GDP-mannose dehydratase polypeptide and application thereof

Abstract

The invention provides a series of GDP-mannose dehydratase polypeptides which can be applied to synthesis of GDP-fucose, and belongs to the technical field of enzyme engineering. The GDP-mannose dehydratase polypeptide comprises a polypeptide of which the amino acid sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% identical to that of a polypeptide shown in SEQ ID NO: 1, or a polypeptide of which the amino acid sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% identical to that of a polypeptide M1-13. The GDP-mannose dehydratase polypeptide disclosed by the invention has relatively high catalytic activity.

Description

Technical Field

[0001] This invention relates to a series of GDP-mannose dehydrating enzyme polypeptides, which can be applied to the synthesis of GDP-fucose or human milk oligosaccharides, 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 HMOs and have been proven to play an important role in the brain, gut, 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. The biosynthesis of 2'-FL and 3-FL was initially achieved through enzymatic catalysis. In 2000, Albermann et al., through exogenous expression and purification of GDP-mannose-4,6-dehydratase (GMD) and GDP-fucose synthase (WcaG) from *Escherichia coli* K12, converted GDP-mannose to GDP-fucose in vitro. GDP-fucose then synthesized fucosyllactose under the catalysis of fucotransferase. GDP-fucose is a key precursor for the synthesis of fucosyllactose. They also found that the activity of GMD could be inhibited by GDP-fucose, thus GMD is a critical rate-limiting enzyme. Therefore, it is necessary to study GMD enzymes (GDP-mannose-4,6-dehydratase) to improve the synthesis efficiency and conversion rate of fucosyllactose. Modification of key enzymes is crucial for improving catalytic efficiency. Summary of the Invention

[0005] One object of the present invention is to provide a GDP-mannose dehydratase polypeptide (GMD) and its application in the synthesis of fucoidan lactose, so as to improve the production efficiency of fucoidan lactose.

[0006] The GDP-mannose dehydratase polypeptide, also known as GDP-D-mannose-4,6-dehydratase or GDP-mannose-6-dehydrogenase, belongs to the lyase family and has the activity of catalyzing the conversion of GDP-mannose to GDP-4-keto-6-deoxymannose. As one of the key enzymes in GDP-fucose synthesis, the GDP-mannose dehydratase polypeptide also participates in the metabolism of fructose and mannose.

[0007] Technical solution:

[0008] The present invention provides the following technical features, and the combination of one or more of the following technical features constitutes the technical solution of the present invention.

[0009] The applicant has discovered a GDP-mannose dehydratase from Bacillus smithii in nature, named BsGMD, with its amino acid sequence shown in SEQ ID NO:1 and its nucleotide sequence shown in SEQ ID NO:6. It has the activity of catalyzing the conversion of GDP-mannose to GDP-4-keto-6-deoxymannose, and can be applied to the synthesis of GDP-fucose.

[0010] A comparison with the NCBI database revealed that the GDP-mannose dehydratase with the amino acid sequence shown in SEQ ID NO:1 was most similar to that of GDP-mannose dehydratase derived from *Escherichia coli* (as of November 15, 2023). The two enzymes shared only 69.03% 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-mannose dehydratase from *Escherichia coli* is shown in SEQ ID NO:2.

[0011] The inventors of this application have performed a series of modifications on the GDP-mannose dehydratase polypeptide with the amino acid sequence shown in SEQ ID NO:1, and screened and obtained a series of derivative peptides M1-M13 with enhanced catalytic activity in converting GDP-mannose to GDP-4-keto-6-deoxymannose. The derivative peptides M1-M13 are dehydratase polypeptides formed by substitution of one or more amino acid residues / fragments in the polypeptide with the amino acid sequence shown in SEQ ID NO:1.

[0012] This application describes exemplary substitutions or combinations of substitutions for polypeptides with amino acid sequences as shown in SEQ ID NO:1.

[0013] Specifically, in a first aspect, this application provides a series of GDP-mannose dehydratase polypeptides for synthesizing fucoidylated lactose, wherein the GDP-mannose dehydratase polypeptides are selected from polypeptides (BsGMD) with amino acid sequences as shown in SEQ ID NO:1 and

[0014] BsGMD-derived polypeptide M1-13. The derived polypeptide M1-13 is obtained by modifying at least one of the following sites in the amino acid sequence shown in SEQ ID NO:1: positions 69-72, 118-119, 123-124, 176, 203-205, 213-217, 257-264, 273-276, 288, 304-312, 339-443, and 365.

