Gdp-fucose synthetase polypeptides and uses thereof

Abstract

The present application relates to a series of GDP-fucose synthetase polypeptides, which can be applied to the synthesis of human milk oligosaccharides (HMOs) precursor GDP-fucose, belonging to the field of enzyme engineering technology. In certain embodiments, the polypeptide having catalytic GDP-4-keto-6-deoxymannose converted into GDP-fucose comprises an amino acid sequence which is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical to SEQ ID NO: 1 and / or to the polypeptide M1-14. The present application provides a series of polypeptides for synthesizing GDP-fucose.

Description

Technical Field

[0001] This invention relates to a series of GDP-fucose synthase polypeptides, which can be applied to the synthesis of GDP-fucose, a precursor of human milk oligosaccharides (HMOs), and belongs to the field of enzyme engineering technology. Background Technology

[0002] Human milk oligosaccharides (HMOs) are a unique and diverse mixture of oligosaccharides found in human milk, and are the third largest solid component of breast milk after fat and lactose. Among them, 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) are important components of human milk oligosaccharides, and have been proven to play an important role in the brain, intestines, and growth and development of infants and young children.

[0003] 2'-FL and 3-FL have been approved by the US FDA, the EU and other countries and regions as raw materials for infant formula and can be added to infant formula, general food, dietary supplements and / or medical food.

[0004] The synthesis of fucoidan is primarily achieved through chemical, enzymatic, or microbial cell factory methods. While chemical synthesis has made significant progress, the process involves repeated and complex protection and deprotection of the fucoidan molecule, resulting in low yields and high production costs. Achieving large-scale synthesis of 2'-FL and 3-FL using chemical methods remains extremely challenging. Microbial synthesis of fucoidan is currently a feasible method for large-scale production. Furthermore, microbially synthesized fucoidan is functionally identical to naturally occurring fucoidan and has not caused adverse reactions in clinical trials. Compared to chemical synthesis, microbial synthesis is safer and faster, avoiding the introduction of large amounts of toxic reagents. It also allows for the direct production of 2'-FL and 3-FL using food-grade microorganisms, significantly increasing product safety.

[0005] The biosynthesis of 2'-FL and 3-FL was initially achieved through enzyme catalysis. In 2000, Albermann et al. expressed and purified GDP-mannose-4,6-dehydrase and GDP-fucose synthase from Escherichia coli K12, and converted GDP-mannose into GDP-fucose in vitro. GDP-fucose is the most critical precursor for the synthesis of 2'-FL and 3-FL.

[0006] GDP-fucose can be produced in vitro via a biocatalytic (enzymatic) process (APMIS 2006, 114, 539–548). For example, Zhao et al. (Nature Protocols. 2010, 5(4): 636–646) reported the enzymatic synthesis of GDP-fucose from L-fucose and guanosine triphosphate using the bifunctional enzyme L-fucose pyrophosphorylase (FKP). Wittmann et al. (The Journal of Organic Chemistry. 1997, 62, 2144–2147) described the synthesis of GDP-fucose from fucose-1-phosphate and guanosine 5'-monophosphate-morpholinate using phosphoramide chemistry. Tonetti et al. (Journal of Biological Chemistry)

[0007] In 1996, 271(44), 27274–27279, the homodimeric NADP(H)-binding protein FX was reported, which significantly catalyzed the combined epimerase and NADPH-dependent reductase activities to convert GDP-4-keto-6-D-deoxymannose to GDP-L-fucose. Sullivan et al. (Journal of Biological Chemistry 1998, 273(14), 8193–8202) described the in vitro preparation of GDP-fucose from GDP-mannose using recombinant human GDP-mannose 4,6-dehydratase and FX protein. Lau et al. reported the biosynthesis of GDP-L-fucose from GDP-D-mannose and investigated the enzyme involved in the synthesis, GDP-fucose synthase. This enzyme converts GDP-4-keto-6-deoxy-D-mannose to GDP-L-fucose (Journal of the American Chemical Society. 2008, 130, 17593-17602). Rexer et al. reported a one-pot synthesis of GDP-mannose from mannose via a multi-enzyme cascade reaction of glucokinase (Glk), phosphomannose mutase (ManB), mannose-1-phosphoguanylate transferase (ManC), inorganic pyrophosphatase (PmPpA), and 1-domain polyphosphatase 2 (1D-Ppk2) expressed in Escherichia coli (Biotechnology and Bioengineering. 2018, 115, 192-205).

[0008] In summary, it is necessary to study GDP-fucose synthase to improve the synthesis efficiency of 2'FL and 3-FL. Modification of key enzymes is crucial for improving their catalytic efficiency. Summary of the Invention

[0009] Objective: To provide a GDP-fucose synthase polypeptide (GDP-L-fucose synthase, WcaG) and its application in the synthesis of fucoidan-lactose, thereby improving the production efficiency of fucoidan-lactose. The GDP-fucose synthase polypeptide, also known as GDP-mannose-4,6-dehydratase or GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, possesses catalytic activity in the production of GDP-fucose from GDP-4-keto-6-deoxy-mannose and belongs to the oxidoreductase family. As a key enzyme in the synthesis of GDP-fucose, which is a precursor to fucoidan-lactose.

[0010] The GDP-fucose synthase mentioned herein may be derived from sources including, but not limited to, Bacillus paralicheniformis, Escherichia coli, Plasmodium knowlesi, Halyomorpha halys, Zootermopsis nevadensis, Lingula anatina, and Caenorhabditis elegans.

[0011] Technical solution:

[0012] The applicant has discovered a GDP-fucose synthase from Bacillus subtilis in nature, named BsWcaG, with its amino acid sequence as shown in SEQ ID NO:1 and its nucleotide sequence as shown in SEQ ID NO:6. It has the activity of catalyzing the production of GDP-fucose from GDP-4-keto-6-deoxymannose and can be applied to the synthesis of GDP-fucose.

