A glycosyltransferase polypeptide and uses thereof

Abstract

The present application relates to a series of glycosyltransferase polypeptides, belonging to the technical field of enzyme engineering. The present application provides an amino acid sequence as shown in SEQ ID NO:1 alpha-1,2-transferring enzyme polypeptide and its derivative polypeptide M1-M20, the polypeptide M1-M20, which is obtained by substituting part of amino acids in at least one of the following positions of the amino acid sequence shown in SEQ ID NO:1: 37th, 40-42nd, 46th, 49th, 58th, 77th, 84th, 91st, 108th, 118th, 128th, 154th, 165-174th, 177-178th, 184th, 197-198th, 200th, 203th, 217th, 288th. The polypeptide shown in SEQ ID NO:1 has the ability to catalyze the synthesis of LNFP I, and the polypeptide M1-M20 improves the ability to catalyze the synthesis of LNFP I.

Description

Technical Field

[0001] This invention relates to a series of glycosyltransferase polypeptides that can be used in the synthesis of human milkoligosaccharides (HMOs), and belongs to the field of enzyme engineering technology. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third solid component of breast milk, playing a vital role in regulating immunity, aiding brain development, and modulating gut microbiota, thus contributing to infant growth and development. Over the past decade, several HMOs, including lactose-N-trisaccharide, lactose-N-tetrasaccharide, and 2'-fucosylated lactose, have been approved by the U.S. Food and Drug Administration. The commercial value of HMOs in the food industry has driven the demand for their industrial-scale production. Currently, metabolic engineering microbial synthesis is the main route for industrial production of HMOs. When constructing HMO-producing strains, three important factors should be considered: promoting donor supply, introducing specific glyconucleotide-dependent glycosyltransferases, and enhancing the synthesis of the corresponding glyconucleotides.

[0003] Lacto-N-fucopentaose I (LNFP I) is the fifth most abundant sugar in breast milk, thus its industrial production has attracted widespread attention. LNFP I has a relatively complex structure, and its synthesis via lactosylation involves three steps: lactose → lactose-N-trisaccharide II (LNT II) → lactose-N-tetrasaccharide (LNT) → lactose-N-fucopentaose I (LNFP I). These three steps are catalyzed by three glycosyltransferases: β-N-acetylglucosamine transferase (β-GlcNAcT), β1,3-galactosyltransferase (β1,3-GalT), and α1,2-fucosyltransferase (α1,2-FucT), using 5′-bisphosphate (UDP)-N-acetylglucosamine (GlcNAc), UDP-galactose, and guanosine 5′-bisphosphate (GDP)-L-fucose as glycosylation donors, respectively. To construct a complete pathway for LNFP I synthesis, these three glycosyltransferases must be introduced and the synthesis pathways of the corresponding sugars (LNT II, ​​LNT, and LNFP I) nucleotides must be enhanced. LNTII and LNT are the indirect and direct precursors of LNFP I microbial synthesis, respectively, and the microbial synthesis of LNT II and LNT has been well-studied. However, research on the in vivo or in vitro synthesis of LNFP I is limited.

[0004] International patent WO2022243314A discloses a method for synthesizing a blend of human lactose oligosaccharides (HMOs) containing LNFP I. The method includes providing genetically engineered cells capable of producing at least two HMOs, wherein the cells contain a heterologous β-1,3-N-acetyl-glucosamine transferase protein (source: Neisseria meningitidis et al.) and a heterologous α-1,2-fucosyltransferase protein (source: Methylobacter tundripaludum or Sideroxydanslithotrophicus). Chinese patent 202280035296.3 discovers an α-1,2-fucosyltransferase (Smob, from Aspergillus motiformis) that is superior to previously disclosed α-1,2-fucosyltransferases in the biosynthetic production of LNFP I, reducing the technical problems of byproduct formation and improving LNFP I titer and yield. In the HMOs blend synthesized by strain MP1 (FutC), the LNFP I:2'FL content ratio was almost 50%:50%, while in strain MP2 (FucT54) it became almost 70%:30%, and in strain MP3 (Smob) it even became 90%:10%. Chinese Patent 202280035349.1 discloses a method for synthesizing HMOs blends with LNFP I and LNT as the main compounds, wherein the cells used contain heterologous β-1,3-N-acetyl-glucosyltransferase protein and heterologous α-1,2-fucosyltransferase protein (from motile Aspergillus thiocarpa).

[0005] In summary, although researchers have made improvements in the production of fucosylated HMOs and discovered many fucosyltransferases, thereby increasing product yield, reducing the generation of byproducts while achieving high production of LNFP I remains an urgent technical problem to be solved. Summary of the Invention

[0006] Objective of the invention: To provide an α-1,2-fucosyltransferase polypeptide and its application in the synthesis of fucosyl lactose, so as to improve the production efficiency of fucosyl lactose.

[0007] Preferably, the fucoidyl lactose includes, but is not limited to, lactose-N-fucopentaose I (LNFP I).

[0008] The α-1,2-fucosyltransferase polypeptide described in this application is capable of transferring fucose residues from a donor substrate to an acceptor molecule, wherein the acceptor molecule is lacto-N-tetraose (LNT).

[0009] Technical solution:

[0010] Firstly, the applicant provides an α-1,2-fucosyltransferase polypeptide, named CsFut-wt, which is derived from Clostridium sp. C8-1-8, NCBI:

[0011] WP_160674431.1), the amino acid sequence is shown in SEQ ID NO:1.

[0012] The α-1,2-fucosyllactase polypeptide has the activity of catalyzing the synthesis of LNFP I, while having a low side activity of producing the undesirable synthesis of 2'-fucosyllactose (2'-FL).

[0013] The α-1,2-fucosyltransferase polypeptide with the amino acid sequence shown in SEQ ID NO:1, when compared with the NCBI database, showed 29.7% similarity to the α-1,2-fucosyltransferase (named HpFut-wt, amino acid sequence shown in SEQ ID NO:2) derived from Helicobacter pylori 26695 (NCBI: ARB16053.1); and 45.1% similarity to the α-1,2-fucosyltransferase (named FsFut-wt, amino acid sequence shown in SEQ ID NO:3) derived from Francisella sp. FSC1006 (NCBI: WP_040008276.1), making it the most similar.