[0015] Preferably, the modification is a mutation, and the mutation is selected from any of the following mutations:

[0016] 1) In the amino acid sequence shown in SEQ ID NO:1, the amino acid fragment at positions 69-72 is replaced with ASIR by EKIK, thereby obtaining polypeptide M1;

[0017] 2) In the amino acid sequence of polypeptide M1, the amino acid fragment at positions 118-119 is replaced by LD, thereby obtaining polypeptide M2;

[0018] 3) In the amino acid sequence of polypeptide M2, the amino acid fragment at positions 123-124 is replaced by EK with QD, thereby obtaining polypeptide M3;

[0019] 4) In the amino acid sequence of polypeptide M3, the amino acid fragment at position 176 is replaced with D instead of N, thus obtaining polypeptide M4;

[0020] 5) In the amino acid sequence of polypeptide M4, the amino acid fragment at positions 203-205 is replaced by IQL with RDI, thereby obtaining polypeptide M5;

[0021] 6) In the amino acid sequence of polypeptide M5, the amino acid residues at positions 213-217 are replaced by LDTLY with KKVLK, thereby obtaining polypeptide M6;

[0022] 7) In the amino acid sequence of polypeptide M6, the amino acid fragment at positions 257-264 is replaced by FTVREAVE with YTVKEIVK, thereby obtaining polypeptide M7;

[0023] 8) In the amino acid sequence of polypeptide M7, the amino acid fragment at positions 273-276 is replaced by ELEF with ALRW, thereby obtaining polypeptide M8;

[0024] 9) In the amino acid sequence of polypeptide M8, the amino acid fragment at position 288 is replaced by K with E, thereby obtaining polypeptide M9;

[0025] 10) In the amino acid sequence of polypeptide M9, amino acid residues at positions 304-312 are replaced by ILEVDPSFL with VLKVDPAFR, thereby obtaining polypeptide M10.

[0026] 11) In the amino acid sequence of polypeptide M10, the amino acid fragment at positions 339-443 is replaced by FEEMM with IEEMI, thereby obtaining polypeptide M11;

[0027] 12) In the amino acid sequence of polypeptide M11, the amino acid fragment at positions 352-360 is replaced by KVAEEYAEK with AVAEKYAEL, thereby obtaining polypeptide M12.

[0028] 13) In the amino acid sequence of polypeptide M12, the amino acid residue at position 365 is replaced by Y with E, thereby obtaining polypeptide M13.

[0029] In some embodiments, the polypeptide having enhanced catalytic conversion of GDP-mannose to GDP-4-keto-6-deoxymannose comprises a polypeptide having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to that of polypeptide M1-13, and comprises an amino acid substitution mutation that is functionally equivalent to the schemes described in 1)-13) 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)-13) above.

[0030] Secondly, this application provides a polynucleotide encoding the GDP-mannose dehydratase polypeptide as described in the first aspect above.

[0031] Polynucleotides encoding GDP-mannose dehydratase 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.

[0032] Thirdly, this application provides a nucleic acid construct comprising the polynucleotides described in the second aspect above.

[0033] 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 cell.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] In one specific embodiment, the expression cassette contains all the elements necessary for expressing the GDP-mannose dehydratase 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.

[0038] 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.

[0039] 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.

[0040] In some embodiments, the host cell includes, but is not limited to, natural strains or genetically modified strains such as bacteria, yeast, and mold.

[0041] More preferably, the host cell includes, but is not limited to, natural or genetically modified strains of Escherichia sp., Bacillus sp., Kluyveromyces sp., etc.

[0042] More preferably, the host cell includes, but is not limited to, Saccharomyces cerevisiae, Kluyveromyces marxianus, Kluyveromyces lactis, and Yarrowia lipolytica.

[0043] More preferably, the host cell, the Escherichia sp., includes, but is not limited to, the genetically engineered E. coli BL21(DE3).

[0044] In a sixth aspect, this application provides an enzyme agent or enzyme composition comprising the polypeptide as described in the first aspect above.

