Gdp-mannose dehydratase polypeptides and application thereof

Abstract

Provided are a series of GDP-mannose dehydratase polypeptides, which comprise derivative polypeptides M1-M13 of a polypeptide having an amino acid sequence as represented by SEQ ID NO: 1, and which belong to the technical field of biological genetic engineering. Further provided are gene-modified cells containing heterologous nucleic acids of polypeptides M1-M13. The GDP-mannose dehydratase has relatively high catalytic activity, and the gene-modified cells have activity of de novo synthesis of fucosyllactose. The present technical solution has positive significance for the industrialized production of human milk oligosaccharides. and a method of the present application is eco-friendly, highly efficient, and sustainable, and facilitates being applied to industrialized large-scale production.

Description

A GDP-mannose dehydratase polypeptide and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application No. 202311640201.3, filed with the State Intellectual Property Office of China on November 30, 2023, entitled “A GDP-mannose dehydratase polypeptide and its application”, and claims priority to Chinese patent application No. 202311694307.1, filed with the State Intellectual Property Office of China on December 8, 2023, entitled “Kluyveromyces genetically modified cells and their applications”, and claims priority to Chinese patent application No. 202311740753.1, filed with the State Intellectual Property Office of China on December 15, 2023, entitled “Saccharomyces cerevisiae genetically modified cells”, 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-mannose dehydratase polypeptides, which can be applied to the synthesis of GDP-fucose or human milk oligosaccharides and belong to the field of enzyme 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. Fucosyllactose is a key component of HMOs, particularly 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL). These components have been shown to play a significant role in the brain, intestinal tract, and growth and development of infants. 2'-FL and 3-FL have been approved by the US FDA, the EU, and other countries and regions as ingredients in infant formula and can be added to infant formula, regular foods, dietary supplements, and / or medical foods.

[0006] Fucosyllactose is typically synthesized chemically, enzymatically, or in microbial cell factories. While chemical synthesis has made significant progress, the cumbersome process requires repeated protection and deprotection of the fucosyllactose molecule, resulting in low product yields and high production costs. Microbial synthesis of fucosyllactose is currently a feasible method for large-scale production, and microbially synthesized fucosyllactose is functionally identical to natural fucosyllactose. In 2000, Albermann et al. exogenously expressed and purified GDP-mannose-4,6-dehydratase (GMD) and GDP-fucose synthase (WcaG) from Escherichia coli K12. They subsequently converted GDP-mannose to GDP-fucose in vitro, which was then catalyzed by fucotransferase to synthesize fucosyllactose. GDP-fucose is a key precursor for the synthesis of fucosyllactose. They also discovered that GMD activity can be inhibited by GDP-fucose, indicating that GMD is a key rate-limiting enzyme. Therefore, it is necessary to study the GMD enzyme (GDP-mannose-4,6-dehydratase) to improve the efficiency and conversion of fucosyllactose synthesis. Modification of key enzymes is crucial for improving their catalytic efficiency. Summary of the Invention

[0007] The present invention aims to provide a GDP-mannose dehydratase polypeptide (GDP-mannose dehydratase, GMD) and a genetically modified cell containing the GDP-mannose dehydratase polypeptide gene. The GDP-mannose dehydratase polypeptide and the genetically modified cell thereof can improve the production efficiency of fucosyllactose.

[0008] The GDP-mannose dehydratase polypeptide, also known as GDP-D-mannose-4,6-dehydratase or GDP-mannose-6-dehydrogenase, catalyzes the conversion of GDP-mannose to GDP-4-keto-6-deoxymannose. As a key enzyme in GDP-fucose synthesis, the GDP-mannose dehydratase polypeptide also participates in the metabolism of fructose and mannose.

[0009] Technical solution:

[0010] In the first aspect, the applicant discovered a GDP-mannose dehydratase (BsGMD) from the natural bacterium Bacillus smithii. The enzyme, named BsGMD, has an amino acid sequence as shown in SEQ ID NO:1. BsGMD catalyzes the conversion of GDP-mannose to GDP-4-keto-6-deoxymannose and can be used in the synthesis of GDP-fucose. Preferably, the nucleotide sequence of the GDP-mannose dehydratase shown in SEQ ID NO:1 is shown in SEQ ID NO:6.

[0011] Comparison with the NCBI database reveals that the amino acid sequence of the GDP-mannose dehydratase shown in SEQ ID NO: 1 is most similar to the GDP-mannose dehydratase from Escherichia coli (as of November 15, 2023). The amino acid sequences of the two enzymes share only 69.03% 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-mannose dehydratase from Escherichia coli is shown in SEQ ID NO: 2.

[0012] The inventors of the present application have conducted a series of modifications to the GDP-mannose dehydratase polypeptide with the amino acid sequence set forth in SEQ ID NO: 1, screening and obtaining a series of derivative peptides M1-M13 with enhanced activity in catalyzing the conversion of GDP-mannose to GDP-4-keto-6-deoxymannose. These derivative peptides M1-M13 are dehydratase polypeptides derived from the polypeptide with the amino acid sequence set forth in SEQ ID NO: 1 by replacing one or more amino acid residues or fragments.

[0013] Specifically, the GDP-mannose dehydratase derivative polypeptides M1-M13 described in the present application are obtained by modifying the amino acid sequence shown in SEQ ID NO: 1 at least one of positions 69-72, 118-119, 123-124, 176, 203-205, 213-217, 257-264, 273-276, 288, 304-312, 339-343, and 365.

[0014] Preferably, the modification is a mutation, and the mutation is selected from any one of the following:

[0015] 1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid fragment at positions 69-72 is replaced by EKIK with ASIR, thereby obtaining polypeptide M1;

[0016] 2) In the amino acid sequence of polypeptide M1, the amino acid fragment at positions 118-119 is replaced from IE to LD, thereby obtaining polypeptide M2;

[0017] 3) In the amino acid sequence of polypeptide M2, the amino acid fragment at positions 123-124 is replaced by QD to EK, thereby obtaining polypeptide M3;

[0018] 4) In the amino acid sequence of polypeptide M3, the amino acid fragment at position 176 is substituted from N to D, thereby obtaining polypeptide M4;

[0019] 5) In the amino acid sequence of polypeptide M4, the amino acid fragment at positions 203-205 is replaced by RDI with IQL, thereby obtaining polypeptide M5;

[0020] 6) In the amino acid sequence of polypeptide M5, amino acid residues 213-217 are replaced by KKVLK to LDTLY, thereby obtaining polypeptide M6;

[0021] 7) In the amino acid sequence of polypeptide M6, the amino acid fragment at positions 257-264 was replaced by FTVREAVE to YTVKEIVK, thereby obtaining polypeptide M7;

[0022] 8) In the amino acid sequence of polypeptide M7, the amino acid fragment at positions 273-276 is replaced by ALRW to ELEF, thereby obtaining polypeptide M8;

[0023] 9) In the amino acid sequence of polypeptide M8, the amino acid fragment at position 288 is replaced by E to K, thereby obtaining polypeptide M9;

[0024] 10) In the amino acid sequence of polypeptide M9, amino acid residues 304-312 are replaced by ILEVDPSFL to VLKVDPAFR, thereby obtaining polypeptide M10;

[0025] 11) In the amino acid sequence of polypeptide M10, the amino acid fragment at positions 339-343 is replaced by FEEMM to IEEMI, thereby obtaining polypeptide M11;

[0026] 12) In the amino acid sequence of polypeptide M11, the amino acid fragment at positions 352-360 is replaced by KVAEEYAEK to AVAEKYAEL, thereby obtaining polypeptide M12;

[0027] 13) In the amino acid sequence of polypeptide M12, the amino acid residue at position 365 is substituted from E to Y, thereby obtaining polypeptide M13.

