Kluyveromyces gene-modified cells and uses thereof

Abstract

The present application relates to a kind of GDP-mannose dehydratase polypeptide-containing kloeckera gene modification cell, it can be applied to the synthesis of human milk oligosaccharide, belong to synthetic biology field.The technical scheme of the present application is a kind of gene modification cell, it includes the recombinant nucleic acid sequence of polypeptide with the activity of GDP-mannose dehydratase polypeptide (GDP-mannose dehydratase).The polypeptide with catalyzing GDP-mannose is converted into GDP-4-ketone-6-deoxymannose, it includes amino acid sequence with SEQ ID NO:1, SEQ ID NO:2 And / or with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical amino acid sequence of polypeptide M1-M13.The present application provides a kind of kloeckera gene modification cell with the activity of synthesizing fucosyllactose.

Description

Technical Field

[0001] This invention relates to Kluyveromyces gene-modified cells containing GDP-mannose dehydrating enzyme polypeptide, which can be used for the synthesis of human milk oligosaccharides (HMOs), and belongs to the field of bioengineering technology. Background Technology

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

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

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

[0005] The biosynthesis of 2'-FL and 3-FL was initially achieved through enzymatic catalysis. In 2000, Albermann et al., through exogenous expression and purification of GDP-mannose-4,6-dehydratase and GDP-fucose synthase from *Escherichia coli* K12, converted GDP-D-mannose to GDP-fucose in vitro. GDP-fucose is the most crucial precursor in the synthesis of 2'-FL and 3-FL. They also found that the activity of GDP-mannose-4,6-dehydratase could be inhibited by GDP-fucose, thus making GDP-mannose-4,6-dehydratase a critical rate-limiting enzyme. Therefore, it is necessary to study GDP-mannose-4,6-dehydratase to improve the synthesis efficiency and conversion rate of 2'-FL and 3-FL. Modification of key enzymes is crucial for improving catalytic efficiency.

[0006] Saccharomyces cerevisiae strains are recognized as safe microorganisms, characterized by their non-pyrogenicity, lack of toxicity, and high food safety, while also reducing subsequent isolation and purification costs. Furthermore, Saccharomyces cerevisiae is widely used industrially. However, it cannot utilize lactose or transport it into the cell. Although scientists have introduced lactose transporters into Saccharomyces cerevisiae, the yield of 2'-fucosylated lactose synthesized by the recombinant strain is only 0.5 g / L (Yuet al., Microb Cell Fact, 2018, 17:101. DOI:10.1186 / s12934-018-0947-2), which is insufficient to meet the needs of industrial production.

[0007] Chinese patents 202010187309.1 and 202010187632.9 synthesize 2'-FL using recombinant Saccharomyces cerevisiae, which includes GDP-mannose-dehydratase, GDP-fucose synthase, and α-1,2-fucotransferase, with yields of 2.9 g / L and 3.8 g / L, respectively.

[0008] As the importance of HMOs to the human body is gradually recognized, their social demand will gradually increase. In order to meet social needs, it is necessary to optimize the key factors of the fucose-lactose synthesis pathway to improve the efficiency of fucose synthesis and meet social needs. Summary of the Invention

[0009] Objective of the invention: To provide a gene-modified cell and its application in the synthesis of fucoidan, which has de novo fucoidan synthesis activity. The fucoidan includes, but is not limited to, 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL).

[0010] Technical solution:

[0011] A genetically modified cell containing a recombinant nucleic acid sequence encoding a GDP-mannose dehydratase (GMD) polypeptide.

[0012] The GDP-mannose dehydratase polypeptide, also known as GDP-D-mannose-4,6-dehydratase or GDP-mannose-6-dehydrogenase, belongs to the lyase family and has the activity of catalyzing the conversion of GDP-mannose to GDP-4-keto-6-deoxymannose. The GDP-mannose dehydratase polypeptide is one of the key enzymes in GDP-fucose synthesis.

[0013] The heterologous GDP-mannose dehydratase polypeptide gene sources include, but are not limited to, *Escherichia coli*, *Caenorhabditis elegans*, human *Homo sapiensis*, *Arabidopsis thaliana*, *Dictyostelium discoideum*, *Mus musculus*, *Drosophila melanogaster*, *Sinorhizobium fredii*, *Pandoraea vervacti*, and *C. bimastigotes*.

[0014] Natural or genetically modified strains of bacteria such as *Caenorhabditis briggsae*, *Candidatus Curtissbacteria*, *Pseudomonas sp.*, *Clostridium sp.*, *Cricetulus griseus*, *Arthrobacter siccitolerans*, *Paraburkholderia piptadeniae*, or *Bacillus smithii*.

[0015] Preferably, the GDP-mannose dehydratase polypeptide is a GDP-mannose dehydratase derived from Bacillus smithii, which is excavated from nature, named BsGMD, and its amino acid sequence is shown in SEQ ID NO:1 and its nucleotide sequence is shown in SEQ ID NO:7.

[0016] A comparison with the NCBI database revealed that the GDP-mannose dehydratase with the amino acid sequence shown in SEQ ID NO:1 was most similar to that of GDP-mannose dehydratase derived from *Escherichia coli* (as of November 15, 2023). The two enzymes shared only 69.03% amino acid sequence similarity, indicating that SEQ ID NO:1 does not have a high degree of similarity to known enzymes. The amino acid sequence of the GDP-mannose dehydratase from *Escherichia coli* is shown in SEQ ID NO:2.

[0017] In some embodiments, the GDP-mannose dehydratase polypeptide comprises a polypeptide whose amino acid sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of the polypeptide shown in SEQ ID NO:1 or SEQ ID NO:2, or polypeptides M1-M13. Wherein, polypeptides M1-M13 are derived peptides of the polypeptide whose amino acid sequence is as shown in SEQ ID NO:1.

[0018] The genetically modified cells include, but are not limited to, GDP-mannose dehydratase polypeptides whose amino acid sequences are identical to those of polypeptides shown in SEQ ID NO:1 or SEQ ID NO:2, or whose amino acid sequences are at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to those of polypeptides M1-M13.

[0019] Preferably, the GDP-mannose dehydratase polypeptide comprises the polypeptide (BsGMD) with the amino acid sequence shown in SEQ ID NO:1 and its derived peptides M1-M13. The derived peptides M1-M13 are dehydratase polypeptides formed by substitution of one or more amino acid residues / fragments in the polypeptide with the amino acid sequence shown in SEQ ID NO:1.

[0020] Preferably, the derived peptide M1-M13 is obtained by modifying at least one of the following sites in the amino acid sequence shown in SEQ ID NO:1: positions 69-72, 118-119, 123-124, 176, 203-205, 213-217, 257-264, 273-276, 288, 304-312, 339-443, and 365.

