Gdp-fucose synthase polypeptides and application thereof

Abstract

A series of GDP-fucose synthase polypeptides and gene-modified cells comprising heterologous nucleic acid sequences of said polypeptides, which can be applied in the synthesis of human fucosyllactose, and belong to the technical field of biological genetic engineering. Provided are derivative peptides A1-A14 of the polypeptide represented by SEQ ID NO: 1; A1-A14 are obtained via modification of at least one site among 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 of the amino acid sequence represented by SEQ ID NO: 1. Further provided are gene-modified cells, comprising heterologous nucleic acid sequences of polypeptides A1-A14. The polypeptides or the gene-modified cells improve fucosyllactose production efficiency.

Description

GDP-fucose synthase polypeptide and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application No. 202311635360.4, filed with the State Intellectual Property Office of China on November 30, 2023, entitled “GDP-fucose synthase polypeptides and their applications”, and claims priority to Chinese patent application No. 202411481555.2, filed with the State Intellectual Property Office of China on October 23, 2024, entitled “Genetically modified cells producing fucosyllactose”, the entire contents of which are incorporated by reference into the present invention and constitute a part of the present invention for all purposes. Technical Field

[0003] The present invention relates to a series of GDP-fucose synthase polypeptides and genetically modified cells containing heterologous nucleic acid sequences of the GDP-fucose synthase polypeptides, which can be applied to the synthesis of human milk oligosaccharides (HMOs) and belong to the field of biogenetic engineering technology. Background Art

[0004] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0005] Human milk oligosaccharides (HMOs) are a unique and diverse mixture of oligosaccharides found in human milk. They 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 key components of HMOs and have been shown to play an important role in the brain, intestinal tract, and growth and development of infants.

[0006] 2'-FL and 3'-FL have been approved as raw materials for infant formula by the U.S. FDA, the European Union, Australia, New Zealand, Canada and other countries and regions, and can be added to infant formula, regular food, dietary supplements and / or medical food.

[0007] Fucosyllactose is typically synthesized chemically, enzymatically, or in microbial cell factories. While chemical synthesis has made significant progress, the cumbersome process requires multiple protection and deprotection steps, resulting in low product yields and high production costs. Biosynthesis of fucosyllactose is currently a feasible method for large-scale production.

[0008] The biosynthesis of 2'-FL and 3-FL was first achieved through enzyme catalysis. In 2000, Albermann et al. exogenously expressed and purified GDP-mannose-4,6-dehydratase 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.

[0009] Microbial synthesis of fucosyllactose is currently a feasible method for large-scale production. Furthermore, microbially synthesized fucosyllactose is functionally identical to natural fucosyllactose and does not cause adverse reactions in clinical trials. Compared with chemical synthesis, microbial synthesis is safer and faster. Directly utilizing food-safe microbial fucosyllactose can enhance product safety. Therefore, research on GDP-fucose synthase is necessary to improve the efficiency of fucosyllactose synthesis. Summary of the Invention

[0010] The purpose of the invention is to provide a GDP-fucose synthase polypeptide (GDP-L-fucose synthase, WcaG) and a genetically modified cell containing the GDP-fucose synthase polypeptide gene. The GDP-fucose synthase polypeptide and the genetically modified cell thereof can improve the production efficiency of fucosyllactose.

[0011] The GDP-fucose synthase polypeptide described herein, also known as GDP-L-fucose synthase (GDP-mannose-4,6-dehydratase) or GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, catalyzes the conversion of GDP-4-keto-6-deoxymannose to GDP-fucose and belongs to the oxidoreductase family. The GDP-fucose synthase polypeptide is a key enzyme in the synthesis of GDP-fucose, a precursor of fucosyllactose.

[0012] Technical solution:

[0013] The applicant discovered a GDP-fucose synthase from Bacillus subtilis in nature, named BsWcaG, whose amino acid sequence is shown in SEQ ID NO: 1. It has the activity of catalyzing the production of GDP-fucose from GDP-4-keto-6-deoxymannose and can be used in the synthesis of GDP-fucose.

[0014] Comparison with the NCBI database reveals that the amino acid sequence of the GDP-fucose synthase polypeptide shown in SEQ ID NO: 1 is most similar to the GDP-fucose synthase from Escherichia coli (as of November 15, 2023). The amino acid sequences of the two polypeptides share only 64.06% identity, indicating that the amino acid sequence similarity between SEQ ID NO: 1 and known enzymes is not high. The amino acid sequence of the GDP-fucose synthase from Escherichia coli is shown in SEQ ID NO: 2.

[0015] The inventors of this application conducted a series of modifications to the GDP-fucose synthase polypeptide set forth in SEQ ID NO: 1, screening and obtaining a series of derivative peptides A1-A14 with enhanced activity in catalyzing the conversion of GDP-4-keto-6-deoxymannose to GDP-fucose. These derivative peptides A1-A14 are GDP-fucose synthase polypeptides derived from the polypeptide set forth in SEQ ID NO: 1 by replacing one or more amino acid residues or fragments thereof.

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

[0017] Specifically, in a first aspect, the present application provides a series of GDP-fucose synthase polypeptides, wherein the GDP-fucose synthase polypeptides are selected from derivative peptides A1-A14 of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 1. The derivative polypeptides A1-A14 are obtained by modifying at least one of 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 of the amino acid sequence as shown in SEQ ID NO: 1.

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

[0019] 1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid fragment at positions 20-29 is replaced by YEQLKQRGDIEIVA to KAQLEQDGNVELV, thereby obtaining polypeptide A1;

[0020] 2) In the amino acid sequence of polypeptide A1, the amino acid fragment at positions 40-53 is replaced by NLLDADAVRAFFAA to DLLDEKKVKDFFAK, thereby obtaining polypeptide A2;

[0021] 3) In the amino acid sequence of polypeptide A2, the amino acid fragment at positions 75-81 is replaced by NPEGFYT to KPKEFYE, thereby obtaining polypeptide A3;

[0022] 4) In the amino acid sequence of polypeptide A3, the amino acid fragment at position 95 is replaced from L to A, thereby obtaining polypeptide M4;

[0023] 5) In the amino acid sequence of polypeptide A4, the amino acid segment at positions 148-158 is replaced by ADYNNKYGADY to EKYNNEKGLDA, thereby obtaining polypeptide A5;

[0024] 6) In the amino acid sequence of polypeptide A5, the amino acid residue at position 193 is substituted from K to A, thereby obtaining polypeptide A6;

[0025] 7) In the amino acid sequence of polypeptide A6, the amino acid fragment at position 215 is substituted from Y to N, thereby obtaining polypeptide A7;

[0026] 8) In the amino acid sequence of polypeptide A7, the amino acid segment at positions 219-221 is replaced by EAS to DAA, thereby obtaining polypeptide A8;

[0027] 9) In the amino acid sequence of polypeptide A8, the amino acid fragment at positions 227-232 was replaced by QDQAAV to LDEAAR, thereby obtaining polypeptide A9;

[0028] 10) In the amino acid sequence of polypeptide A9, amino acid residues 239-241 are replaced by DNH to TNS, thereby obtaining polypeptide A10;

[0029] 11) In the amino acid sequence of polypeptide A10, amino acid residues 259-264 are substituted from KIAEVV to TIAEVT, thereby obtaining polypeptide A11;

[0030] 12) In the amino acid sequence of polypeptide A11, amino acid residues 301-302 are substituted from IV to LR, thereby obtaining polypeptide A12;

[0031] 13) In the amino acid sequence of polypeptide A12, the amino acid residue at position 306 is substituted from K to A, thereby obtaining polypeptide A13;

[0032] 14) In the amino acid sequence of polypeptide A13, amino acid residues 310-316 were substituted from EVMEKNK to AVMQANL, thereby obtaining polypeptide A14.

[0033] Preferably, the nucleotide sequence of the polypeptide shown in SEQ ID NO: 1 is shown in SEQ ID NO: 9.

[0034] In certain embodiments, the polypeptide having improved catalytic activity for converting GDP-4-keto-6-deoxymannose to GDP-fucose comprises an amino acid sequence having at least 65% or greater identity to SEQ ID NO: 1 and / or the amino acid sequence set forth in any one of polypeptides A1-A14, and these polypeptides comprise certain amino acid substitution mutations that are functionally equivalent to the mutations in the aforementioned schemes 1)-14). In certain embodiments, the polypeptide having improved catalytic activity for converting GDP-4-keto-6-deoxymannose to GDP-fucose comprises an amino acid sequence set forth in any one of polypeptides A1-A14 and is functionally equivalent to the polypeptides having the amino acid sequence set forth in polypeptides A1-A14.

[0035] In a second aspect, the present application provides a polynucleotide encoding the GDP-fucose synthase polypeptide as described in the first aspect above.

[0036] 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. A variety of methods can be used to express expression vectors encoding the polypeptides presented herein. Methods for preparing recombinant nucleic acids, expressing, and isolating expression products are known and described in the Examples.

[0037] In a third aspect, the present application provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above.

[0038] The nucleic acid construct preferably further comprises one or more regulatory sequences operably linked thereto, and the regulatory sequences can direct the production of the polypeptide in an appropriate expression host.

