GDP-fucose synthase polypeptide and application thereof

Abstract

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

Description

Technical Field

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

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

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

[0004] Fucosyllactose is synthesized by chemical, enzymatic or microbial cell factory methods. Although chemical synthesis has made great progress, the fucosyllactose molecules need to be repeatedly protected and deprotected during the synthesis process. The process is cumbersome, resulting in low product yield and high production costs. It is still very difficult to truly achieve the large-scale synthesis of 2'-FL and 3-FL using chemical synthesis. The synthesis of fucosyllactose using microorganisms is currently a feasible method for large-scale production of fucosyllactose. Moreover, the fucosyllactose synthesized by microorganisms is functionally the same as natural fucosyllactose, and clinical trials will not cause adverse reactions. Compared with chemical synthesis methods, microbial synthesis methods are safer and faster. There is no need to introduce a large amount of toxic reagents during the synthesis process. 2'-FL and 3-FL can be directly produced using food safety-grade microorganisms, which greatly increases the safety of the product.

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

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

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

[0008] In summary, it is necessary to study GDP-fucose synthase to improve the synthesis efficiency of 2'FL and 3-FL. The modification of key enzymes is crucial to improve the catalytic efficiency of enzymes. Summary of the invention

[0009] Purpose of the invention: Provide a GDP-fucose synthase polypeptide (GDP-L-fucose synthase, WcaG) and its application in the synthesis of fucosyllactose to improve the production efficiency of fucosyllactose. The GDP-fucose synthase polypeptide, 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 (GDP-4-Keto-6-deoxy-D-mannose-3,5 epimerase-4-reductase), has the activity of catalyzing GDP-4-keto-6-deoxymannose to generate GDP-fucose, and belongs to the oxidoreductase family. The GDP-fucose synthase polypeptide is a key enzyme in the synthesis of GDP-fucose, and the GDP-fucose is a precursor of fucosyllactose.

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

[0011] Technical solution:

[0012] 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, and whose nucleotide sequence is shown in SEQ ID NO: 6. It has the activity of catalyzing GDP-4-keto-6-deoxymannose to generate GDP-fucose, and can be used in the synthesis of GDP-fucose.

[0013] After comparison with the NCBI database, the GDP-fucose synthase polypeptide with the highest similarity to the amino acid sequence of SEQ ID NO: 1 is the GDP-fucose synthase from Escherichia coli (as of November 15, 2023), and the amino acid sequences of the two are only 64.06% identical, indicating that the amino acid sequence similarity of SEQ ID NO: 1 and the known enzymes is not high. The amino acid sequence of the GDP-fucose synthase from Escherichia coli is shown in SEQ ID NO: 2.

[0014] The inventors of the present application have carried out a series of modifications on the GDP-fucose synthase polypeptide as shown in SEQ ID NO: 1, and screened and obtained a series of derivative peptides with improved activity in catalyzing the conversion of GDP-4-keto-6-deoxymannose into GDP-fucose.

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

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

[0017] Specifically, in the first aspect, the present application provides a series of GDP-fucose synthase polypeptides, wherein the GDP-fucose synthase polypeptides are selected from the polypeptides with amino acid sequences as shown in SEQ ID NO: 1 and its derivative peptides M1-14, and the polypeptides shown in SEQ ID NO: 2. The derivative polypeptides M1-14 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 shown in SEQ ID NO: 1.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0034] In certain embodiments, the polypeptide having improved catalytic conversion of GDP-4-keto-6-deoxymannose to GDP-fucose comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical amino acid sequence to SEQ ID NO: 1 and / or to polypeptide M1-14, and comprises an amino acid substitution mutation functionally equivalent to the scheme described in 1)-14) above. In certain embodiments, the substitution mutation comprises a mutation to a charged residue; in certain embodiments, the substitution mutation comprises a mutation to a basic residue. In certain embodiments, the substitution mutation comprises a mutation homologous to the amino acid sequence described in 1)-14) above.