[0021] Preferably, the modification is a mutation, and the mutation is selected from any of the following mutations:

[0022] 1) In the amino acid sequence shown in SEQ ID NO:1, the amino acid fragment at positions 69-72 is replaced with ASIR by EKIK, thereby obtaining polypeptide M1;

[0023] 2) In the amino acid sequence of polypeptide M1, the amino acid fragment at positions 118-119 is replaced by LD, thereby obtaining polypeptide M2;

[0024] 3) In the amino acid sequence of polypeptide M2, the amino acid fragment at positions 123-124 is replaced by EK with QD, thereby obtaining polypeptide M3;

[0025] 4) In the amino acid sequence of polypeptide M3, the amino acid fragment at position 176 is replaced with D instead of N, thereby obtaining polypeptide M4;

[0026] 5) In the amino acid sequence of polypeptide M4, the amino acid fragment at positions 203-205 is replaced by IQL with RDI, thereby obtaining polypeptide M5;

[0027] 6) In the amino acid sequence of polypeptide M5, the amino acid residues at positions 213-217 are replaced by LDTLY with KKVLK, thereby obtaining polypeptide M6;

[0028] 7) In the amino acid sequence of polypeptide M6, the amino acid fragment at positions 257-264 is replaced by FTVREAVE with YTVKEIVK, thereby obtaining polypeptide M7;

[0029] 8) In the amino acid sequence of polypeptide M7, the amino acid fragment at positions 273-276 is replaced by ELEF with ALRW, thereby obtaining polypeptide M8;

[0030] 9) In the amino acid sequence of polypeptide M8, the amino acid fragment at position 288 is replaced by K with E, thereby obtaining polypeptide M9;

[0031] 10) In the amino acid sequence of polypeptide M9, amino acid residues at positions 304-312 are replaced by ILEVDPSFL with VLKVDPAFR, thereby obtaining polypeptide M10.

[0032] 11) In the amino acid sequence of polypeptide M10, the amino acid fragment at positions 339-443 is replaced by FEEMM with IEEMI, thereby obtaining polypeptide M11;

[0033] 12) In the amino acid sequence of polypeptide M11, the amino acid fragment at positions 352-360 is replaced by KVAEEYAEK with AVAEKYAEL, thereby obtaining polypeptide M12.

[0034] 13) In the amino acid sequence of polypeptide M12, the amino acid residue at position 365 is replaced by Y with E, thereby obtaining polypeptide M13.

[0035] Preferably, the GDP-mannose dehydratase polypeptide further comprises an amino acid substitution mutation functionally equivalent to the schemes described in 1)-13) above. In some embodiments, the substitution mutation comprises a mutation to a charged residue; in some embodiments, the substitution mutation comprises a mutation to a basic residue. In some embodiments, the substitution mutation comprises a mutation homologous to the amino acid sequence described in 1)-13) above.

[0036] The method for producing GDP-mannose dehydrating enzyme peptides as described above includes:

[0037] (1) Transformed host cells are cultured under conditions suitable for expressing the polypeptide;

[0038] (2) The polypeptide is recovered.

[0039] In some embodiments, the host cell includes, but is not limited to, natural strains or genetically modified strains such as bacteria, yeast, and mold;

[0040] More preferably, the host cell includes, but is not limited to, natural strains or genetically modified strains such as Escherichia sp., Bacillus sp., and Kluyveromyces sp.;

[0041] Further preferably, the host cell, the yeast cell, includes, but is not limited to, natural or genetically modified strains such as *Saccharomyces cerevisiae*, *Cloveromyces cerevisiae*, *Yarrowia lipolytica*, *Saccharomyces paradoxus*, *Saccharomyces babyus*, *Saccharomyces pastorianus*, *Saccharomyces cariocas*, *Saccharomyces mikatae*, and *Saccharomyces kudriavzevii*, or other nucleic acid constructs capable of transforming the polypeptides and / or derived peptides M1-M13 shown in SEQ ID NO:1 and SEQ ID NO:2. Preferably, the yeast cell is selected from *Cloveromyces cerevisiae* cells; more preferably, it is selected from *Cloveromyces lactis* cells and *Cloveromyces marx* cells.

[0042] More preferably, the host cell is the genetically engineered Escherichia coli E.coli BL21(DE3).

[0043] In a specific implementation, step (1) includes: firstly, introducing a nucleic acid construct or recombinant expression vector containing a polypeptide encoding GDP-mannose dehydratase as described 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.

[0044] In a specific implementation, step (2) includes the steps of separating and purifying the polypeptide from the culture.

[0045] Host cells can be cultured in nutrient media suitable for peptide production using methods known in the art. For example, cells can be cultured by shake-flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a laboratory or industrial fermenter in a suitable medium and under conditions that allow for peptide expression and / or isolation. The culture occurs using procedures known in the art in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media can be purchased commercially or prepared according to publicly available compositions.

[0046] In some embodiments, preferably, the genetically modified cells include, but are not limited to, yeast genetically modified cells.

[0047] Preferably, the yeast gene cells include, but are not limited to, Kluyveromyces gene-modified cells;

[0048] More preferably, the Kluyveromyces gene-modified cells include, but are not limited to, Kluyveromyces lactis gene-modified cells and Kluyveromyces marxosa gene-modified cells.

[0049] Furthermore, in some embodiments, in addition to the heterologous nucleic acid encoding the aforementioned dehydrating enzyme, the genetically modified yeast gene cell may also include one or more heterologous nucleic acids encoding GDP-L-fucose synthase, alpha-1,2-fucosyltransferase, or alpha-1,3-fucosyltransferase. The genetically modified yeast gene cell is capable of de novo synthesis of fucoidan lactose using lactose and glucose, or glycerol, or sucrose as carbon sources.

[0050] The α-1,2-fucosyltransferase gene is derived from, but is not limited to, natural or genetically modified strains of Helicobacter pylori, Thermophilic Chlorella, Escherichia coli, Caenorhabditis elegans, Schistosoma mansoni, Bacillus cereus, Pseudopedobater saltans, Helicobacter mustelae, Bacillus fragilis, Bacteroides vulgatus, Bacteroides fragilis, or Bacillus smithii.

[0051] The α-1,3-fucosyltransferase gene is derived from, but is not limited to, European wild boar (Sus scrofa), green monkey (Chlorocebus sabaeus), chimpanzee (Pan troglodytes), Helicobacter pylori, Akkermansia muciniphila, Bacteroides fragilis, and maize (Zea mays), Escherichia coli, western lowland gorilla (Gorilla gorilla gorilla), rhesus monkey (Macaca mulatta), rabbit (Oryctolagus cuniculus), Borneo orangutan (Pongo pygmaeus), brown rat (Rattus norvegicus), Akkermansia muciniphila, gibbon (Hylobates lar), and domestic cattle (Bos Natural or genetically modified strains of bacteria such as *Helicobacter hepaticus*, *Hylobatesagilis*, *Eulemur fulvus*, *Helicobacter hepaticus*, and *Azospirillum brasilense*.