[0045] The enzyme or enzyme composition preferably contains one or more of the polypeptides and polypeptides M1-13 with the amino acid sequence shown in SEQ ID NO:1 of the present invention.

[0046] Depending on the reaction substrate and product, the enzyme or composition may also contain bifunctional enzymes beneficial to the synthesis of fucose-syllactose, such as L-fucokinase / GDP-L-fucosepyrophosphorylase (FKP), glucokinase (Glk), mannose-phosphate mutase (ManB), mannose-1-phosphoguanyltransferase (ManC), GDP-L-Fucose synthase (WcaG), etc.

[0047] In a seventh aspect, this application provides a method for producing the polypeptide as described in the first aspect above, comprising:

[0048] (1) Transformed host cells are cultured under conditions suitable for expressing the GDP-mannose dehydratase polypeptide; the transformed host cells are as described in aspect five above; and

[0049] (2) The GDP-mannose dehydrating enzyme polypeptide was recovered.

[0050] In a specific implementation, step (1) includes: firstly, introducing a nucleic acid construct or recombinant expression vector containing a polypeptide encoding the GDP-mannose dehydratase 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-mannose dehydratase polypeptide.

[0051] In a specific implementation, step (2) includes the steps of separating and purifying the GDP-mannose dehydratase polypeptide from the culture.

[0052] 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.

[0053] The GDP-mannose dehydratase 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.

[0054] In addition, GDP-mannose dehydratase 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.

[0055] Eighthly, this application provides the use of the GDP-mannose dehydrating enzyme polypeptide as described in the first aspect above, the transformed host cell as described in the fifth aspect, or the enzyme agent or enzyme composition as described in the sixth aspect in the synthesis of GDP-4-keto-6-deoxymannose;

[0056] Preferably, the application also includes applications with GDP-fucose and / or fucose-based lactose;

[0057] Preferably, the fucoidyl lactose includes 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL).

[0058] 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-mannose dehydratase peptide and GDP-fucose synthase described in this application to exert their enzymatic activity.

[0059] In one embodiment, the method includes: adding one or more of the GDP-mannose dehydrating enzyme peptides described in this application to a reaction system containing GDP-mannose, wherein the dehydrating enzyme peptides contact the substrate GDP-mannose to synthesize GDP-4-keto-6-deoxymannose; then, GDP-4-keto-6-deoxymannose is converted into GDP-fucose by GDP-fucose synthase; furthermore, GDP-fucose can react with lactose in the reaction system under the catalysis of α-1,2-fucotransferase or α-1,3-fucotransferase to synthesize and obtain the desired fucose-based lactose.

[0060] Preferably, the application involves preparing the GDP-mannose dehydratase 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-mannose dehydratase polypeptide listed above can be isolated, and GDP-fucose or fucoidyl lactose can be synthesized in a suitable system, optionally followed by further purification. Specifically, this includes:

[0061] a) Using the polypeptides described in the first aspect above or the enzymes or enzyme compositions described in the sixth aspect above, catalyze the synthesis of GDP-4-keto-6-deoxymannose from the substrate GDP-mannose;

[0062] b) GDP-4-keto-6-deoxymannose is converted into GDP-fucose under the catalysis of GDP-fucose synthase and NADPH enzyme.

[0063] 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.

[0064] Optional, also including:

[0065] c) GDP-fucose reacts with lactose to synthesize fucosyl lactose under the catalysis of α-1,2-fucotransferase or α-1,3-fucotransferase.

[0066] The sources of the α-1,2-fucotransferase or α-1,3-fucotransferase include, but are not limited to: Helicobacter pylori, Thermophilic chlorella, Escherichia coli, mammals, Caenorhabditis elegans, Schistosoma mansoni, Bacillus cereus, Pseudopedobater saltans, Helicobacter mustelae, Bacillus fragilis, Bacteroides vulgaris, or Bacillus smithii, etc.

[0067] d) Optionally, the steps of purifying and / or recovering GDP-fucose or fucose-based lactose.