[0035] In certain embodiments, the polypeptide having the ability to catalyze the conversion of GDP-4-keto-6-deoxymannose to GDP-fucose comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical amino acid sequence to SEQ ID NO: 1 and / or to polypeptide M1-14, and comprises an amino acid substitution mutation that is functionally equivalent to the scheme described in 1)-14) above. In certain embodiments, the substitution mutation comprises a mutation to a charged residue; in certain embodiments, the substitution mutation comprises a mutation to a basic residue. In certain embodiments, the substitution mutation comprises a mutation homologous to the amino acid sequence described in 1)-14) above.

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

[0037] Polynucleotides encoding GDP-fucose synthase polypeptides can be prepared using recombinant DNA techniques known in the art. These methods include, for example, cloning, recombination, in vitro synthesis, in vitro amplification and / or other available methods. A variety of methods can be used to express expression vectors encoding polypeptides presented in the present invention. Methods for preparing recombinant nucleotides, expressing and isolating expression products are known and described in the Examples.

[0038] In a third aspect, the present application provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above.

[0039] 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 expression host.

[0040] 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.

[0041] 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.

[0042] A variety of kits are commercially available for purifying plasmids and other related nucleic acids from cells. Any isolated and / or purified nucleic acid can be further manipulated to produce other nucleic acids for transfection of cells, integration into related vectors to infect organisms for expression, etc. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters that can be used to regulate expression of specific target nucleic acids, and the vector optionally contains a universal expression cassette.

[0043] In a specific embodiment, the expression cassette comprises all elements for expressing the GDP-fucose synthase polypeptide, including elements necessary for transcription and translation in the host cell, for example, the expression cassette comprises a promoter and a terminator, and the promoter and the terminator are not particularly limited and may be promoters and terminators known in the art that can achieve expression of the variant.

[0044] 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.

[0045] 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.

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

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

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

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

[0050] 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.

[0051] 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.

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

[0053] 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 guanylate transferase (Mannose-1-phosphoguanyltransferase, ManC), GDP-mannose dehydratase (GDP-mannose-4,6-dehydratase, GMD), etc., which are beneficial to the synthesis of fucosyllactose.

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

[0055] (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

[0056] (2) Recovering the GDP-fucose synthase polypeptide.

[0057] In a specific embodiment, the step (1) comprises: firstly 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 expressing the polypeptide; then, culturing the engineered host cell and inducing it to express the GDP-fucose synthase polypeptide.

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

[0059] Host cells can be cultivated in a nutrient medium suitable for producing polypeptides using methods known in the art. For example, cells can be cultured by shaking bottles, or in a suitable medium and under conditions that allow polypeptide expression and / or separation, small-scale or large-scale fermentations (including continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) can be carried out in a laboratory or industrial fermentor tank to cultivate cells. Cultivation is to use procedures known in the art, which occur in a suitable nutrient medium, and the medium comprises carbon and nitrogen sources and inorganic salts. Suitable medium can be purchased through commercial channels, or prepared according to disclosed compositions.

[0060] 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.

[0061] In addition, the GDP-fucose synthase polypeptide can be purified by various procedures known in the art to obtain substantially pure polypeptides, 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.

[0062] In an eighth 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;

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

[0064] The application, suitable reaction conditions include: the presence of a suitable reaction substrate, the presence of a suitable polypeptide, or the presence of basic auxiliary factors 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 the present application, but the reaction conditions must enable the GDP-fucose synthase polypeptide described in the present application to exert its enzymatic activity.

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

[0066] Preferably, the application is to prepare the GDP-fucose synthase polypeptide in a cell-free expression system, such as but not limited to the PURExpress system (NEB) or in a host organism (such as but not limited to Escherichia coli or Saccharomyces cerevisiae), after which the GDP-fucose synthase polypeptide listed above can be isolated and GDP-fucose or fucosyllactose can be synthesized in a suitable system, and optionally further purified. Specifically, it includes:

[0067] Using the polypeptide as described in the first aspect above or the enzyme or enzyme composition as described in the sixth aspect above, catalyzing the substrate GDP-4-keto-6-deoxymannose to synthesize GDP-fucose;

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

[0069] Optionally, a step of purifying and / or recovering GDP-fucose is also included.