[0039] In a fourth aspect, the present application provides an expression vector comprising the polynucleotide as described in the second aspect above, or comprising the nucleic acid construct as described in the third aspect above.

[0040] The expression vector has a nucleotide or nucleic acid construct according to an embodiment of the present invention operably linked to a regulatory sequence capable of achieving expression of the DNA fragment, such as a promoter region.

[0041] In a fifth aspect, the present application provides a transformed host cell, which is transformed with the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above.

[0042] In some embodiments, the transformed host cell is a cell transformed with the polynucleotide described in the second aspect or the nucleic acid construct described in the third aspect.

[0043] In some embodiments, the host cell includes but is not limited to natural strains or genetically modified strains of bacteria, yeast, mold, etc.; preferably, the host cell includes but is not limited to natural strains or genetically modified strains of Escherichia sp., Bacillus sp., Kluyveromyces sp., etc.;

[0044] Further preferably, the host cell is the genetically engineered bacterium E. coli BL21 (DE3); or other host cells capable of transforming the polynucleotide described in the second aspect, the nucleic acid construct described in the third aspect, or the expression vector described in the fourth aspect.

[0045] In a sixth aspect, the present application provides an enzyme agent or enzyme composition, which comprises the polypeptide as described in the first aspect above.

[0046] Preferably, the enzyme or enzyme composition contains one or more of polypeptides A1-A14.

[0047] Depending on the reaction substrate and product, the enzyme agent or composition may also contain enzymes that are beneficial to the synthesis of fucosyllactose, such as the bifunctional enzyme L-fucose pyrophosphorylase (L fucokinase / GDP-L-fucosepyrophosphorylase, FKP), glucokinase (glucokinase, Glk), mannose-phosphate mutase (mannose-phosphate mutase, ManB), mannose-1-phosphate guanylyltransferase (ManC), GDP-mannose dehydratase (GDP-mannose-4,6-dehydratase, GMD), etc.

[0048] In a seventh aspect, the present application provides a method for producing the polypeptide as described in the first aspect above, comprising:

[0049] (1) culturing a transformed host cell under conditions suitable for expressing the GDP-fucose synthase polypeptide; the transformed host cell is as described in the fifth aspect above; and

[0050] (2) Recovering the GDP-fucose synthase polypeptide.

[0051] In a specific embodiment, the step (1) comprises: first, introducing a nucleic acid construct or a recombinant expression vector encoding the GDP-fucose synthase polypeptide as described in the first aspect above into a host cell to construct an engineered host cell (i.e., a transformed host cell) that expresses the polypeptide; then, culturing the engineered host cell and inducing it to express the GDP-fucose synthase polypeptide.

[0052] In a specific embodiment, the step (2) includes the steps of isolating and purifying the GDP-fucose synthase polypeptide from the culture.

[0053] Can use methods known in the art, in the nutrient medium that is suitable for producing polypeptide, cultivate host cell.For example, can pass through shake flask culture, or in applicable substratum and under the condition of allowing polypeptide expression and / or separation, carry out small-scale or large-scale fermentation (comprising continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) in laboratory or industrial fermentor tank and cultivate cell.Cultivation is to use program known in the art, occurs in applicable nutrient medium, and described substratum comprises carbon and nitrogen source and inorganic salt.Suitable substratum can be purchased through commercial channels, or according to disclosed composition preparation.

[0054] The GDP-fucose synthase polypeptide can be recovered from the culture using methods known in the art. For example, the variant can be recovered from the nutrient medium by a variety of conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In addition, the GDP-fucose synthase polypeptide can be purified by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography), electrophoresis procedures (e.g., preparative isoelectric focusing), differential solubility methods (e.g., ammonium sulfate precipitation), SDS-PAGE, salting out, and the like.

[0055] In an eighth aspect, the present application provides a genetically modified cell comprising a heterologous nucleic acid sequence encoding any one of the polypeptides A1-A14 described in the first aspect.

[0056] Preferably, the genetically modified cells include but are not limited to yeast genetically modified cells, preferably Saccharomyces cerevisiae genetically modified cells and Kluyveromyces genetically modified cells.

[0057] Furthermore, in some embodiments, the genetically modified yeast gene cell, in addition to containing the heterologous nucleic acid encoding the above-mentioned GDP-fucose synthase (GDP-L-fucose synthase, referred to as WCAG), also includes one or more heterologous nucleic acids encoding GDP-mannose dehydratase polypeptide (GDP-mannose dehydratase, referred to as GMD), α-1,2-fucosyltransferase polypeptide (alpha-1,2-fucosyltransferase, referred to as FutC) or α-1,3-fucosyltransferase polypeptide (alpha-1,3-fucosyltransferase, referred to as FutA).

[0058] The α-1,2-fucosyltransferase polypeptide gene may be derived from natural strains or genetically modified strains of Helicobacter pylori, Thermophilic Chlorella, Escherichia coli, Bacucilius cereus, Pseudopedobater saltans, Bacillus fragilis, Bacteroides vulgatus, Bacteroides fragilis, and Bacillus smithii. Preferably, any of the α-1,2-fucosyltransferase polypeptides described in Chinese Patent No. 202311372479.7, entitled "A Fucosyltransferase Polypeptide and Its Application" is used.

[0059] The α-1,3-fucosyltransferase polypeptide gene may be derived from natural cells or genetically modified cells such as Helicobacter pylori, Akkermansia muciniphila, Bacteroides fragilis, Zea mays, and Escherichia coli. Preferably, any of the α-1,3-fucosyltransferase polypeptides described in Chinese Patent No. 202410206745.7, entitled "Fucosyltransferase Polypeptide and Its Application" is used.

[0060] The GDP-mannose dehydratase polypeptide gene source includes, but is not limited to, natural strains or genetically modified strains such as Escherichia coli, Caenorhabditis elegans, Homo sapiens, Arabidopsis, Dictyostelium discoideum, or Bacillus smithii. Preferably, any of the dehydratase polypeptides described in Chinese Patent No. 202311640201.3, entitled "A GDP-mannose dehydratase polypeptide and its application" is used.

[0061] In some embodiments, the genetically modified cell is selected from a genetically modified cell of Saccharomyces cerevisiae, which further comprises a heterologous nucleic acid encoding a lactose permease. The lactose permease is a lactose transporter responsible for transferring lactose from the extracellular to the intracellular. In some embodiments, the lactose permease source includes but is not limited to natural strains or genetically modified strains such as Neurospora crassa, Neofusicoccum parvum, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Helicobacter pylori and Escherichia coli.

[0062] In some embodiments, the genetically modified Saccharomyces cerevisiae cell further comprises a heterologous nucleic acid encoding a transporter polypeptide that exports fucosyllactose, wherein the heterologous nucleic acid encoding the transporter polypeptide is integrated into the genome of the yeast cell.

[0063] In some embodiments, the transporter polypeptide for exporting fucosyllactose from the genetically modified cell of Saccharomyces cerevisiae is a transporter polypeptide for exporting 2'-fucosyllactose or 3-fucosyllactose, and its source includes but is not limited to natural strains or genetically modified strains such as Escherichia coli, Kluyveromyces marxianus, Kluyveromyces lactis, and Neurospora crassa.

[0064] In one embodiment, a preferred technical solution is: the starting strain of the genetically modified cell is Saccharomyces cerevisiae SctgtP8 CCTCC NO: M20231127.

[0065] In certain embodiments, the genetically modified cell is selected from Kluyveromyces genetically modified cells, which also include a deletion or destruction of the β-galactosidase gene (lac4) of the starting strain. Compared with the starting strain, the expression of the β-galactosidase gene in the Kluyveromyces genetically modified cells is reduced, and therefore, the Kluyveromyces genetically modified cells of the present invention reduce the consumption of lactose. When the Kluyveromyces genetically modified cells are Kluyveromyces lactis genetically modified cells, the nucleotide sequence Genbank numbering M84410.1 and the amino acid sequence Genbank numbering AAA35265.1 of the β-galactosidase lac4 gene of the Kluyveromyces genetically modified cells are; when the Kluyveromyces genetically modified cells are Kluyveromyces marxianus genetically modified cells, the nucleotide sequence Genbank numbering XM_022818497.1 and the amino acid sequence Genbank numbering XP_022675157.1 of the β-galactosidase gene lac4 gene of the Kluyveromyces genetically modified cells are

[0066] In one embodiment, a preferred technical solution is: the starting strain of the Kluyveromyces lactis genetically modified cells is K. lactis DSM70799; the starting strain of the Kluyveromyces marxianus genetically modified cells is K. marxianus DMKU3-1042. The starting strains of K. lactis and K. marxianus are readily commercially available.

[0067] The present invention also provides a genetically modified cell containing a molecular marker. The amino acid sequence of the molecular marker is shown in SEQ ID NO: 4. Preferably, the nucleotide sequence of the molecular marker of the genetically modified cell containing the molecular marker is shown in SEQ ID NO: 10.

[0068] Preferably, the present invention also provides a method for preparing genetically modified cells, wherein the method uses yeast as a starting strain and integrates a heterologous α-1,2-fucosyltransferase gene or a heterologous α-1,3-fucosyltransferase gene, a heterologous GDP-fucose synthase gene, and a heterologous GDP-mannitol dehydratase gene into the yeast genome.