[0068] In one embodiment, at least one of the GDP-mannose dehydrating enzyme peptides described in this application is combined with GDP-fucose synthase, GDP-mannose, and NADP. + A mixture of NADPH| and Tris-HCl buffer (such as Tris-HCl or HEPES) is prepared. The mixture is incubated at a specific temperature (e.g., 35°C, 37°C, or 40°C) for a specific period of time (e.g., 24 hours), during which GDP-mannose is converted to GDP-fucose by the GDP-mannose dehydratase and GDP-fucose synthase; then, fucosyllactose (2'-FL or 3-FL) is synthesized under the catalysis of α-1,2-fucotransferase or α-1,3-fucotransferase; GDP-fucose or fucosyllactose can be isolated and purified by methods known in the art. At the end of the reaction or after isolation and / or purification, the yield of GDP-fucose or fucosyllactose is determined by HPLC.

[0069] The application of the GDP-mannose dehydrating enzyme polypeptide of the present invention uses GDP-mannose as a substrate. GDP-fucose is produced by the catalytic reaction of GDP-mannose dehydrating enzyme and GDP-fucose synthase. Fucosyl lactose is then synthesized under the catalysis of α-1,2-fucotransferase or α-1,3-fucotransferase.

[0070] In the above steps, the reaction product is a solution of GDP-fucose or fucoidan, which can be clarified by conventional methods. Preferably, the solution containing GDP-fucose or fucoidan can be clarified by centrifugation, flocculation, decantation, and / or filtration.

[0071] Preferably, substantially all proteins, as well as amino acids, RNA, and DNA, are removed from the solution containing GDP-fucose or fucosyllactose (preferably after clarification). In this step, proteins and related impurities can be removed from the solution containing GDP-fucose or fucosyllactose in a conventional manner. Preferably, proteins, salts, byproducts, colors, and other related impurities are removed from the mixture containing GDP-fucose or fucosyllactose 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.

[0072] 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.

[0073] 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.

[0074] Proteins and / or protein sequences are “homological” 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.

[0075] 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.

[0076] The term "fucosyllactose" includes 2'-fucosyllactose (2'-FL) or 3-fucosyllactose (3-FL).

[0077] Beneficial effects:

[0078] This application provides a series of GDP-mannose dehydrating enzyme polypeptides, including polypeptides with amino acid sequences as shown in SEQ ID NO:1 and polypeptide M1-13. These GDP-mannose dehydrating enzymes are important enzymes in the biosynthesis of GDP-fucose. This invention provides important biological resources for the biosynthesis of GDP-fucose and fucoidan. The GDP-mannose dehydrating enzymes 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 fucoidan.

[0079] 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

[0080] Figure 1 HPLC chromatogram of GDP-fucose standard.

[0081] Figure 2 HPLC chromatogram of the polypeptide M7 catalytic reaction solution in Example 2.

[0082] Figure 3 HPLC chromatogram of .2'-FL standard.

[0083] Figure 4 HPLC chromatogram of .3-FL standard.

[0084] Figure 5 HPLC analysis chromatogram of the fermentation broth for the synthesis of 2'-fucosylated lactose using BS-M13 in Example 3.

[0085] Figure 6 HPLC analysis chromatogram of the fermentation broth for synthesizing 3-fucosylated lactose using BS-M13-1 in Example 4. Detailed Implementation

[0086] 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.

[0087] 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.

[0088] Unless otherwise specified, the experimental methods described in the following specific embodiments are generally performed according to conventional molecular biology methods and conditions in the art, which are fully explained in the literature; all materials, reagents, etc., are commercially available unless otherwise specified.

[0089] 2'-FL and 3-FL standards were produced by ELICITYL, France. GDP-fucose standard was purchased from Aladdin.

[0090] The enzymes involved in the following examples are as follows:

[0091] GDP-mannose synthase (WcaG), microbial source: Escherichia coli (E. col), amino acid sequence: SEQ ID NO:3.

[0092] α-1,2-fucosyltransferase (FutC), derived from Helicobacter pylori, has the amino acid sequence shown in SEQ ID NO:4.

[0093] α-1,3-fucosyltransferase (futA), derived from Helicobacter pylori, has the amino acid sequence shown in SEQ ID NO:5.

[0094] GDP Fucose HPLC Detection Method:

[0095] 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.

[0096] HPLC method for determining fucoidan-lactose content:

[0097] 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℃.

[0098] 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.

[0099] Example 1. Expression of the polypeptides shown in SEQ ID NO:1 and SEQ ID NO:2 and polypeptide M1-13 in Escherichia coli.