[0013] A comparison with the NCBI database revealed that the GDP-fucose synthase polypeptide with the amino acid sequence shown in SEQ ID NO:1 has the highest similarity to that of GDP-fucose synthase from *Escherichia coli* (as of November 15, 2023). The two enzymes share only 64.06% amino acid sequence similarity, indicating that SEQ ID NO:1 does not have a high degree of similarity to known enzymes. The amino acid sequence of the GDP-fucose synthase from *Escherichia coli* is shown in SEQ ID NO:2.

[0014] The inventors of this application have carried out a series of modifications to the GDP-fucose synthase polypeptide shown in SEQ ID NO:1, and screened and obtained a series of derivative peptides with enhanced catalytic activity in converting GDP-4-keto-6-deoxymannose to GDP-fucose.

[0015] M1-M14. The derived peptides M1-M14 are GDP-fucose synthase peptides formed by replacing one or more amino acid residues and / or fragments in the polypeptide with the amino acid sequence shown in SEQ ID NO:1.

[0016] This application describes exemplary substitutions or combinations of substitutions for polypeptides with amino acid sequences as shown in SEQ ID NO:1.

[0017] Specifically, in a first aspect, this application provides a series of GDP-fucose synthase polypeptides, wherein the GDP-fucose synthase polypeptides are selected from the polypeptide with the amino acid sequence shown in SEQ ID NO:1 and its derived peptides M1-14, as well as the polypeptide shown in SEQ ID NO:2. The derived peptide M1-14 is obtained by modifying at least one site from the following positions of the amino acid sequence shown in SEQ ID NO:1: positions 20-29, 40-53, 75-81, 95, 148-158, 193, 215, 219-221, 227-232, 239-241, 259-264, 301-302, 306, and 310-316.

[0018] Preferably, the modification is a mutation, and the mutation is selected from any of the following mutations:

[0019] 1) In the amino acid sequence shown in SEQ ID NO:1, the amino acid fragment at positions 20-29 is composed of...

[0020] YEQLKQRGDIEIVA was replaced with KAQLEQDGNVELV, thereby obtaining polypeptide M1;

[0021] 2) In the amino acid sequence of polypeptide M1, the amino acid fragment at positions 40-53 is replaced by NLLDADAVRAFFAA with DLLDEKKVKDFFAK, thereby obtaining polypeptide M2;

[0022] 3) In the amino acid sequence of polypeptide M2, the amino acid fragment at positions 75-81 is replaced by NPEGFYT with KPKEFYE, thereby obtaining polypeptide M3;

[0023] 4) In the amino acid sequence of polypeptide M3, the amino acid fragment at position 95 is replaced by A instead of L, thus obtaining polypeptide M4;

[0024] 5) In the amino acid sequence of polypeptide M4, the amino acid fragment at positions 148-158 is replaced by ADYNNKYGADY with EKYNNEKGLDA, thereby obtaining polypeptide M5;

[0025] 6) In the amino acid sequence of polypeptide M5, the amino acid residue at position 193 is replaced by A instead of K, thus obtaining polypeptide M6;

[0026] 7) In the amino acid sequence of polypeptide M6, the amino acid fragment at position 215 is replaced by N instead of Y, thereby obtaining polypeptide M7;

[0027] 8) In the amino acid sequence of polypeptide M7, the amino acid fragment at positions 219-221 is replaced by DAA with EAS, thereby obtaining polypeptide M8;

[0028] 9) In the amino acid sequence of polypeptide M8, the amino acid fragment at positions 227-232 is replaced by LDEAAR with QDQAAV, thereby obtaining polypeptide M9;

[0029] 10) In the amino acid sequence of polypeptide M9, amino acid residues at positions 239-241 are replaced with TNS by replacing DNH, thereby obtaining polypeptide M10.

[0030] 11) In the amino acid sequence of polypeptide M10, the amino acid residues at positions 259-264 are replaced by KIAEVV with TIAEVT, thereby obtaining polypeptide M11;

[0031] 12) In the amino acid sequence of polypeptide M11, the amino acid residues at positions 301-302 are replaced by IV with LR, thereby obtaining polypeptide M12.

[0032] 13) In the amino acid sequence of polypeptide M12, the amino acid residue at position 306 is replaced by A instead of K, thus obtaining polypeptide M13;

[0033] 14) In the amino acid sequence of polypeptide M13, the amino acid residues at positions 310-316 are replaced by EVMEKNK with AVMQANL, thereby obtaining polypeptide M14.

[0034] In some embodiments, the polypeptide having enhanced catalytic conversion of GDP-4-keto-6-deoxymannose to GDP-fucose comprises a polypeptide having an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to that of polypeptide M1-14, and comprises an amino acid substitution mutation that is functionally equivalent to the schemes described in 1)-14) above. In some embodiments, the substitution mutation comprises a mutation to a charged residue; in some embodiments, the substitution mutation comprises a mutation to a basic residue. In some embodiments, the substitution mutation comprises a mutation homologous to the amino acid sequence described in 1)-14) above.

[0035] In some embodiments, the polypeptide having the catalytic conversion of GDP-4-keto-6-deoxymannose to GDP-fucose comprises a polypeptide having an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to that of polypeptide M1-14, and comprises an amino acid substitution mutation that is functionally equivalent to the schemes described in 1)-14) above. In some embodiments, the substitution mutation comprises a mutation to a charged residue; in some embodiments, the substitution mutation comprises a mutation to a basic residue. In some embodiments, the substitution mutation comprises a mutation homologous to the amino acid sequence described in 1)-14) above.