[0069] Preferably, the method for preparing the genetically modified cells comprises the following steps:

[0070] (1) Cultivate the starting strain;

[0071] (2) constructing an expression cassette and introducing a heterologous α-1,2-fucosyltransferase gene or α-1,3-fucosyltransferase gene into the starting strain;

[0072] (3) constructing an expression cassette and introducing a heterologous GDP-fucose synthase gene into the starting strain;

[0073] Preferably, the GDP-fucose synthase is selected from any one of A1-A14.

[0074] (4) constructing an expression cassette and introducing a heterologous GDP-mannose dehydratase gene into the starting strain;

[0075] (5) Construct a molecular marker expression cassette and introduce the marker gene into the starting strain.

[0076] Optionally, in the above method for preparing genetically modified cells, the steps are not in any particular order.

[0077] Preferably, the preparation method further comprises the step of (6) recovering the genetically modified cells;

[0078] In one embodiment, the starting strain is Saccharomyces cerevisiae, and the preparation method further comprises:

[0079] (7) constructing an expression cassette and introducing a transporter polypeptide gene for exporting fucosyllactose into the starting strain;

[0080] (8) Construct an expression cassette and introduce a heterologous lactose permease gene into the starting strain.

[0081] In one embodiment, the starting strain is a Kluyveromyces cell, and the preparation method further comprises deleting or disrupting the β-galactosidase gene (lac4).

[0082] Preferably, the genetically modified cells in the preparation method described above include, but are not limited to, yeast genetically modified cells; further preferably, include, but are not limited to, Saccharomyces cerevisiae genetically modified cells, Kluyveromyces lactis genetically modified cells, and Kluyveromyces marxianus genetically modified cells. Further preferably, the genetically modified cells are selected from Saccharomyces cerevisiae CCTCC NO: M20231127 (Saccharomyces cerevisiae SctgtP8), Kluyveromyces lactis DSM70799, and Kluyveromyces marxianus DMKU3-1042.

[0083] In a ninth aspect, the present application provides the use of the GDP-fucose synthase polypeptide described in the first aspect, the transformed host cell described in the fifth aspect, or the enzyme or enzyme composition described in the sixth aspect in synthesizing GDP-fucose and / or fucosyllactose. Preferably, the fucosyllactose includes but is not limited to 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose (DFL), lactose-N-fucopentaose I, and lactose-N-difucohexaose I.

[0084] For such applications, suitable reaction conditions include: the presence of a suitable reaction substrate, a suitable polypeptide, or essential cofactors such as monovalent or divalent ions, a pH value within an appropriate range, a suitable temperature, etc. It is not necessary to meet the optimal value of every factor affecting the polypeptide described in this application, but the reaction conditions must enable the GDP-fucose synthase polypeptide described in this application to exert its enzymatic activity.

[0085] In one embodiment, the method comprises: adding any one of the GDP-fucose synthase polypeptides A1-A14 described herein to a reaction system, which reacts with the substrate GDP-4-keto-6-deoxymannose in the reaction system to generate GDP-fucose.

[0086] The transformed host cell described in the fifth aspect can also synthesize fucosyllactose de novo.

[0087] Optionally, GDP-fucose can react with lactose to synthesize 2'-fucosyllactose or 3-fucosyllactose under the catalysis of α-1,2-fucotransferase or α-1,3-fucotransferase.

[0088] In one embodiment, at least one of the GDP-fucose synthase polypeptides described herein is mixed with GDP-4-keto-6-deoxymannose, NADPH, 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 this period, GDP-4-keto-6-deoxymannose is catalyzed by the GDP-fucose synthase to convert it into GDP-fucose. The 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 GDP-fucose concentration in the solution is determined by HPLC chromatography.

[0089] Alternatively, a GDP-mannose dehydratase polypeptide is added to a reaction system containing GDP-mannose, and contacts the substrate GDP-mannose in the reaction system to synthesize GDP-4-keto-6-deoxymannose; GDP-4-keto-6-deoxymannose is synthesized into the required GDP-fucose under the action of the GDP-fucose synthase described in this application; GDP-fucose reacts with lactose in the reaction system under the catalysis of α-1,2-fucotransferase or α-1,3-fucotransferase to synthesize and obtain the required fucosylated lactose 2'-FL or 3-FL.

[0090] In one embodiment, the method can synthesize 2'-FL or 3-FL using GDP-mannose as a substrate. Specifically, the method includes:

[0091] (1) GDP-mannose dehydratase catalyzes the synthesis of GDP-4-keto-6-deoxymannose from the substrate GDP-mannose;

[0092] (2) Using the polypeptide described in the first aspect or the enzyme or enzyme composition described in the sixth aspect, catalyzing the conversion of the substrate GDP-4-keto-6-deoxymannose into GDP-fucose.

[0093] More preferably, it also includes:

[0094] (3) Under the catalysis of α-1,2-fucotransferase or α-1,3-fucotransferase, GDP-fucose reacts with lactose to synthesize fucosyllactose.

[0095] In the above steps, the reaction product is a solution containing GDP-fucose or fucosyllactose, which can be clarified in a conventional manner. Preferably, the solution containing GDP-fucose or fucosyllactose can be clarified by centrifugation, flocculation, decantation, and / or filtration. 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). During 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, color, 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, etc. GDP-fucose or fucosyllactose is further separated from the reaction solution and further purified using techniques known in the art, such as evaporation, lyophilization, crystallization, precipitation, and / or drying, or spray drying.

[0096] In a tenth aspect, the present application provides the use of the genetically modified cells described in the eighth aspect above in synthesizing fucosyllactose.

[0097] Preferably, the fucosyllactose includes but is not limited to 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose (DFL), lactose-N-fucopentaose I and lactose-N-difucohexaose I.

[0098] For such applications, suitable reaction conditions include the presence of a suitable reaction substrate, a suitable polypeptide, or essential cofactors such as monovalent or divalent ions, a pH value within an appropriate range, a suitable temperature, etc. It is not necessary to meet the optimal value of every factor affecting the polypeptides described herein, but the reaction conditions must enable the GDP-mannose dehydratase polypeptide and GDP-fucose synthase described herein to exert their enzymatic activity.

[0099] In some embodiments, the culture medium can be any culture medium that can maintain the growth and vitality of the genetically modified cells that produce fucosyllactose. In some embodiments, the culture medium can also include appropriate salts, minerals, metals or other nutrients. In some embodiments, the carbon source and nutrients necessary for each cell growth are added to the culture medium in an incremental or continuous manner.

[0100] The present application also provides a method for preparing fucosyllactose, wherein the preparation method is to obtain fucosyllactose by fermentation of one or more of the genetically modified cells described in the present application.

[0101] Preferably, the use of the genetically modified cells in synthesizing fucosyllactose or the method for preparing fucosyllactose as described above is to synthesize fucosyllactose de novo using lactose and one or more of glucose, glycerol, and sucrose as carbon sources.

[0102] The term "polypeptide" refers to any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to short chains (commonly referred to as peptides, oligopeptides, and oligomers) and long chains (commonly referred to as proteins). "Polypeptides" include those modified by natural processes (such as processing and other post-translational modifications), as well as by chemical modification techniques. These modification methods are described in basic textbooks and in more detail in monographs, as well as in a large amount of research literature, and are well known to technicians. Beneficial effects:

[0103] This application provides a series of GDP-fucose synthase polypeptides, including polypeptides A1-A14 derived from the amino acid sequence of the polypeptide set forth in SEQ ID NO: 1. The GDP-fucose synthase is an important enzyme in the biosynthesis of GDP-fucose. The technology of the present invention provides an important biological resource for the bioproduction of GDP-fucose and fucosyllactose. The GDP-fucose synthases described herein often have high catalytic activity, and the specific activity of the enzyme is far higher than that of the prior art. The present invention provides an excellent enzyme resource for the bioproduction of GDP-fucose or fucosyllactose.

[0104] The technical solution of the present application has positive significance for the industrial production of human milk oligosaccharides. The method is green, efficient, and sustainable, is conducive to the application of industrial large-scale production, and has important practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute undue limitations thereon. The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0106] Figure 1. HPLC chromatogram of GDP-fucose standard.

[0107] Figure 2. HPLC spectrum of the reaction solution catalyzed by polypeptide A1 in Example 2.

[0108] Figure 3. HPLC spectrum of the reaction solution catalyzed by polypeptide A10 in Example 2.

[0109] Figure 4. HPLC profile of 2'-FL standard.

[0110] Figure 5. HPLC profile of 3-FL standard.

[0111] Figure 6. HPLC analysis of the fermentation broth of BS-A14 continuous fed-batch synthesis of 2'-fucosyllactose in Example 9.

[0112] Figure 7. HPLC analysis of the fermentation broth of BS-A14-1 continuously fed-batch synthesis of 3-fucosyllactose in Example 9. DETAILED DESCRIPTION

[0113] The experimental methods used in the following examples are conventional methods unless otherwise specified; all materials, reagents, etc. are commercially available unless otherwise specified. The present invention is further described in detail below through examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Modifications or substitutions to the details and forms of the technical solution without departing from the structural concept and scope of use of the present invention fall within the scope of protection of the present invention.

[0114] Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art. In general, the nomenclature used in this specification and the experimental methods described below are well known and commonly used in the art.