[0100] 1. Using the polypeptides shown in SEQ ID NO:1 (nucleotide sequence shown in SEQ ID NO:6) and SEQ ID NO:2 as templates, gene coding sequences of the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2 were synthesized respectively, and finally constructed into the PET32a vector to obtain the plasmids PET32a-BsGMD-wt and PET32a-EcGMD-wt.

[0101] 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-M13) were constructed using a point mutation kit.

[0102] 3. Transform the above recombinant plasmids into Escherichia coli BL21(DE3) according to the following steps:

[0103] 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-M13 recombinant plasmid (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 cells were incubated at 37℃ and 100 rpm for 1 h. Then, 100 μL of the bacterial solution was spread on an LB plate containing ampicillin (100 μg / mL) and incubated at 37℃ for 12 h. Single colonies were picked for colony PCR to screen for positive transformants.

[0104] 4. Cultivate positive transformants, then extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the pET32a-M1-M13 recombinant plasmid was successfully introduced into E. coli.

[0105] 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.

[0106] 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.

[0107] 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 and polypeptide M1-13 were obtained. For details, please refer to Table 1.

[0108] Table 1. Correspondence between GDP-mannose dehydratase polypeptides and their amino acid sequence numbers

[0109]

[0110]

[0111] Example 2. Determination of the ability of each polypeptide (SEQ ID NO:1, SEQ ID NO:2) and polypeptide M1-13 obtained in Example 1 as catalysts for the synthesis of GDP-Fucose.

[0112] GDP-mannose was synthesized using GDP-mannose as a substrate, the purified polypeptides shown in SEQ ID NO:1 and SEQ ID NO:2 obtained in Example 1, and polypeptide M1-13, along with GDP-fucose synthase as an enzyme catalyst. The specific procedure is as follows:

[0113] The samples were prepared in solutions containing 100 μM GDP-mannose, 50 mM Tris-HCl (pH 7.0), and 1 mM NADP. + In a system containing 1 mM NADPH, add 0.2 mg / mL of the purified polypeptide or polypeptide M1-13 (SEQ ID NO:1 or SEQ ID NO:2 obtained in Example 1) and 0.2 mg / mL of GDP-fucose synthase. Mix well and react at 30°C for 6 hours. Terminate the reaction and purify the polypeptide using gel column chromatography.

[0114] The standard and reaction solution were analyzed using the aforementioned analytical method. HPLC analysis showed that the peak elution time (rt) of the GDP-fucose standard was 13.3 min. The reaction solution of the peptide described in Example 1 also showed 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.

[0115] The concentration of GDP-fucose in the catalytic reaction solution was detected using the method described above, and the results are recorded in Table 2.

[0116] Table 2. Studies on the catalytic synthesis of GDP-fucose by various peptides

[0117]

[0118]

[0119] Table 2 Data Explanation:

[0120] (1) The polypeptides shown in SEQ ID NO:1 and SEQ ID NO:2 and their derivative peptide M1-13 all have the activity of synthesizing GDP-4-keto-6-deoxymannose with GDP-mannose; and then GDP-fucose is synthesized under the action of GDP-fucose synthase.

[0121] (2) The catalytic activity of peptides M5-M9 and M11-M13 in the synthesis of GDP-fucose was more than 1.5 times higher than that of the peptide shown in SEQ ID NO:1.

[0122] Example 3. Synthesis of 2'-fucosylated lactose using Escherichia coli BL21 as a chassis.

[0123] 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 PET32a-M1-HpFutC and PET32a-M13-HpFutC into Escherichia coli chassis cells BL21ΔlacZΔlonΔwcaJ by electroporation, thereby constructing strains EC-M1 and EC-M13.

[0124] Methods for culturing Escherichia coli EC-M1 and EC-M13 to synthesize 2'-fucosylated lactose:

[0125] Escherichia coli strains EC-M1 and EC-M13 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, 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 2′-FL 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.

[0126] Table 3

[0127] Gene-modified cells 2'-FL yield, g / L EC-M1 2.2 EC-M13 4.5

[0128] Example 4. Synthesis of 3-fucosylated lactose using Escherichia coli BL21 as a chassis.

[0129] 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-HpFutA and PET32a-M13-HpFutA into E. coli chassis cells BL21ΔlacZΔlonΔwcaJ by electroporation, thereby constructing strains EC-M1-1 and EC-M13-1.