[0052] The heterologous GDP-fucose synthase gene is derived from, but is not limited to, *Escherichia coli* and *mice*. Mus musculus Natural or genetically modified strains of bacteria such as Homo sapiens, Marinobacter alaris, Sinorhizobium fredii, Citrobacter, Pongo abelii, Caenorhabditis elegans, Candidatus Staskawiczbacteria, Azorhizobium caulinodans, or Candictus nitrospira nitrificans are used.

[0053] In some embodiments, the Kluyveromyces gene-modified cells further include at least a portion of the β-galactosidase gene (lac4) required for lactose metabolism in the starting strain, which is missing or destroyed. Therefore, the Kluyveromyces gene-modified cells of this application can introduce lactose without consuming lactose. Compared to the starting strain, the expression level of the β-galactosidase gene in the Kluyveromyces gene-modified cells is reduced; therefore, the Kluyveromyces gene-modified cells of this invention reduce lactose consumption. When the Kluyveromyces gene-modified cells are Kluyveromyces lactis gene-modified cells, the nucleic acid sequence of its β-galactosidase lac4 gene is Genebank number M84410.1, and the amino acid sequence is Genebank number AAA35265.1; when the Kluyveromyces gene-modified cells are Kluyveromyces marxosa gene-modified cells, the nucleic acid sequence of its β-galactosidase gene lac4 gene is Genebank number XM_022818497.1, and the amino acid sequence is Genebank number XP_022675157.1.

[0054] The Kluyveromyces gene-modified cells of this invention can achieve high yields of fucosylated lactose without the introduction of heterologous transport proteins. Preferably, the Kluyveromyces lactis and Kluyveromyces marxoiris are used.

[0055] In one embodiment, a preferred technical solution is that the starting strain of the Kluyveromyces lactis gene-modified cells is Kluyveromyces lactis DSM70799; and the starting strain of the Kluyveromyces marxianus gene-modified cells is Kluyveromyces marxianus DMKU3-1042. As described above, the Kluyveromyces lactis and Kluyveromyces marxianus starting strains can be readily purchased commercially or from culture collection centers.

[0056] The Kluyveromyces gene-modified cells, compared to the starting strain, have been genetically engineered to include increased intracellular GDP-fucose and fucoidan production capacity.

[0057] Preferably, the Kluyveromyces gene-modified cells have been genetically engineered and have the following characteristics:

[0058] 1) Express at least one of the genes encoding GDP-mannose dehydratase and GDP-fucose synthase.

[0059] 2) Expressing genes encoding α-1,2-fucosyltransferase polypeptide or α-1,3-fucosyltransferase polypeptide.

[0060] 3) Disruption of the galactosidase gene lac4.

[0061] Optionally, the amino acid sequence of the GDP-mannose dehydratase is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to that of the polypeptide shown in SEQ ID NO:1 or to polypeptides M1-M13.

[0062] The GDP-fucose synthase, α-1,2-fucosyltransferase polypeptide, and α-1,3-fucosyltransferase polypeptide may be genetically modified enzymes or wild-type enzymes.

[0063] The present invention also provides a gene-modified cell containing a molecular marker. The molecular marker has the amino acid sequence shown in SEQ ID NO:4.

[0064] Preferably, the molecular marker-containing gene-modified cell has a molecular marker nucleotide sequence as shown in SEQ ID NO:8.

[0065] The present invention also provides a method for preparing gene-modified cells, wherein the method uses Kluyveromyces as the starting strain and integrates heterologous α-1,2-fucosyltransferase gene or α-1,3-fucosyltransferase gene, heterologous GDP-fucose synthase gene, and heterologous GDP-mannose dehydrase gene into the Kluyveromyces genome, respectively.

[0066] Preferably, the GDP-mannose dehydratase gene includes, but is not limited to, genes of GDP-mannose dehydratase polypeptides whose amino acid sequences are at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequences of the polypeptide or polypeptide M1-M13 shown in SEQ ID NO:1.

[0067] Preferably, the gene-modified cells in the preparation method are Kluyveromyces gene-modified cells; more preferably, they are Kluyveromyces lactis gene-modified cells or Kluyveromyces marxosa gene-modified cells.

[0068] More preferably, the starting strain of Kluyveromyces lactis is K. lacticDSM70799, and the starting strain of Kluyveromyces marxianus is K. marxianusDMKU3-1042.

[0069] Preferably, the method for preparing the Kluyveromyces gene-modified cells includes the following steps:

[0070] (1) Cultivate the starting strain;

[0071] Preferably, the starting strain includes, but is not limited to, natural strains or genetically modified strains of Kluyveromyces cells;

[0072] Preferably, the Kluyveromyces cells are natural strains or genetically modified strains of Kluyveromyces lactis (K. lactic) or Kluyveromyces marxianus.

[0073] Preferably, the starting strain is selected from Kluyveromyces lactis cells K. lacticDSM70799;

[0074] Preferably, the starting strain is selected from Kluyveromyces marxianus cells DMKU3-1042.

[0075] (2) An expression cassette was constructed and a heterologous GDP-mannose dehydrase gene was introduced into Kluyveromycetes cells;

[0076] Preferably, the GDP-mannose dehydratase comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, or any one of polypeptides M1-M13;

[0077] (3) Destroy the β-galactosidase gene lac4 in the strain obtained in step (1);

[0078] (4) Construct an expression cassette and introduce a heterologous GDP-fucose synthase gene into Kluyveromycetes cells;

[0079] (5) Construct an expression cassette and introduce a heterologous α-1,2-fucosyltransferase gene or α-1,3-fucosyltransferase gene into Kluyveromycetes cells.

[0080] Optionally, the recombinant construction technology of Kluyveromyces gene-modified cells further includes:

[0081] (6) Construct a molecular marker expression cassette and introduce the marker gene into Kluyveromycetes cells.

[0082] Preferably, it further includes (7) the step of recovering the Kluyveromyces gene-modified cells.

[0083] Alternatively, the steps described above for preparing Kluyveromyces gene-modified cells are not sequential.

[0084] In some embodiments of the present invention, a method for preparing fucoidosyl lactose is provided, wherein the preparation method synthesizes fucoidosyl lactose through fermentation of gene-modified cells as described in this application.

[0085] Preferably, the fucoidyl lactose in the preparation method includes, but is not limited to, 2'-fucosyl lactose or 3-fucosyl lactose;

[0086] Preferably, the gene-modified cells in the preparation method include, but are not limited to, yeast gene-modified cells;

[0087] More preferably, the yeast gene-modified cells in the preparation method include, but are not limited to, Kluyveromyces gene-modified cells;

[0088] More preferably, the Kluyveromyces gene-modified cells in the preparation method include, but are not limited to, Kluyveromyces lactis gene-modified cells or Kluyveromyces marxosa gene-modified cells.