[0028] In certain embodiments, the polypeptide having improved activity in catalyzing the conversion of GDP-mannose to GDP-4-keto-6-deoxymannose comprises an amino acid sequence having at least 70% or greater identity to the amino acid sequence set forth in SEQ ID NO: 1 and / or any one of polypeptides M1-M13, and these polypeptides comprise certain amino acid substitution mutations that are functionally equivalent to the mutations in the aforementioned schemes 1)-13). In certain embodiments, the polypeptide having improved activity in catalyzing the synthesis of GDP-4-keto-6-deoxymannose comprises an amino acid sequence as set forth in SEQ ID NO: 1 and / or any one of polypeptides M1-M13, and is functionally equivalent to the polypeptide having the amino acid sequence as set forth in SEQ ID NO: 1 and / or polypeptides M1-M13.

[0029] In a second aspect, the present application provides a polynucleotide encoding the GDP-mannose dehydratase polypeptide as described in the first aspect above.

[0030] Polynucleotides encoding GDP-mannose dehydratase polypeptides can be prepared using recombinant DNA techniques known in the art, including, for example, cloning, recombination, in vitro synthesis, in vitro amplification, and / or other available methods.

[0031] In a third aspect, the present application provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above.

[0032] 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 cell.

[0033] 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.

[0034] The vector has a nucleotide or nucleic acid construct according to an embodiment of the present invention operably linked to a regulatory sequence capable of achieving expression of the DNA fragment, such as a promoter region.

[0035] 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.

[0036] In some embodiments, the transformed host cell is transformed with the polynucleotide described in the second aspect or the nucleic acid construct described in the third aspect.

[0037] In some embodiments, the host cell includes but is not limited to natural strains or genetically modified strains of bacteria, yeast, mold, etc.

[0038] In some embodiments, the preferred host cell is Escherichia coli, 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. The host cell is preferably selected from Escherichia coli, Bacillus, and yeast; the yeast is selected from, for example, Saccharomyces cerevisiae, Kluyveromyces, and Yarrowia lipolytica; further preferably, the host cell is the genetically engineered E. coli BL21 (DE3).

[0039] 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.

[0040] The enzyme or enzyme composition preferably contains the polypeptide of the present invention having an amino acid sequence as shown in SEQ ID NO: 1 and / or one or more of polypeptides M1-M13.

[0041] Depending on the reaction substrate and product, the enzyme or composition may also contain bifunctional enzymes such as L-fucose pyrophosphorylase (L fucokinase / GDP-L-fucosepyrophosphorylase, FKP), glucokinase (glucokinase, Glk), mannose-phosphate mutase (mannose-phosphate mutase, ManB), mannose-1-phosphate guanylyltransferase (ManC), GDP-fucose synthase (GDP-L-Fucose Synthase, WcaG), etc., which are beneficial for the synthesis of fucosyllactose.

[0042] In a seventh aspect, the present application provides a method for producing the polypeptide as described in the first aspect above, comprising:

[0043] (1) culturing a transformed host cell under conditions suitable for expressing the GDP-mannose dehydratase polypeptide; the transformed host cell is as described in the fifth aspect above; and

[0044] (2) Recovering the GDP-mannose dehydratase polypeptide.

[0045] In a specific embodiment, step (1) comprises: first, introducing a nucleic acid construct or a recombinant expression vector encoding the GDP-mannose dehydratase 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-mannose dehydratase polypeptide.

[0046] In a specific embodiment, the step (2) includes the steps of isolating and purifying the GDP-mannose dehydratase polypeptide from the culture.

[0047] 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.

[0048] The GDP-mannose dehydratase 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.

[0049] In addition, the GDP-mannose dehydratase polypeptide can be purified by various procedures known in the art to obtain substantially pure polypeptide, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography), electrophoresis procedures (e.g., preparative isoelectric focusing), differential solubility methods (e.g., ammonium sulfate precipitation), SDS-PAGE, salting out, etc., or a combination thereof; further preferably, purification can be performed by Ni column affinity chromatography.

[0050] In an eighth aspect, the present application provides a genetically modified cell comprising a heterologous nucleic acid sequence encoding the derivative polypeptides M1-M13 described in the first aspect.

[0051] Preferably, the genetically modified cells include but are not limited to yeast genetically modified cells. Preferably, they include but are not limited to Saccharomyces cerevisiae genetically modified cells and Kluyveromyces genetically modified cells.

[0052] Preferably, the genetically modified cells include, but are not limited to, genetically modified cells of Saccharomyces cerevisiae, Pichia kluyveri, Kluyveromyces lactis, Kluyveromyces polysporus, Kluyveromyces dobzhanskii, Kluyveromyces thermotolerans, Kluyveromyces yarrowii, or Kluyveromyces marxianus. Further preferably, the Kluyveromyces genetically modified cells include, but are not limited to, genetically modified cells of Saccharomyces cerevisiae, Kluyveromyces lactis, and Kluyveromyces marxianus.

[0053] The genetically modified cells have the activity of synthesizing fucosyllactose de novo. The fucosyllactose includes but is not limited to 2'-fucosyllactose, 3-fucosyllactose, synthetic difucosyllactose (DFL), lactose-N-fucopentaose I (Lacto-N-fucopentaose I), and lactose-N-difucohexaose I (Lacto-N-difucohexaose I).

[0054] The genetically modified cells can synthesize fucosyllactose de novo using lactose and one or more selected from glucose, glycerol and sucrose as carbon sources (ie, relying on the genetically modified cells' own metabolic system to achieve a complete synthesis pathway).

[0055] Furthermore, in some embodiments, in addition to the heterologous nucleic acid encoding the above-mentioned dehydratase, the genetic cell may also contain one or more heterologous nucleic acids encoding GDP-L-fucose synthase, alpha-1,2-fucosyltransferase, or alpha-1,3-fucosyltransferase.

[0056] The heterologous GDP-L-fucose synthase (wcag) gene is from Escherichia coli. Other suitable sources of GDP-L-fucose synthase include, for example, but are not limited to, natural or genetically modified cells such as Marinobacter salarius, Sinorhizobium fredii NGR234, Candidatus Staskawiczbacteria bacterium, Azorhizobium caulinodans, or Candictus Nitrospira nitrificans.

[0057] The α-1,2-fucosyltransferase gene may be derived from natural cells or genetically modified cells such as Helicobacter pylori, Thermophilic Chlorella, Escherichia coli, Bacucilius cereus, Pseudopedobater saltans, Bacillus fragilis, Bacteroides vulgatus, Bacteroides fragilis, or Bacillus smithii.

[0058] The sources of the α-1,3-fucosyltransferase gene include but are not limited to natural cells or genetically modified cells such as Helicobacter pylori, Bacteroides fragilis, Escherichia coli, Akkermansia muciniphila, Helicobacter hepaticus, and Azospirillum brasilense.

[0059] In some embodiments, the genetically modified cells described herein are selected from genetically modified cells of Saccharomyces cerevisiae, which further comprise a heterologous nucleic acid encoding a lactose permease. The lactose permease is a lactose transport polypeptide responsible for transferring lactose from the extracellular to the intracellular. The lactose permease source includes, but is not limited to, natural cells or genetically modified cells such as Neurospora crassa, Neofusicoccum parvum, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Helicobacter pylori, Rhizobium meliloti, Zymomonas mobilis, or Escherichia coli.