[0014] In another aspect of the present invention, the inventors performed a series of modifications on the polypeptide with the amino acid sequence shown in SEQ ID NO:1, and screened and obtained a series of derived peptides capable of catalyzing the synthesis of LNFP I. The ability of these derived peptides to catalyze the synthesis of LNFP I and their substrate specificity are both improved. The derived peptides are transferase polypeptides formed by replacing one or more amino acid residues / fragments of the amino acid sequence shown in SEQ ID NO:1.

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

[0016] The derived polypeptides described in this application have higher LNFP I synthesis activity and lower side activity compared to known α-1,2-fucosyltransferase polypeptides. When synthesizing LNFP I, these polypeptides have no or low 2'-FL synthesis side activity.

[0017] Specifically, the α-1,2-fucosyltransferase polypeptide capable of synthesizing fucosylated lactose described in this application includes, but is not limited to, polypeptides with amino acid sequences as shown in SEQ ID NO:1 and / or polypeptides M1-M20. Polypeptides M1-M20 are derived peptides of the polypeptide with amino acid sequences as shown in SEQ ID NO:1.

[0018] The derived polypeptides M1-M20 described in this application are obtained by partial amino acid substitution at at least one of the following positions in the amino acid sequence shown in SEQ ID NO:1: positions 37, 40-42, 46, 49, 58, 77, 84, 91, 108, 118, 128, 154, 165-174, 177-178, 184, 197-198, 200, 203, 217, and 288; preferably, the polypeptides are selected from any of the following.

[0019] 1) In the amino acid sequence shown in SEQ ID NO:1, the amino acid residue at position 37 is replaced by F with Y, thereby obtaining polypeptide M1;

[0020] 2) In the amino acid sequence of polypeptide M1, the amino acid fragment at positions 40-42 is replaced by YDL with VSD, thereby obtaining polypeptide M2;

[0021] 3) In the amino acid sequence of polypeptide M2, the amino acid residue at position 46 is replaced by F instead of Y, thereby obtaining polypeptide M3;

[0022] 4) In the amino acid sequence of polypeptide M3, the amino acid residue at position 49 is replaced by D instead of N, thus obtaining polypeptide M4;

[0023] 5) In the amino acid sequence of polypeptide M4, the amino acid residue at position 58 is replaced by I instead of F, thereby obtaining polypeptide M5;

[0024] 6) In the amino acid sequence of polypeptide M5, the amino acid residue at position 77 is replaced by V instead of K, thereby obtaining polypeptide M6;

[0025] 7) In the amino acid sequence of polypeptide M6, the amino acid residue at position 84 is replaced by Y instead of G, thereby obtaining polypeptide M7;

[0026] 8) In the amino acid sequence of polypeptide M7, the amino acid residue at position 91 is replaced by S instead of L, thereby obtaining polypeptide M8;

[0027] 9) In the amino acid sequence of polypeptide M8, the amino acid residue at position 108 is replaced by Y, thereby obtaining polypeptide M9;

[0028] 10) In the amino acid sequence of polypeptide M9, the amino acid residue at position 118 is replaced by Y with F, thereby obtaining polypeptide M10;

[0029] 11) In the amino acid sequence of polypeptide M10, the amino acid residue at position 128 is replaced by E instead of Q, thereby obtaining polypeptide M11;

[0030] 12) In the amino acid sequence of polypeptide M11, the amino acid residue at position 154 is replaced by S instead of A, thereby obtaining polypeptide M12.

[0031] 13) In the amino acid sequence of polypeptide M12, the amino acid fragment at positions 165-174 was replaced by PRYYKTFGSV with DLSANNIHGI, thereby obtaining polypeptide M13.

[0032] 14) In the amino acid sequence of polypeptide M13, the amino acid fragment at positions 177-178 is replaced by LK with EE, thereby obtaining polypeptide M14;

[0033] 15) In the amino acid sequence of polypeptide M14, the amino acid residue at position 184 is replaced by I, thereby obtaining polypeptide M15.

[0034] 16) In the amino acid sequence of polypeptide M15, the amino acid fragment at positions 197-198 is replaced by YI with FV, thereby obtaining polypeptide M16;

[0035] 17) In the amino acid sequence of polypeptide M16, the amino acid residue at position 200 is replaced by S instead of T, thereby obtaining polypeptide M17.

[0036] 18) In the amino acid sequence of polypeptide M17, the amino acid residue at position 203 is replaced by I instead of P, thereby obtaining polypeptide M18.

[0037] 19) In the amino acid sequence of polypeptide M18, the amino acid residue at position 217 is replaced by Y, thereby obtaining polypeptide M19;

[0038] 20) In the amino acid sequence of polypeptide M19, the amino acid residue at position 288 is replaced by K with V, thereby obtaining polypeptide M20.

[0039] In some embodiments, the α-1,2-fucosyltransferase and / or peptides M1-M20 described herein reduce the production of the byproduct 2'-FL. The peptides M1-M20 have enhanced catalytic activity for the synthesis of LNFP I.

[0040] In some embodiments, the polypeptide having enhanced catalytic synthesis LNFP I activity includes, but is not limited to, polypeptides whose amino acid sequences are at least 60% identical to those of the polypeptides shown in SEQ ID NO:1 and / or polypeptides M1-M20.

[0041] Secondly, this application provides a polynucleotide encoding the α-1,2-fucosylation polypeptide as described in the first aspect above.

[0042] Preferably, the α-1,2-fucosyltransferase polypeptide includes, but is not limited to, the polypeptide and / or polypeptide M1-M20 with the amino acid sequence as described in SEQ ID NO:1.

[0043] 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. Various methods can be used to express expression vectors encoding the polypeptides presented in this invention. Methods for preparing recombinant nucleotides, expressing, and isolating expression products are known and described in the examples.

[0044] Thirdly, this application provides a nucleic acid construct comprising the polynucleotides described in the second aspect above.

[0045] The nucleic acid construct preferably further includes one or more regulatory sequences operatively linked thereto, the regulatory sequences being able to guide the production of the peptide in an appropriate expression host.

[0046] Fourthly, this application provides an expression vector comprising the polynucleotides described in the second aspect above, or the nucleic acid constructs described in the third aspect above.

[0047] The vector has a nucleotide or nucleic acid construct according to an embodiment of the invention operably linked to a regulatory sequence, the regulatory sequence being capable of expressing the DNA fragment, such as a promoter region.

[0048] A variety of commercially available kits are available for purifying plasmids and other related nucleotides from cells. Any isolated and / or purified nucleotides can be further manipulated to produce other nucleotides for transfecting cells, integrating into relevant 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 the expression of specific target nucleotides; vectors optionally include a universal expression cassette.