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

[0071] The GDP-mannose dehydratase may be derived from, but is not limited to, Escherichia coli, Caenorhabditis elegans, Homo sapien, Arabidopsis thaliana, Dictyostelium discoidum, Mus musculus, Drosophila melanogaster, Sinorhizobium fredii, Pandoraea vervacti, Caenorhabditis briggsae, Candidatus Curtissbacteria, Pseudomonas sp., Clostridium sp., Cricetulus griseus, Arthrobacter siccitolerans, and Paraburkholderia piptadeniae.

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

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

[0074] In one embodiment, at least one of the GDP-fucose synthase polypeptides described in the present application is mixed with GDP-4-keto-6-deoxymannose, NADPH|, and 50 mM Tris-HCl buffer solution, and incubated at a specific temperature (e.g., 35°C, 37°C, or 40°C) for a specific period of time (e.g., 24 hours), during which GDP-4-keto-6-deoxymannose is catalyzed by the GDP-fucose synthase to be converted into GDP-fucose; GDP-fucose can be separated and purified by methods known in the art. At the end of the reaction or after separation and / or purification, the concentration of GDP-fucose in the solution is determined by HPLC chromatography.

[0075] Alternatively, a GDP-mannose dehydratase polypeptide is added to a reaction system containing GDP-mannose, and the GDP-mannose dehydratase polypeptide 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 the present 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.

[0076] The application of the GDP-fucose synthase polypeptide firstly uses GDP-4-keto-6-deoxymannose (GDP-mannose) as a substrate, adds GMD enzyme and NADP + enzyme, react at room temperature; and then catalyzed by the GDP-fucose synthase to synthesize GDP-fucose.

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

[0078] (1) GDP-mannose dehydratase catalyzes the synthesis of GDP-4-keto-6-deoxymannose from the substrate GDP-mannose;

[0079] (2) using the polypeptide described in the first aspect above or the enzyme or enzyme composition described in the sixth aspect above to catalyze the conversion of the substrate GDP-4-keto-6-deoxymannose into GDP-fucose;

[0080] Further preferably, it also includes:

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

[0082] 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.

[0083] 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, colors and other related impurities are removed from the mixture containing 2'-FL by ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated carbon or carbon treatment, chromatography, ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange), hydrophobic interaction chromatography and / or gel filtration (i.e., size exclusion chromatography), in particular by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography.

[0084] 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, spray drying.

[0085] The term "identity" refers to the identity of an amino acid sequence, and refers to the percentage of two or more sequences or subsequences that are identical or have identical amino acid residues or nucleotides when compared and aligned for maximum correspondence. An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

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

[0087] The term "polypeptide" refers to any peptide or protein comprising two or more amino acids connected 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 recorded in basic textbooks, and in more detail in monographs, as well as in a large amount of research literature, and they are well known to technicians.

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

[0089] Beneficial effects:

[0090] The present application provides a series of GDP-fucose synthase polypeptides, which include a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 and polypeptide M1-14, wherein 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 biopreparation of GDP-fucose and fucosyllactose. The GDP-fucose synthase of the present invention has a high catalytic activity, and the specific activity of the enzyme is much higher than that of the prior art. The present invention provides an excellent enzyme resource for the biopreparation technology of GDP-fucose or fucosyllactose.

[0091] 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

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

[0093] Figure 2 .HPLC spectrum of the catalytic reaction liquid of polypeptide M1 in Example 2.

[0094] Figure 3 .HPLC spectrum of the reaction liquid catalyzed by polypeptide M10 in Example 2.

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

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

[0097] Figure 6 . HPLC analysis of the fermentation broth of BS-M14 continuous feeding synthesis of 2'-fucosyllactose in Example 5.