[0060] Preferably, the genetically modified Saccharomyces cerevisiae cell further comprises a heterologous nucleic acid encoding a transport protein that exports fucosyllactose. The heterologous nucleic acid encoding the transport polypeptide is integrated into the genome of the yeast cell. Preferably, the transport polypeptide that exports fucosyllactose is derived from, but is not limited to, natural cells or genetically modified cells such as Escherichia coli, Kluyveromyces marxianus, Kluyveromyces lactis, and Neurospora crassa.

[0061] In some embodiments, the polypeptide that exports fucosyllactose is selected from the group consisting of a polypeptide that exports 2'-fucosyllactose or 3-fucosyllactose, encoding a transporter polypeptide that exports 2'-fucosyllactose or 3-fucosyllactose.

[0062] In some embodiments, the yeast genetically modified cells are selected from Kluyveromyces genetically modified cells, which further include a deletion or disruption of the β-galactosidase gene (lac4) required for lactose metabolism in at least a portion of the starting strain. The Kluyveromyces engineered bacteria described herein are capable of importing lactose (i.e., transporting lactose into the cell) without consuming lactose. Compared to the starting strain, the expression of the β-galactosidase gene in the Kluyveromyces genetically modified cells is reduced, thereby reducing lactose consumption in the Kluyveromyces engineered bacteria. The Kluyveromyces genetically modified cells described herein do not require the introduction of a heterologous transport polypeptide, and can achieve a higher yield of synthetic fucosylated lactose.

[0063] In some embodiments, the Kluyveromyces genetically modified cell is a Kluyveromyces lactis genetically modified cell, and the nucleic acid sequence of the β-galactosidase lac4 gene thereof has a Genbank number of M84410.1, and an amino acid sequence thereof has a Genbank number of AAA35265.1.

[0064] In some embodiments, the Kluyveromyces genetically modified cell is a Kluyveromyces marxianus genetically modified cell, and the nucleic acid sequence of the β-galactosidase gene lac4 gene thereof has a Genbank number of XM_022818497.1, and an amino acid sequence thereof has a Genbank number of XP_022675157.1.

[0065] Any gene encoding the above enzymes and / or polypeptides, or any other enzymes and / or polypeptides mentioned herein, can be optimized by genetic or protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. This allows those of ordinary skill in the art to optimize enzyme expression and increase activity in yeast.

[0066] In one embodiment, a preferred technical solution is: the starting strain of the genetically modified Saccharomyces cerevisiae cell is Saccharomyces cerevisiae CCTCC NO: M20231127 (Saccharomyces cerevisiae SctgtP8), and the starting strain of Saccharomyces cerevisiae can be easily purchased through commercial channels or culture collection centers.

[0067] In one embodiment, a preferred technical solution is: the starting strain of the genetically modified Kluyveromyces lactis cells is K. lactis DSM70799; the starting strain of the genetically modified Kluyveromyces marxianus cells is K. marxianus DMKU3-1042. The starting strains of K. lactis and K. marxianus as described above can be easily purchased commercially or from culture collections.

[0068] The present invention also provides a genetically modified cell containing a molecular marker. The amino acid sequence of the molecular marker in the genetically modified cell containing the molecular marker is shown in SEQ ID NO: 7. Preferably, the nucleotide sequence of the molecular marker in the genetically modified cell is shown in SEQ ID NO: 10.

[0069] The present invention also provides a method for preparing genetically modified cells. The method uses yeast cells as a starting strain, and integrates a heterologous α-1,2-fucosyltransferase / or α-1,3-fucosyltransferase gene, a heterologous GDP-L-fucose synthase gene (wcaG), and a heterologous GDP-mannitol-4,6-dehydratase gene (gmd) into the Kluyveromyces genome, respectively.

[0070] Preferably, the method for preparing the genetically modified yeast cells specifically comprises the following steps:

[0071] (1) Cultivating yeast starting strains;

[0072] Preferably, the yeast starting strain is selected from Saccharomyces cerevisiae, Kluyveromyces lactis or Kluyveromyces marxianus.

[0073] Preferably, the yeast starting strain is selected from Saccharomyces cerevisiae CCTCC NO: M20231127 (Saccharomyces cerevisiae SctgtP8), Kluyveromyces lactis DSM70799 or Kluyveromyces marxianus DMKU3-1042.

[0074] (2) constructing an expression cassette and introducing a heterologous α-1,2-fucosyltransferase gene or α-1,3-fucosyltransferase gene into yeast cells;

[0075] (3) constructing an expression cassette and introducing the heterologous GDP-L-fucose synthase gene (wcag) into yeast cells;

[0076] (4) constructing an expression cassette and introducing a heterologous GDP-mannitol 4,6 dehydratase (gmd) gene into yeast cells;

[0077] Preferably, the method further comprises the step of (5) recovering the genetically engineered yeast.

[0078] Preferably, the recombinant construction technology of the yeast genetic engineering bacteria also includes:

[0079] (6) Construct a molecular marker expression cassette and introduce the marker gene into yeast cells.

[0080] Preferably, the amino acid sequence of the marker gene is shown as SEQ ID NO: 7. Preferably, the nucleotide sequence of the marker gene is shown as SEQ ID NO: 10.

[0081] Preferably, in the above method for preparing genetically modified yeast cells, the steps are not ordered in any particular order.

[0082] Preferably, the method for preparing the genetically modified Saccharomyces cerevisiae cells further comprises the following steps:

[0083] (a) constructing an expression cassette and introducing a transporter polypeptide gene for exporting fucosyllactose into the starting strain;

[0084] (b) Construct an expression cassette and introduce a heterologous lactose permease gene into the starting strain.

[0085] Preferably, the method for preparing the genetically modified Kluyveromyces cells further comprises the step of destroying the lac4 gene in the strain obtained in step (1).

[0086] In the ninth aspect, the present application provides the use of the GDP-mannose dehydratase 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-4-keto-6-deoxymannose and / or GDP-fucose and / or fucosyllactose.

[0087] Preferably, the application includes application in synthesizing fucosyllactose.

[0088] In one embodiment, the method comprises: adding one or more of the GDP-mannose dehydratase polypeptides described herein to a reaction system containing GDP-mannose, contacting the dehydratase polypeptide with the substrate GDP-mannose to synthesize GDP-4-keto-6-deoxymannose; then, the GDP-4-keto-6-deoxymannose is synthesized into GDP-fucose under the action of GDP-fucose synthase; and subsequently, the 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 fucosyllactose.

[0089] Preferably, the application involves producing 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, Escherichia coli or Saccharomyces cerevisiae). The GDP-mannose dehydratase polypeptide listed above can then be isolated and used to synthesize GDP-fucose or fucosyllactose in a suitable system, and optionally further purified. Specifically, the process includes:

[0090] a) catalyzing the synthesis of GDP-4-keto-6-deoxymannose from the substrate GDP-mannose using the polypeptide described in the first aspect or the enzyme or enzyme composition described in the sixth aspect;

[0091] b) GDP-4-keto-6-deoxymannose is converted into GDP-fucose under the catalysis of GDP-fucose synthase and NADPH enzyme.

[0092] For such applications, suitable reaction conditions include: the presence of a suitable reaction substrate, a suitable polypeptide, or the presence of essential cofactors such as monovalent or divalent ions, a pH value within an appropriate range, a suitable temperature, etc. It is not necessary to meet the optimal value of each factor affecting the polypeptide described in this application, but the reaction conditions must enable the α-1,2-fucosyltransferase polypeptide described in this application to exert its enzymatic activity.

[0093] In one embodiment, the method comprises: adding the α-1,2-fucosyltransferase polypeptide described in the present application to a reaction system, contacting the polypeptide with an acceptor and a fucosyl donor in the reaction system, and performing a catalytic reaction to synthesize the desired fucosylated lactose.