[0036] In a second aspect, this application provides a polynucleotide that encodes the GDP-fucose synthase polypeptide as described in the first aspect above.

[0037] Polynucleotides encoding GDP-fucose synthase 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.

[0038] Thirdly, this application provides a nucleic acid construct comprising the polynucleotides described in the second aspect above.

[0039] 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.

[0040] 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.

[0041] The expression 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.

[0042] 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.

[0043] In one specific embodiment, the expression cassette contains all the elements necessary for expressing the GDP-fucose synthase polypeptide, including elements required 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.

[0044] 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.

[0045] 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.

[0046] In some embodiments, the host cell includes, but is not limited to, natural strains or genetically modified strains such as bacteria, yeast, and mold;

[0047] More preferably, the host cell includes, but is not limited to, natural strains or genetically modified strains such as Escherichia sp., Bacillus sp., and Kluyveromyces sp.;

[0048] More preferably, the host cell includes, but is not limited to, natural or genetically modified strains such as *Saccharomyces cerevisiae*, *Kluyveromyces marxianus*, *Kluyveromyces lactis*, and *Yarrowia lipolytica*.

[0049] More preferably, the host cell is the genetically engineered Escherichia coli E. coli BL21(DE3); or

[0050] Other host cells capable of converting the polynucleotides described in the second aspect above, or the nucleic acid constructs described in the third aspect above, or the expression vectors described in the fourth aspect above.

[0051] In a sixth aspect, this application provides an enzyme agent or enzyme composition comprising the polypeptide as described in the first aspect above.

[0052] The enzyme or enzyme composition preferably contains one or more of the polypeptides and polypeptides M1-14 with the amino acid sequences described in this invention, such as those shown in SEQ ID NO:1 and SEQ ID NO:2.

[0053] Depending on the reaction substrate and product, the enzyme or composition may also contain bifunctional enzymes beneficial to the synthesis of fucoidan, such as L-fucokinase / GDP-L-fucosepyrophosphorylase (FKP), glucokinase (Glk), mannose-phosphate mutase (ManB), mannose-1-phosphoguanyltransferase (ManC), GDP-mannose-4,6-dehydratase (GMD), etc.

[0054] In a seventh aspect, this application provides a method for producing the polypeptide as described in the first aspect above, comprising:

[0055] (1) Transformed host cells are cultured under conditions suitable for expressing the GDP-fucose synthase polypeptide; the transformed host cells are as described in aspect five above; and

[0056] (2) The GDP-fucose synthase polypeptide was recovered.

[0057] In a specific implementation, step (1) includes: firstly, introducing a nucleic acid construct or recombinant expression vector containing a protein encoding the GDP-fucose synthase 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 GDP-fucose synthase polypeptide.

[0058] In a specific implementation, step (2) includes the steps of separating and purifying the GDP-fucose synthase polypeptide from the culture.

[0059] 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.

[0060] The GDP-fucose synthase 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.

[0061] In addition, GDP-fucose synthase peptides can be purified by a variety of procedures known in the art to obtain substantially pure peptides, 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 carried out by Ni column affinity chromatography.

[0062] Eighthly, this application provides the use of the GDP-fucose synthase 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 GDP-fucose and / or fucoidan;

[0063] Preferably, the fucoidyl lactose includes 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL).

[0064] 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 GDP-fucose synthase peptide described in this application to exert its enzymatic activity.

[0065] In one embodiment, the method includes: adding the GDP-fucose synthase polypeptide described in this application to a reaction system, which reacts with the substrate GDP-4-keto-6-deoxymannose in the reaction system to generate GDP-fucose.

[0066] Preferably, the application involves preparing the GDP-fucose synthase 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, *E. coli* or *Saccharomyces cerevisiae*), after which the GDP-fucose synthase polypeptide listed above can be isolated, and GDP-fucose or fucoidosyllactose can be synthesized in a suitable system, optionally with further purification. Specifically, this includes:

[0067] Using the polypeptides described in the first aspect above or the enzymes or enzyme compositions described in the sixth aspect above, the substrate GDP-4-keto-6-deoxymannose is catalyzed to synthesize GDP-fucose;

[0068] Optionally, the GDP-4-keto-6-deoxymannose can be synthesized using GDP-mannose as a substrate and GDP-mannose dehydrating enzyme as a catalyst.

[0069] Optionally, the process also includes steps of purifying and / or recovering GDP-fucose.

[0070] The transformed host cells described in the fifth aspect can also synthesize 2'-fucosylated lactose or 3-fucosylated lactose de novo.

[0071] The sources of the GDP-mannose dehydrating enzyme include, but are not limited to, *Escherichia coli*, *Caenorhabditis elegans*, *Homo sapien*, *Arabidopsis thaliana*, *Dictyostelium discoideum*, *Mus musculus*, *Drosophila melanogaster*, *Sinorhizobium fredii*, *Pandoraea vervacti*, *Caenorhabditis briggsae*, *Candidatus Curtissbacteria*, *Pseudomonas sp.*, *Clostridium sp.*, *Cricetulus griseus*, *Arthrobacter siccitolerans*, and *Paraburkholderia piptadeniae*.

[0072] Optionally, GDP-fucose can be reacted with lactose to synthesize 2'-fucosyllactose or 3-fucosyllactose under the catalysis of α-1,2-fucotransferase or α-1,3-fucotransferase.

[0073] The sources of the α-1,2-fucotransferase or α-1,3-fucotransferase include, but are not limited to: Helicobacter pylori, Synechococcus thermophilus, Escherichia coli, in mammals, Caenorhabditis elegans, Schistosoma mansoni, Bacillus cereus, Pseudopedobatersaltans, Helicobacter ferruginosa, Bacillus fragilis, Bacteroides spp., Bacteroides fragilis, or Bacillus smithii, etc.