[0098] Figure 7 .HPLC analysis of the fermentation broth of BS-M14-1 continuously fed-batch synthesis of 3-fucosyllactose in Example 6. DETAILED DESCRIPTION

[0099] The present invention is further described in detail below by way of 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. It should be noted that, for those of ordinary skill in the art, without departing from the principles of the present invention, the details and forms of the technical solution of the present invention may be modified or replaced in a number of ways, and these modifications or replacements also fall within the scope of protection of the present invention.

[0100] It should be noted that the terms used are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are to describe specific embodiments, rather than to limit the scope of protection of the present invention. If the experimental methods of specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology within the art are generally followed, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: A Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0101] In the following specific embodiments, if the experimental methods for specific conditions are not specified, they are usually based on conventional methods and conditions of molecular biology within the technology of the field, and such techniques and conditions are fully explained in the literature; all materials, reagents, etc., unless otherwise specified, can be obtained from commercial channels.

[0102] 2'-FL and 3-FL standards were produced by ELICITYL, France.

[0103] GDP-fucose standards were purchased from Aladdin.

[0104] In the following examples, the enzymes involved are as follows:

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

[0106] alpha-1,2-fucosyltransferase (FutC), microbial source Helicobacter pylori

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

[0108] GDP-fucose HPLC detection method:

[0109] C18μ Bondapack column (Waters, Milford, MA; 3.9*300 mm, 10-mm particlesize), mobile phase: 0.5 M KH 2 PO 4 of aqueous solution, flow rate: 1 ml / min, column temperature: 30 °C, injection volume: 10 μL, UV detector,

[0110] Fucosyllactose content HPLC detection method:

[0111] 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.

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

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

[0114] 1. Using the polypeptide shown in the amino acid sequence of SEQ ID NO: 1 (nucleotide sequence of SEQ ID NO: 6) or the polypeptide shown in the amino acid sequence of 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 PET32a-BsWcaG-wt plasmids and PET32a-EcWcaG-wt.

[0115] 2. Different mutation sites were obtained by computer-assisted rational design. According to the method of Molecular Cloning Experiment Guide, Primer-BLAST ( Primer designing tool(nih.gov) ) mutant primers were designed and a recombinant plasmid series (pET32a-M1-M14) was constructed using a point mutation kit (Fast Mutagenesis Kit).

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

[0117] Take the prepared Escherichia coli BL21 (DE3) competent cells, place them on ice for 30 minutes to melt, take 100 μL of competent cells and 10 μL of pET32a--M1-M14 recombinant plasmid (concentration 50 ng / μL) respectively, mix them, place them in a 42°C water bath for heat shock for 45 seconds, then immediately cool them in an ice bath for 2 minutes, add 1 mL of fresh LB medium (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, 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 out positive transformants.

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

[0119] 5. Inoculate the correct transformant into LB liquid medium, place 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) inoculation amount and shake and culture at 37°C until OD 600The expression of the expression vector was 0.8, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG), the final concentration of IPTG was 0.1mmol / L, and the induction was carried out at 16℃ for 12h, the rotation speed was 200rpm; after induction of expression, the fermentation broth was centrifuged at 5000r / min for 30min at 4℃ to collect the bacteria; the bacteria were resuspended in 20mM pH7.4 PBS buffer, and ultrasonicated at a frequency of plus on 5s / off 5s for 30min to break the bacteria; the liquid after breaking was centrifuged at 13000×g and 4℃ for 30min to remove cell debris and collect the supernatant.

[0120] 6. The soluble polypeptide sequence was purified by nickel column affinity chromatography. The process was as follows: deionized water was added to the top of the nickel column, and 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, and the sample was fully combined with 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, and finally the target protein was eluted with 5 times the Elution buffer, and the eluate was collected; then, the expression of the target protein was analyzed by SDS-PAGE.

[0121] The results of SDS-PAGE 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. It can be seen that the polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2 and polypeptide M1-14 were obtained. For detailed information, see Table 1.

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

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

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

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

[0126] 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 enzyme was mixed and reacted at 25°C for 8 hours, and the concentration of the substrate obtained by the reaction was measured.