[0089] In some embodiments, the fermentation in the preparation method requires the use of a culture medium, which can be any genetically modified cells capable of maintaining growth and viability to produce fucosylated lactose. In some embodiments, the culture medium may also include appropriate salts, minerals, metals, or other nutrients. In some embodiments, the carbon source and each nutrient necessary for cell growth are added to the culture medium in an incremental or continuous manner.

[0090] The preparation method involves de novo synthesis of fucoidosyllactose using lactose and sucrose, or glycerol, or glucose as carbon sources.

[0091] In some implementations, the culture medium does not contain fucose.

[0092] In some embodiments, the method also includes adjusting the mass ratio of carbon source (such as sucrose) to lactose to adjust the yield of fucose-based lactose.

[0093] Preferably, the fermentation product of the yeast gene-modified cell can be purified by centrifugation, filtration, decolorization, nanofiltration, chromatographic purification, crystallization, recrystallization, etc., to obtain pure fucoidosyl lactose.

[0094] This application also provides a method for preparing fucoidosyl lactose, wherein the preparation method utilizes the gene-modified cells described in this application to ferment and synthesize fucoidosyl lactose using lactose and sucrose, or glycerol, or glucose as carbon sources.

[0095] The term "heterogeneous" refers to polynucleotides, genes, nucleic acids, polypeptides, or enzymes that are derived from or originate from sources other than the host organism species.

[0096] The term "molecular marker" refers to a specific DNA segment that reflects a certain difference in the genome of an individual or population.

[0097] The term "deletion or disruption" refers to the partial or complete modification (e.g., by deletion, insertion, and / or substitution of one or more nucleotides) of the coding region and / or control sequence of a reference gene, such that the expression of the encoded polypeptide is absent (inactivated) or reduced, and / or the enzyme activity of the encoded polypeptide is absent or reduced.

[0098] The term "fucosyl lactose" includes, but is not limited to, 2'-fucosyl lactose or 3-fucosyl lactose.

[0099] Beneficial effects:

[0100] This application provides gene-modified cells containing the nucleotide sequence of the GDP-mannose dehydratase polypeptide, or polypeptides M1-M13, as shown in SEQ ID NO:1. Its beneficial effects are as follows:

[0101] (1) Compared to the original strain, the Kluyveromyces gene-modified cells produced the activity of synthesizing fucosylated lactose.

[0102] (2) Compared with fucoidan produced by Escherichia coli or Escherichia coli gene-modified cells, fucoidan synthesized by recombinant yeast gene-modified cells containing the nucleotide sequence of SEQ ID NO:1 or polypeptide M1-M13 does not contain endotoxins or other allergens, and is safe for consumption, making it more suitable for application in food, health products, infant food and other fields.

[0103] (3) The process of synthesizing fucoidan-based lactose by Kluyveromyces gene modification cells described in this invention is simple and easy to implement, improves production efficiency and reduces production costs.

[0104] This invention provides excellent microbial resources for the bioprocessing of GDP-fucose and fucose-based lactose.

[0105] The technical solution of this application has positive significance for the industrial production of human milk oligosaccharides. This method is green, efficient, and sustainable, and is conducive to large-scale industrial production, thus having important practical value. Attached Figure Description

[0106] Figure 1 HPLC chromatogram of .2'-FL standard.

[0107] Figure 2 HPLC chromatogram of .3-FL standard.

[0108] Figure 3 HPLC chromatogram of KL-M6 fermentation broth in Example 4, rt = 13.43 min.

[0109] Figure 4 HPLC chromatogram of KM-M10-1 fermentation broth in Example 4, rt = 14.16 min.

[0110] Figure 5 .2'-FL standard quality spectrum.

[0111] Figure 6 .3-FL standard quality spectrum.

[0112] Figure 7 The mass spectrum of the substance with rt=13.43 min in the HPLC chromatogram of the fermentation broth of strain KL-M6 in Example 4.

[0113] Figure 8 The mass spectrum of the substance with rt=14.16min in the HPLC chromatogram of the fermentation broth of strain KM-M10-1 in Example 4. Detailed Implementation

[0114] The present invention will be further described in detail below through embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. It should be noted that those skilled in the art can make several modifications or substitutions to the details and form of the technical solutions of the present invention without departing from the principles of the present invention, and these modifications or substitutions also fall within the protection scope of the present invention.

[0115] It should be noted that the terminology used is for describing specific embodiments only and is not intended to limit the exemplary embodiments of the present invention. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, and such techniques and conditions are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0116] Unless otherwise specified, the experimental methods described in the following specific embodiments are generally performed according to conventional methods and conditions in molecular biology within the art, which are fully explained in the literature; all materials, reagents, etc., are commercially available unless otherwise specified.

[0117] This document also provides a method for preparing fucosyllactose. The method includes providing genetically modified yeast cells capable of producing fucosyllactose. The yeast cells include the GDP-mannose dehydratase polypeptide gene described in this application. In some embodiments, the method further includes providing a culture medium and culturing the yeast cells in the culture medium under conditions suitable for yeast cells to produce fucosyllactose.

[0118] The cultivation can be carried out in suitable containers, including but not limited to cell culture plates, culture flasks, or fermenters in suitable culture media. Any suitable fermenter can be used, including but not limited to stirred fermenters, airlift fermenters, bubble fermenters, or any combination thereof. In a specific embodiment using *Kluyveromyces lactis* as the host cell, the strain can be grown in a fermenter. Furthermore, the method can be performed at any scale of fermentation known in the art to support the industrial production of microbial products. The materials and methods used for maintaining or growing cell cultures are well known to those skilled in the art of microbiology or fermentation.

[0119] In some embodiments, the culture medium contains lactose and sucrose, or glucose. In some embodiments, the carbon source in the culture medium is substantially composed of lactose and sucrose, or glucose. Preferably, in some embodiments, the carbon source in the culture medium is composed of lactose and sucrose.

[0120] The abbreviations in the following examples are explained as follows:

[0121] GMD: GDP-mannose dehydratase; GMER: GDP-fucose synthase; futC: α-1,2-fucosyltransferase polypeptide; futA: α-1,3-fucosyltransferase polypeptide.

[0122] In the following example:

[0123] The starting strains of Kluyveromyces lactis include: K. lacticDSM70799; the starting strain of Kluyveromyces marxianus is: K. marxianusDMKU3-1042.

[0124] GDP-mannose dehydratase (GMD): amino acid sequences as shown in SEQ ID NO:1 and SEQ ID NO:2, or polypeptides M1-M13;

[0125] GDP-fucose synthase (GMER) is derived from Escherichia coli (GenBank:WP_000043652.1), and its amino acid sequence is shown in SEQ ID NO:3.