[0094] Preferably, the α-1,2-fucosyltransferase polypeptide is used in the synthesis of fucosyllactose, using GDP-L-fucose and lactose as substrates to catalyze the synthesis and obtain 2'-FL.

[0095] Preferably, the application includes:

[0096] a) catalyzing the synthesis of 2'-FL from substrates GDP-L-fucose and lactose using the polypeptide described in the first aspect or the enzyme or enzyme composition described in the sixth aspect. Further preferably, the method further comprises:

[0097] b) Optionally, a step of purifying and / or recovering 2'-FL.

[0098] The application of the GDP-mannose dehydratase polypeptide of the invention uses GDP-mannose as a substrate, and the GDP-fucose can be produced through the catalytic reaction of the GDP-mannose dehydratase and GDP-fucose synthase; then, fucosyllactose is synthesized under the catalytic action of α-1,2-fucotransferase or α-1,3-fucotransferase.

[0099] In the above steps, the reaction product is a solution of 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.

[0100] Preferably, substantially all proteins, as well as amino acids, RNA and DNA, etc., 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, by-products, color 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), in particular by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography.

[0101] GDP-fucose or fucosyllactose is further separated from the reaction solution and further purified using techniques well known in the art such as evaporation, freeze drying, crystallization, precipitation and / or drying, spray drying.

[0102] In a tenth aspect, the present application provides the use of the genetically modified bacteria described in the eighth aspect above in synthesizing fucosyllactose.

[0103] Preferably, the fucosyllactose includes but is not limited to 2'-fucosyllactose, 3-fucosyllactose, synthetic difucosyllactose (DFL), lactose-N-fucopentaose I and lactose-N-difucohexaose I.

[0104] 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. Beneficial effects:

[0105] The present application provides a series of GDP-mannose dehydratase polypeptides, including polypeptides M1-M13 derived from the amino acid sequence of the polypeptide set forth in SEQ ID NO: 1. The present application also provides genetically modified cells containing heterologous nucleic acids for the polypeptides M1-M13. The GDP-mannose dehydratase enzymes described herein generally have high catalytic activity, and the genetically modified cells have the ability to synthesize fucosyllactose de novo. The technical solution of the present application has positive implications for the industrial production of human milk oligosaccharides. This method is environmentally friendly, efficient, and sustainable, and is conducive to application in large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] 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:

[0107] Figure 1. HPLC chromatogram of GDP-fucose standard.

[0108] Figure 2. HPLC spectrum of the reaction solution catalyzed by polypeptide M7 in Example 2.

[0109] Figure 3. HPLC chromatogram of 2'-FL standard.

[0110] Figure 4. HPLC chromatogram of 3-FL standard.

[0111] FIG5 is a HPLC analysis chart of the fermentation broth in which BS-M13 synthesizes 2′-fucosyllactose in Example 9.

[0112] Figure 6. HPLC analysis of the fermentation broth for synthesizing 3-fucosyllactose by BS-M 13-1 in Example 9. DETAILED DESCRIPTION

[0113] The present invention will be further described in detail below through examples. It should be understood that the specific embodiments described herein are only for the purpose of explaining the present invention and are not intended to limit the present invention. It should be noted that, for those skilled in the art, various modifications or substitutions may be made to the details and forms of the technical solutions of the present invention without departing from the principles of the present invention, and such modifications or substitutions also fall within the scope of protection of the present invention.

[0114] 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.

[0115] 2'-FL and 3-FL standards were produced by ELICITYL, France. GDP-fucose standards were purchased from Aladdin.

[0116] In the following examples, the enzymes involved are as follows:

[0117] GDP-mannose synthase (WcaG), microbial origin: Escherichia coli, amino acid sequence: SEQ ID NO: 3.

[0118] The microbial source of alpha-1,2-fucosyltransferase (FutC) is Helicobacter pylori, and the amino acid sequence is shown in SEQ ID NO: 4.

[0119] The microbial source of alpha-1,3-fucosyltransferase (FutA) is Helicobacter pylori, and the amino acid sequence is shown in SEQ ID NO:5.

[0120] The microbial source of lactose permease is Kluyveromyces lactis, and the amino acid sequence is shown in SEQ ID NO:8.

[0121] The microbial origin of the transport protein for exporting fucosyllactose is Neurospora crassa, and the amino acid sequence is shown in SEQ ID NO:9.

[0122] Example 1. Expression of the polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2, and polypeptides M1-M13 in Escherichia coli.

[0123] 1. Using the amino acid sequence of SEQ ID NO: 1 (nucleotide sequence of SEQ ID NO: 6) and the polypeptide of SEQ ID NO: 2 as templates, the gene coding sequences of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized, respectively, and finally constructed into the pET32a vector to obtain the pET32a-BsGMD-wt plasmids and pET32a-EcGMD-wt.

[0124] 2. Computer-assisted rational design was used to identify different mutation sites. Referring to 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: 6 of pET32a-wt. A Fast Mutagenesis Kit was used to construct a series of recombinant plasmids (13 recombinant plasmids, from pET32a-M1 to pET32a-M13; see Table 1).

[0125] 3. Transform the above recombinant plasmids into Escherichia coli BL21 (DE3) according to the following steps:

[0126] Take the prepared Escherichia coli BL21 (DE3) competent cells, place them on ice for 30 minutes to thaw, take 100 μL of competent cells and 10 μL of pET32a-M1 to pET32a-M13 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.

[0127] 4. Culture positive transformants, extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the recombinant plasmids pET32a-M1 to pET32a-M13 are successfully introduced into E. coli.

[0128] 5. Inoculate the correct transformant into LB liquid medium and culture it on a shaker at 37°C and 200 rpm for 12 hours to obtain seed solution. Then, inoculate the seed solution into fresh LB medium at a 1% (v / v) inoculum and culture it at 37°C with shaking until the OD 600 The expression of the culture medium was 0.8, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG) with a final IPTG concentration of 0.1 mmol / L, and induced at 16°C for 12 h at a speed of 200 rpm; after induction of expression, the fermentation broth was centrifuged at 5000 r / min for 30 min at 4°C to collect the bacteria; the bacteria were resuspended in 20 mM pH 7.4 PBS buffer and ultrasonicated at a frequency of plus on 5s / off 5s for 30 min to break the bacteria; the broken liquid was centrifuged at 13000×g and 4°C for 30 min to remove cell debris, and the supernatant was collected.

[0129] 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.

[0130] The SDS-PAGE results showed that the genetically engineered bacteria had obvious specific expression bands after induction, and the molecular weight of the bands was basically consistent with the expected molecular weight of 35.3 kDa. Therefore, it can be seen that the polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2 and polypeptide M1-13 were obtained. For detailed information, please see Table 1.

[0131] Table 1. Corresponding numbers of GDP-mannose dehydratase polypeptides and their amino acid sequences

[0132] Example 2. Determination of the ability of the polypeptides obtained in Example 1 (polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 2) and polypeptides M1-M13 to serve as catalysts for the synthesis of GDP-fucose.

[0133] GDP-fucose was synthesized using GDP-mannose as a substrate, the purified polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 2 and polypeptides M1-M13 obtained in Example 1, and GDP-fucose synthase as enzyme catalysts. The specific procedure is as follows:

[0134] The cells were cultured in a 50 mM Tris-HCl (pH 7.0) solution containing 100 μM GDP-mannose, 1 mM NADP- + In a system of 1 mM NADPH, 0.2 mg / mL of the purified polypeptide represented by SEQ ID NO: 1 or SEQ ID NO: 2 or one of polypeptides M1-M13 obtained in Example 1 and 0.2 mg / mL GDP-fucose synthase were added, mixed, reacted at 30° C. for 6 hours, terminated the reaction, and purified by gel column method.