[0074] In one embodiment, at least one of the GDP-fucose synthase peptides described in this application is mixed with GDP-4-keto-6-deoxymannose, NADPH1, and a 50 mM Tris-HCl buffer solution, 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 which GDP-4-keto-6-deoxymannose is catalyzed by the GDP-fucose synthase to convert to GDP-fucose; GDP-fucose can be separated and purified by methods known in the art. At the end of the reaction or after separation and / or purification, the concentration of GDP-fucose in the solution is determined by HPLC.

[0075] Alternatively, GDP-mannose dehydratase polypeptide is added to a reaction system containing GDP-mannose, which contacts the substrate GDP-mannose in the reaction system to synthesize GDP-4-keto-6-deoxymannose; GDP-4-keto-6-deoxymannose, under the action of the GDP-fucose synthase described in this application, synthesizes the desired GDP-fucose; GDP-fucose, under the catalysis of α-1,2-fucotransferase or α-1,3-fucotransferase, reacts with lactose in the reaction system to synthesize and obtain the desired fucosylated lactose 2'-FL or 3-FL.

[0076] The application of the GDP-fucose synthase polypeptide involves first using GDP-4-keto-6-deoxymannose as a substrate, and then adding GMD enzyme and NADP. + The enzyme reacts at room temperature; then, GDP-fucose is synthesized by the catalytic reaction of the GDP-fucose synthase.

[0077] In one embodiment, the method can synthesize 2'-FL or 3-FL using GDP-mannose as a substrate. Specifically, it includes:

[0078] (1) GDP-4-keto-6-deoxymannose was synthesized from the substrate GDP-mannose by GDP-mannose dehydratase catalysis;

[0079] (2) Using the polypeptide described in the first aspect above or the enzyme or enzyme composition described in the sixth aspect above, the substrate GDP-4-keto-6-deoxymannose is catalyzed to be converted into GDP-fucose.

[0080] Further preferred options include:

[0081] c) GDP-fucose reacts with lactose to synthesize fucosyllactose under the catalysis of α-1,2-fucotransferase or α-1,3-fucotransferase.

[0082] In the above steps, the reaction product is a solution containing GDP-fucose or fucoidan, which can be clarified using conventional methods. Preferably, the solution containing GDP-fucose or fucoidan can be clarified by centrifugation, flocculation, decantation, and / or filtration.

[0083] Preferably, substantially all proteins, as well as amino acids, RNA, and DNA, are removed from the solution containing GDP-fucose or fucosyl lactose (preferably after clarification). In this step, proteins and related impurities can be removed from the solution containing GDP-fucose or fucosyl lactose in a conventional manner. Preferably, proteins, salts, byproducts, colors, and other related impurities are removed from the mixture containing 2'-FL by ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated carbon or carbon treatment, chromatography, ion exchange chromatography (such as, but not limited to, cation exchange, anion exchange, mixed-bed ion exchange), hydrophobic interaction chromatography, and / or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, and more particularly by ion exchange chromatography, hydrophobic interaction chromatography, or ligand exchange chromatography.

[0084] GDP-fucose or fucose-based lactose is further separated from the reaction solution using purification steps known in the art, such as evaporation, lyophilization, crystallization, precipitation and / or drying, spray drying.

[0085] 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.

[0086] Proteins and / or protein sequences are “homologous” when they are naturally or artificially derived from a common ancestral protein or protein sequence. Similarly, nucleic acids and / or nucleic acid sequences are homologous when they are naturally or artificially derived from a common ancestral nucleic acid or nucleic acid sequence.

[0087] 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 through chemical modification techniques. These modification methods are documented in basic textbooks and, more detailed in monographs, as well as in a large body of research literature, and are well-known to those skilled in the art.

[0088] The term "fucosyl lactose" includes 2'-fucosyl lactose (2'-FL) or 3-fucosyl lactose (3-FL).

[0089] Beneficial effects:

[0090] This application provides a series of GDP-fucose synthase polypeptides, including polypeptides with amino acid sequences as shown in SEQ ID NO:1 and polypeptide M1-14. These GDP-fucose synthases are important enzymes in the biosynthesis of GDP-fucose. This invention provides important biological resources for the biosynthesis of GDP-fucose and fucosyllactose. The GDP-fucose synthases described in this invention generally exhibit high catalytic activity, and their specific activity is significantly higher than that of existing technologies. This invention provides excellent enzyme resources for the biosynthesis of GDP-fucose or fucosyllactose.

[0091] 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

[0092] Figure 1 HPLC chromatogram of GDP-fucose standard.

[0093] Figure 2 HPLC chromatogram of the polypeptide M1 catalytic reaction solution in Example 2.

[0094] Figure 3 HPLC chromatogram of the polypeptide M10 catalytic reaction solution in Example 2.

[0095] Figure 4 HPLC chromatogram of .2'-FL standard.

[0096] Figure 5HPLC chromatogram of .3-FL standard.

[0097] Figure 6 HPLC analysis chromatogram of the fermentation broth for the continuous fed synthesis of 2'-fucosylated lactose using BS-M14 in Example 5.

[0098] Figure 7 HPLC analysis chromatogram of the fermentation broth for the continuous fed synthesis of 3-fucosylated lactose using BS-M14-1 in Example 6. Detailed Implementation

[0099] The present invention will be further described in detail below through embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. It should be noted that those skilled in the art can make several modifications or substitutions to the details and form of the technical solutions of the present invention without departing from the principles of the present invention, and these modifications or substitutions also fall within the protection scope of the present invention.

[0100] 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.

[0101] 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.

[0102] The 2'-FL and 3-FL standards are manufactured by ELICITYL, a French company.

[0103] GDP-fucose standard was purchased from Aladdin.