[0126] The α-1,2-fucosyltransferase polypeptide (futC) was derived from Helicobacter pylori (GenBank: AAC99764.1), and its amino acid sequence is shown in SEQ ID NO:5.

[0127] The α-1,3-fucosyltransferase polypeptide (futA) is derived from Helicobacter pylori (WP_000487428.1), and its amino acid sequence is shown in SEQ ID NO:6.

[0128] The 2'-FL and 3-FL standards are manufactured by ELICITYL, a French company.

[0129] In the following embodiments, the artificial amino acid sequence of the encryption tag was synthesized by a bioengineering company, and the artificial amino acid sequence of the encryption tag is shown in SEQ ID NO:4.

[0130] Example 1. Expression of the polypeptides and polypeptides M1-M13 shown in SEQ ID NO:1 and SEQ ID NO:2 in Escherichia coli.

[0131] 1. Using the amino acid sequence as shown in SEQ ID NO:1 (nucleotide sequence as shown in SEQ ID NO:7) or the polypeptide shown in SEQ ID NO:2 as templates, the gene coding sequences shown in SEQ ID NO:1 and SEQ ID NO:2 were synthesized respectively, and finally constructed into the PET32a vector to obtain the plasmids PET32a-BSGMD-WT and PET32a-ECGMD-WT.

[0132] 2. By utilizing computer-aided rational design, different mutation sites were obtained. Referring to the methods in *Molecular Cloning: A Laboratory Manual*, and based on the sequence of SEQ ID NO:7 of PET32a-wt, Primer-BLAST was used. Primer designing tool(nih.gov) Mutant primers were designed, and a series of recombinant plasmids (pET32a-M1-M13) were constructed using a point mutation kit.

[0133] 3. Transform the above recombinant plasmids into Escherichia coli BL21(DE3) according to the following steps:

[0134] Prepared Escherichia coli BL21(DE3) competent cells were thawed on ice for 30 min. 100 μL of competent cells were mixed with 10 μL of pET32a--M1-M13 recombinant plasmid (concentration 50 ng / μL) and heat-shocked in a 42℃ water bath for 45 s. Immediately afterward, the mixture was cooled on ice for 2 min. 1 mL of fresh LB medium (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, plate with 1.5% agar powder) was added and the cells were incubated at 37℃ and 100 rpm for 1 h. Then, 100 μL of the bacterial solution was spread on an LB plate containing ampicillin (100 μg / mL) and incubated at 37℃ for 12 h. Single colonies were picked for colony PCR to screen for positive transformants.

[0135] 4. Cultivate positive transformants, then extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the pET32a-M1-M13 recombinant plasmid was successfully introduced into E. coli.

[0136] 5. Inoculate the correct transformants into LB liquid medium and incubate at 37°C and 200 rpm on a shaker for 12 hours to obtain the seed culture; then inoculate the seed culture into fresh LB medium at a 1% (v / v) inoculation rate and incubate at 37°C with shaking until OD reaches 100%. 600 The concentration was 0.8, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mmol / L. Induction was performed at 16℃ for 12 h at 200 rpm. After induction, the fermentation broth was centrifuged at 5000 r / min for 30 min at 4℃ to collect the cells. The cells were resuspended in 20 mM pH 7.4 PBS buffer and sonicated at a frequency of plus on 5 s / off 5 s for 30 min to lyse the cells. The lysed liquid was centrifuged at 13000×g at 4℃ for 30 min to remove cell debris and the supernatant was collected.

[0137] 6. Soluble peptide sequences were purified using nickel column affinity chromatography as follows: Deionized water was added to the top of the nickel column, and after natural elution, elution was performed with 5 volumes of binding buffer. Crude enzyme solution filtered through a 0.45 μm membrane was then loaded onto the column, and the sample was allowed to fully bind to the nickel column at a flow rate of 1.5 mL / min. After the sample dried, impurities were removed by a continuous gradient elution with 5 column volumes of washing buffer. Finally, the target protein was eluted with 5 volumes of elution buffer, and the eluent was collected. The expression of the target protein was then analyzed using SDS-PAGE.

[0138] SDS-PAGE results showed that the gene-modified cells had obvious specific expression bands after induction, and the molecular weight of the bands was basically consistent with the expected molecular weight of 35.3 kDa. Therefore, the polypeptides shown in SEQ ID NO:1 and SEQ ID NO:2 and polypeptides M1-M13 were obtained. For details, please refer to Table 1.

[0139] Table 1. Correspondence between GDP-mannose dehydratase polypeptides and their amino acid sequence numbers or nomenclature

[0140] Recombinant plasmid GDP-mannose dehydratase polypeptide nomenclature / amino acid sequence PET32a-ECGMD-WT SEQ ID NO:2 PET32a-BSGMD-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

[0141] Example 2. Construction of gene-modified cells of Kluyveromyces lactis.

[0142] (1) Construct a lactate-Kluyvedet modified cell expression cassette.

[0143] 1) Construction of the KL-ΔLAC4 knockout box.

[0144] Referring to the applicant's prior patent: 202211453172.5, invention title: recombinant yeast and its application, constructing a KL-ΔLAC4 knockout box.

[0145] 2) Construction of the KL-ΔLAC4::FutC / KL-ΔLAC4::FutA expression box.

[0146] Using the Kluyveromyces lactis genome as a template, the upstream and downstream homologous arm sequences lac4 up and lac4-do, the promoter sequence tef1, and the terminator sequence adh1 were amplified. Using the synthesized FutC and FutA sequences as templates, the target gene sequences FutC and FutA were amplified. Using the expression cassette KL-ΔLAC4 as a template, the G418+loxp sequence was amplified. The lac4 up, tef1, FutC / FutA, adh1, G418+loxp, and lac4-do were fused by sequential fusion PCR of the two fragments. Finally, using primers lac4-upF and lac4-doR as primers (Table 2) and the fusion PCR system as a template, the expression cassette KL-ΔLAC4::FutC / KL-ΔLAC4::FutA was amplified by PCR.

[0147] Table 2

[0148]

[0149] 3) Construction of the KL-ΔXK::GMER expression box.

[0150] Using the Kluyveromyces lactis genome as a template, the upstream and downstream homologous arm sequences xk-up and xk-do of the xylose 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 an existing plasmid in the laboratory as a template, the G418+loxp sequence was amplified. Through a two-fragment successive fusion PCR method, xyl1 up, pgk1, gmer, tdh3, G418+loxp, and xyl1-do were fused. Finally, using primers xK-upF and xK-doR as primers (Table 3), and using the fusion PCR system as a template, the gmer expression cassette KL-ΔXK::GMER was amplified by PCR.

[0151] Table 3

[0152]

[0153] 4) Construction of expression boxes from KL-ΔGK::BSGMD-WT, KL-ΔGK::BSGMD-M1 to KL-ΔGK::BSGMD-M13, KL-ΔGK::ECGMD-WT.