[0135] HPLC detection method of GDP-fucose:

[0136] 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.

[0137] HPLC detection method for fucosyllactose content:

[0138] Detection conditions: Chromatographic column model: Shodex Asahipak NH2P-50 4E, mobile phase: 65% acetonitrile in water, flow rate: 0.5 ml / min, column temperature: 35°C, injection volume: 10 μL, evaporative light detector, evaporation temperature 75°C, nebulization temperature 45°C.

[0139] 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.

[0140] The standard and reaction solution were tested using the aforementioned analytical method. HPLC analysis results showed that the GDP-fucose standard exhibited a peak elution time (RT) of 13.3 min. The reaction solutions of the polypeptide described in Example 1 all exhibited a strong absorption peak around 13.3 min, consistent with the peak elution time of the GDP-fucose standard, indicating that the polypeptide-catalyzed reaction in Example 1 produced GDP-fucose.

[0141] The GDP-fucose concentration in the catalytic reaction solution was detected as above, and the results are recorded in Table 2.

[0142] Table 2. Study on the synthesis of GDP-fucose catalyzed by various peptides

[0143] Table 2 Data Description:

[0144] (1) The polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 2 and their derivative peptides M1-M13 all have the activity of synthesizing GDP-4-keto-6-deoxymannose with GDP-mannose; and then synthesizing GDP-fucose under the action of GDP-fucose synthase.

[0145] (2) The activity of polypeptides M5-M9 and M11-M13 in catalyzing the synthesis of GDP-fucose was increased by more than 1.5 times compared with the polypeptide represented by SEQ ID NO: 1.

[0146] Example 3. Construction of genetically modified Saccharomyces cerevisiae cells.

[0147] (1) Construction of SC-ΔARO3::FutC-WCAG / SC-ΔARO3::FutA-WCAG expression cassette

[0148] 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 wcaG target gene sequence was amplified using the synthesized wcaG as a template; with reference to the applicant's prior patent (CN202211453172.5, invention name: recombinant yeast and its application) in KL- The ΔLAC4 knockout cassette was used as a template to amplify the G418+loxp sequence. Aro3-up, TDH3p, FutC / FutA, PGK1t, TEF1p, WCAG, ADH1t, G418+loxp, and aro3-do were fused by successive fusion PCR of two fragments. Finally, primers ARO3-F and ARO3-R were used as primers (Table 3) and the fusion PCR system was used as a template to amplify the SC-ΔARO3::FutC-WCAG / SC-ΔARO3::FutA-WCAG expression cassette.

[0149] Table 3. Plasmids and primers

[0150] (2) Construction of SC-ΔTRP3::CDT2-LAC12 expression cassette

[0151] 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 as primers (Table 4) and the fusion PCR system as a template.

[0152] Table 4. Plasmids and primers

[0153] (3) Construction of expression cassettes of SC-ΔGK::BSGMD-WT, SC-ΔGK::BSGMD-M1 to SC-ΔGK::BSGMD-M13, SC-ΔGK::ECGMD-WT, SC-ΔGK::CEGMD-WT, SC-ΔGK::ATGMD-WT, and SC-ΔGK::CAGMD-WT.

[0154] 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, respectively; using the SC-ΔTRP3::CDT2-LAC12 expression cassette as a template, a resistance screening marker containing G418 resistance and loxp sites was amplified; using PET32a-BSGMD-WT, PET32a-BSGMD-M1 to PET32a-BSGMD-M13, PET32a-ECGMD-WT, PET32a-CEGMD-WT, PET32a-ATGMD-WT, and PET32a-CAGMD-WT as templates, PCR amplification was performed to obtain BSGMD-WT and mutant sequences, as well as ECGMD-WT, CEGMD-WT, ATGMD-WT, and CAGMD-WT gene sequences. ; By using two fragments in successive fusion PCR, gk-up, CUP1p, BSFutGMD-WT and mutant sequence / ECGMD-WT / ECFut-wtCEGMD-WT / ATGMD-WT / CAGMD-WT gene sequences, GPMt, G418+loxp and gk-do were fused. Finally, primers GK-F and GK-R were used as primers (Table 5), and the fusion PCR system was used as a template. PCR amplification obtained the expression cassettes SC-ΔGK::BSGMD-WT, SC-ΔGK::BSGMD-M1 to SC-ΔGK::BSGMD-M13, SC-ΔGK::ECGMD-WT, SC-ΔGK::CEGMD-WT, SC-ΔGK::ATGMD-WT, SC-ΔGK::CAGMD-WT for the next step of constructing recombinant strains.

[0155] Primers were used to construct expression cassettes for SC-ΔGK::BSGMD-WT, SC-ΔGK::BSGMD-M1 to SC-ΔGK::BSGMD-M13, SC-ΔGK::ECGMD-WT, SC-ΔGK::CEGMD-WT, SC-ΔGK::ATGMD-WT, and SC-ΔGK::CAGMD-WT.

[0156] Table 5. Plasmids and primers

[0157] (4) Transformation of the recombinant expression cassette and verification of the recombinant strain.

[0158] The expression cassettes constructed in (1) were respectively transferred into the starting strain Saccharomyces cerevisiae CCTCC NO: M20231127 cells. The specific method is:

[0159] 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.

[0160] Meanwhile, boil 1 mL of salmon sperm DNA for 5 min and quickly place on ice to prepare single-stranded DNA.

[0161] 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 50 μL of 5-10 μg / 50 μL plasmid DNA in water. 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.

[0162] 3) Verification: The correspondence between the recombinant strains and their genotypes is shown in Tables 6 and 7. To verify the correctness of these 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.

[0163] Table 6. Saccharomyces cerevisiae engineered strains and their genotypes (α-1,2-fucosyltransferase gene)

[0164] Molecular marker genes (amino acid sequence as shown in SEQ ID NO: 7, nucleotide sequence as shown in SEQ ID NO: 10) were introduced into the recombinant strains described in Table 6 to obtain strains SC-1-J to SC-7-J, and SC-M1-J to SC-M13-J, respectively.

[0165] Table 7. Saccharomyces cerevisiae engineered strains and their genotypes (α-1,3-fucosyltransferase gene)

[0166] Molecular marker genes (amino acid sequence as shown in SEQ ID NO: 7, nucleotide sequence as shown in SEQ ID NO: 10) were introduced into the recombinant strains described in Table 7 to obtain strains SC-1-1-J to SC-7-1-J, and SC-M1-1-J to SC-M13-1-J.

[0167] Verification of Molecular Marker Genes: 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 with the same size as the knockout or expression cassette is obtained after PCR amplification, the strain is considered correct; otherwise, the strain is considered a false positive. For tag verification, PCR amplification using primers is performed and further verified by sequencing; otherwise, the strain is considered a false positive.

[0168] Example 4. Construction of genetically modified Kluyveromyces lactis cells.

[0169] (1) Construct a Kluyveromycin-modified cell expression cassette.

[0170] 1) Construction of the KL-ΔLAC4 knockout cassette.

[0171] Referring to the applicant's prior patent (CN202211453172.5, invention name: Recombinant yeast and its application), a KL-ΔLAC4 knockout cassette was constructed.

[0172] 2) Construction of KL-ΔLAC4::FutC / KL-ΔLAC4::FutA expression cassette.