[0104] The enzymes involved in the following examples are as follows:

[0105] GDP-mannose dehydratase (GMD), microbial source: Escherichia coli (E. col), amino acid sequence: SEQ ID NO:3.

[0106] α-1,2-fucosyltransferase (FutC), derived from Helicobacter pylori.

[0107] (Helicobacter pylori), amino acid sequence as shown in SEQ ID NO:4. α-1,3-fucosyltransferase (futA), microbial source Helicobacter pylori, amino acid sequence as shown in SEQ ID NO:5.

[0108] GDP Fucose HPLC Detection Method:

[0109] C18μBondapack column (Waters, Milford, MA; 3.9*300mm, 10-mm particle size), mobile phase: 0.5M KH₂PO₄ aqueous solution, flow rate: 1ml / min, column temperature: 30℃, injection volume: 10μL, UV detector.

[0110] HPLC method for determining fucoidan-lactose content:

[0111] 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℃.

[0112] Under the above conditions, the rt for GDP-fucose standard was 13.3 min, the rt for 2'-FL standard was 13.43 min, and the rt for 3-FL standard was 14.16 min.

[0113] Example 1. Expression of GDP-fucose synthase polypeptide and polypeptide M1-14 as shown in SEQ ID NO:1 and SEQ ID NO:2 in Escherichia coli.

[0114] 1. Using the polypeptide with the amino acid sequence as shown in SEQ ID NO:1 (nucleotide sequence as shown in SEQ ID NO:6) or the polypeptide with the amino acid sequence as shown in SEQ ID NO:2 as a template, synthesize the polypeptides shown in SEQ ID NO:1 and SEQ ID NO:2, and finally construct them into the PET32a vector to obtain the plasmids PET32a-BsWcaG-wt and PET32a-EcWcaG-wt.

[0115] 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:6 of PET32a-wt, Primer-BLAST was used. Primer designing tool(nih.gov) Mutant primers were designed, and a series of recombinant plasmids (pET32a-M1-M14) were constructed using a point mutation kit.

[0116] 3. Transform the above recombinant plasmids into Escherichia coli BL21(DE3) according to the following steps:

[0117] 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-M14 recombinant plasmid (concentration 50 ng / μL) and heat-shocked in a 42℃ water bath for 45 s. Immediately afterward, the cells were 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 cells were incubated at 37℃ and 100 rpm for 1 h. Then, 100 μL of the bacterial culture 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.

[0118] 4. Cultivate positive transformants, then extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the pET32a-M1-M14 recombinant plasmid was successfully introduced into E. coli.

[0119] 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%. 600The 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.

[0120] 6. Soluble peptide sequences 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 membrane 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.

[0121] 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 and SEQ ID NO:2, as well as polypeptide M1-14, were obtained. For details, please refer to Table 1.

[0122] Table 1. Correspondence between GDP-fucose synthase polypeptides and their amino acid sequence numbers

[0123] Recombinant plasmid GDP-fucose synthase amino acid sequence or polypeptide nomenclature PET32a-EcWcaG-WT SEQ ID NO:2 PET32a-BsWcaG-WT SEQ ID NO:1 PET32a-M1 M1 PET32a-M2 M2 PET32a-M3 M3 PET32a-M4 M4 PET32a-M5 M5 PET32a-M6 M6 PET32a-M7 M7 PET32a-M8 M8 PET32a-M9 M9 PET32a-M10 M10 PET32a-M11 M11 PET32a-M12 M12 PET32a-M13 M13 PET32a-M14 M14

[0124] Example 2. Determination of the ability of each polypeptide obtained in Example 1 (GDP-fucose synthase polypeptide and polypeptide M1-14 shown in SEQ ID NO:1 and SEQ ID NO:2) to catalyze the synthesis of GDP-fucose.

[0125] GDP-mannose was synthesized using GDP-mannose as a substrate, one of the polypeptides or polypeptides M1-14 shown in SEQ ID NO:1 or SEQ ID NO:2 obtained in Example 1 (purified), and GDP-mannose dehydratase as an enzyme catalyst. The specific procedure is as follows:

[0126] To prepare the substrate GDP-4-keto-6-deoxymannose required for the above peptide reaction, firstly, 5 mM GDP-mannose and 0.2 mg / ml of the GMD enzyme obtained in Example 1, and 5 mM NADP were respectively added. + Mix the enzyme thoroughly and react at 25°C for 8 hours. Measure the concentration of the substrate obtained from the reaction.

[0127] Then, add 100 μM GDP-4-keto-6-deoxymannose, 50 mM Tris-HCl (pH 7.0), and 1 mM NADPH enzyme to 0.2 mg / mL of the purified polypeptides shown in SEQ ID NO:1 and SEQ ID NO:2 obtained in Example 1 or one of polypeptides M1-14, mix well, react at 30 °C for 6 hours, terminate the reaction, and purify it by gel column method.

[0128] The catalytic reaction solution was analyzed using the aforementioned analytical method. HPLC analysis showed that the reaction solution of the peptide described in Example 1 exhibited a strong absorption peak around 13.3 min, consistent with the elution time of the GDP-fucose standard, indicating that the catalytic reaction of the peptide described in Example 1 produced GDP-fucose.

[0129] The concentration of GDP-fucose in the reaction solution was detected by the HPLC method described above, and the results are recorded in Table 2.

[0130] Table 2. Studies on the catalytic synthesis of GDP-fucose by various peptides

[0131] amino acid sequence number of the polypeptide GDP-Fucose production, μM SEQ ID NO:1 53 M1 62 M2 63 M3 65 M4 69 M5 71 M6 72 M7 70 M8 73 M9 75 M10 80 M11 81 M12 84 M13 90 M14 95 SEQ ID NO:2 40

[0132] Table 2 Data Explanation:

[0133] (1) The polypeptides shown in SEQ ID NO:1 and SEQ ID NO:2 described in this application, as well as polypeptide M1-14, have the activity of synthesizing GDP-fucose.