[0154] 2. Using Kluyveromyces lactis DSM70799 genomic DNA as a template, PCR amplification was performed using primers galk up-F and galk up-R, and galk down-F and galk down-R, to obtain upstream and downstream homologous arms, respectively. Using loxp-F and loxp-R as primers and G418 resistance plasmid as a template, resistance selection markers containing G418 resistance and loxp sites were amplified. Using PET32a-BSGMD-WT, PET32a-M1 to PET32a-M13, and PET32a-ECGMD-WT as templates, the original and mutant sequences of the GMD gene were obtained by PCR amplification. Using Kluyveromyces lactis genome as a template, the promoter tdh3 and terminator sequence tef of the transferase were obtained by PCR amplification. Using KL-PF and KL-PR as primers and PUC19 plasmid as a template, the plasmid vector sequence was obtained by PCR amplification. Fusion PCR was used to combine the upstream homologous arm, promoter, target gene (original and mutant sequences), terminator, G418+loxp, and downstream homologous arm to obtain expression cassettes ΔGK::BSGMD-WT, ΔGK::BSGMD-M1 to ΔGK::BSGMD-M13, and ΔGK::ECGMD-WT. These expression cassettes were then ligated to plasmid vector sequences using an ABclonal technique to obtain plasmids PUC-KL-WT, PUC-KL-M1 to PUC-KL-M13, and PUC-KL-ECGMD-WT, each carrying one of the expression cassettes ΔGK::BSGMD-WT, ΔGK::ECGMD-WT, ΔGK::BSGMD-M1 to ΔGK::BSGMD-M13.

[0155] Using primers galk up-F and galk down-R as templates, and PUC-KL-WT, PUC-KL-M1 to PUC-KL-M13, and PUC-KL-ECGMD-WT as templates, expression cassettes KL-ΔGK::BSGMD-WT, KL-ΔGK::BSGMD-M1 to KL-ΔGK::BSGMD-M13, and KL-ΔGK::ECGMD-WT were amplified and used for the construction of recombinant strains in the next step.

[0156] Table 4. Primers for constructing KL-ΔGK::BSGMD-WT, KL-ΔGK::BSGMD-M1, KL-ΔGK::BSGMD-M13, and KL-ΔGK::ECGMD-WT expression cassettes

[0157]

[0158] (2) Transformation of recombinant expression cassettes and verification of recombinant strains.

[0159] The expression cassettes constructed in (1) above were transformed into the starting strain Kluyveromyces lactis K. lacticDSM70799 cells. The specific method is as follows:

[0160] 1) First, prepare competent yeast cells: Streak a small amount of frozen yeast strain onto a plate of solid culture medium and incubate upside down at 30°C for 2 days. Pick a single yeast colony and place it in 50 mL of liquid culture medium, incubate at 30°C and 220 rpm until OD reaches 100%. 600 The pH should be between 0.8 and 1.5. Collect the bacterial 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 precipitate, centrifuge at 12000 rpm for 30 s, and discard the supernatant. Add 400 μL of 100 mM lithium chloride buffer again, resuspend the precipitate, and obtain competent yeast cells. Aliquot into 50 μL tubes for transformation.

[0161] Meanwhile, boil 1 mL of salmon sperm DNA for 5 minutes, then quickly place it on an ice bath to prepare single-stranded DNA.

[0162] 2) Transformation: Centrifuge the prepared competent yeast cells and remove residual lithium chloride solution using Tips. For each transformation, add the following in order: 50% PEG3350 (240 μL); 1M LiCl (36 μL); 2 mg / mL single-stranded Salmon sperm DNA (25 μL); 5–10 μg / 50 μL plasmid DNA aqueous solution (50 μL), vortex vigorously until the precipitated cells are completely and evenly distributed; incubate at 30°C for 30 min; heat shock at 42°C for 20–25 min; centrifuge at 8000 rpm for 10 min, and collect the yeast cells; then, resuspend the yeast in 500 μL of liquid medium and incubate at 30°C on a shaker; after 1–4 h, take 25–100 μL of the bacterial suspension and spread it on selective medium plates, and incubate upside down at 30°C.

[0163] 3) Verification: The correspondence between recombinant strains and their genotypes is shown in Table 5-6. To verify the correctness of the above strains, we extracted the genomes of the transformants and the original strains, and performed PCR amplification on the genomes using the corresponding knockout or expression cassette primers. If a unique band was obtained after PCR amplification and its size matched that of the knockout or expression cassette, the strain was considered correct; otherwise, the strain was considered a false positive.

[0164] Table 5. Genetically modified cells of Kluyveromyces lactis and their genotypes (transferase is α-1,2-fucosyltransferase gene)

[0165] Microbial cells genotype K.lacticDSM70799 wt KL-1 K.lacticDSM70799;ΔLAC4::FutC KL-2 KL-1;KL-ΔXK::GMER KL-3 KL-2;KL-ΔGK::BSGMD-WT KL-4 KL-2;KL-ΔGK::ECGMD-WT KL-M1 KL-2;KL-ΔGK::BSGMD-M1 KL-M2 KL-2;KL-ΔGK::BSGMD-M2 KL-M3 KL-2;KL-ΔGK::BSGMD-M3 KL-M4 KL-2;KL-ΔGK::BSGMD-M4 KL-M5 KL-2;KL-ΔGK::BSGMD-M5 KL-M6 KL-2;KL-ΔGK::BSGMD-M6 KL-M7 KL-2;KL-ΔGK::BSGMD-M7 KL-M8 KL-2;KL-ΔGK::BSGMD-M8 KL-M9 KL-2;KL-ΔGK::BSGMD-M9 KL-M10 KL-2;KL-ΔGK::BSGMD-M10 KL-M11 KL-2;KL-ΔGK::BSGMD-M11 KL-M12 KL-2;KL-ΔGK::BSGMD-M12 KL-M13 KL-2;KL-ΔGK::BSGMD-M13

[0166] Encryption tags were introduced into the recombinant strains listed in Table 5, resulting in a total of 17 strains, including KL-XJ and KL-YJ. Where: X represents values ​​1-4; Y represents polypeptides M1-M13.