[0173] 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 lac4up, tef1, FutC / FutA, adh1, G418+loxp, and lac4-do were fused by successive fusion PCR of two fragments. Finally, using primers lac4-upF and lac4-doR as primers (Table 8), the expression cassette KL-ΔLAC4::FutC / KL-ΔLAC4::FutA was amplified by PCR using the fusion PCR system as a template.

[0174] Table 8

[0175] 3) Construction of KL-ΔXK::GMER expression cassette.

[0176] Using the Kluyveromyces lactis genome as a template, the upstream and downstream homology arm sequences xk-up and xk-do of the xylulose kinase XKb coding region, the promoter sequence pgk1, and the terminator sequence tdh3 were amplified; using the synthesized gmer sequence as a template, the GMER target gene sequence was amplified; using the existing plasmid in the laboratory as a template, the G418+loxp sequence was amplified, and by sequential fusion PCR of two fragments, xyl1 up, pgk1, gmer, tdh3, G418+loxp, and xyl1-do were fused. Finally, using primers xK-upF and xK-doR as primers (Table 9), the fusion PCR system was used as a template to amplify the gmer expression cassette KL-ΔXK::GMER.

[0177] Table 9

[0178] 4) Construction of KL-ΔGK::BSGMD-WT, KL-ΔGK::BSGMD-M1 to KL-ΔGK::BSGMD-M13, and KL-ΔGK::ECGMD-WT expression cassettes.

[0179] Using the genomic DNA of Kluyveromyces lactis DSM70799 as a template, PCR amplification was performed with primers galk up-F and galk up-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 the G418 resistance plasmid as a template, a resistance screening marker containing G418 resistance and loxp sites was amplified; using PET32a-BSGMD-WT, PET32a-M1 to PET32a-M13 and PET32a-ECGMD-WT as templates, the original sequence and mutant sequence of the GMD gene were amplified by PCR; using the Kluyveromyces lactis genome as a template, the promoter tdh3 and terminator sequence tef of the transferase were amplified by PCR, respectively; using KL-PF and KL-PR as primers 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 by PCR to obtain ΔGK::BSGMD-WT, ΔGK::BSGMD-M1 to ΔGK::BSGMD-M13 expression cassettes, and ΔGK::ECGMD-WT; the expression cassettes were connected to the plasmid vector sequence by ABclonal to obtain plasmids PUC-KL-WT, PUC-KL-M1 to PUC-KL-M13, and PUC-KL-ECGMD-WT carrying the ΔGK::BsGMD-WT, ΔGK::ECGMD-WT, ΔGK::BSGMD-M1 to ΔGK::BSGMD-M13 expression cassettes, respectively.

[0180] Using primers galk up-F and galk down-R as templates (Table 10), and PUC-KL-WT, PUC-KL-M1 to PUC-KL-M13, and PUC-KL-ECGMD-WT as templates, the KL-ΔGK::BSGMD-WT, KL-ΔGK::BSGMD-M1 to KL-ΔGK::BSGMD-M13 expression cassettes, and KL-ΔGK::ECGMD-WT were amplified for the next step of constructing recombinant strains.

[0181] Table 10. Primers and sequences

[0182] (2) Transformation of the recombinant expression cassette and verification of the recombinant strain.

[0183] The expression cassettes constructed in (1) above were respectively transferred into the starting strain Kluyveromyces lactis DSM70799 cells. The specific method is:

[0184] 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.

[0185] Meanwhile, boil 1 mL of salmon sperm DNA for 5 min and quickly place on ice to prepare single-stranded DNA.

[0186] 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 50 μL of 5-10 μg / 50 μL plasmid DNA in water. 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.

[0187] 3) Verification: The correspondence between the recombinant strains and their genotypes is shown in Tables 11-12. 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 with the size of the knockout or expression cassette is obtained after PCR amplification, the strain is considered correct; otherwise, the strain is considered a false positive.

[0188] Table 11. Genetically modified Kluyveromyces lactis cells and their genotypes (transferase is α-1,2-fucosyltransferase gene)

[0189] Molecular marker genes (amino acid sequence as shown in SEQ ID NO: 7, nucleotide sequence as shown in SEQ ID NO: 10) were introduced into the recombinant strains described in Table 11 to obtain a total of 17 strains, including KL-1-J to KL-4-J and KL-M1-J to KL-M13-J.

[0190] Table 12. Genetically modified Kluyveromyces lactis cells and their genotypes (transferase is α-1,3-fucosyltransferase gene)

[0191] Molecular marker genes (amino acid sequence as shown in SEQ ID NO: 7, nucleotide sequence as shown in SEQ ID NO: 10) were introduced into the recombinant strains described in Table 12 to obtain a total of 17 strains, including KL-1-1-J to KL-4-1-J, and KL-M1-1-J to KL-M13-1-J.

[0192] Example 5. Construction of genetically modified Kluyveromyces marxianus cells.

[0193] (1) Construct a Max-Kluyveromycin-modified cell expression cassette.

[0194] 1) Construction of the KM-ΔLAC4 knockout cassette.

[0195] The KM-ΔLAC4 knockout cassette was constructed with reference to the applicant's prior patent (CN202211453172.5, invention name: Recombinant yeast and its application).

[0196] 2) Construction of KM-ΔLAC4::FutC / KM-ΔLAC4::FutA expression cassette.

[0197] 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 were used as primers (Table 13) and the fusion PCR system was used as a template to obtain the FutC and FutA expression cassettes KM-ΔLAC4::FutC and KM-ΔLAC4::FutA by PCR amplification.

[0198] Table 13

[0199] 3) Construction of KM-ΔXK::GMER expression cassette.

[0200] Using the Kluyveromyces marxianus genome as a template, the upstream and downstream homology arm sequences km-xk up and km-xk-do of Kluyveromyces marxianus xylulose kinase were amplified. Using the KM-ΔXK::GMER expression cassette as a template, the GMER expression cassette and G418+loxp site were amplified. By three-fragment fusion PCR, km-xk up, GMD expression cassette+G418+loxp and km-xk-do were fused. Finally, primers xk-upF1 and xk-doR1 were used as primers (Table 14) and the fusion PCR system was used as a template to obtain the gmer expression cassette KM-ΔXK::GMER.

[0201] Table 14

[0202] 4) Construction of KM-ΔGK::BSGMD-WT, KM-ΔGK::BSGMD-M1, KM-ΔGK::BSGMD-M13, and KM-ΔGK::ECGMD-WT expression cassettes.

[0203] 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 were used for PCR amplification to obtain the upstream and downstream homology arms, respectively. Using KL-ΔGK::BSGMD-wt, KL-ΔGK::BSGMD-M1 to KL-ΔGK::BSGMD-M13 expression cassettes, and KL-ΔGK::ECGMD-wt expression cassette as templates, PCR amplified the BSGMD-wt / GMD mutant expression cassette / ECGMD+G418+loxp sequence, and fused the upstream and downstream homology arms and the BSGMD-wt / GMD mutant expression cassette / ECGMD expression cassette+G418+loxp sequence by fusion PCR. Down-R1 was used as a template (Table 15), and KM-ΔGK::BSGMD-WT, KM-ΔGK::BSGMD-M1 to KM-ΔGK::BSGMD-M13, and KM-ΔGK::ECGMD-WT expression cassettes were amplified for the next step of constructing recombinant strains.

[0204] Table 15.

[0205] (2) Transformation of recombinant plasmid and verification of recombinant strain:

[0206] 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.

[0207] The correspondence between the recombinant strains and their genotypes is shown in Tables 16-17. To verify the correctness of the above strains, the genomes of the transformants and the original strains 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 matched the knockout or expression cassette, the strain was considered correct; otherwise, the strain was considered a false positive.