[0134] (2) Compared with the polypeptide shown in SEQ ID NO:1, the ability of polypeptide M1-14 to synthesize GDP-fucose is improved, especially M13 and M14, which are improved by more than 1.7 times.

[0135] Example 3. Synthesis of 2'-fucosylated lactose using Escherichia coli BL21 as a chassis.

[0136] References: 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.) constructed strain BL21ΔlacZΔlonΔwcaJ, and then transformed the above-constructed PET32a-M1-HpFutC and PET32a-M14-HpFutC into Escherichia coli chassis cells BL21ΔlacZΔlonΔwcaJ by electroporation, thereby constructing strains EC-M1 and EC-M14.

[0137] Methods for culturing Escherichia coli EC-M1 and EC-M14 to synthesize 2'-fucosylated lactose:

[0138] Escherichia coli strains EC-M1 and EC-M14 were cultured 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, 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 OD... 600 When the yield 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 sequentially to supplement fucosylated lactose production. Simultaneously, ampicillin was added to the culture medium to a final concentration of 100 μg / ml. After completion, the culture medium was boiled for 10 min, centrifuged, and the supernatant was used to analyze the 2'-FL yield. The results are recorded in Table 3.

[0139] Table 3

[0140] Gene-modified cells 2'-FL yield, g / L EC-M1 2.1 EC-M14 4.7

[0141] Example 4. Synthesis of 3-fucosylated lactose using Escherichia coli BL21 as a chassis.

[0142] References: 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.) constructed strain BL21ΔlacZΔlonΔwcaJ, and then transferred the above-constructed strain or PET32a-M1-HpFutA and PET32a-M14-HpFutA into E. coli chassis cells BL21ΔlacZΔlonΔwcaJ via electroporation to obtain strains EC-M1-1 and EC-M14-1.

[0143] Methods for culturing Escherichia coli EC-M1-1 and EC-M14-1 to synthesize 3-fucosylated lactose:

[0144] Strains EC-M1-1 and EC-M14-1 were used respectively, and 3-fucosylated lactose was synthesized by fermentation using the method described in Example 3 for culturing Escherichia coli EC-M1 and EC-M14 to produce 2'-fucosylated lactose. After the fermentation was completed, the culture medium was boiled for 10 min, centrifuged, and the supernatant was collected to detect the yield of 3-FL. The results are recorded in Table 4.

[0145] Table 4

[0146] Gene-modified cells 3-FL yield, g / L EC-M1-1 2.0 EC-M14-1 3.8

[0147] Example 5. Synthesis of 2'-fucosylated lactose using Bacillus BStgtP8 (CCTCC NO: M 20231126) as a chassis.

[0148] Bacillus subtilis chassis cells were constructed, referring to Zhang et al. (Zhang,Q.,Liu,Z.,Xia,H.,Huang,Z.,Zhu,Y.,Xu,L.,Liu,Y.,Li,J.,Du,G.,Lv,X.,&Liu,L.(2022).Engineered Bacillus subtilis for the de novo production of 2'-fucosyllactose.Microbial cellfactories,21(1),110.) to construct Bacillus subtilis chassis cells BSP43-manB-P43-manC-P43-HpFutC-P43-GMD-P43-lacY. Simultaneously, P43-M1 and P43-M14 expression cassettes were constructed, and the expression cassettes were transformed into the manP gene of BSP43-manB-P43-manC-P43--HpFutC-P43-GMD-P43-lacY, respectively, to obtain strains BS-M1 and BS-M14.

[0149] Methods for culturing strains BS-M1 and BS-M14 to synthesize 2'-fucosylated lactose:

[0150] Bacillus subtilis strains BS-M1 and BS-M14 were cultured 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. Te 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 mL / 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 and 10 g / L, respectively. After fermentation, the culture was boiled for 10 min, centrifuged, and the supernatant was used to determine the 2'-FL yield. The results are recorded in Table 5.

[0151] Table 5

[0152] Gene-modified cells 2'-FL yield, g / L BS-M1 1.6 BS-M14 3.2

[0153] The above process was scaled up to a 1L fermenter, with continuous feeding for fermentation. The sucrose concentration in the fermentation broth was controlled within the range of 17g / L to 22g / L, and the lactose concentration within the range of 10g / L to 20g / L. Fermentation was terminated after 30 hours of continuous feeding. After the fermentation was completed, the culture broth was boiled for 10 minutes, centrifuged, and the supernatant was collected to detect the 2'-FL content. The results are recorded in Table 6.

[0154] Table 6

[0155] Gene-modified cells 2'-FL yield, g / L BS-M1 15.6 BS-M14 23.2

[0156] Example 6. Synthesis of 3-fucosyl lactose using Bacillus BStgtP8 (CCTCC NO: M 20231126) as a chassis.

[0157] Bacillus subtilis chassis cells were constructed, referring to Zhang et al. (Zhang,Q.,Liu,Z.,Xia,H.,Huang,Z.,Zhu,Y.,Xu,L.,Liu,Y.,Li,J.,Du,G.,Lv,X.,&Liu,L.(2022).Engineered Bacillus subtilis for the de novo production of 2'-fucosyllactose.Microbial cellfactories,21(1),110.) to construct Bacillus subtilis chassis cells BSP43-manB-P43-manC-P43-HpFutA-P43-GMD-P43-lacY. Simultaneously, P43-M1 and P43-M14 expression cassettes were constructed, and the expression cassettes were transformed into the manP gene of BSP43-manB-P43-manC-P43-HpFutA-P43-GMD-P43-lacY, respectively, to obtain strains BS-M1-1 and BS-M14-1.