[0049] In one specific embodiment, the expression cassette contains all the elements necessary for expressing the α-1,2-fucosyltransferase polypeptide, including elements essential for transcription and translation in the host cell. For example, the expression cassette includes a promoter and a terminator, which are not particularly limited and can be promoters and terminators known in the art that enable expression of the variant.

[0050] Fifthly, this application provides a transformed host cell that has been 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.

[0051] In some embodiments, the transformed host cell is one in which a polynucleotide as described in the second aspect above, or a nucleic acid construct as described in the third aspect above, has been transformed.

[0052] In some embodiments, the host cell includes, but is not limited to, natural and / or genetically modified cells of prokaryotic and eukaryotic cells.

[0053] Preferably, the prokaryotic host cell includes, but is not limited to, natural cells and / or genetically modified cells such as bacterial cells and archaea. The bacterial host cell is not particularly limited in principle; it can be eubacteria (Gram-positive or Gram-negative) or archaea, as long as it allows for genetic manipulation to insert a target gene and can be cultured on a manufacturing scale. Preferably, the host cell has the characteristic of allowing culture to high cell densities.

[0054] Preferably, the bacterial host cell includes, but is not limited to, *Citrobacter freundii*, *Pantoeacitrea*, or *Xanthomonas campestris*. Bacteria of the genus *Bacillus* sp., including *Bacillus subtilis*, *Bacillus licheniformis*, *Bacillus coagulans*, *Bacillus thermophilus*, *Bacillus laterosporus*, *Bacillus megaterium*, *Bacillus mycoides*, *Bacillus pumilus*, *Bacillus lentus*, *Bacillus cereus*, and *Bacillus circulans*, can also be used, either naturally occurring or genetically modified.

[0055] Preferably, the bacterial host cells include, but are not limited to, natural or genetically modified cells of Enterococcus sp., Bifidobacterium sp., Sporolactobacillus sp., Micromomospora sp., Micrococcus sp., and Rhodococcus sp.

[0056] Preferably, the Enterococcus sp. includes, but is not limited to, natural or genetically modified cells of Enterococcus faecium and Enterococcus thermophiles.

[0057] Preferably, the Bifidobacterium sp. includes, but is not limited to, natural or genetically modified cells of Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum.

[0058] Preferably, the bacterial host cell includes, but is not limited to, natural or genetically modified cells of Escherichia sp.

[0059] More preferably, the bacterial host cells include, but are not limited to, Escherichia coli BL21(DE3) or Escherichia coli K12 cells.

[0060] Preferably, the eukaryotic host cell includes, but is not limited to, natural cells and / or genetically modified cells of yeast.

[0061] Preferably, the yeast host cell includes, but is not limited to, natural or genetically modified cells of Komagataella phaffii, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

[0062] Preferably, the eukaryotic host cell includes, but is not limited to, filamentous fungi such as Aspergillus sp., Fusarium sp., or Trichoderma sp., with exemplary species being natural or genetically modified cells of Aspergillus nige, Aspergillus nidulans, Aspergillus oryzae, Fusarium solani, etc.

[0063] In a sixth aspect, this application provides an enzyme agent or enzyme composition comprising one or more of the α-1,2-fucosyltransferase polypeptides as described in the first aspect above.

[0064] Preferably, the α-1,2-fucosyltransferase polypeptide includes, but is not limited to, one or more of the polypeptides and / or polypeptides M1-M20 whose amino acid sequences are shown in SEQ ID NO:1.

[0065] In a seventh aspect, this application provides a method for producing the α-1,2-fucosyltransferase polypeptide as described in the first aspect above, comprising:

[0066] (1) Transformed host cells are cultured under conditions suitable for expression of the polypeptide; the transformed host cells are as described in the fifth aspect above; and

[0067] (2) The polypeptide is recovered.

[0068] In a specific implementation, step (1) includes: firstly, introducing a nucleic acid construct or recombinant expression vector containing the α-1,2-fucosyltransferase 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.

[0069] The α-1,2-fucosyltransferase polypeptide includes, but is not limited to, polypeptides with amino acid sequences as shown in SEQ ID NO:1 and / or polypeptides M1-M20.

[0070] Optionally, step (2) may further include the step of separating and purifying the polypeptide from the culture.

[0071] Host cells can be cultured in nutrient media suitable for peptide production using methods known in the art. For example, cells can be cultured by shake-flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-by-feed fermentation, or solid-state fermentation) in a laboratory or industrial fermenter in a suitable medium and under conditions that allow for peptide expression and / or isolation. The culture occurs using procedures known in the art in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media can be purchased commercially or prepared according to publicly available compositions.

[0072] The polypeptide can be recovered from the culture using methods known in the art. For example, variants can be recovered from nutrient media through a variety of routine procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0073] In addition, peptides can be purified to obtain substantially pure peptides using a variety of procedures known in the art, including but not limited to chromatographic methods (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography), electrophoretic procedures (e.g., preparative isoelectric point focusing), differential solubility methods (e.g., ammonium sulfate precipitation), SDS-PAGE, salting out, and combinations thereof; more preferably, purification can be performed by Ni column affinity chromatography.

[0074] Eighthly, this application provides the use of the α-1,2-fucosyltransferase polypeptide as described in the first aspect above, the transformed host cell as described in the fifth aspect, or the enzyme or enzyme composition as described in the sixth aspect in the synthesis of fucoidan; preferably, the fucoidan includes, but is not limited to, LNFP I.

[0075] In some embodiments, the α-1,2-fucosyltransferase polypeptide includes, but is not limited to, polypeptides and / or polypeptides M1-M20 with amino acid sequences as shown in SEQ ID NO:1.

[0076] For the application described herein, suitable reaction conditions include: the presence of a suitable reaction substrate, a suitable peptide, or essential cofactors such as monovalent or divalent ions, a suitable pH range, and a suitable temperature. It is not necessary to meet the optimal values ​​for every factor affecting the peptide described in this application, but the reaction conditions must allow the α-1,2-fucosyltransferase peptide described in this application to exert its enzymatic activity.

[0077] In a ninth aspect, this application provides a method for synthesizing fucosylated oligosaccharides, the method comprising using an α-1,2-fucosylation polypeptide to transfer fucose residues from a donor substrate to an acceptor molecule.

[0078] Preferably, the fucoidylated oligosaccharide includes, but is not limited to, LNFP I.