[0167] Table 6. Genetically modified cells of Kluyveromyces lactis and their genotypes (transferase is α-1,3-fucosyltransferase gene)

[0168] Microbial cells genotype K.lacticDSM70799 WT KL-1-1 K.lacticDSM70799;ΔLAC4::FutA KL-2-1 KL-1;KL-ΔXK::GMER KL-3-1 KL-2;KL-ΔGK::BSGMD-wt KL-4-1 KL-2;KL-ΔGK::ECGMD-WT KL-M1-1 KL-2;KL-ΔGK::BSGMD-M1 KL-M2-1 KL-2;KL-ΔGK::BSGMD-M2 KL-M3-1 KL-2;KL-ΔGK::BSGMD-M3 KL-M4-1 KL-2;KL-ΔGK::BSGMD-M4 KL-M5-1 KL-2; KL-ΔGK::BSGMD-M5 KL-M6-1 KL-2; KL-ΔGK::BSGMD-M6 KL-M7-1 KL-2; KL-ΔGK::BSGMD-M7 KL-M8-1 KL-2; KL-ΔGK::BSGMD-M8 KL-M9-1 KL-2; KL-ΔGK::BSGMD-M9 KL-M10-1 KL-2; KL-ΔGK::BSGMD-M10 KL-M11-1 KL-2; KL-ΔGK::BSGMD-M11 KL-M12-1 KL-2; KL-ΔGK::BSGMD-M12 KL-M13-1 KL-2; KL-ΔGK::BSGMD-M13

[0169] Encryption tags were introduced into the recombinant strains listed in Table 6, resulting in 17 strains, including KL-X-1-J and KL-Y-1-J. Where: X represents values ​​1-4; Y represents polypeptides M1-M13.

[0170] Example 3. Construction of gene-modified cells of Max Kluyveromyces.

[0171] (1) Construct the Max Kluwer modified cell expression cassette.

[0172] 1) Construction of the KM-ΔLAC4 knockout box.

[0173] Referring to the applicant's prior patent: 202211453172.5, invention title: Recombinant yeast and its application in constructing a KM-ΔLAC4 knockout box.

[0174] 2) Construction of the KM-ΔLAC4::FutC / KM-ΔLAC4::FutA expression box.

[0175] Using the Kluyveromyces martensii genome as a template, the upstream and downstream homologous 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. The km-lac4 up, FutC or FutA expression cassette + G418+loxp and km-lac4-do were fused by three fragment fusion PCR. Finally, using primers lac4-upF1 and lac4-doR1 as primers (Table 7) and the fusion PCR system as a template, the FutC and FutA expression cassettes KM-ΔLAC4::FutC and KM-ΔLAC4::FutA were amplified by PCR.

[0176] Table 7

[0177]

[0178] 3) Construction of the KM-ΔXK::GMER expression box.

[0179] Using the genome of *Kluyveromyces martensii* as a template, the upstream and downstream homologous arm sequences of *Kluyveromyces martensii* xylulokine, km-xk up and km-xk-do, were amplified. Using the KM-ΔXK::GMER expression cassette as a template, the GMER expression cassette and the G418+loxp site were amplified. The km-xk up, GMD expression cassette + G418+loxp and km-xk-do were fused by three-fragment fusion PCR. Finally, using primers xk-upF1 and xk-doR1 as primers (Table 8) and the fusion PCR system as a template, the gmer expression cassette KM-ΔXK::GMER was amplified by PCR.

[0180] Table 8

[0181]

[0182] 4) Construction of expression boxes KM-ΔGK::BSGMD-WT, KM-ΔGK::BSGMD-M1, KM-ΔGK::BSGMD-M13, KM-ΔGK::ECGMD-WT.

[0183] Using genomic DNA from *Kluyveromyces marxianus* DMKU3-1042 as a template, PCR amplification was performed using primers galk up-F1 and galk up-R1, as well as galk down-F1 and galk down-R1, to obtain upstream and downstream homologous arms, respectively. Using KL-ΔGK::BSGMD-wt, KL-ΔGK::BSGMD-M1 to KL-ΔGK::BSGMD-M13 expression cassettes, and KL-ΔGK::ECGMD-wt expression cassettes as templates, the BSGMD-wt / GMD mutant expression cassette was amplified by PCR.

[0184] The / ECGMD+G418+loxp sequence was used to fuse the upstream and downstream homologous arms and the BSGMD-wt / GMD mutant expression cassette / ECGMD expression cassette+G418+loxp sequence using fusion PCR. Using the fusion system as a template and primers galk up-F1 and galk down-R1 as templates, KM-ΔGK::BSGMD-WT, KM-ΔGK::BSGMD-M1 to KM-ΔGK::BSGMD-M13 and KM-ΔGK::ECGMD-WT expression cassettes were amplified and used in the next step of constructing recombinant strains.

[0185] Table 9. Primers for constructing the KM-ΔGK::BSGMD-WT, KM-ΔGK::BSGMD-M1 to KM-ΔGK::BSGMD-M13 expression cassettes, and the KM-ΔGK::ECGMD-WT expression cassette.

[0186]

[0187]

[0188] (2) Transformation of recombinant plasmids and verification of recombinant strains:

[0189] The expression cassette constructed in (1) above was transformed into the starting strain Kluyveromyces marxianus DMKU3-1042 cells. The specific method is as described in Part (2) of Example 2, Transformation of Recombinant Expression Cassette and Verification of Recombinant Strains.

[0190] The correspondence between recombinant strains and their genotypes is shown in Table 10-11. To verify the correctness of the above strains, we extracted the genomes of the transformants and the original strains, and performed PCR amplification on the genomes using the corresponding knockout or expression cassette primers. If a unique band was obtained after PCR amplification and its size matched that of the knockout or expression cassette, the strain was considered correct; otherwise, the strain was considered a false positive.

[0191] Table 10. Genetically modified cells of Kluyveromyces martensii and their genotypes (transferase is α-1,2-fucosyltransferase gene)

[0192] Strain Genotype K. marxianus DMKU3-1042 wt KM-1 K. marxianus DMKU3-1042; ΔLAC4::FutC KM-2 KM-1; KM-ΔXK::GMER KM-3 KM-2; KM-ΔGK::BSGMD-WT KM-4 KM-2; KM-ΔGK::ECGMD-WT KM-M1 KM-2; KM-ΔGK::BSGMD-M1 KM-M2 KM-2; KM-ΔGK::BSGMD-M2 KM-M3 KM-2; KM-ΔGK::BSGMD-M3 KM-M4 KM-2; KM-ΔGK::BSGMD-M4 KM-M5 KM-2; KM-ΔGK::BSGMD-M5 KM-M6 KM-2; KM-ΔGK::BSGMD-M6 KM-M7 KM-2; KM-ΔGK::BSGMD-M7 KM-M8 KM-2; KM-ΔGK::BSGMD-M8 KM-M9 KM-2; KM-ΔGK::BSGMD-M9 KM-M10 KM-2; KM-ΔGK::BSGMD-M10 KM-M11 KM-2; KM-ΔGK::BSGMD-M11 KM-M12 KM-2; KM-ΔGK::BSGMD-M12 KM-M13 KM-2; KM-ΔGK::BSGMD-M13

[0193] Encryption tags were introduced into the recombinant strains listed in Table 10, resulting in a total of 17 recombinant strains, including KM-XJ and KM-YJ. Where: X represents values ​​1-4; Y represents polypeptides M1-M13.