[0127] Then, GDP-4-keto-6-deoxymannose with a final concentration of 100 μM, 50 mM Tris-HCl (pH 7.0), and 1 mM NADPH enzyme were added to 0.2 mg / mL of the purified polypeptides shown in SEQ ID NO: 1 and SEQ ID NO: 2 or one of the polypeptides M1-14 obtained in Example 1, respectively, mixed, reacted at 30° C. for 6 hours, terminated the reaction, and purified by gel column method.

[0128] The catalytic reaction liquid was detected according to the above analysis method. The HPLC analysis results showed that the reaction liquid of the polypeptide described in Example 1 showed a strong absorption peak around 13.3 min, which was consistent with the peak time of GDP-fucose (GDP-Fucose) standard, indicating that the catalytic reaction of the polypeptide described in Example 1 generated GDP-fucose (GDP-Fucose).

[0129] 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.

[0130] Table 2. Study on the synthesis of GDP-Fucose catalyzed by various peptides

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

[0132] Table 2 Data Description:

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

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

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

[0136] 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.) constructed strain BL21ΔlacZΔlonΔwcaJ, and the above-mentioned constructions PET32a-M1-HpFutC and PET32a-M14-HpFutC were transferred into the Escherichia coli chassis cells BL21ΔlacZΔlonΔwcaJ by electroporation, thereby constructing strains EC-M1 and EC-M14.

[0137] Method for culturing Escherichia coli EC-M1 and EC-M14 to synthesize 2'-fucosyllactose:

[0138] E. coli strains EC-M1 and EC-M14 were cultured on LB solid medium at 37°C for 10-12 hours, and single colonies were inoculated into 20 mL liquid LB and cultured in 250 mL shake flasks at 37°C and 220 rpm for 10-12 hours. Each strain was inoculated into 5 mL LB, and when the strain reached stationary phase, 1 mL of the culture was inoculated into 100 mL LB medium containing 36 g / L glucose (or glycerol) as a carbon source for growth in 500 mL shake flasks. When OD 600 When the pH reached about 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 production of fucosyllactose. At the same time, the medium needed to be supplemented with ampicillin at a final concentration of 100 μg / ml. After the end, the culture was boiled for 10 minutes, and the supernatant was centrifuged to detect the 2'-FL production. The results are recorded in Table 3.

[0139] Table 3

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

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

[0142] 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.) constructed strain BL21ΔlacZΔlonΔwcaJ, and the above construction or PET32a-M1-HpFutA and PET32a-M14-HpFutA were transferred into Escherichia coli chassis cells BL21ΔlacZΔlonΔwcaJ by electroporation to construct strains EC-M1-1 and EC-M14-1.

[0143] Method for culturing Escherichia coli EC-M1-1 and EC-M14-1 to synthesize 3-fucosyllactose:

[0144] The strains EC-M1-1 and EC-M14-1 were respectively used to ferment and synthesize 3-fucosyllactose according to the method of culturing Escherichia coli EC-M1 and EC-M14 to produce 2'-fucosyllactose as described in Example 3. After the culture was boiled for 10 minutes, the supernatant was centrifuged and the 3-FL production was detected. The results are recorded in Table 4.

[0145] Table 4

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

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

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

[0149] Method for culturing strains BS-M1 and BS-M14 to synthesize 2'-fucosyllactose:

[0150] Bacillus subtilis strains BS-M1 and BS-M14 were cultured on LB solid medium at 37°C for 10-12 hours, and single colonies were inoculated into 20 mL liquid LB and cultured in 250 mL shake flasks at 37°C and 220 rpm for 10-12 hours. Te seed culture was further inoculated into 30 mL fermentation medium at a rate of 10% and cultured in 250 mL shake flasks at 37°C and 220 rpm for 72 hours. A medium with the following composition was used during shake flask fermentation: 6 g / L trypsin, 12 g / L yeast extract, 12.5 g / L K 2 HPO 4 ·3H 2 O, 2.5 g / L KH 2 PO 4 and 10 mL / L of trace metal solution (composition: 4 g / L FeSO 4 7H 2 O, 4 g / L CaCl 2 , 1g / L MnSO4·H 2 O, 0.2g / LNaMoO 4 ·2H 2 O, 0.2g / LZnSO 4 7H 2 O, 0.1 g / L AlCl 3 6H 2 O, 0.1g / LCuCl2 ·2H 2 O, and 0.05 g / L H 3 BO 4 Sucrose and lactose were sterilized and added to the sterilized shake flask to a final concentration of 20 and 10 g / L, respectively. After the culture was boiled for 10 min, the supernatant was centrifuged and the 2'-FL yield was detected. The results are recorded in Table 5.