[0115] It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments and are not intended to limit the scope of protection of the present invention. In the following embodiments, if no specific experimental conditions are specified, the experimental methods are generally based on conventional molecular biology methods and conditions within the skill of the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual, or follow the conditions recommended by the manufacturer.

[0116] In the following specific embodiments, if the experimental methods for specific conditions are not specified, they are generally based on conventional methods and conditions of molecular biology within the art, and such techniques and conditions are fully explained in the literature; all materials, reagents, etc., unless otherwise specified, can be obtained from commercial channels.

[0117] Explanation of the abbreviations in the examples: GMD: GDP-mannose dehydratase; WCAG: GDP-fucose synthase; FutC: α-1,2 fucosyltransferase polypeptide; FutA: α-1,3 fucosyltransferase polypeptide; lac12: lactose permease; CDT2: transporter polypeptide that exports fucosyllactose.

[0118] Also provided herein is a method for producing fucosyllactose. The method comprises providing a genetically modified yeast cell capable of producing fucosyllactose. The yeast cell comprises a gene for a GDP-fucose synthase polypeptide as described herein. In some embodiments, the method further comprises providing a culture medium and culturing the yeast cell in the culture medium under conditions suitable for the yeast cell to produce fucosyllactose.

[0119] Cultivation can be carried out in suitable containers, including but not limited to carrying out in suitable culture medium in cell culture plates, culture bottles or fermentor tanks. Any suitable fermentor tank can be used, including but not limited to stirred fermentor tanks, airlift fermentor tanks, bubble fermentor tanks or its any combination. In addition, the method can be carried out with the fermentation of any scale known in the art to support the industrial production of microbial products. The materials and methods used for cell culture maintenance or growth are well known to those skilled in the art in microbiology or fermentation science fields.

[0120] In some embodiments, the culture medium comprises lactose and sucrose, or glucose. In some embodiments, the carbon source in the culture medium consists essentially of lactose and sucrose, or glucose. Preferably, in some embodiments, the carbon source in the culture medium consists of lactose and sucrose.

[0121] In the following examples, the enzymes involved are as follows:

[0122] GDP-fucose synthase (WCAG): amino acid sequence as shown in SEQ ID NO: 1, or SEQ ID NO: 2, or polypeptides A1-A14.

[0123] GDP-mannose dehydratase (GMD): The amino acid sequence is shown in SEQ ID NO: 3, which is derived from Escherichia coli. Alternatively, the dehydratase described in Chinese Patent 202311640201.3, entitled "A GDP-mannose dehydratase polypeptide and its application," may be selected.

[0124] α-1,2-fucosyltransferase polypeptide (FutC, alpha-1,2-fucosyltransferase) is recorded in Chinese patent 202311372479.7, the invention name of which is "A fucosyltransferase polypeptide and its application". It is recorded as a mutant in α-1,2-fucosyltransferase polypeptide, and its amino acid sequence is shown in SEQ ID NO: 5, which is an artificial sequence.

[0125] α-1,3-fucosyltransferase polypeptide (FutA) is described in Chinese Patent No. 202410206745.7, entitled “Fucosyltransferase polypeptide and its application”. It is described as a mutant of α-1,3-fucosyltransferase polypeptide, and its amino acid sequence is shown in SEQ ID NO: 6, which is an artificial sequence.

[0126] Lactose permease (lac12) is derived from Kluyveromyces lactis, and its amino acid sequence is shown in SEQ ID NO: 7. Fucosyllactose export transporter polypeptide (CDT2) is derived from Neurospora crassa, and its amino acid sequence is shown in SEQ ID NO: 8.

[0127] 2'-FL and 3-FL standards were produced by ELICITYL, France.

[0128] GDP-fucose standards were purchased from Aladdin.

[0129] GDP-fucose HPLC detection method: C18μ Bondapack column (Waters, Milford, MA; 3.9*300 mm, 10-mm particle size), mobile phase: 0.5 M KH2PO4 in water, flow rate: 1 ml / min, column temperature: 30°C, injection volume: 10 μL, UV detector.

[0130] HPLC detection method for fucosyllactose content: Detection conditions: chromatographic column model: Shodex Asahipak NH2P-504E, mobile phase: 65% acetonitrile aqueous solution, flow rate: 0.5 mL / min, column temperature: 35°C, injection volume: 10 μL, evaporative light detector, evaporation temperature 75°C, and nebulization temperature 45°C.

[0131] Under the above assay conditions, the rt of the GDP-fucose standard was 13.3 min, the rt of the 2'-FL standard was 13.43 min, and the rt of the 3-FL standard was 14.16 min.

[0132] Example 1. Expression of the GDP-fucose synthase polypeptides shown in SEQ ID NO: 1 and SEQ ID NO: 2 and polypeptides A1-A14 in Escherichia coli.

[0133] 1. Using the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 (nucleotide sequence as shown in SEQ ID NO: 9) or the polypeptide with an amino acid sequence as shown in SEQ ID NO: 2 as a template, the polypeptides shown in SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized and finally constructed into the pET32a vector to obtain the pET32a-BsWcaG-wt plasmids and pET32a-EcWcaG-wt.

[0134] 2. Computer-assisted rational design was used to obtain different mutation sites. Referring to the methods in the Molecular Cloning Manual, primers for mutants were designed using Primer-BLAST (Primer designing tool (nih.gov)) based on the sequence of SEQ ID NO: 9 of pET32a-wt. Recombinant plasmid series (pET32a-A1 to pET32a-A14, see Table 1) were constructed using the Fast Mutagenesis Kit.

[0135] 3. Transform the above recombinant plasmids into Escherichia coli BL21 (DE3) according to the following steps:

[0136] Take the prepared Escherichia coli BL21 (DE3) competent cells, place them on ice for 30 minutes to thaw, take 100 μL of competent cells and 10 μL of pET32a-A1 to pET32a-A14 recombinant plasmids (concentration 50 ng / μL), respectively, mix them, place them in a 42°C water bath for 45 seconds, then immediately cool them in an ice bath for 2 minutes, add 1 mL of fresh LB medium (LB medium: 1.0% peptone, 0.5% yeast extract, 1.0% NaCl, and 1.5% agar powder on the plate), and resuscitate and culture at 37°C and 100 rpm for 1 hour. Then, take 100 μL of the bacterial solution and spread it on an LB plate containing ampicillin (100 μg / mL). After culturing in a 37°C constant temperature incubator for 12 hours, pick a single colony for colony PCR to screen positive transformants.

[0137] 4. Culture positive transformants, extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the recombinant plasmids pET32a-A1 to pET32a-A14 are successfully introduced into E. coli.

[0138] 5. Inoculate the correct transformant into LB liquid medium and culture it on a shaker at 37°C and 200 rpm for 12 hours to obtain seed solution. Then, inoculate the seed solution into fresh LB medium at a 1% (v / v) inoculum and culture it at 37°C with shaking until the OD 600 The expression of the culture medium was 0.8, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG) with a final IPTG concentration of 0.1 mmol / L, and induced at 16°C for 12 h at a speed of 200 rpm; after induction of expression, the fermentation broth was centrifuged at 5000 r / min for 30 min at 4°C to collect the bacteria; the bacteria were resuspended in 20 mM pH 7.4 PBS buffer and ultrasonicated at a frequency of plus on 5s / off 5s for 30 min to break the bacteria; the broken liquid was centrifuged at 13000×g and 4°C for 30 min to remove cell debris and collect the supernatant.

[0139] 6. The soluble polypeptide sequence was purified using nickel column affinity chromatography. The process was as follows: deionized water was added to the top of the nickel column. After natural elution, it was eluted with 5 volumes of Binding buffer. The crude enzyme solution filtered through a 0.45 μm filter membrane was then loaded onto the column. The sample was fully bound to the nickel column at a flow rate of 1.5 mL / min. After the sample was dried, it was continuously eluted with 5 column volumes of Washing buffer to remove impurities. Finally, the target protein was eluted with 5 times the volume of Elution buffer and the eluate was collected. Then, the expression of the target protein was analyzed by SDS-PAGE.

[0140] The SDS-PAGE results showed that the genetically engineered bacteria had obvious specific expression bands after induction, and the molecular weight of the bands was basically consistent with the expected molecular weight of 35.3 kDa. Therefore, it can be seen that the polypeptides represented by SEQ ID NO: 1, SEQ ID NO: 2 and polypeptides A1-A14 were obtained. The relevant information is shown in Table 1.

[0141] Table 1. Corresponding numbers of GDP-fucose synthase polypeptides and their amino acid sequences

[0142] Example 2. Determination of the ability of the polypeptides obtained in Example 1 (GDP-fucose synthase polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 2, and polypeptides A1-A14) to catalyze the synthesis of GDP-fucose.

[0143] GDP-fucose was synthesized using GDP-mannose as a substrate, the purified polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2, or one of polypeptides A1-A14, obtained in Example 1, and GDP-mannose dehydratase as an enzyme catalyst. The specific procedure is as follows:

[0144] In order to prepare the substrate GDP-4-keto-6-deoxymannose required for the above polypeptide reaction, first, 5 mM GDP-mannose and 0.2 mg / ml of the GMD enzyme obtained in Example 1, 5 mM NADP + The enzymes were mixed and reacted at 25°C for 8 hours, and the concentration of GDP-4-keto-6-deoxymannose obtained by the reaction was measured.