[0130] Methods for culturing Escherichia coli EC-M1-1 and EC-M13-1 to synthesize 3-fucosyllactose:

[0131] 3-fucosyllactose was synthesized from Escherichia coli strains EC-M1-1 and EC-M13-1, respectively, following the method for synthesizing 2'-fucosyllactose using strains EC-M1 and EC-M13 as described in Example 3. After synthesis, the culture medium was boiled for 10 minutes, centrifuged, and the supernatant was collected to detect the yields of 2'-FL and 3-FL. The results are recorded in Table 4.

[0132] Table 4

[0133] Gene-modified cells 3-FL yield, g / L EC-M1-1 1.8 EC-M13-1 3.6

[0134] Example 5. Synthesis of 2'-fucosylated lactose using Bacillus BStgtP8 (CCTCC NO: M 20231126) as a chassis.

[0135] 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-wcaG-P43-lacY. Simultaneously, P43-M1 and P43-M13 expression cassettes were constructed, and the expression cassettes were transformed into the manP gene of BSP43-manB-P43-manC-P43--HpFutC-P43-wcaG-P43-lacY, respectively, to obtain BS-M1 and BS-M13 strains.

[0136] Methods for culturing Bacillus subtilis BS-M1 and BS-M13 to synthesize 2'-fucosylated lactose:

[0137] Bacillus subtilis strains BS-M1 and BS-M13 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.

[0138] Table 5

[0139] Gene-modified cells 2'-FL yield, g / L BS-M1 1.9 BS-M13 3.8

[0140] 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 for fucoidan lactose content. The results were: BS-M1 fermentation broth: 2'-FL content 18.8g / L; BS-M13 fermentation broth: 2'-FL content 28.1g / L.

[0141] Example 6. Synthesis of 3-fucosyl lactose using Bacillus BStgtP8 (CCTCC NO: M 20231126) as a chassis.

[0142] 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-wcaG-P43-lacY. Simultaneously, P43-M1 and P43-M13 expression cassettes were constructed, and the expression cassettes were transformed into the manP gene of BSP43-manB-P43-manC-P43-HpFutA-P43-wcaG-P43-lacY, respectively, to obtain strains BS-M1-1 and BS-M13-1.

[0143] Methods for culturing Bacillus subtilis BS-M1-1 and BS-M13-1 to synthesize 3-fucosylated lactose:

[0144] 3-fucosylated lactose was synthesized using Bacillus subtilis strains BS-M1-1 and BS-M13-1, following the method described in Example 5 for synthesizing 2'-fucosylated lactose using strains BS-M1 and BS-M13. After synthesis, 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 6.

[0145] Table 6

[0146] Gene-modified cells 3-FL yield, g / L BS-M1-1 1.4 BS-M13-1 2.8

[0147] 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 analyzed for fucosylated lactose content. The results were: 14.5g / L in BS-M1-1 fermentation broth and 21.3g / L in S-M13-1 fermentation broth.

[0148] Example 7. Synthesis of 2'-fucosylated lactose using Saccharomyces cerevisiae SctgtP8 (CCTCC NO: M20231127) as the chassis.

[0149] 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.), and SC-Δgal80-Pgal1-lac12-Pgal1-HpFutC-wcaG and

[0150] The plasmids SC-Δgal80-Pgal1-lac12-Pgal1-HpFutA-wcaG were constructed, and pRS305-Pgal-M1 and pRS305-Pgal-M13 were constructed. These plasmids were then transformed into SC-Δgal80-Pgal1-lac12-Pgal1-HpFutC-wcaG, respectively, to obtain strains SC-M1 and SC-M13.

[0151] Methods for culturing Saccharomyces cerevisiae strains SC-M1 and SC-M13 to synthesize 2'-fucosylated lactose:

[0152] Saccharomyces cerevisiae strains SC-M1 and SC-M13 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 (October Expiratory Scale) was 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 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 product, and the 2'-FL yield in the supernatant was measured. The results are recorded in Table 7.

[0153] Table 7

[0154]

[0155] Example 8. Synthesis of 3-fucosyllactose using Saccharomyces cerevisiae SctgtP8 (CCTCC NO: M20231127) as a chassis.