[0151] Table 5

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

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

[0154] Table 6

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

[0156] Example 6. Synthesis of 3-fucosyllactose using Bacillus subtilis BStgtP8 (CCTCC NO: M 20231126) as the chassis.

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

[0158] Method for culturing strains BS-M1-1 and BS-M14-1 to synthesize 3-fucosyllactose:

[0159] E. coli strains EC-M1-1 and EC-M14-1 were respectively fermented to synthesize 3-fucosyllactose according to the method for producing 2'-fucosyllactose by strains BS-M1 and BS-M14 described in Example 5. After the fermentation, the culture solution was boiled for 10 minutes, and the supernatant was centrifuged to detect the 3-FL production. The results are recorded in Table 7.

[0160] Table 7

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

[0162] The above process was simultaneously scaled up to a 1L fermenter, and continuous feeding fermentation was performed. The sucrose content in the fermentation broth was controlled within the range of 17g / L to 22g / L; the lactose concentration was controlled within the range of 10g / L to 20g / L. The fermentation was terminated after continuous feeding for 30 hours. After the fermentation was completed, the culture broth was boiled for 10 minutes, and the supernatant was centrifuged to detect the 3-FL content. The results are recorded in Table 8.

[0163] Table 8

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

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

[0166] The Saccharomyces cerevisiae chassis cells were constructed according to Xu et al. (Xu, M., Meng, X., Zhang, W., Shen, Y., & Liu, W. (2021). Improved production of 2'-fucosyllactose in engineered Saccharomyces cerevisiae expressing a putative α-1,2-fucosyltransferase from Bacillus cereus. Microbial cell factories, 20(1), 165.), and SC-Δgal80-Pgal1-lac12-Pgal1-HpFutA-GMD was constructed. The pRS305-Pgal-M1 and pRS305-Pgal-M14 plasmids were constructed and transferred into SC-Δgal80-Pgal1-lac12-Pgal1-HpFutC-GMD, respectively, to obtain strains SC-M1 and SC-M14.

[0167] Method for culturing strains SC-M1 and SC-M14 to synthesize 2'-fucosyllactose:

[0168] Take Saccharomyces cerevisiae SC-M1 and SC-M14 respectively, and let them grow rapidly until they enter the late logarithmic phase or the stable phase. Streak the strains in solid culture medium such as YDP, culture at 30℃ for 2-3 days, pick a single colony and inoculate it in 1.5mL YPD liquid culture medium, and culture it at 30℃ and 200rpm overnight. Then inoculate it in a 50mL liquid culture medium shake flask at a 2% inoculation rate, and culture it at 30℃ and 200rpm until OD 600 =1, and inoculated into 1.5L YPD medium (yeast extract powder 10g / L, peptone 20g / L, glucose 20g / L) (3L volume fermenter) at an inoculum of 2%, and after culturing for 5-6h, began to flow sucrose (mother liquor concentration 50%), and the sucrose addition flow rate was controlled at 8mL / h. At the same time, lactose (mother liquor concentration was 40%) was added to maintain the final lactose concentration of 15g / L. The total fermentation time was 72h. After 72h of fermentation, the fermentation liquid was centrifuged, and the supernatant and precipitate were collected respectively. The precipitate was crushed by a high-pressure homogenizer, boiled and centrifuged to remove protein, and the supernatant was combined to obtain the final fermentation product of yeast. The 2'-FL yield in the supernatant was detected, and the results were recorded in Table 9.