[0145] Then, 0.2 mg / mL of the purified polypeptides represented by SEQ ID NO: 1, SEQ ID NO: 2, or one of polypeptides A1-A14 obtained in Example 1 were added to 100 μM GDP-4-keto-6-deoxymannose, 50 mM Tris-HCl (pH 7.0), and 1 mM NADPH enzyme at a final concentration, mixed, and reacted at 30° C. for 6 hours. The reaction was terminated and purified using a gel column method.

[0146] The reaction mixture of the catalytic reaction was analyzed using the aforementioned analytical method. HPLC analysis results showed that the reaction mixtures of the polypeptide described in Example 1 all exhibited a strong absorption peak around 13.3 min, which coincided with the peak elution time of a GDP-fucose standard, indicating that the polypeptide-catalyzed reaction in Example 1 produced GDP-fucose.

[0147] The GDP-fucose concentration in the reaction solution was detected by the above-mentioned HPLC detection method, and the results are recorded in Table 2.

[0148] Table 2. Study on the synthesis of GDP-fucose catalyzed by various peptides

[0149] Table 2 Data Description:

[0150] (1) The polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 2, as well as polypeptides A1-A14 described in the present application, have the activity of synthesizing GDP-fucose.

[0151] (2) Compared with the polypeptide shown in SEQ ID NO: 1, the ability of polypeptides A1-A14 to synthesize GDP-fucose was improved, especially A13 and A14, which increased by more than 1.7 times.

[0152] Example 3. Construction of genetically modified Saccharomyces cerevisiae cells.

[0153] (1) Construction of SC-ΔARO3::FutC-EcGMD-WT and SC-ΔARO3::FutA-EcGMD-WT expression cassettes

[0154] The upstream and downstream homology arm sequences aro3-up and aro3-do, the promoter sequence TDH3p and the terminator sequence PGK1t, the promoter sequence TFF1p and the terminator sequence GPMt were amplified using the Saccharomyces cerevisiae genome as a template respectively; the FutC and FutA target gene sequences were amplified using the synthesized FutC and FutA sequences as templates; the GMD target gene sequence was amplified using the synthesized WCAG as a template; referring to the KL-ΔL in the applicant's prior patent (CN202211453172.5, invention name: Recombinant yeast and its application) The AC4 knockout cassette was used as a template to amplify the G418+loxp sequence. Aro3-up, TDH3p, FutC / FutA, PGK1t, TEF1p, GMD, ADH1t, G418+loxp and aro3-do were fused by successive fusion PCR of two fragments. Finally, primers ARO3-F and ARO3-R (Table 3) were used as templates to amplify the fusion PCR system to obtain the SC-ΔARO3::FutC-EcGMD-WT and SC-ΔARO3::FutA-EcGMD-WT expression cassettes.

[0155] Table 3. Primers and sequences

[0156] (2) Construction of SC-ΔTRP3::CDT2-LAC12 expression cassette

[0157] The upstream and downstream homology arm sequences trp3-up and trp3-do of the TRP3 coding region, the promoter sequence TFF1p and the terminator sequence CYC1t, the terminator sequence GPMt and the promoter sequence TPILp were amplified using the Saccharomyces cerevisiae genome as a template, respectively. The CDT2 target gene sequence was amplified using the synthesized CDT2 sequence as a template. The lac12 gene was amplified using the Kluyveromyces lactis genome as a template. The G418+loxp sequence was amplified using SC-ΔARO3::GMD-WCAG as a template. Trp3-up, TFF1p, CDT2, CYC1t, GPMt, lac12, TPILp, G418+loxp and trp3-do were fused by successive fusion PCR of two fragments. Finally, the expression cassette SC-ΔTRP3::CDT2-LAC12 was amplified by PCR using primers TRP3-F and TRP3-R (Table 4) and the fusion PCR system as a template.

[0158] Table 4. Primers and sequences

[0159] (3) Construction of SC-ΔGK::WCAG, SC-ΔGK::WCAG-A1 to SC-ΔGK::WCAG-A14, and SC-ΔGK::ECWCAG expression cassettes

[0160] Using Saccharomyces cerevisiae genomic DNA as a template, PCR amplification was performed to obtain the upstream and downstream homology arms gk-up and gk-down, the promoter sequence CUP1p, and the terminator sequence GPMt. Using the SC-ΔTRP3::CDT2-LAC12 expression cassette as a template, a resistance selection marker containing G418 resistance and loxp sites was amplified. Using PET32a-WCAG, PET32a-WCAG-A1 to PET32a-WCAG-A14, and PET32a-EC WCAG as templates, PCR amplification was performed to obtain the BS WCAG and mutant sequences, as well as the EC WCAG gene sequence; by successive fusion PCR of two fragments, gk-up, CUP1p, BSFWCAG-WT and mutant sequence ECWCAG-WT gene sequences, GPMt, G418+loxp and gk-do were fused, and finally primers GK-F and GK-R (Table 5) were used as templates to amplify the fusion PCR system to obtain the expression cassettes SC-ΔGK::BSWCAG-WT, SC-ΔGK::BSWCAG-A1 to SC-ΔGK::BSWCAG-A14, SC-ΔGK::ECWCAG-WT for the next step of constructing the recombinant strain.

[0161] Table 5. Primers and sequences.

[0162] (4) Transformation of the recombinant expression cassette and verification of the recombinant strain.

[0163] The expression cassettes constructed in (1), (2) and (3) above were respectively transferred into the starting strain Saccharomyces cerevisiae CCTCC NO: M20231127 cells. The specific method is:

[0164] 1) Prepare competent yeast cells: Take a small amount of frozen yeast strain and streak it on a solid culture medium plate, invert and culture at 30℃ for 2 days. Pick a single yeast colony in 50mL liquid culture medium and culture at 30℃, 220rpm until OD 600 The nitric oxide concentration (DNA saturation) should be between 0.8 and 1.5. Collect the cells, wash with 25 mL of sterile water, centrifuge at 1500 × g for 10 min at room temperature, and discard the supernatant. Add 1 mL of 100 mM lithium chloride buffer, resuspend the pellet, centrifuge at 12,000 rpm for 30 s, and discard the supernatant. Add 400 μL of 100 mM lithium chloride buffer again, resuspend the pellet, and obtain competent yeast cells. Aliquot 50 μL / tube for transformation.

[0165] Meanwhile, boil 1 mL of salmon sperm DNA for 5 min and quickly place on ice to prepare single-stranded DNA.

[0166] 2) Transformation: Centrifuge the competent yeast prepared above and remove any residual lithium chloride solution with a tip. For each transformation, add the following solution in the following order: 50% PEG3350 (240 μL); 1 M LiCl (36 μL); 2 mg / mL single-stranded salmon sperm DNA (25 μL); and 5-10 μg / 50 μL plasmid DNA in water (50 μL). Vortex vigorously until the precipitated yeast cells are completely distributed. Incubate in a 30°C waterbath for 30 min. Heat shock the cells in a 42°C waterbath for 20-25 min. Centrifuge at 8000 rpm for 10 min and harvest the yeast cells. Resuspend the yeast in 500 μL of liquid culture medium and incubate on a shaker at 30°C. After 1-4 h, spread 25-100 μL of the culture medium onto selective culture plates and incubate them upside down at 30°C.

[0167] 3) Verification: The correspondence between the recombinant strains and their genotypes is shown in Table 6. To verify the correctness of the above strains, the genomes of the transformants and the original strain were extracted and PCR amplified using primers corresponding to the knockout or expression cassette. If a single band was obtained after PCR amplification and the size was consistent with the knockout or expression cassette, the strain was considered correct; otherwise, the strain was considered a false positive.

[0168] The starting strain of the genetically modified cells is Saccharomyces cerevisiae SctgtP8 (hereinafter referred to as SctgtP8) CCTCC NO: M20231127.

[0169] Table 6

[0170] Molecular markers (amino acid sequence as shown in SEQ ID NO: 4, nucleotide sequence as shown in SEQ ID NO: 10) were introduced into the recombinant strains described in the above table to obtain SC-1-J to SC-4-J, SC-A1-J to SC-A14-J strains, and SC-1-1-J to SC-4-1-J, SC-A1-1-J to SC-A14-1-J strains, respectively.

[0171] Verification of the molecular marker gene: To verify the correctness of the above strains, the genomes of the above transformants and the original strain were extracted and PCR amplified using primers corresponding to the knockout cassette or expression cassette. If a single band with a size consistent with the knockout cassette or expression cassette was obtained after PCR amplification, the strain was considered correct; otherwise, the strain was considered a false positive. For tag verification, PCR amplification was performed using primers and further verified by sequencing; otherwise, the strain was considered a false positive.

[0172] Example 4. Construction of genetically modified Kluyveromyces lactis cells.

[0173] The starting strain of Kluyveromyces lactis engineered bacteria is Kluyveromyces lactis DSM70799.

[0174] (1) Construct an expression cassette for Kluyveromyces lactis gene-modified cells.

[0175] 1) Construction of the KL-ΔLAC4 knockout cassette.