[0079] The present application describes an α-1,2-fucosyltransferase, including but not limited to polypeptides and / or polypeptides M1-M20 with amino acid sequences as shown in SEQ ID NO:1, which can transfer fucose residues from donor substrates to acceptor molecules. Its specificity for LNT as an acceptor molecule is higher than its specificity for lactose as an acceptor molecule.

[0080] The α-1,2-fucosyltransferase is capable of transferring the fucosyl moiety from a donor molecule to the Gal moiety of a receptor. It is capable of transferring fucose residues from a donor substrate to a receptor molecule, wherein the receptor molecule includes, but is not limited to, LNT, and the donor molecule includes, but is not limited to, GDP-fucose.

[0081] In one embodiment, the method includes: adding the α-1,2-fucosyltransferase polypeptide described in this application to a reaction system, contacting it with the acceptor and fucose donor in the reaction system to catalyze a reaction to synthesize the desired fucosylated lactose.

[0082] Preferably, the application described in the eighth aspect and / or the method described in the ninth aspect use LNT and GDP-fucose as substrates and one or more of the polypeptides and / or polypeptides M1-M20 with the amino acid sequence described in the first aspect of this application as shown in SEQ ID NO:1 as catalysts to catalyze the synthesis and obtain LNFP I.

[0083] Preferably, the application described in the eighth aspect and / or the method described in the ninth aspect includes preparing the polypeptide and / or polypeptide M1-M20 of the first aspect amino acid sequence as shown in SEQ ID NO:1 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, *E. coli* or *Saccharomyces cerevisiae*), after which the polypeptide can be isolated and optionally further purified. Specifically, this includes:

[0084] a) Catalyzing and / or fermenting the synthesis of LNFP I using the polypeptide and / or polypeptide M1-M20 with the amino acid sequence as described in the first aspect above, or the host cells transformed as described in the fifth aspect, or the enzyme or enzyme composition as described in the sixth aspect above, or

[0085] The catalyst uses LNT and GDP-fucose as its reaction substrates.

[0086] The fermentation includes the synthesis of LNFP I by fermentation of the host cells transformed as described in the fifth aspect in a suitable culture medium.

[0087] Further preferred options include:

[0088] b) Optionally, the steps of purifying and / or recovering LNFP I.

[0089] In one embodiment, at least one of the polypeptides and / or polypeptides M1-M20 with the amino acid sequence described in this application as shown in SEQ ID NO:1 is mixed with LNT, a buffer solution (such as Tris-HCl or HEPES), and GDP-fucose. The mixture is incubated at a specific temperature (such as 35°C, 37°C, or 40°C) for a specific period of time (e.g., 24 hours), during which GDP-fucose and LNT are catalyzed by the enzyme to synthesize LNFP I. LNFP I is then isolated and purified using methods known in the art. At the end of the reaction or after isolation and / or purification, the yield of LNFP I is determined by HPLC.

[0090] The application of the polypeptide and / or polypeptides M1-M20 with the amino acid sequence shown in SEQ ID NO:1 of the present invention, through the enzyme-catalyzed reaction of substrate LNT and GDP-fucose, can generate LNFP I, accompanied by the generation of 2'-FL with reduced content, which is beneficial for the separation and purification of reaction products.

[0091] In this step, the mixture containing LNFP I can be clarified by conventional methods. Preferably, the mixture containing LNFP I is clarified by centrifugation, flocculation, decantation, and / or filtration.

[0092] Preferably, substantially all proteins, as well as amino acids, RNA, and DNA, are removed from the mixture containing LNFP I (preferably after clarification). In this step, proteins and related impurities can be removed from the mixture containing LNFP I in a conventional manner. Preferably, proteins, salts, byproducts, colors, and other related impurities are removed from the mixture containing LNFP I 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), particularly by chromatography, and more particularly by ion exchange chromatography, hydrophobic interaction chromatography, or ligand exchange chromatography.

[0093] LNFP I was further separated from the reaction mixture and purified using techniques known in the art, such as evaporation, lyophilization, crystallization, precipitation and / or drying, and spray drying.

[0094] In a tenth aspect, fucoidylated oligosaccharides produced according to the methods of the eighth and ninth aspects are provided.

[0095] Preferably, the fucoidylated oligosaccharide includes, but is not limited to, LNFP I.

[0096] Eleventhly, the use of the fucoidylated oligosaccharide obtained according to the ninth aspect in the preparation of nutritional compositions is provided.

[0097] In a twelfth aspect, a nutritional composition is provided, which contains at least one fucoidan-based oligosaccharide as described in the tenth aspect.

[0098] The term "identity" refers to the similarity of amino acid sequences, specifically the percentage of two or more identical sequences or subsequences, or sequences containing the same amino acid residues or nucleotides, when compared and aligned for maximum correspondence. An example of an algorithm suitable for determining percentage sequence identity and sequence similarity is the BLAST algorithm. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information.

[0099] The term "polypeptide" refers to any peptide or protein comprising two or more amino acids linked together by peptide bonds or modified peptide bonds. "Polypeptide" can refer to both short chains (commonly called peptides, oligopeptides, and oligomers) and long chains (commonly called proteins). "Polypeptides" include those modified through natural processes (such as processing and other post-translational modifications) and those modified by chemical modification techniques. These modifications are well documented in basic textbooks and, in more detail, in monographs and a wealth of research literature, and are well known to those skilled in the art.

[0100] The term "host cell" is defined as a cell that has been transformed or transfected by an exogenous polynucleotide sequence or is capable of being transformed or transfected by an exogenous polynucleotide sequence, thereby containing at least one non-naturally occurring sequence in the host cell.

[0101] Beneficial effects:

[0102] This application provides a polypeptide with the amino acid sequence shown in SEQ ID NO:1 and polypeptides M1-M20. The polypeptide shown in SEQ ID NO:1 has the ability to catalyze the synthesis of LNFP I, and polypeptides M1-M20 enhance the ability of the polypeptide shown in SEQ ID NO:1 to catalyze the synthesis of LNFP I; and improve its substrate specificity, wherein the catalytic reaction of polypeptides M1-M20 produces little or no 2'-FL byproduct. Furthermore, this application also provides polynucleotides and related nucleic acid products encoding the polypeptide, transformed host cells, enzymes containing the polypeptide, and methods for catalyzing the synthesis of LNFP I using the polypeptide as a catalyst.

