; Further preferably, the host cell includes but is not limited to natural strains or genetically modified strains such as Saccharomyces cerevisiae, Kluyveromyces marxianus, Kluyveromyces lactis, and Yarrowia lipolytica. Further preferably, the host cell includes but is not limited to the genetically engineered Escherichia coli BL21 (DE3). 7 . An enzyme agent or enzyme composition comprising one or more of the α-1,3-fucosyltransferase polypeptides according to claim 1 .

8. The method for producing the α-1,3-fucosyltransferase polypeptide according to any one of claims 1 to 2, characterized in that: The steps include: (1) culturing the transformed host cell under conditions suitable for expressing the α-1,3-fucosyltransferase 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 α-1,3-fucosyltransferase polypeptide according to any one of claims 1 to 2, or the transformed host cell according to claim 6, or the enzyme or enzyme composition according to claim 7 in synthesizing fucosyllactose; Preferably, the fucosyllactose includes but is not limited to 3-fucosyllactose; The application, preferably, is to catalyze the synthesis and obtain 3-FL using GDP-L-fucose and lactose as substrates; Said application is preferably to produce fucosyllactose in a cell-free expression system or in a host organism.

10. A method for preparing 3-fucosyllactose, characterized in that: The steps include: a) using the α-1,3-fucosyltransferase polypeptide according to any one of claims 1 to 2, or the transformed host cell according to claim 6, or the enzyme agent or enzyme composition according to claim 7 as a catalyst, and substrates including but not limited to GDP-L-fucose and lactose; Further preferably, the preparation method further comprises: b) optionally, a step of purifying and / or recovering 3-fucosyllactose.

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