[0158] Methods for synthesizing 3-fucosylated lactose by culturing strains BS-M1-1 and BS-M14-1:

[0159] Escherichia coli strains EC-M1-1 and EC-M14-1 were used to synthesize 3-fucosylated lactose by fermentation using the method described in Example 5 for the production of 2'-fucosylated lactose by strains BS-M1 and BS-M14. After the fermentation was completed, the culture medium was boiled for 10 min, centrifuged, and the supernatant was collected to detect the yield of 3-FL. The results are recorded in Table 7.

[0160] Table 7

[0161] Gene-modified cells 3-FL yield, g / L BS-M1-1 1.5 BS-M14-1 3.0

[0162] 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, and 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 collected to determine the 3-FL content. The results are recorded in Table 8.

[0163] Table 8

[0164] Gene-modified cells 3-FL yield, g / L BS-M1-1 14.6 BS-M14-1 20.2

[0165] Example 7. Synthesis of 2'-fucosylated lactose using Saccharomyces cerevisiae SctgtP8 (CCTCC NO: M20231127) as the chassis.

[0166] The construction of Saccharomyces cerevisiae chassis cells was referenced by 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-HpFutA-GMD was constructed, and pRS305-Pgal-M1 and pRS305-Pgal-M14 plasmids were constructed. These plasmids were then transformed into SC-Δgal80-Pgal1-lac12-Pgal1-HpFutC-GMD to obtain strains SC-M1 and SC-M14, respectively.

[0167] Methods for culturing strains SC-M1 and SC-M14 to synthesize 2'-fucosylated lactose:

[0168] Saccharomyces cerevisiae SC-M1 and SC-M14 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, 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 5-6 hours of cultivation, sucrose (50% mother liquor concentration) was added at a flow rate of 8mL / h, and lactose (40% mother liquor concentration) was added simultaneously, maintaining 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 product, and the 2'-FL yield in the supernatant was measured. The results are recorded in Table 9.

[0169] Table 9

[0170]

[0171] Example 8. Synthesis of 3-fucosyllactose using Saccharomyces cerevisiae SctgtP8 (CCTCC NO: M20231127) as a chassis.

[0172] 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-HpFutA-GMD was constructed, and pRS305-Pgal-M1 and pRS305-Pgal-M14 plasmids were constructed. These plasmids were then transformed into SC-Δgal80-Pgal1-lac12-Pgal1-HpFutA-GMD, respectively, to obtain strains SC-M1-1 and SC-M14-1.

[0173] Methods for synthesizing 3-fucosylated lactose by culturing strains SC-M1-1 and SC-M14-1:

[0174] Saccharomyces cerevisiae SC-M1-1 and SC-M14-1 were fermented to synthesize 3-fucosylated lactose (3-FL) according to the method for producing 2'-fucosylated lactose by strains BS-M1 and BS-M14 described in Example 7. After fermentation, the culture medium was boiled for 10 minutes, centrifuged, and the supernatant was collected to detect the yield of 3-FL. The results are recorded in Table 10.

[0175] Table 10

[0176] Gene-modified cells 3-FL yield, g / L SC-M1-1 16.6 SC-M14-1 21.5

[0177] Example 9. Synthesis of 2'-fucosylated lactose using Kluyveromyces oryzae HLLWF3 (CCTCC NO: M2022118) as a chassis.

[0178] 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. The strain was obtained using the construction and transformation method described at https: / / doi.org / 10.1021 / acs.jafc.3c01779. KL-ΔLAC4::HpFutA-ΔXK::GMD, and simultaneously construct expression boxes ΔGK::Ptef1-M1 and ΔGK::Ptef1-M14, respectively, and then transfer the expression boxes into... Strain KL-ΔLAC4 ::HpFutC-ΔXK::GMD, yielding strains KL-M1 and KL-M14.

[0179] Methods for synthesizing 2'-fucosylated lactose by culturing strains KL-M1 and KL-M14:

[0180] Kluyveromyces kluyveromyces KL-M1 and KL-M14 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, 2% of each yeast extract was inoculated into 1L of YPD medium (10g / L yeast extract, 20g / L peptone, 20g / L glucose) in a 3L fermenter. After 6-7 hours of cultivation, 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. The total fermentation time was 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and the supernatant and precipitate were collected separately. The precipitate was 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, and the 2'-FL content in the supernatant was detected. The results are recorded in Table 11.

[0181] Table 11

[0182] Gene-modified cells 2'-FL yield, g / L KL-M1 21.0 KL-M14 28.6

[0183] Example 10. Synthesis of 3-fucosyllactose using Kluyveromyces HLLWF3 (CCTCC NO: M2022118) as a chassis.

[0184] 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. The strain was obtained using the construction and transformation method described at https: / / doi.org / 10.1021 / acs.jafc.3c01779. KL-ΔLAC4::HpFutA-ΔXK::GMD was used to construct expression cassettes ΔGK::Ptef1-M1 and ΔGK::Ptef1-M14. The expression cassettes were then transformed into KL-ΔLAC4::HpFutA-ΔXK::GMD to obtain strains KL-M1-1 and KL-M14-1, respectively.

[0185] Methods for synthesizing 3-fucosylated lactose by culturing strains KL-M1-1 and KL-M14-1:

[0186] Kluyveromyces kluyveromyces KL-M1-1 and KL-M14-1 were fermented to synthesize 3-fucosylated lactose, following the method described in Example 9 for the production of 2'-fucosylated lactose by strains KL-M1 and KL-M14. After fermentation, the culture medium was boiled for 10 minutes, centrifuged, and the supernatant was collected to detect the yield of 3-FL. The results are recorded in Table 11.