[0103] The technical solution of this application has positive significance for the industrial production of human milk oligosaccharides. This method is green, efficient, and sustainable, and is conducive to large-scale industrial production, thus having important practical value. Attached Figure Description

[0104] Figure 1 HPLC chromatograms of .2'-FL, LNT, LNFP I standards and M18 peptide reaction solution. Detailed Implementation

[0105] The present invention will be further described in detail below through embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Modifications or substitutions to the details and form of the technical solution without departing from the structural concept and scope of use of the present invention shall fall within the protection scope of the present invention.

[0106] It should be noted that the terminology used is for describing specific embodiments only and is not intended to limit the exemplary embodiments of the present invention. Furthermore, 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, and such techniques and conditions are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0107] Unless otherwise specified, the experimental methods described in the following specific embodiments are generally performed according to conventional methods and conditions in molecular biology within the art, which are fully explained in the literature; all materials, reagents, etc., are commercially available unless otherwise specified.

[0108] LNFP I and 2'-FL standards are manufactured by ELICITYL, a French company.

[0109] Example 1. Expression of the polypeptide and polypeptides M1-M20 as shown in SEQ ID NO:1 in Escherichia coli.

[0110] 1. Using the amino acid sequence shown in SEQ ID NO:1 as a template, a polypeptide with the amino acid sequence shown in SEQ ID NO:1 was synthesized and finally constructed into the PET32a vector to obtain the PET32a-wt plasmid.

[0111] 2. By utilizing computer-aided rational design, different mutation sites were obtained. Referring to the methods in *Molecular Cloning: A Laboratory Manual*, and based on the sequence of SEQ ID NO:1 of PET32a-wt, Primer-BLAST was used. Primer designingtool (nih.gov) Mutant primers were designed, and a series of recombinant plasmids (pET32a-M1 to pET32a-M20) were constructed using a point mutation kit.

[0112] 3. Transform the above recombinant plasmids into Escherichia coli BL21(DE3) according to the following steps:

[0113] Prepared Escherichia coli BL21(DE3) competent cells were thawed on ice for 30 min. 100 μL of competent cells were mixed with 10 μL of pET32a-M1 to pET32a-M20 recombinant plasmids (concentration 50 ng / μL) and heat-shocked in a 42℃ water bath for 45 s. Immediately afterward, the mixture was cooled on ice for 2 min. 1 mL of fresh LB medium (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, plate with 1.5% agar powder) was added, and the mixture was incubated at 37℃ and 100 rpm for 1 h. Then, 100 μL of the bacterial solution was spread on LB plates containing ampicillin (100 μg / mL) and incubated at 37℃ for 12 h. Single colonies were picked for colony PCR to screen for positive transformants.

[0114] 4. Cultivate positive transformants, then extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the pET32a-M1 to pET32a-M20 recombinant plasmids were successfully introduced into E. coli.

[0115] 5. Inoculate the correct transformants into LB liquid medium and incubate at 37°C and 200 rpm on a shaker for 12 hours to obtain the seed culture; then inoculate the seed culture into fresh LB medium at a 1% (v / v) inoculation rate and incubate at 37°C with shaking until OD reaches 100%. 600 The concentration was 0.8, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mmol / L. Induction was performed at 16℃ for 12 h at 200 rpm. After induction, the fermentation broth was centrifuged at 5000 r / min for 30 min at 4℃ to collect the cells. The cells were resuspended in 20 mM pH 7.4 PBS buffer and sonicated at a frequency of plus on 5 s / off 5 s for 30 min to lyse the cells. The lysed liquid was centrifuged at 13000×g at 4℃ for 30 min to remove cell debris and the supernatant was collected.

[0116] 6. Soluble peptides were purified using nickel column affinity chromatography as follows: Deionized water was added to the top of the nickel column, and after natural elution, elution was performed with 5 volumes of binding buffer. Crude enzyme solution filtered through a 0.45 μm filter was then loaded onto the column, and the sample was allowed to fully bind to the nickel column at a flow rate of 1.5 mL / min. After the sample dried, impurities were removed by a continuous gradient elution with 5 column volumes of washing buffer. Finally, the target protein was eluted with 5 volumes of elution buffer, and the eluent was collected. The expression of the target protein was then analyzed using SDS-PAGE.

[0117] 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, the polypeptides shown in SEQ ID NO:1-3 and polypeptides M1-M20 were obtained. For details, please refer to Table 1.

[0118] Table 1. Correspondence between α-1,2-fucosyltransferase polypeptides and their amino acid sequence numbers

[0119]

[0120]

[0121] Example 2. Determination of the ability of each polypeptide obtained in Example 1 (polypeptides shown in SEQ ID NO:1-SEQ ID NO:3 and polypeptides M1-M20) to catalyze the synthesis of LNFP I.

[0122] Using one of the peptides or peptides M1-M20 (purified as shown in SEQ ID NO:1-SEQ ID NO:3 obtained in Example 1) as a catalyst, and GDP-fucose and LNT as reaction substrates, LNFP I was synthesized, respectively. The specific procedure is as follows:

[0123] In a system containing 15 mM GDP-fucose, 15 mM LNT, 10 mM MnCl2, and 25 mM Tris-HCl (pH 7.5), 0.2 mg / mL of the purified polypeptides SEQ ID NO:1-SEQ ID NO:3 or polypeptides M1-M20 obtained in Example 1 were added, mixed, and reacted at 37°C for 6 hours. The reaction was then terminated, and the polypeptides were purified by gel column chromatography.

[0124] Identification of catalytically synthesized products:

[0125] HPLC detection method: Detection conditions: Column type: Shodex Asahipak NH2P-50 4E, mobile phase: 65% acetonitrile aqueous solution, flow rate: 0.5 mL / min, column temperature: 35℃, injection volume: 10 μL, evaporative light detector, evaporation temperature: 75℃, nebulization temperature: 45℃.

[0126] The LC-MS analysis conditions are as follows:

[0127] Chromatographic column type: 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℃, mobile phase: acetonitrile:water

[0128] =65:35; ESI-MS mode, molecular weight scan range 100–800.

[0129] The standard and reaction solution were analyzed using the aforementioned analytical method. HPLC analysis showed that the peak elution time (rt) of the LNFP I standard was 21.40 min. The reaction solution of the peptide described in Example 1 showed a strong absorption peak around 21.4 min, consistent with the elution time of the LNFP I standard, indicating that the catalytic reaction of the peptide described in Example 1 generated LNFP I.

[0130] Additionally: the HPLC chromatogram of the 2'-FL standard showed a rt of 17.67 min, and the reaction solution of the peptide shown in SEQ ID NO:1 showed a weak peak at rt of 17.67 min; the reaction solutions of peptides M1-M20 showed no peak or even smaller peaks at this position.