[0156] 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-wcaG was constructed, and pRS305-Pgal-M1 and pRS305-Pgal-M13 plasmids were constructed. These plasmids were then transformed into SC-Δgal80-Pgal1-lac12-Pgal1-HpFutA-wcaG, respectively, to obtain strains SC-M1-1 and SC-M13-1.

[0157] Methods for culturing Saccharomyces cerevisiae strains SC-M1-1 and SC-M13-1 to synthesize 3-fucosylated lactose:

[0158] Saccharomyces cerevisiae strains SC-M1-1 and SC-M13-1 were used to synthesize 3-fucosylated lactose using the method described in Example 7 for culturing strains SC-M1 and SC-M13 to synthesize 2'-fucosylated lactose. 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 fermentation product of the yeast. The yield of 3-FL in the supernatant was detected, and the results are recorded in Table 8.

[0159] Table 8

[0160] Gene-modified cells 3-FL yield, g / L SC-M1-1 20.6 SC-M13-1 19.8

[0161] Example 9. Synthesis of 2'-fucosylated lactose using Kluyveromyces oryzae HLLWF3 (CCTCC NO: M2022118) as a chassis.

[0162] 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 construction and transformation method described at https: / / doi.org / 10.1021 / acs.jafc.3c01779 was used to obtain strain KL- ΔLAC4 ::HpFutC-ΔXK::WcaG, and simultaneously construct expression cassettes ΔGK::Ptef1-M1 and ΔGK::Ptef1-M13. The expression cassettes were then transformed into KL-ΔLAC4::HpFutA-ΔXK::WcaG, respectively, to obtain strains KL-M1 and KL-M13.

[0163] Methods for culturing Kluyveromyces strains KL-M1 and KL-M13 to synthesize 2'-fucosylated lactose:

[0164] Kluyveromyces strains KL-M1 and KL-M13 were streaked onto solid media such as YDP and incubated at 30°C for 2-3 days. Single colonies were then picked and 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 (dose elongation) reached. 600 =1, and 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 9.

[0165] Table 9

[0166] Gene-modified cells 2'-FL yield, g / L KL-M1 21.3 KL-M13 34.6

[0167] Example 10. Synthesis of 3-fucosyllactose using Kluyveromyces HLLWF3 (CCTCC NO: M2022118) as a chassis.

[0168] 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 construction and transformation method described at https: / / doi.org / 10.1021 / acs.jafc.3c01779 was used to obtain strain KL- ΔLAC4::HpFutA-ΔXK::WcaG was constructed, and expression cassettes ΔGK::Ptef1-M1 and ΔGK::Ptef1-M13 were constructed. The expression cassettes were then transformed into KL-ΔLAC4::HpFutA-ΔXK::WcaG, respectively, to obtain strains KL-M1-1 and KL-M13-1.

[0169] Methods for culturing Kluyveromyces strains KL-M1-1 and KL-M13-1 to synthesize 3-fucosyllactose:

[0170] Kluyveromyces strains KL-M1-1 and KL-M13-1 were used to synthesize 3-fucosylated lactose using the method described in Example 9 for culturing KL-M1-1 and KL-M13-1 strains. 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. The 3-FL content in the supernatant was detected, and the results are recorded in Table 10.

[0171] Table 10

[0172] Gene-modified cells 3-FL yield, g / L KL-M1-1 19.8 KL-M13-1 32.5

[0173] This invention discloses a series of GDP-mannose dehydratase 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.

[0174] 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-mannose dehydratase polypeptide comprising a polypeptide having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of the polypeptide shown in SEQ ID NO:1; or a polypeptide having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of polypeptide M1-13; Preferably, the GDP-mannose dehydratase polypeptide has the amino acid sequence shown in SEQ ID NO:

1.