[0187] Table 11

[0188] Gene-modified cells 3-FL yield, g / L KL-M1-1 18.8 KL-M14-1 24.6

[0189] This invention discloses a series of GDP-fucose synthase 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.

[0190] 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.

Claims

1. A GDP-fucose synthase polypeptide, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The mutant of the GDP-fucose synthase polypeptide as described in claim 1, selected from any of the following mutations: 1) In the amino acid sequence shown in SEQ ID NO:1, the amino acid fragment at positions 20-33 is replaced by YEQLKQRGDIEIVA with KAQLEQDGNVELV, thereby obtaining polypeptide M1; 2) Based on the mutation of peptide M1, the amino acid fragment at positions 40-53 of the amino acid sequence shown in SEQ ID NO:1 was replaced by NLLDADAVRAFFAA with DLLDEKKVKDFFAK, thereby obtaining peptide M2; 3) Based on the mutation of peptide M2, the amino acid fragment at positions 75-81 of the amino acid sequence shown in SEQ ID NO:1 was replaced with NPEGFYT and then with KPKEFYE to obtain peptide M3; 4) Based on the mutation of polypeptide M3, the amino acid fragment at position 95 of the amino acid sequence shown in SEQ ID NO:1 is replaced with A by replacing L, thereby obtaining polypeptide M4; 5) Based on the mutation of peptide M4, the amino acid fragment at positions 148-158 of the amino acid sequence shown in SEQ ID NO:1 was replaced by ADYNNKYGADY with EKYNNEKGLDA, thereby obtaining peptide M5; 6) Based on the mutation of polypeptide M5, the amino acid residue at position 193 of the amino acid sequence shown in SEQ ID NO:1 is replaced by A instead of K, thereby obtaining polypeptide M6; 7) Based on the mutation of polypeptide M6, the amino acid fragment at position 215 of the amino acid sequence shown in SEQ ID NO:1 is replaced with N by replacing Y, thereby obtaining polypeptide M7; 8) Based on the mutation of peptide M7, the amino acid fragment at positions 219-221 of the amino acid sequence shown in SEQ ID NO:1 is replaced with DAA by replacing EAS, thereby obtaining peptide M8; 9) Based on the mutation of peptide M8, the amino acid fragment at positions 227-232 of the amino acid sequence shown in SEQ ID NO:1 was replaced with LDEAAR by replacing QDQAAV, thereby obtaining peptide M9; 10) Based on the mutation of peptide M9, the amino acid residues at positions 239-241 of the amino acid sequence shown in SEQ ID NO:1 were replaced with TNS by replacing DNH, thereby obtaining peptide M10; 11) Based on the mutation of peptide M10, the amino acid residues at positions 259-264 of the amino acid sequence shown in SEQ ID NO:1 were replaced by KIAEVV with TIAEVT, thereby obtaining peptide M11; 12) Based on the mutation of polypeptide M11, the amino acid residues at positions 301-302 of the amino acid sequence shown in SEQ ID NO:1 were replaced by LR instead of IV, thereby obtaining polypeptide M12; 13) Based on the mutation of polypeptide M12, the amino acid residue at position 306 of the amino acid sequence shown in SEQ ID NO:1 is replaced by A instead of K, thereby obtaining polypeptide M13; 14) Based on the mutation of peptide M13, the amino acid residues at positions 310-316 of the amino acid sequence shown in SEQ ID NO:1 were replaced by AVMQANL with EWMEKNK, thereby obtaining peptide M14.

3. A polynucleotide encoding the GDP-fucose synthase polypeptide of claim 1 or a mutant of the GDP-fucose synthase polypeptide of claim 2.

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

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

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

7. The transformed host cell as described in claim 6, characterized in that, The host cells used for the transformation are selected from natural strains or genetically modified strains of bacteria, yeast, and mold.

8. The transformed host cell as described in claim 6, characterized in that, The host cells for the transformation are selected from Escherichia coli (Escherichia coli). Escherichia sp.), Bacillus spp. Bacillus sp.), Kluyveromyces (sp.), Kluyveromyces genus ( Kluyveromyces Natural or genetically modified strains of sp.

9. The transformed host cell as described in claim 6, characterized in that, The host cells for the transformation were selected from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Kluyveromycin ( Kluyveromyces marxianus Kluyveromycin (lactic acid yeast) Kluyveromyces lactis ), Yarrowia lipolytica ( Yarrowia lipolytica ( ) natural strains or genetically modified strains.

10. The transformed host cell as described in claim 6, characterized in that, The host cells used for the transformation were selected from genetically engineered Escherichia coli. E. coli BL21(DE3).

11. An enzyme preparation comprising one or more of the GDP-fucose synthase polypeptide of claim 1 or a mutant of the GDP-fucose synthase polypeptide of claim 2.

12. The use of the GDP-fucose synthase polypeptide of claim 1 or a mutant of the GDP-fucose synthase polypeptide of claim 2, or the transformed host cell of claim 6, or the enzyme of claim 11 in the synthesis of GDP-fucose or fucoidan.

13. The application as described in claim 12, wherein, The fucoidyl lactose is selected from 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL).

14. The application as described in claim 12, preparing fucoidosyl lactose in a cell-free expression system or in a host organism.

15. A method for preparing GDP-fucose, characterized in that, Includes the following steps: a) Using the GDP-fucose synthase polypeptide of claim 1 or a mutant of the GDP-fucose synthase polypeptide of claim 2, or the host cell transformed according to any one of claims 6-10, or the enzyme of claim 11 as a catalyst, to catalyze the synthesis of GDP-fucose from the substrate GDP-4-keto-6-deoxymannose.

16. The preparation method according to claim 15, characterized in that, Also includes: The step of catalyzing the synthesis of GDP-4-keto-6-deoxymannose from the substrate GDP-mannose using GDP-mannose dehydratase as a catalyst.

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