[0131] The LC-MS analysis conditions are shown above, and the analysis results show:

[0132] The product components corresponding to the HPLC peak near rt = 21.40 min were isolated from the reaction solution and analyzed by LC-MS. The results showed that their molecular weight was consistent with that of the LNFP I standard and within the allowable error range of the theoretical molecular weight of LNFP I (853.8). This proves that the peptides shown in SEQ ID NO:1 and peptides M1-M20 successfully synthesized LNFP I. This indicates that the peptides shown in SEQ ID NO:1 and peptides M1-M20 derived from Clostridium sp. can replace the previously reported α-1,2-fucosyltransferases derived from Helicobacter pylori or Francisella sp. FSC1006 for the synthesis of LNFP I.

[0133] The yields of LNFP I and 2'-FL in the fermentation broth were determined using the HPLC analysis method described above, and the results are recorded in Table 2.

[0134] Table 2. Studies on the catalytic synthesis of LNFP I by various peptides

[0135]

[0136]

[0137] Table 2 Data Explanation:

[0138] (1) α-1,2-fucosyltransferase derived from Clostridium sp. C8-1-8

[0139] The ability of (α-1,2-fucosyltransferase) to catalyze the synthesis of LNFP I is lower than that of the α-1,2-fucosyltransferase (α-1,2-fucosyltransferase) derived from Helicobacter pylori and Francisella sp. FSC1006 disclosed in the prior art.

[0140] (2) α-1,2-fucosyltransferase derived from Clostridium sp. C8-1-8

[0141] Both α-1,2-fucosyltransferase, derived from Helicobacter pylori and α-1,2-fucosyltransferase derived from Francisella sp.FSC1006, produce the byproduct 2'-FL during the catalytic synthesis of LNFP I.

[0142] (3) In the reaction solutions of the derived peptides M1-M2, M5, M13, M14 and M18, the byproduct 2'-FL was generated.

[0143] (4) The ability of M1-M20 to catalyze the synthesis of LNFP I is improved compared with the peptide shown in SEQ ID NO:1, especially M6-M20, which has a more than 5-fold increase in the ability to synthesize LNFP I.

[0144] Example 3. Synthesis of LNFP I using Escherichia coli (BL21(DE3)) as a chassis

[0145] References Huang et al. (Huang, D., Yang, K., Liu, J., Xu, Y., Wang, Y., Wang, R., Liu, B., &

[0146] 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.) Strains BL21ΔlacZΔlonΔwcaJ were constructed, and pRSF-lgtA-wbgO was introduced into *E. coli* to construct strain EC. Further, the constructed PET32a-M1 and PET32a-M20 were electroporated into *E. coli* chassis cells EC to construct EC-M1 and EC-M20.

[0147] Methods for culturing Escherichia coli to produce LNFP I:

[0148] Escherichia coli strains EC-M1 and EC-M20 were cultured separately on LB solid medium at 37°C for 10–12 hours. Single colonies were then inoculated into 20 mL of liquid LB medium and cultured in a 250 mL shake flask at 37°C and 220 rpm for 10–12 hours. Each strain was then inoculated into 5 mL of LB medium. When the strains reached the stationary phase, another 1 mL of culture was inoculated into 100 mL of LB medium containing 36 g / L glucose (or glycerol) as a carbon source and grown in a 500 mL shake flask. When OD... 600 When the concentration 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 GDP-fucose was added sequentially to supplement LNFP I production. Simultaneously, ampicillin and kanamycin were added to the culture medium to a final concentration of 100 μg / mL and 25 μg / mL, respectively. After completion, the culture medium was boiled for 10 min, centrifuged, and the supernatant was used to analyze the LNFP I and 2'-FL yields. The results are recorded in Table 3.

[0149] Table 3

[0150]

[0151] Example 4. Synthesis of LNFP I using Bacillus Subtilis BStgtP8 as a chassis.

[0152] Bacillus subtilis chassis cells were constructed according to the method described by 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.). The Bacillus subtilis chassis cells BSP43-manB-P43-manC-P43-gmd-P43-wcaG-P43-lgtA-P43-wbgo were constructed. Simultaneously, P43-M1 and P43-M20 expression cassettes were constructed and transferred into…

[0153] BSP43-manB-P43-manC-P43-gmd-P43-wcaG-P43-lgtA-P43-wbgo was inserted into the manP gene to construct BS-M1 and BS-M20.

[0154] Bacillus subtilis strains BS-M1 and BS-M20 were cultured on LB solid medium at 37°C for 10–12 hours. Single colonies were inoculated into 20 mL of liquid LB medium and cultured in a 250 mL shake flask at 37°C and 220 rpm for 10–12 hours. The seed cultures were further inoculated into 30 mL of fermentation medium at a rate of 10% and cultured in a 250 mL shake flask at 37°C and 220 rpm for 72 hours. The culture medium used in the shake-flask fermentation process had the following composition: 6 g / L trypsin, 12 g / L yeast extract, 12.5 g / L K₂HPO₄·3H₂O, 2.5 g / L KH₂PO₄, and 10 g / L trace metal solution (composition: 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 sterilized shake flasks to final concentrations of 20 g / L and 10 g / L, respectively. After the fermentation reaction was completed, the culture medium was boiled for 10 minutes, and the supernatant was collected by centrifugation to detect the yields of LNFP I and 2'-FL. The results are recorded in Table 4.

[0155] Table 4

[0156]

[0157] The above process was scaled up to a 1L fermenter, with continuous feeding fermentation. Sucrose concentration in the fermentation broth was controlled within the range of 17g / L to 22g / L; lactose concentration within the range of 10g / L to 20g / L. Fermentation was terminated after 30 hours of continuous feeding. After completion, the culture broth was boiled for 10 minutes, centrifuged, and the supernatant was analyzed to determine the LNFP I content: 1.8 mg / mL in BS-M1 fermentation broth; and 7.8 mg / mL in BS-M20 fermentation broth.

[0158] Example 5. Synthesis of LNFP I using Saccharomyces cerevisiae SctgtP8 as a chassis.

[0159] The construction of Saccharomyces cerevisiae chassis cells was referenced in 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.). SC-Δgal80-Pgal1-lac12-Pgal1-gmd-wcaG was constructed, and further construction was performed.