[0208] Table 16. Genetically modified Kluyveromyces marxianus cells and their genotypes (transferase is α-1,2-fucosyltransferase gene)

[0209] Molecular marker genes (amino acid sequence as shown in SEQ ID NO: 7, nucleotide sequence as shown in SEQ ID NO: 10) were introduced into the recombinant strains described in Table 16 to obtain a total of 17 recombinant strains, including KM-1-J to KM-4-J and KM-M1-J to KM-M13-J.

[0210] Table 17. Genetically modified Kluyveromyces marxianus cells and their genotypes (transferase is α-1,3-fucosyltransferase gene)

[0211] Molecular marker genes (amino acid sequence as shown in SEQ ID NO: 7, nucleotide sequence as shown in SEQ ID NO: 10) were introduced into the recombinant strains described in Table 17 to obtain a total of 17 recombinant strains, including KM-1-1-J to KM-4-1-J, and KM-M1-1-J to KM-M13-1-J.

[0212] Verification of marker genes: To verify the correctness of the above strains, extract the genomes of the transformants and the original strain and perform PCR amplification using primers corresponding to the knockout or expression cassette. If a single band with the same size as the knockout or expression cassette is obtained after PCR amplification, the strain is considered correct; otherwise, the strain is considered a false positive. For tag verification, perform PCR amplification using primers and further verify by sequencing; otherwise, the strain is considered a false positive.

[0213] Example 6. The genetically modified cells obtained in Examples 3-5 were fermented to synthesize fucosyllactose.

[0214] Yeast was cultured with glucose as the carbon source. The genetically modified cells obtained in Examples 3-5 were taken respectively, and the genetically modified yeast cells were allowed to grow rapidly until the growth entered the late logarithmic phase or the stable phase. The strains were streaked and cultured in solid culture media such as YDP, 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 h. After 72 h of fermentation, samples were taken and boiled for 10 minutes. After cooling, the supernatant was assayed for 2'-FL and 3-FL content.

[0215] HPLC detection method: Detection conditions: chromatographic column model: Shodex Asahipak NH2P-50 4E, mobile phase: 65% acetonitrile in water, flow rate: 0.5 ml / min, column temperature: 35°C, injection volume: 10 μL, evaporative light detector, evaporation temperature 75°C, and nebulization temperature 45°C.

[0216] LC-MS analysis conditions are as follows:

[0217] Chromatographic column model: Shodex Asahipak NH2P-50 4E, detector: UV detector (Hitachi Chromaster), detection wavelength: 210 nm, injection volume: 10 μL, flow rate: 0.5 mL / min, column temperature: 35°C, mobile phase: acetonitrile:water = 65:35; ESI-MS mode, molecular weight scan range: 100-800.

[0218] The standard product and the reaction solution were tested according to the above analytical method. The HPLC analysis results showed:

[0219] (1) The peak time rt of the 2'-FL standard product is 13.43 min; the HPLC analysis spectrum of the 3-FL standard product has a rt of 14.16 min.

[0220] (2) The yeast gene-modified cells introduced with the α-1,2-fucosyltransferase gene obtained in Example 3-5 (SC-3, SC-4, KL-3, KL-4, KM-3, KM-4 and strains introduced with the M1-M13 genes, as well as the above strains introduced with the marker genes) showed a strong absorption peak at around 13.43 min, which was consistent with the peak time of the 2'-FL standard, indicating that 2'-FL was produced in the fermentation broth of the gene-modified cells obtained in Example 3-5.

[0221] (3) The yeast gene-modified cells introduced with the α-1,3-fucosyltransferase gene obtained in Example 3-5 (SC-3-1, SC-4-1, KL-3-1, KL-4-1, KM-3-1, KM-4-1 and strains introduced with the M1-M13 genes, as well as the above strains introduced with the marker genes) showed a strong absorption peak at around 14.16 min, which was consistent with the peak time of the 3-FL standard, indicating that 3-FL was produced in the fermentation broth of the gene-modified cells obtained in Example 2.

[0222] The LC-MS analysis conditions are shown above, and the analysis results show:

[0223] 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.

[0224] 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.

[0225] 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 18-20.

[0226] Table 18. Research on the synthesis of 2'-FL and 3-FL by Saccharomyces cerevisiae CCTCC NO: M20231127 gene-modified cells

[0227] Fermentation experiments found that the ability of strains SC-1-J to SC-7-J and SC-M1-J to SC-M13-J to synthesize 2'-FL after the introduction of the marker gene was comparable to that of the corresponding strains before the addition of the marker gene; the ability of strains SC-1-1-J to SC-7-1-J and SC-M1-1-J to SC-M13-1-J to synthesize 3-FL after the introduction of the marker gene was comparable to that of the corresponding strains before the addition of the marker gene.

[0228] The data in Table 18 illustrate that the genetically modified cells of Saccharomyces cerevisiae of the present invention have the activity of synthesizing 2'-FL or 3-FL.

[0229] Table 19. Study on the synthesis of fucosyllactose by Kluyveromyces lactis gene-modified cells obtained in Example 4

[0230] Fermentation experiments found that the ability of strains KL-1-J to KL-4-J and KL-M1-J to KL-M13-J to synthesize 2'-FL after the introduction of the marker gene was comparable to that of the corresponding strains before the addition of the marker gene; the ability of strains KL-1-1-J to KL-4-1-J and KL-M1-1-J to KL-M13-1-J to synthesize 3-FL after the introduction of the marker gene was comparable to that of the corresponding strains before the addition of the marker gene.

[0231] Table 20. Study on the synthesis of fucosyllactose by Kluyveromyces maximus gene-modified cells obtained in Example 5

[0232] Fermentation experiments found that the ability of strains KM-1-J to KL-4-J and KM-M1-J to KM-M13-J to synthesize 2'-FL after the introduction of the marker gene 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-M1-1-J to KM-M13-1-J to synthesize 3-FL after the introduction of the marker gene was comparable to that of the corresponding strains before the addition of the marker gene.

[0233] The data in Tables 19-20 indicate that the Kluyveromycin gene-modified cells of the present invention have the activity of synthesizing 2'-FL or 3-FL.

[0234] As shown in the above experiments, the introduction of the marker gene does not affect the fermentation activity of the yeast gene-modified cells obtained in Examples 3-5 in synthesizing 2'-FL or 3-FL.

[0235] Example 7. Yeast genetically modified cells SC-M10, SC-M10-1, SC-M10-J, SC-M10-J-1, KL-4-J, KL-4-1-J, KL-M1-J, KL-M1-1-J, KL-M13-J, KL-M13-1-J, KM-4-J, KM-4-1-J, KM-M1-J, KM-M1-1-J, KM-M13-J, KM-M13-1-J were taken, and the strains were streaked and cultured on solid culture media such as YDP. After culturing at 30°C for 2-3 days, single colonies were picked and inoculated into 1.5 mL YPD liquid culture medium and cultured at 30°C and 200 rpm overnight. Subsequently, they were inoculated into 50 mL liquid culture medium shake flasks at a 2% inoculum size and cultured at 30°C and 200 rpm until 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 h of culture, CuSO4 was added to control the final Cu ion concentration to approximately 0.1 mM. Sucrose (50% mother liquor concentration) was then added at a rate of 8 mL / h. Simultaneously, lactose (40% mother liquor concentration) was added to maintain a final lactose concentration of 15 g / L. The total fermentation duration was 72 h. After 72 h of fermentation, the fermentation broth was centrifuged, and the supernatant and precipitate were collected. 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 reported in Table 21.

[0236] Table 21

[0237] Example 8. Synthesis of 2'-fucosyllactose or 3-fucosyllactose using Escherichia coli BL21 as a chassis.