2. The GDP-mannose dehydratase polypeptide as described in claim 1, obtained by modifying at least one of the following sites in the amino acid sequence shown in SEQ ID NO:1: positions 69-72, 118-119, 123-124, 176, 203-205, 213-217, 257-264, 273-276, 288, 304-312, 339-443, 352-360, and 365; Preferably, the modification is a mutation, and the mutation is 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 69-72 is replaced with ASIR by EKIK, thereby obtaining polypeptide M1; 2) In the amino acid sequence of polypeptide M1, the amino acid fragment at positions 118-119 is replaced by LD, thereby obtaining polypeptide M2; 3) In the amino acid sequence of polypeptide M2, the amino acid fragment at positions 123-124 is replaced by EK with QD, thereby obtaining polypeptide M3; 4) In the amino acid sequence of polypeptide M3, the amino acid fragment at position 176 is replaced with D instead of N, thereby obtaining polypeptide M4; 5) In the amino acid sequence of polypeptide M4, the amino acid fragment at positions 203-205 is replaced by IQL with RDI, thereby obtaining polypeptide M5; 6) In the amino acid sequence of polypeptide M5, the amino acid residues at positions 213-217 are replaced by LDTLY with KKVLK, thereby obtaining polypeptide M6; 7) In the amino acid sequence of polypeptide M6, the amino acid fragment at positions 257-264 is replaced by FTVREAVE with YTVKEIVK, thereby obtaining polypeptide M7; 8) In the amino acid sequence of polypeptide M7, the amino acid fragment at positions 273-276 is replaced by ELEF with ALRW, thereby obtaining polypeptide M8; 9) In the amino acid sequence of polypeptide M8, the amino acid fragment at position 288 is replaced by K with E, thereby obtaining polypeptide M9; 10) In the amino acid sequence of polypeptide M9, amino acid residues at positions 304-312 are replaced by ILEVDPSFL with VLKVDPAFR, thereby obtaining polypeptide M10. 11) In the amino acid sequence of polypeptide M10, the amino acid fragment at positions 339-443 is replaced by FEEMM with IEEMI, thereby obtaining polypeptide M11; 12) In the amino acid sequence of polypeptide M11, the amino acid fragment at positions 352-360 is replaced by KVAEEYAEK with AVAEKYAEL, thereby obtaining polypeptide M12. 13) In the amino acid sequence of polypeptide M12, the amino acid residue at position 365 is replaced by Y with E, thereby obtaining polypeptide M13.

3. A polynucleotide encoding the GDP-mannose dehydratase polypeptide of any one of claims 1-2.

4. A nucleic acid construct comprising the polynucleotide as described in claim 3; The nucleic acid construct preferably further includes one or more regulatory sequences operatively linked thereto, the regulatory sequences guiding the production of the polypeptide in an appropriate expression host cell.

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; Preferably, the nucleic acid construct as described in claim 5, or the host cell as described in claim 6, is characterized in that, The host cells include, but are not limited to, natural strains or genetically modified strains such as bacteria, yeast, and mold; More preferably, the host cell includes, but is not limited to, natural strains or genetically modified strains such as Escherichia sp., Bacillus sp., and yeast sp. 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*. More preferably, the host cell includes, but is not limited to, the genetically engineered E. coli BL21(DE3).

7. An enzyme agent or enzyme composition comprising one or more of the GDP-mannose dehydrating enzyme polypeptides according to any one of claims 1-2.

8. The method for producing the GDP-mannose dehydrating enzyme polypeptide according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Transformed host cells were cultured under conditions suitable for expression of the GDP-mannose dehydratase polypeptide; and (2) The polypeptide is recovered. Preferably, step (1) includes: firstly, introducing a nucleic acid construct or recombinant expression vector containing a polypeptide encoding the polypeptide described in the first aspect above into a host cell to construct an engineered host cell expressing the polypeptide; then, culturing the engineered host cell and inducing it to express the polypeptide. Preferably, step (2) includes the steps of separating and purifying the polypeptide from the culture.

9. The use of the GDP-mannose dehydrating enzyme polypeptide of any one of claims 1-2, or the transformed host cell of claim 6, or the enzyme or enzyme composition of claim 7 in the synthesis of GDP-4-keto-6-deoxymannose; The application, preferably, uses GDP-mannose as a substrate; The preferred application is the preparation of fucoidosyl lactose in a cell-free expression system or in a host organism.

10. A method for preparing GDP-4-keto-6-deoxymannose, characterized in that, Includes the following steps: a) Using the GDP-mannose dehydrating enzyme polypeptide of any one of claims 1-2, or the host cell transformed according to claim 6, or the enzyme or enzyme composition of claim 7 as a catalyst, to catalyze the synthesis of GDP-4-keto-6-deoxymannose from the substrate GDP-mannose; More preferably, the preparation method further includes: b) optionally, the step of purifying and / or recovering GDP-fucose.

Images