[0160] Δxyl1::Pgal1-lgtA-Pgal1-sumo-wbgo was used to obtain chassis cells SC, and pRS305-Pgal-M1 and pRS305-Pgal-M20 plasmids were constructed and transformed into SC to obtain strains SC-M1 and SC-M20.

[0161] Saccharomyces cerevisiae SC-M1 and SC-M20 were taken and allowed to grow rapidly until they entered the late logarithmic or stationary phase. The strains were streaked onto solid media such as YDP and incubated at 30°C for 2-3 days. Single colonies were then inoculated into 1.5 mL of LYPD liquid medium and incubated overnight at 30°C with shaking at 200 rpm. Subsequently, 2% of each colony was inoculated into 50 mL shake flasks of liquid medium and incubated at 30°C with shaking at 200 rpm until OD (occurrence depth) reached. 600=1, and 2% of each yeast extract was inoculated into 1.5L of YPD medium (10g / L yeast extract, 20g / L peptone, 20g / L glucose) in a 3L fermenter. After culturing for 5-6 hours, sucrose (50% mother liquor concentration) was added at a flow rate of 8mL / h, and lactose (40% mother liquor concentration) was added simultaneously to maintain a final lactose concentration of 15g / L for a total fermentation time of 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and the supernatant and precipitate were collected separately. The precipitate was homogenized using a high-pressure homogenizer, and the protein was removed by boiling and centrifugation. The supernatants were combined to obtain the final yeast fermentation products. The yields of LNFP I and 2'-FL in the supernatant were detected, and the results are recorded in Table 5.

[0162] Table 5

[0163] Gene-modified cells LNFP I production, mg / mL 2'-FL yield, mg / mL SC-M1 2.1 0.41 SC-M20 8.5 Not detected

[0164] Example 6. Synthesis of LNFP I using Kluyveromyces lactis (Kluyveromyces lactis HLLWF3) as a chassis.

[0165] 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.

[0166] The strain was obtained using the construction and transformation method described at https: / / doi.org / 10.1021 / acs.jafc.3c01779.

[0167] KL-ΔLAC4 The expression cassette Δxyl1::lgtA-wbgo was further transformed into Kluyveromyces lactis to obtain the target chassis cell KL. The constructed expression cassettes Δxyl2::Ptef1-M1 and Δxyl2::Ptef1-M20 were then transformed into strain KL to obtain strains KL-M1 and KL-M20, respectively.

[0168] Kluyveromyces kluyveromyces KL-M1 and KL-M20 were taken and allowed to grow rapidly until they entered the late logarithmic or stationary phase. The strains were streaked onto solid media such as YDP and incubated at 30°C for 2-3 days. Single colonies were then inoculated into 1.5 mL of LYPD liquid medium and incubated overnight at 30°C with shaking at 200 rpm. Subsequently, 2% of each colony was inoculated into 50 mL shake flasks of liquid medium and incubated at 30°C with shaking at 200 rpm until OD (occurrence limit) was reached. 600 =1, and 2% of each culture medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) were inoculated into 1 L of YPD medium (3 L fermenter). After 6-7 h of culture, sucrose (50% mother liquor concentration) was added at a flow rate of 8 mL / h, and lactose (40% mother liquor concentration) was added simultaneously 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 homogenized using a high-pressure homogenizer, and the protein was removed by boiling and centrifugation. The supernatants were combined to obtain the final yeast fermentation product. The contents of LNFP I and 2'-FL in the supernatant were detected, and the results are recorded in Table 6.

[0169] Table 6

[0170] Gene-modified cells LNFP I production, mg / mL 2'-FL yield, mg / mL KL-M1 1.8 0.35 KL-M20 14.4 Not detected

[0171] This invention discloses a series of α-1,2-fucosyltransferase polypeptides, their preparation methods and applications, the DNA molecules encoding these polypeptides, vectors, and host cells. Those skilled in the art can refer to the content of this invention and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0172] While the invention has been described in considerable detail by way of illustration and example for purposes of clarity, it will be apparent to those skilled in the art that any equivalent aspects or modifications may be made. Therefore, this specification and the embodiments should not be construed as limiting the scope of the invention.

[0173] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An α-1,2-fucosyltransferase polypeptide, characterized in that, Selected from any of the following mutant peptides, 1) In the amino acid sequence shown in SEQ ID NO:1, the amino acid residue at position 37 is replaced by F with Y, thereby obtaining polypeptide M1; 2) In the amino acid sequence of polypeptide M1, amino acid residues at positions 40-42 are replaced by YDL with VSD, thereby obtaining polypeptide M2; 3) In the amino acid sequence of polypeptide M2, the amino acid residue at position 46 is replaced by F instead of Y, thereby obtaining polypeptide M3; 4) In the amino acid sequence of polypeptide M3, the amino acid residue at position 49 is replaced by D instead of N, thus obtaining polypeptide M4; 5) In the amino acid sequence of polypeptide M4, the amino acid residue at position 58 is replaced by I instead of F, thereby obtaining polypeptide M5; 6) In the amino acid sequence of polypeptide M5, the amino acid residue at position 77 is replaced by V instead of K, thus obtaining polypeptide M6; 7) In the amino acid sequence of polypeptide M6, the amino acid residue at position 84 is replaced by Y instead of G, thus obtaining polypeptide M7; 8) In the amino acid sequence of polypeptide M7, the amino acid residue at position 91 is replaced by S instead of L, thus obtaining polypeptide M8; 9) In the amino acid sequence of polypeptide M8, the amino acid residue at position 108 is replaced by Y, thereby obtaining polypeptide M9; 10) In the amino acid sequence of polypeptide M9, the amino acid residue at position 118 is replaced by Y with F, thereby obtaining polypeptide M10; 11) In the amino acid sequence of polypeptide M10, the amino acid residue at position 128 is replaced by E instead of Q, thus obtaining polypeptide M11; 12) In the amino acid sequence of polypeptide M11, the amino acid residue at position 154 is replaced by S instead of A, thereby obtaining polypeptide M12. 13) In the amino acid sequence of polypeptide M12, the amino acid residues at positions 165-174 are replaced by PRYYKTFGSV with DLSANNIHGI, thereby obtaining polypeptide M13. 14) In the amino acid sequence of polypeptide M13, the amino acid residues at positions 177-178 are replaced by LK with EE, thereby obtaining polypeptide M14; 15) In the amino acid sequence of polypeptide M14, the amino acid residue at position 184 is replaced by I, thereby obtaining polypeptide M15; 16) In the amino acid sequence of polypeptide M15, amino acid residues at positions 197-198 are replaced with FV by replacing YI, thereby obtaining polypeptide M16; 17) In the amino acid sequence of polypeptide M16, the amino acid residue at position 200 is replaced by S instead of T, thereby obtaining polypeptide M17. 18) In the amino acid sequence of polypeptide M17, the amino acid residue at position 203 is replaced by I instead of P, thereby obtaining polypeptide M18. 19) In the amino acid sequence of polypeptide M18, the amino acid residue at position 217 is replaced by Y, thereby obtaining polypeptide M19; 20) In the amino acid sequence of polypeptide M19, the amino acid residue at position 288 is replaced by K with V, thereby obtaining polypeptide M20.