[0238] 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 engineering, 41, 23–38.) The strain BL21ΔlacZΔlonΔwcaJ was constructed, and the above-constructed PET32a-M1-HpFutC, PET32a-M1-HpFutA, PET32a-M13-HpFutC and PET32a-M13-HpFutA were respectively transformed into the Escherichia coli chassis cells BL21ΔlacZΔlonΔwcaJ by electroporation, thereby constructing the EC-M1, EC-M1-1, EC-M13 and EC-M13-1 strains.

[0239] Method for culturing Escherichia coli EC-M1, EC-M1-1, EC-M13, and EC-M13-1 to synthesize 2'-fucosyllactose or 3-fucosyllactose:

[0240] E. coli strains EC-M1, EC-M1-1, EC-M133, and EC-M13-1 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 2′-FL production. At the same time, the culture medium was supplemented with ampicillin to a final concentration of 100 μg / mL. After completion, the culture was boiled for 10 minutes, centrifuged, and the supernatant was collected for analysis of 2′-FL or 3-FL production. The results are recorded in Table 22.

[0241] Table 22

[0242] Example 9. Synthesis of 2'-fucosyllactose or 3-fucosyllactose using Bacillus sp. BStgtP8 (CCTCC NO: M 20231126) as a chassis.

[0243] 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.) The Bacillus subtilis chassis cells BSP43-manB-P43-manC-P43-HpFutC-P43-wcaG-P43-lacY and BSP43-manB-P43-manC-P43-HpFutA-P43-wcaG-P43-lacY were constructed, and the P43-M1 and P43-M13 expression cassettes were constructed. The expression cassettes were transferred into BSP43-manB-P43-manC-P43-HpFutC-P43-wcaG-P43-lacY and BSP43-manB-P43-manC-P43-HpFutA-P43-wcaG-P43-lacY, respectively, and inserted into the manP gene, thereby constructing the BS-M1, BS-M13, BS-M1-1 and BS-M13-1 strains.

[0244] Method for culturing Bacillus subtilis BS-M1, BS-M13, BS-M1-1 and BS-M13-1 to synthesize 2'-fucosyllactose or 3-fucosyllactose:

[0245] Bacillus subtilis strains BS-M1, BS-M13, BS-M1-1, and BS-M13-1 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, a culture medium with the following composition 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 23.

[0246] Table 23

[0247] The above process was simultaneously scaled up to a 1L fermenter using continuous fed-batch fermentation. Sucrose 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 fucosyllactose content. The results were: 18.8g / L for 2'-FL in the BS-M1 fermentation broth; 28.1g / L for the BS-M13 fermentation broth; 14.5g / L for 3-FL in the BS-M1-1 fermentation broth; and 21.3g / L for the S-M13-1 fermentation broth.

[0248] The present invention discloses a series of GDP-mannose dehydratase 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.

Claims

1. A GDP-mannose dehydratase polypeptide, characterized in that The amino acid sequence shown in SEQ ID NO: 1 is modified at least one of positions 69-72, 118-119, 123-124, 176, 203-205, 213-217, 257-264, 273-276, 288, 304-312, 339-343, 352-360 and 365; 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 69-72 is replaced by EKIK with ASIR to obtain polypeptide M1; 2) In the amino acid sequence of polypeptide M1, the amino acid fragment at positions 118-119 is replaced from IE to LD to obtain polypeptide M2; 3) In the amino acid sequence of polypeptide M2, the amino acid fragment at positions 123-124 is replaced by QD to EK to obtain polypeptide M3; 4) In the amino acid sequence of polypeptide M3, the amino acid fragment at position 176 is replaced from N to D to obtain polypeptide M4; 5) In the amino acid sequence of polypeptide M4, the amino acid fragment at positions 203-205 is replaced by RDI to IQL to obtain polypeptide M5; 6) In the amino acid sequence of polypeptide M5, the amino acid fragment at positions 213-217 was replaced by KKVLK to LDTLY to obtain polypeptide M6; 7) In the amino acid sequence of polypeptide M6, the amino acid fragment at positions 257-264 was replaced by FTVREAVE to YTVKEIVK to obtain polypeptide M7; 8) In the amino acid sequence of polypeptide M7, the amino acid fragment at positions 273-276 was replaced by ALRW to ELEF to obtain polypeptide M8; 9) In the amino acid sequence of polypeptide M8, the amino acid fragment at position 288 is replaced from E to K to obtain polypeptide M9; 10) In the amino acid sequence of polypeptide M9, the amino acid fragment at positions 304-312 is replaced by ILEVDPSFL to VLKVDPAFR to obtain polypeptide M10; 11) In the amino acid sequence of polypeptide M10, the amino acid fragment at positions 339-343 is replaced by FEEMM to IEEMI to obtain polypeptide M11; 12) In the amino acid sequence of polypeptide M11, the amino acid fragment at positions 352-360 was replaced by KVAEEYAEK to AVAEKYAEL to obtain polypeptide M12; 13) In the amino acid sequence of polypeptide M12, the amino acid residue at position 365 was replaced from E to Y, thereby obtaining polypeptide M13.

2. A polynucleotide encoding the GDP-mannose dehydratase polypeptide of 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 an appropriate expression host cell. 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, or the nucleic acid construct according to claim 3, or the expression vector according to claim 4; Preferably, the host cell comprises a natural cell selected from bacteria, yeast and mold or a genetically modified cell thereof; Further preferably, the host cell comprises a natural cell or a genetically modified cell thereof selected from the group consisting of Escherichia sp., Bacillus sp. and yeast sp.; Further preferably, the host cell comprises a natural cell selected from Saccharomyces cerevisiae, Kluyveromyces marxianus, Kluyveromyces lactis and Yarrowia lipolytica or a genetically modified cell thereof; Further preferably, the host cell is the genetically engineered Escherichia coli BL21 (DE3).

6. An enzyme agent or enzyme composition comprising at least one GDP-mannose dehydratase polypeptide according to claim 1.

7. A genetically modified cell comprising a heterologous nucleic acid sequence encoding the GDP-mannose dehydratase polypeptide of claim 1.

8. A method for producing a GDP-mannose dehydratase polypeptide, characterized in that: The steps include: (1) culturing the transformed host cell to express the GDP-mannose dehydratase polypeptide; and (2) recovering the GDP-mannose dehydratase polypeptide; Preferably, the transformed host cell is as described in claim 5; Preferably, the step (1) comprises: firstly introducing a nucleic acid construct or an expression vector comprising a polynucleotide encoding the GDP-mannose dehydratase polypeptide according to claim 1 into a host cell to construct a transformed host cell; then, culturing the transformed host cell and inducing it to express the GDP-mannose dehydratase polypeptide; Preferably, the step (2) comprises: a step of isolating and purifying the GDP-mannose dehydratase polypeptide from the culture.

9. Use of the GDP-mannose dehydratase polypeptide according to claim 1, or the transformed host cell according to claim 5, or the enzyme agent or enzyme composition according to claim 6 in synthesizing GDP-4-keto-6-deoxymannose; The application, preferably, uses GDP-mannose as a substrate; The use is preferably for producing fucosyllactose in a cell-free expression system or in a host organism.

10. Use of the genetically modified cell according to claim 7 in synthesizing fucosyllactose; Preferably, the fucosyllactose includes 2'-fucosyllactose, 3-fucosyllactose, synthetic difucosyllactose, lactose-N-fucopentose I and lactose-N-difucohexaose I; Preferably, the application uses lactose and one or more selected from glucose, glycerol and sucrose as carbon sources to synthesize fucosyllactose de novo.

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