[0176] Referring to the applicant's prior patent CN202211453172.5, invention name: Recombinant yeast and its application, a KL-ΔLAC4 knockout cassette was constructed.

[0177] 2) Construction of KL-ΔLAC4::FutC / KL-ΔLAC4::FutA expression cassette.

[0178] Using the Kluyveromyces lactis genome as a template, the upstream and downstream homology arm sequences lac4 up and lac4-do, the promoter sequence tef1, and the terminator sequence adh1 were amplified; the synthesized FutC and FutA sequences were used as templates to amplify the FutC and FutA target gene sequences; using the expression cassette KL-ΔLAC4 as a template, the G418+loxp sequence was amplified, and lac4 up, tef1, FutC / FutA, adh1, G418+loxp, and lac4-do were fused by successive fusion PCR of two fragments. Finally, the expression cassette KL-ΔLAC4::FutC / KL-ΔLAC4::FutA was amplified by PCR using the primers lac4-upF and lac4-doR (Table 7) and the fusion PCR system as a template.

[0179] Table 7

[0180] 3) Construction of the KL-ΔXK::GMD expression cassette.

[0181] Using the Kluyveromyces lactis genome as a template, the upstream and downstream homology arm sequences xk-up and xk-do of the xylulose kinase XK coding region, the promoter sequence pgk1, and the terminator sequence tdh3 were amplified. Using the synthetic GMD sequence as a template, the GMD target gene sequence was amplified. Using KL-ΔLAC4 as a template, the G418+loxp sequence was amplified. By sequential fusion PCR of two fragments, xyl1 up, pgk1, gmd, tdh3, G418+loxp, and xyl1-do were fused. Finally, primers xK-upF and xK-doR (Table 8) were used as a template to amplify the gmd expression cassette KL-ΔXK::GMD using the fusion PCR system as a template.

[0182] Table 8

[0183] 4) Construction of KL-ΔGK::BSWCAG-WT, KL-ΔGK::BSWCAG-A1 to KL-ΔGK::BSWCAG-A14, KL-ΔGK::ECWCAG-WT expression cassettes.

[0184] Using the genomic DNA of Kluyveromyces lactis DSM70799 as a template, PCR amplification was performed with primers galkup-F and galkup-R as well as galk down-F and galk down-R to obtain the upstream and downstream homology arms, respectively; using loxp-F and loxp-R as primers and KL-ΔLAC4 as a template, a resistance selection marker containing G418 resistance and loxp sites was amplified; using PET32a-BSWCAG-WT, PET32a-A1 to PET32a-A14 and PET32a-ECWCAG-WT as templates, the original sequence and mutant sequence of the GMD gene were amplified by PCR; using the genome of Kluyveromyces lactis as a template, the promoter tdh3 and terminator sequence tef of the transferase were amplified by PCR, respectively; using primers KL-PF and KL-PR and the PUC19 plasmid as a template, the plasmid vector sequence was amplified by PCR. The upstream homology arm, promoter, target gene (original sequence and mutant sequence), terminator, G418+loxp and downstream homology arm were fused PCR to obtain ΔGK::BSWCAG-WT, ΔGK::BSWCAG-A1 to ΔGK::BSWCAG-A14 expression cassettes, and ΔGK::ECWCAG-WT; the expression cassettes were connected to the plasmid vector sequence by ABclonal to obtain plasmids PUC-KL-WT, PUC-KL-A1 to PUC-KL-A14, and PUC-KL-ECWCAG-WT carrying the ΔGK::BSWCAG-WT, ΔGK::ECWCAG-WT, and ΔGK::BSWCAG-A1 to ΔGK::BSWCAG-A14 expression cassettes, respectively.

[0185] Using primers galkup-F and galkdown-R as templates (Table 9), PUC-KL-WT, PUC-KL-A1 to PUC-KL-A14, and PUC-KL-ECWCAG-WT as templates, KL-ΔGK::BSWCAG-WT, KL-ΔGK::BSWCAG-A1 to KL-ΔGK::BSWCAG-A14 expression cassettes, and KL-ΔGK::ECWCAG-WT were amplified for the next step of constructing recombinant strains.

[0186] Table 9. Primers and sequences

[0187] (2) Transformation of the recombinant expression cassette and verification of the recombinant strain.

[0188] The expression cassettes constructed in (1) above were respectively transferred into the starting strain Kluyveromyces lactis DSM70799 cells. The specific method is:

[0189] 1) Prepare competent yeast cells: Take a small amount of frozen yeast strain and streak it on a solid culture medium plate, invert and culture at 30℃ for 2 days. Pick a single yeast colony in 50mL liquid culture medium and culture at 30℃, 220rpm until OD 600 The nitric oxide concentration (DNA saturation) should be between 0.8 and 1.5. Collect the cells, wash with 25 mL of sterile water, centrifuge at 1500 × g for 10 min at room temperature, and discard the supernatant. Add 1 mL of 100 mM lithium chloride buffer, resuspend the pellet, centrifuge at 12,000 rpm for 30 s, and discard the supernatant. Add 400 μL of 100 mM lithium chloride buffer again, resuspend the pellet, and obtain competent yeast cells. Aliquot 50 μL / tube for transformation.

[0190] Meanwhile, boil 1 mL of salmon sperm DNA for 5 min and quickly place on ice to prepare single-stranded DNA.

[0191] 2) Transformation: Centrifuge the competent yeast prepared above and remove any residual lithium chloride solution with a tip. For each transformation, add the following solution in the following order: 50% PEG3350 (240 μL); 1 M LiCl (36 μL); 2 mg / mL single-stranded salmon sperm DNA (25 μL); and 5-10 μg / 50 μL plasmid DNA in water (50 μL). Vortex vigorously until the precipitated yeast cells are completely distributed. Incubate in a 30°C waterbath for 30 min. Heat shock the cells in a 42°C waterbath for 20-25 min. Centrifuge at 8000 rpm for 10 min and harvest the yeast cells. Resuspend the yeast in 500 μL of liquid culture medium and incubate on a shaker at 30°C. After 1-4 h, spread 25-100 μL of the culture medium onto selective culture plates and incubate them upside down at 30°C.

[0192] 3) Verification: The correspondence between the recombinant strains and their genotypes is shown in Table 10. To verify the correctness of the above strains, the genomes of the transformants and the original strain were extracted and PCR amplified using primers corresponding to the knockout or expression cassette. If a single band was obtained after PCR amplification and the size was consistent with the knockout or expression cassette, the strain was considered correct; otherwise, the strain was considered a false positive.

[0193] Table 10. Engineered Kluyveromyces lactis strains and their genotypes.

[0194] Molecular markers (amino acid sequence as shown in SEQ ID NO: 4, nucleotide sequence as shown in SEQ ID NO: 10) were introduced into the recombinant strains described in the above table to obtain KL-1-J to KL-4-J, KL-A1-J to KL-A14-J strains, and KL-1-1-J to KL-4-1-J, KL-A1-1-J to KL-A14-1-J strains, respectively.

[0195] Example 5. Construction of genetically modified Kluyveromyces marxianus cells

[0196] The starting strain was Kluyveromyces marxianus DMKU3-1042.

[0197] (1) Construct a Kluyveromycin-McK2 gene-modified cell expression cassette.

[0198] 1) Construction of the KM-ΔLAC4 knockout cassette.

[0199] Referring to the applicant's prior patent CN202211453172.5, invention name: Recombinant yeast and its application, a KM-ΔLAC4 knockout cassette was constructed.

[0200] 2) Construction of KM-ΔLAC4::FutC / KM-ΔLAC4::FutA expression cassette.

[0201] Using the Kluyveromyces marxianus genome as a template, the upstream and downstream homology arm sequences km-lac4 up and km-lac4-do were amplified. Using the KL-ΔLAC4::FutC / KL-ΔLAC4::FutA expression cassette as a template, the FutC and FutA expression cassettes and the G418+loxp site were amplified. By three-fragment fusion PCR, km-lac4 up, FutC or FutA expression cassette + G418+loxp and km-lac4-do were fused. Finally, primers lac4-upF1 and lac4-doR1 (Table 11) were used with the fusion PCR system as a template to obtain the FutC and FutA expression cassettes KM-ΔLAC4::FutC and KM-ΔLAC4::FutA.

[0202] Table 11

[0203] 3) Construction of the KM-ΔXK::GMD expression cassette.

[0204] Using the Kluyveromyces marxianus genome as a template, the upstream and downstream homology arm sequences km-xk up and km-xk-do of the Kluyveromyces marxianus xylulose kinase were amplified. Using the KL-ΔXK::GMD expression cassette as a template, the GMD expression cassette and G418+loxp site were amplified. By three-fragment fusion PCR, km-xkup, GMD expression cassette+G418+loxp and km-xk-do were fused. Finally, primers xk-upF1 and xk-doR1 (Table 12) were used with the fusion PCR system as a template to obtain the gmd expression cassette KM-ΔXK::GMD by PCR amplification.

[0205] Table 12

[0206] 4) Construction of KM-ΔGK::BSWCAG-WT, KM-ΔGK::BSWCAG-A1, KM-ΔGK::BSWCAG-A14, and KM-ΔGK::ECWCAG-WT expression cassettes.