[0169] Table 9

[0170]

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

[0172] The Saccharomyces cerevisiae chassis cells were constructed according to Xu et al. (Xu, M., Meng, X., Zhang, W., Shen, Y., & Liu, W. (2021). Improved production of 2'-fucosyllactose in engineered Saccharomyces cerevisiae expressing a putative α-1,2-fucosyltransferase from Bacillus cereus. Microbial cell factories, 20(1), 165.), and SC-Δgal80-Pgal1-lac12-Pgal1-HpFutA-GMD was constructed. The pRS305-Pgal-M1 and pRS305-Pgal-M14 plasmids were constructed and transferred into SC-Δgal80-Pgal1-lac12-Pgal1-HpFutA-GMD, respectively, to obtain strains SC-M1-1 and SC-M14-1.

[0173] Method for culturing strains SC-M1-1 and SC-M14-1 to synthesize 3-fucosyllactose:

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

[0175] Table 10

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

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

[0178] The Kluyveromyces lactis chassis cells were constructed according to the reference Li et al. (Li, F., Ma, W., Liu, L., Niu, K., Liu, D., Yin, W., Zhang, X., Han, L., & Fang, X. (2023). Reprogramming the Metabolic Network in Kluyveromyces lactis with a Transcriptional Switch for De NovoLacto-N-biose Synthesis. Journal of agricultural and food chemistry, 71(23), 9031–9039. https: / / doi.org / 10.1021 / acs.jafc.3c01779) construction and transformation methods to obtain strains KL-ΔLAC4::HpFutA-ΔXK::GMD, and simultaneously constructed the expression cassettes ΔGK::Ptef1-M1 and ΔGK::Ptef1-M14, and transferred the expression cassettes into Strain KL-ΔLAC4 ::HpFutC-ΔXK::GMD to obtain KL-M1 and KL-M14 strains.

[0179] Method for culturing strains KL-M1 and KL-M14 to synthesize 2'-fucosyllactose:

[0180] Take Kluyveromyces KL-M1 and KL-M14 respectively, and let them grow rapidly until they enter the late logarithmic phase or the stable phase. Streak the strains in solid culture medium such as YDP, culture at 30℃ for 2-3 days, pick a single colony and inoculate it in 1.5mL YPD liquid culture medium, and culture it at 30℃ and 200rpm overnight. Then inoculate it in a 50mL liquid culture medium shake flask at a 2% inoculation rate, and culture it at 30℃ and 200rpm until OD600 =1, and inoculated into 1L YPD medium (yeast extract powder 10g / L, peptone 20g / L, glucose 20g / L) (3L volume fermenter) at an inoculum of 2%, and after culturing for 6-7h, began to flow sucrose (mother liquor concentration 50%), and the sucrose addition flow rate was controlled at 8mL / h, and lactose (mother liquor concentration was 40%) was added at the same time to maintain the final lactose concentration of 15g / L, and the total fermentation time was 72h. After 72h of fermentation, the fermentation liquid was centrifuged, and the supernatant and precipitate were collected respectively. The precipitate was crushed by a high-pressure homogenizer, boiled and centrifuged to remove the protein, and the supernatant was combined to obtain the final fermentation product of the yeast, and the 2'-FL content in the supernatant was detected. The results are recorded in Table 11.

[0181] Table 11

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

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

[0184] The Kluyveromyces lactis chassis cells were constructed according to the reference Li et al. (Li, F., Ma, W., Liu, L., Niu, K., Liu, D., Yin, W., Zhang, X., Han, L., & Fang, X. (2023). Reprogramming the Metabolic Network in Kluyveromyces lactis with a Transcriptional Switch for De NovoLacto-N-biose Synthesis. Journal of agricultural and food chemistry, 71(23), 9031–9039. https: / / doi.org / 10.1021 / acs.jafc.3c01779) construction and transformation methods to obtain strains KL-ΔLAC4::HpFutA-ΔXK::GMD, and the expression cassettes ΔGK::Ptef1-M1 and ΔGK::Ptef1-M14 were simultaneously constructed and the expression cassettes were transferred into KL-ΔLAC4::HpFutA-ΔXK::GMD to obtain the KL-M1-1 and KL-M14-1 strains, respectively.