2. A polynucleotide encoding the α-1,2-fucosyltransferase polypeptide of claim 1.

3. A nucleic acid construct comprising the polynucleotide as described in claim 2.

4. An expression vector comprising the polynucleotide of claim 2, or comprising the nucleic acid construct of claim 3.

5. A transformed host cell, characterized in that, It is transformed into the polynucleotide as described in claim 2, or the nucleic acid construct as described in claim 3, or the expression vector as described in claim 4.

6. The host cell as described in claim 5, characterized in that, The host cell is selected from natural or genetically modified prokaryotic and eukaryotic cells.

7. The host cell as described in claim 6, characterized in that, The prokaryotic cells were selected from Citrobacter freundii (Citrobacter freundii). Citrobacter freundii ), Lemon Pantothecin ( Pantoea citrea ) or Xanthomonas oryzae ( Xanthomonas campestris ).

8. The host cell as described in claim 6, characterized in that, The prokaryotic cells are selected from natural cells of bacteria or archaea, or from genetically modified cells.

9. The host cell as described in claim 8, characterized in that, The bacterial cells are selected from Enterococcus genus ( Enterococcus sp.), Bifidobacterium spp. Bifidobacterium sp.), Lactobacillus spp. Sporolactobacillus sp.), Micromonospora ( Micromomospora sp.), Micrococcus ( Micrococcus sp.), Rhodococcus genus ( Rhodococcus Natural or genetically modified cells (sp.).

10. The host cell as described in claim 9, characterized in that, The Enterococcus spp. ( Enterococcus sp.) was selected from Enterococcus faecalis ( Enterococcus faecium ) and thermophilic enterococci ( Enterococcus thermophilus ( ) natural cells or genetically modified cells.

11. The host cell as described in claim 8, characterized in that, The bacterial cells were selected from Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus coagulans ( Bacillus coagulans thermophilic Bacillus (Bacillus thermophilus ), Bacillus laterosporus ( Bacillus laterosporus ), Bacillus megaterium ( Bacillus megaterium ), Bacillus mycosisviridae ( Bacillus mycoides) Bacillus pumilus ( Bacillus pumilus ), Bacillus tarda ( Bacillus lentus ), Bacillus cereus ( Bacillus cereus) and Bacillus circularis ( Bacillus circulans ( ) natural cells or genetically modified cells.

12. The host cell as described in claim 9, characterized in that, The Bifidobacterium genus ( Bifidobacterium sp.) was selected from Bifidobacterium longum (sp.) Bifidobacterium longum ), Bifidobacterium infantis ( Bifidobacterium infantis ) and Bifidobacterium ( Bifidobacterium bifidum ( ) natural cells or genetically modified cells.

13. The host cell as described in claim 6, characterized in that, The eukaryotic cells are selected from natural yeast cells or genetically modified cells.

14. The host cell as described in claim 13, characterized in that, The yeast cells were selected from *Favora kojiro* yeast. Komagataella phaffii Kluyveromycin (lactic acid yeast) Kluyveromyces lactis ), Max Kluyveromycin ( Kluyveromyces marxianus ), Yarrowia lipolytica ( Yarrowia lipolytica Pichia pastoris () Pichia pastoris ) and brewer's yeast ( Saccharomyces cerevisiae ( ) natural cells or genetically modified cells.

15. The host cell as described in claim 6, characterized in that, The eukaryotic cells were selected from filamentous fungi.

16. The host cell as described in claim 15, characterized in that, The filamentous fungus is selected from the genus Aspergillus ( Aspargillus sp.), Fusarium genus ( Fusarium sp.) or Trichoderma ( Trichoderma sp.).

17. The host cell as described in claim 15, characterized in that, The filamentous fungus is selected from Aspergillus niger ( Aspergillus niger Aspergillus nidus ( ) Aspergillus nidulans Aspergillus oryzae ( Aspergillus oryzae Fusarium solani (Fusarium solani ( ) natural cells or genetically modified cells.

18. An enzyme preparation comprising one or more of the α-1,2-fucosyltransferase polypeptides of claim 1.

19. The method for producing the α-1,2-fucosyltransferase polypeptide according to claim 1, characterized in that, Includes the following steps: (1) Transformed host cells were cultured under conditions suitable for expression of the α-1,2-fucosyltransferase polypeptide; and (2) The polypeptide is recovered.

20. The method for producing the α-1,2-fucosyltransferase polypeptide as described in claim 19, characterized in that, Step (1) includes: firstly, introducing a host cell containing the nucleic acid construct or recombinant expression vector encoding the claimed 3 into the host cell to construct an engineered host cell expressing the polypeptide; then, culturing the engineered host cell and inducing it to express the polypeptide.

21. The use of the α-1,2-fucosyltransferase polypeptide of claim 1, or the host cell transformed according to any one of claims 5-17, or the enzyme of claim 18 in the synthesis of fucoidan.

22. The application as described in claim 21, characterized in that, The fucosyl lactose is selected from lactose-N-fucopentose I.

23. The application as described in claim 21, characterized in that, Using the α-1,2-fucosyltransferase polypeptide of claim 1 or the enzyme of claim 18 as a catalyst, and GDP-L-fucose and LNT as substrates, lactose-N-fucopentose I is synthesized.

24. The application as described in claim 21, characterized in that, Lactose-N-fucopentose I is synthesized by fermentation of host cells transformed according to any one of claims 5-17 using lactose as a substrate.

25. A method for preparing lactose-N-fucopentose I, characterized in that, Includes the following steps: a) Using the α-1,2-fucosyltransferase polypeptide of claim 1 or the enzyme of claim 18 as a catalyst, catalyze the synthesis of lactose-N-fucopentose I from the substrate GDP-L-fucose and LNT, or Lactose-N-fucopentose I is synthesized by fermentation of host cells transformed according to claims 5-17 using lactose as a substrate.

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