[0207] Using Kluyveromyces marxianus DMKU3-1042 genomic DNA as a template, primers galk up-F1 and galk up-R1 as well as galk down-F1 and galk down-R1 (Table 13) were used for PCR amplification to obtain the upstream and downstream homology arms, respectively; using KL-ΔGK::BSWCAG-wt, KL-ΔGK::BSWCAG-A1 to KL-ΔGK::BSWCAG-A14 expression cassettes, and KL-ΔGK::ECWCAG-wt expression cassette as templates, PCR amplified the BSWCAG-wt / WCAG mutant expression cassette / ECWCAG+G418+loxp sequence, and fused the upstream and downstream homology arms and the BSWCAG-wt / BSWCAG mutant expression cassette / ECWCAG expression cassette+G418+loxp sequence using the fusion PCR method, respectively; using the fusion system as a template, primers galk up-F1 and galk up-R1 were used to amplify the upstream and downstream homology arms, respectively; Down-R1 was used as a template to amplify the KM-ΔGK::BSWCAG-WT, KM-ΔGK::BSWCAG-A1 to KM-ΔGK::BSWCAG-A14, and KM-ΔGK::ECWCAG-WT expression cassettes for the next step of constructing recombinant strains.

[0208] Table 13. Primers and sequences

[0209] (2) Transformation of recombinant plasmid and verification of recombinant strain:

[0210] The expression cassette constructed in (1) above was transformed into the starting strain K. marxianus DMKU3-1042 cells. The specific method is as described in Example 2, Part (2), Transformation of the recombinant expression cassette and Verification of the recombinant strain.

[0211] The correspondence between the recombinant strains and their genotypes is shown in Table 14. To verify the correctness of the above strains, the genomes of the transformants and the original strain were extracted and PCR amplified using primers corresponding to the knockout or expression cassette. If a single band was obtained after PCR amplification and the size was consistent with the knockout or expression cassette, the strain was considered correct; otherwise, the strain was considered a false positive.

[0212] Table 14

[0213] Molecular markers (amino acid sequence as shown in SEQ ID NO: 4, nucleotide sequence as shown in SEQ ID NO: 10) were introduced into the recombinant strains described in the above table to obtain strains KM-1-J to KM-4-J, KM-A1-J to KM-A14-J, and KM-1-1-J to KM-4-1-J, KM-A1-1-J to KM-A14-1-J, respectively.

[0214] Example 6. The genetically modified cells obtained in Examples 3-5 were fermented to synthesize fucosyllactose.

[0215] Yeast was cultured with glucose as the carbon source, and the yeast gene-modified cells obtained in Examples 3-5 were taken respectively, and the yeast gene-modified cells were allowed to grow rapidly to the end of the logarithmic phase or the stationary phase. The strains were streaked and cultured in solid culture media such as YPD, and after culturing at 30°C for 2-3 days, single colonies were picked and inoculated into 1.5mL YPD liquid culture medium, and cultured at 30°C and 200rpm overnight. Subsequently, 2% of the inoculation amount was inoculated into 50mL liquid culture medium shake flasks, and cultured at 30°C and 200rpm until the OD 600 = 1, lactose, 3% (w / v) sucrose, and 0.1 mM CuSO4 were added to a final concentration of 10 g / L, and the culture was shaken at 30°C and 200 rpm for a total fermentation time of 72 hours. After 72 hours of fermentation, samples were taken and boiled for 10 minutes. The final yeast fermentation product was obtained by centrifugation, and the 2'-FL and 3-FL contents in the supernatant were assayed.

[0216] HPLC detection method: Detection conditions: chromatographic column model: Shodex Asahipak NH2P-504E, mobile phase: 65% acetonitrile in water, flow rate: 0.5 mL / min, column temperature: 35°C, injection volume: 10 μL, evaporative light detector, evaporation temperature 75°C, and nebulization temperature 45°C.

[0217] LC-MS analysis conditions are as follows:

[0218] Chromatographic column model: Shodex Asahipak NH2P-504E, detector: UV detector (Hitachi Chromaster), detection wavelength: 210 nm, injection volume: 10 μL, flow rate: 0.5 mL / min, column temperature: 35°C, mobile phase: acetonitrile:water = 65:35; ESI-MS mode, molecular weight scan range: 100-800.

[0219] The standard product and the reaction solution were tested according to the above analytical method. The HPLC analysis results showed:

[0220] (1) The peak time rt of 2'-FL standard is 13.43 min; the HPLC analysis spectrum of 3-FL standard is rt 14.16 min.

[0221] (2) In the fermentation broth of the 2'-FL-producing yeast gene-modified cells obtained in Example 3-5, a strong absorption peak appeared at around 13.43 min, which was consistent with the peak time of the 2'-FL standard. This indicates that 2'-FL was produced in the fermentation broth of the gene-modified cells obtained in Example 1-3. The yield is detailed in Tables 13-15.

[0222] (3) In the fermentation broth of the 3-FL-producing yeast genetically modified cells obtained in Example 3-5, a strong absorption peak appeared near 14.16 min, which was consistent with the peak time of the 3-FL standard. This indicates that 3-FL was produced in the fermentation broth of the genetically modified cells obtained in Example 1-3. The yield is detailed in Tables 15-17.

[0223] The LC-MS analysis conditions are shown above, and the analysis results show:

[0224] 2'-FL and 3-FL are compounds with the same molecular formula but different structures, and are called isomers. They have the same molecular weight.

[0225] The products of the reaction solution with HPLC chromatographic peaks near rt = 13.43 min and rt = 14.16 min were analyzed by LC-MS, and the MH value was 487.17, which was consistent with the mass spectrum results of the 2'-FL and 3-FL standards shown, and was within the allowable error range with the theoretical molecular weight of 2'-FL and 3-FL of 488.44.

[0226] The yields of 2'-FL and 3-FL in the fermentation broth were detected by the above-mentioned HPLC analysis method, and the results are recorded in Tables 13-15.

[0227] Table 15

[0228] Fermentation experiments also found that the ability of strains SC-1-J to SC-4-J and SC-A1-J to SC-A14-J to synthesize 2'-FL was comparable to that of the corresponding strains before the addition of the marker gene; similarly, the ability of strains SC-1-1-J to SC-4-1-J and SC-A1-1-J to SC-A14-1-J to synthesize 3-FL was comparable to that of the corresponding strains before the addition of the marker gene.

[0229] Table 16

[0230] Fermentation experiments found that the ability of strains KL-1-J to KL-7-J and KL-A1-J to KL-A14-J to synthesize 2'-FL was comparable to that of the corresponding strains before the addition of the marker gene; similarly, the ability of strains KL-1-1-J to KL-7-1-J and KL-A1-1-J to KL-A14-1-J to synthesize 3-FL was comparable to that of the corresponding strains before the addition of the marker gene.

[0231] Table 17

[0232] In addition, fermentation experiments found that the ability of strains KM-1-J to KL-4-J and KM-A1-J to KM-A14-J to synthesize 2'-FL was comparable to that of the corresponding strains before the addition of the marker gene; the ability of strains KM-1-1-J to KM-4-1-J and KM-A1-1-J to KM-A14-1-J to synthesize 3-FL was comparable to that of the corresponding strains before the addition of the marker gene.

[0233] Example 7. Genetically modified cells SC-A12-J, SC-A12-1-J, KL-A12-J, KL-A12-1-J, KM-A12-J, and KM-A12-1-J were taken and streaked on YPD solid medium. After culturing at 30°C for 2-3 days, single colonies were picked and inoculated into 1.5 mL YPD liquid medium and cultured at 30°C and 200 rpm overnight. Subsequently, 2% of the inoculum was inoculated into 50 mL liquid medium shake flasks and cultured at 30°C and 200 rpm until the OD 600= 1, and inoculated at a 2% inoculum into 1 L of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) (3 L fermentor). After 6-7 hours of culture, CuSO4 was added to control the final Cu ion concentration to approximately 0.1 mM. Sucrose (50% mother liquor concentration) was added at a rate of 8 mL / h. Lactose (40% mother liquor concentration) was also added to maintain a final lactose concentration of 15 g / 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 disrupted using a high-pressure homogenizer, boiled, and centrifuged to remove protein. The supernatants were combined to obtain the final yeast fermentation product. The 2'-FL and 3-FL contents in the supernatants were assayed. The results are recorded in Table 18.

[0234] Table 18

[0235] Although the present invention has been described in considerable detail by way of illustration and example for the purpose of clear understanding, it will be clear to those skilled in the art that any modification of the polypeptide equivalents or the recombination of genetically modified cells can be implemented. Therefore, this specification and examples should not be interpreted as limiting the scope of the invention.

[0236] Example 8. Synthesis of 2'-fucosyllactose using Escherichia coli BL21 as a chassis.

[0237] Reference 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.) The strain BL21ΔlacZΔlonΔwcaJ was constructed, and the above-mentioned constructs PET32a-A1-HpFutC, PET32a-A14-HpFutC, PET32a-A1-HpFutA and PET32a-A14-HpFutA were respectively transformed into the Escherichia coli chassis cells BL21ΔlacZΔlonΔwcaJ by electroporation, thereby constructing strains EC-A1, EC-A14, EC-A1-1 and EC-A-1.