[0185] Method for culturing strains KL-M1-1 and KL-M14-1 to synthesize 3-fucosyllactose:

[0186] Kluyveromyces KL-M1-1 and KL-M14-1 were respectively fermented to synthesize 3-fucosyllactose according to the method for producing 2'-fucosyllactose by strains KL-M1 and KL-M14 described in Example 9. After the fermentation, the culture solution was boiled for 10 minutes, and the supernatant was centrifuged to detect the 3-FL production. The results are recorded in Table 11.

[0187] Table 11

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

[0189] The present invention discloses a series of GDP-fucose synthase polypeptides, preparation methods and applications thereof, 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 present invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all 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.

[0190] Although the present invention has been described in considerable detail by way of illustration and example for purposes of clear understanding, it will be apparent to one of ordinary skill 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 present invention.

Claims

1. A GDP-fucose synthase polypeptide comprising an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical amino acid sequence to the polypeptide shown in SEQ ID NO: 1; or a polypeptide having an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to polypeptide M1-14; Preferably, the amino acid sequence of the GDP-fucose synthase polypeptide is as shown in SEQ ID NO:

1.

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

3. A polynucleotide encoding the GDP-fucose synthase polypeptide according to any one of claims 1-2.

4. A nucleic acid construct comprising the polynucleotide according to claim 3; 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.

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

6. A transformed host cell transformed with the polynucleotide according to claim 3, or the nucleic acid construct according to claim 4, or the expression vector according to claim 5; Preferably, the nucleic acid construct according to claim 5 or the host cell according to claim 6 is characterized in that: The host cells include but are not limited to natural strains or genetically modified strains such as bacteria, yeast, and mold; Further preferably, the host cell includes but is not limited to natural strains or genetically modified strains such as Escherichia sp., Bacillus sp., Kluyveromyces sp., etc.; Further preferably, the host cell includes but is not limited to natural strains or genetically modified strains such as Saccharomyces cerevisiae, Kluyveromyces marxianus, Kluyveromyces lactis, and Yarrowia lipolytica; Further preferably, the host cell includes but is not limited to the genetically engineered Escherichia coli BL21 (DE3).

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

8. The method for producing the GDP-fucose synthase polypeptide according to any one of claims 1 to 2, characterized in that: The steps include: (1) culturing the transformed host cell under conditions suitable for expressing the GDP-fucose synthase polypeptide; and (2) Recovering the polypeptide. Preferably, the step (1) comprises: firstly introducing a nucleic acid construct or a recombinant expression vector encoding the polypeptide as described in the first aspect above into a host cell to construct an engineered host cell expressing the polypeptide; then, culturing the engineered host cell and inducing it to express the polypeptide. Preferably, the step (2) includes the steps of separating and purifying the polypeptide from the culture.

9. Use of the GDP-fucose synthase polypeptide according to any one of claims 1 to 2, or the transformed host cell according to claim 6, or the enzyme agent or enzyme composition according to claim 7 in synthesizing GDP-fucose or fucosyllactose; The fucosyllactose includes 2'-fucosyllactose (2'-fucosyllactose, 2'-FL) and 3-fucosyllactose (3-fucosyllactose, 3-FL); 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. A method for preparing GDP-fucose, characterized in that: The steps include: a) using the GDP-fucose synthase polypeptide according to any one of claims 1 to 2, or the transformed host cell according to claim 6, or the enzyme agent or enzyme composition according to claim 7 as a catalyst to catalyze the synthesis of GDP-fucose from the substrate GDP-4-keto-6-deoxymannose; Further preferably, the preparation method further comprises: b) optionally, a step of purifying and / or recovering GDP-fucose; optionally, the preparation method further comprises: a step of using GDP-mannose dehydratase as a catalyst to catalyze the synthesis of GDP-4-keto-6-deoxymannose from the substrate GDP-mannose.

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