[0238] Method for culturing Escherichia coli EC-A1 and EC-A14 to synthesize 2'-fucosyllactose:

[0239] E. coli strains EC-A1, EC-A14, EC-A1-1, and EC-A-1 were cultured on LB solid medium at 37°C for 10-12 hours, and a single colony was inoculated into 20 mL of liquid LB and cultured in a 250 mL shake flask at 37°C and 220 rpm for 10-12 hours. Each strain was inoculated into 5 mL of LB, and when the strain reached stationary phase, 1 mL of culture was inoculated into 100 mL of LB medium containing 36 g / L glucose (or glycerol) as a carbon source for growth in a 500 mL shake flask. When the OD 600 When the pH reached approximately 0.6, 0.1 mM IPTG was added at 25°C for induction. After 2 hours and 10 hours of additional culture, 5 g / L lactose was added to supplement the culture medium for fucosyllactose production. At the same time, ampicillin was supplemented to a final concentration of 100 μg / mL. After 10 minutes of induction, the culture medium was boiled and centrifuged for 10 minutes. The supernatant was then collected and analyzed for 2'-FL production. The results are recorded in Table 19.

[0240] Table 19

[0241] Example 9. Synthesis of 2'-fucosyllactose using Bacillus sp. BStgtP8 (CCTCC NO: M 20231126) as a chassis.

[0242] The Bacillus subtilis chassis cells were constructed according 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 cell factories, 21(1), 110.) to construct the Bacillus subtilis chassis cells BSP43-manB-P43-manC-P43-HpFutC-P43-GMD-P43-lacY and BSP43-manB-P43-manC-P43-HpFutA-P43-GMD-P43-lacY. At the same time, the P43-A1 and P43-A14 expression cassettes were constructed and transferred into BSP43-manB-P43-manC-P43--HpFutC-P43-GMD-P43-lacY and BSP43-manB-P43-manC-P43-HpFutA-P43-GMD-P43-lacY and inserted into the manP gene, respectively, thereby constructing the BS-A1, BS-A14, BS-A1-1 and BS-A14-1 strains.

[0243] Method for culturing strains BS-A1, BS-A14, BS-A1-1 and BS-A14-1 to synthesize 2'-fucosyllactose:

[0244] Bacillus subtilis strains BS-A1, BS-A14, BS-A1-1, and BS-A14-1 were grown on LB solid medium at 37°C for 10–12 hours. A single colony was inoculated into 20 mL of liquid LB in a 250 mL shake flask and incubated at 37°C and 220 rpm for 10–12 hours. The Te seed culture was further inoculated into 30 mL of fermentation medium at a rate of 10% and incubated in a 250 mL shake flask at 37°C and 220 rpm for 72 hours. During shake flask fermentation, the following culture medium was used: 6 g / L trypsin, 12 g / L yeast extract, 12.5 g / L K₂HPO₄·3H₂O, 2.5 g / L KH₂PO₄, and 10 mL / L trace metal solution (composed of 4 g / L FeSO₄·7H₂O, 4 g / L CaCl₂, 1 g / L MnSO₄·H₂O, 0.2 g / L NaMoO₄·2H₂O, 0.2 g / L ZnSO₄·7H₂O, 0.1 g / L AlCl₃·6H₂O, 0.1 g / L CuCl₂·2H₂O, and 0.05 g / L H₃BO₄). Sterilized sucrose and lactose were added to the sterilized shake flasks to final concentrations of 20 and 10 g / L, respectively. Following fermentation, the culture was boiled for 10 minutes, and the supernatant was centrifuged and analyzed for 2'-FL production. The results are reported in Table 20.

[0245] Table 20

[0246] The above process was scaled up to a 1L fermenter using continuous fed-batch fermentation. Sucrose concentration in the fermentation broth was controlled between 17g / L and 22g / L, and lactose concentration was maintained between 10g / L and 20g / L. Fermentation was terminated after 30 hours of continuous feeding. The culture broth was boiled for 10 minutes, and the supernatant was centrifuged and analyzed for 2'-FL content. The results are recorded in Table 21.

[0247] Table 21

[0248] The present invention discloses a series of GDP-fucose synthase polypeptides, their preparation methods and applications, DNA molecules encoding the polypeptides, vectors, and host cells. Those skilled in the art can refer to the content of the present invention and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0249] Although the present invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that any equivalent aspects or modifications may be implemented. Therefore, the present description and examples should not be construed as limiting the scope of the invention.

Claims

1. A GDP-fucose synthase polypeptide, characterized in that The amino acid sequence of SEQ ID NO: 1 is modified by modifying at least one of 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; Preferably, the modification is a mutation, and the mutation is selected from any one of the following: 1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid fragment at positions 20-29 is replaced by YEQLKQRGDIEIVA to KAQLEQDGNVELV to obtain polypeptide A1; 2) In the amino acid sequence of polypeptide A1, the amino acid fragment at positions 40-53 was replaced by NLLDADAVRAFFAA to DLLDEKKVKDFFAK to obtain polypeptide A2; 3) In the amino acid sequence of polypeptide A2, the amino acid fragment at positions 75-81 is replaced by NPEGFYT to KPKEFYE to obtain polypeptide A3; 4) In the amino acid sequence of polypeptide A3, the amino acid fragment at position 95 is replaced from L to A to obtain polypeptide A4; 5) In the amino acid sequence of polypeptide A4, the amino acid fragment at positions 148-158 is replaced by ADYNNKYGADY to EKYNNEKGLDA to obtain polypeptide A5; 6) In the amino acid sequence of polypeptide A5, the amino acid residue at position 193 is replaced from K to A to obtain polypeptide A6; 7) In the amino acid sequence of polypeptide A6, the amino acid fragment at position 215 is replaced from Y to N to obtain polypeptide A7; 8) In the amino acid sequence of polypeptide A7, the amino acid fragment at positions 219-221 is replaced by EAS to DAA to obtain polypeptide A8; 9) In the amino acid sequence of polypeptide A8, the amino acid fragment at positions 227-232 was replaced by QDQAAV to LDEAAR to obtain polypeptide A9; 10) In the amino acid sequence of polypeptide A9, the amino acid residues at positions 239-241 are replaced by DNH to TNS to obtain polypeptide A10; 11) In the amino acid sequence of polypeptide A10, the amino acid residues at positions 259-264 are replaced by KIAEVV to TIAEVT to obtain polypeptide A11; 12) In the amino acid sequence of polypeptide A11, the amino acid residues at positions 301-302 are replaced by IV to LR to obtain polypeptide A12; 13) In the amino acid sequence of polypeptide A12, the amino acid residue at position 306 is replaced from K to A to obtain polypeptide A13; 14) In the amino acid sequence of polypeptide A13, the amino acid residues at positions 310 to 316 were replaced by EVMEKNK to AVAQANL to obtain polypeptide A14.

2. A polynucleotide encoding the GDP-fucose synthase polypeptide according to claim 1.

3. A nucleic acid construct comprising the polynucleotide according to claim 2; The nucleic acid construct preferably further comprises one or more regulatory sequences operably linked thereto, and the regulatory sequences can direct the production of the polypeptide in a suitable host cell for expression. 4 . An expression vector comprising the polynucleotide according to claim 2 , or comprising the nucleic acid construct according to claim 3 .

5. A transformed host cell transformed with the polynucleotide according to claim 2, the nucleic acid construct according to claim 3, or the expression vector according to claim 4.

6. The transformed host cell according to claim 5, characterized in that It is selected from natural strains of bacteria, yeasts and molds or genetically modified strains thereof; Further preferably, it is selected from natural strains of Escherichia sp., Bacillus sp. and Kluyveromyces sp. or genetically modified strains thereof; Further preferably, it is selected from natural strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, Kluyveromyces lactis and Yarrowia lipolytica or genetically modified strains thereof; More preferably, it is genetically engineered Escherichia coli BL21 (DE3). 7 . An enzyme agent or enzyme composition comprising at least one GDP-fucose synthase polypeptide according to claim 1 .

8. The method for producing the GDP-fucose synthase polypeptide according to claim 1, characterized in that: The steps include: (1) culturing the transformed host cell to express the GDP-fucose synthase; and (2) recovering the polypeptide; The transformed host cell is as described in claim 5 or 6; Preferably, the step (2) includes the steps of separating and purifying the polypeptide from the culture.

9. A genetically modified cell comprising a heterologous nucleic acid sequence encoding the GDP-fucose synthase polypeptide of claim 1; wherein: The GDP-fucose synthase polypeptide is at least one of polypeptides A1 to A14; Preferably, the genetically modified cells are selected from yeast genetically modified cells, Preferably, the yeast genetically modified cells are selected from the group consisting of Saccharomyces cerevisiae genetically modified cells, Preferably, the yeast genetically modified cells are selected from Kluyveromyces lactis genetically modified cells and Kluyveromyces marxianus genetically modified cells.

10. Use of the GDP-fucose synthase polypeptide according to claim 1, or the transformed host cell according to claim 5 or 6, or the enzyme agent or enzyme composition according to claim 7, or the genetically modified cell according to claim 9 in synthesizing fucosyllactose; Preferably, the fucosyllactose is selected from 2'-fucosyllactose and 3-fucosyllactose.

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