[0194] Table 11. Genetically modified cells of Kluyveromyces martensii and their genotypes (transferase is α-1,3-fucosyltransferase gene)

[0195] Strain Genotype K. marxianus DMKU3-1042 wt KM-1-1 K. marxianus DMKU3-1042; ΔLAC4::FutA KM-2-1 KM-1; KM-ΔXK::GMER KM-3-1 KM-2; KM-ΔGK::BSGMD-WT KM-4-1 KM-2; KM-ΔGK::ECGMD-WT KM-M1-1 KM-2; KM-ΔGK::BSGMD-M1 KM-M2-1 KM-2; KM-ΔGK::BSGMD-M2 KM-M3-1 KM-2; KM-ΔGK::BSGMD-M3 KM-M4-1 KM-2; KM-ΔGK::BSGMD-M4 KM-M5-1 KM-2;KM-ΔGK::BSGMD-M5 KM-M6-1 KM-2;KM-ΔGK::BSGMD-M6 KM-M7-1 KM-2;KM-ΔGK::BSGMD-M7 KM-M8-1 KM-2;KM-ΔGK::BSGMD-M8 KM-M9-1 KM-2;KM-ΔGK::BSGMD-M9 KM-M10-1 KM-2;KM-ΔGK::BSGMD-M1 KM-M11-1 KM-2;KM-ΔGK::BSGMD-M1 KM-M12-1 KM-2;KM-ΔGK::BSGMD-M1 KM-M13-1 KM-2;KM-ΔGK::BSGMD-M1

[0196] Encryption tags were introduced into the recombinant strains listed in Table 11, resulting in a total of 17 strains, including KM-X-1-J and KM-Y-1-J. Where: X represents values ​​1-4; Y represents polypeptides M1-M13.

[0197] Validation of marker genes: To verify the correctness of the above strains, we extracted the genomes of the transformants and the original strains, and performed PCR amplification on the genomes using the corresponding knockout or expression cassette primers. If a unique band was obtained after PCR amplification, and its size matched that of the knockout or expression cassette, the strain was considered correct; otherwise, it was considered a false positive. For tag validation, PCR amplification was performed using primers, followed by sequencing for verification; otherwise, the strain was considered a false positive.

[0198] Example 4. Study on the synthesis of fucoidan-based lactose by gene modification in Kluyveromyces oryzae cells.

[0199] Yeast was cultured using glucose as the carbon source. Gene-modified cells obtained in Examples 2-3 were taken and allowed to grow rapidly until they entered the late logarithmic or stationary phase. The strains were streaked onto solid media such as YDP and cultured at 30°C for 2-3 days. Single colonies were then inoculated into 1.5 mL of LYPD liquid medium and cultured overnight at 30°C with shaking at 200 rpm. Subsequently, 2% of the culture was inoculated into 50 mL shake flasks of liquid medium and cultured at 30°C with shaking at 200 rpm until OD (out of growth) was reached. 600 =1, add lactose to a final concentration of 10 g / L and sucrose 3% (w / v), and culture at 30℃ with shaking at 200 rpm for a total fermentation time of 72 h. After 72 h of fermentation, take samples and boil for 10 min, centrifuge to obtain the final fermentation product of yeast, and detect the 2'-FL content in the supernatant.

[0200] HPLC detection method: Detection conditions: Column type: Shodex Asahipak NH2P-50 4E, mobile phase: 65% acetonitrile aqueous solution, flow rate: 0.5 ml / min, column temperature: 35℃, injection volume: 10 μL, evaporative light detector, evaporation temperature: 75℃, nebulization temperature: 45℃.

[0201] The LC-MS analysis conditions are as follows:

[0202] Column type: 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 ℃, mobile phase: acetonitrile:water = 65:35; ESI-MS mode, molecular weight scan range 100–800.

[0203] The standard and reaction solution were analyzed using the aforementioned analytical method. HPLC analysis results showed:

[0204] (1) The peak elution time rt of 2'-FL standard was 13.43 min; the peak elution time rt of 3-FL standard was 14.16 min.

[0205] (2) The Kluyveromyces gene-modified cells (KL-3, KL-4, KM-3, KM-4 and strains with M1-M13 genes introduced, as well as the above strains with marker genes introduced) obtained in Examples 2-3 showed a strong absorption peak around 13.43 min, which was consistent with the peak time of the 2'-FL standard, indicating that 2'-FL was generated in the fermentation broth of the gene-modified cells obtained in Examples 2-3.

[0206] (3) The Kluyveromyces gene-modified cells (KL-3-1, KL-4-1, KM-3-1, KM-4-1 and strains with M1-M13 genes introduced, as well as the above strains with introduced marker genes) obtained in Examples 2-3 showed a strong absorption peak around 14.16 min, which was consistent with the peak time of 3-FL standard, indicating that 3-FL was generated in the fermentation broth of the gene-modified cells obtained in Examples 2-3.

[0207] The LC-MS analysis conditions are shown above, and the analysis results show:

[0208] 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.

[0209] The products at rt=13.43 min and rt=14.16 min of the HPLC chromatographic peaks of the reaction solution were analyzed by LC-MS. The MH value was 487.17, which was consistent with the results of the mass spectra of the 2'-FL and 3-FL standards and was within the allowable error range of the theoretical molecular weight of 2'-FL and 3-FL, 488.44.

[0210] The yields of 2'-FL and 3-FL in the fermentation broth were determined using the HPLC analysis method described above, and the results are recorded in Table 12-15.

[0211] Table 12. Study on the synthesis of fucoidosyllactose in lactate-modified cells obtained in Example 2.

[0212]

[0213]

[0214] Table 13. Study on the synthesis of fucoidosyllactose in Max Kluwer gene-modified cells obtained in Example 3.

[0215]

[0216] Table 14. Study on the synthesis of fucoidosyllactose in lactate-modified cells obtained in Example 2.

[0217]

[0218]

[0219] Table 15. Study on the synthesis of fucoidosyllactose in Max Kluwer gene-modified cells obtained in Example 3.

[0220]

[0221] Table 12-15 Data Explanation:

[0222] (1) Lactate Kluyveromyces and Max Kluyveromyces originating strains, as well as the genetically modified cells KL-1, KL-2, KM-1 and KM-2, cannot synthesize 2'-FL; KL-3 and KM-3 produce 2'-FL; KL-1-1, KL-2-1, KM-1-1 and KM-2-1 cannot synthesize 3-FL; KL-3-1 and KM-3-1 produce 2'-FL.

[0223] Note: Lactate Kluyvei / Max Kluyvei cells do not have the ability to synthesize 2'-FL on their own, and the introduction of GMD and GMER genes also cannot achieve the synthesis of 2'-FL or 3-FL. The generation of 2'-FL or 3-FL was detected in the gene-modified cells described in this application.

[0224] (2) The Kluyvei gene-modified cells described in this invention have the activity to synthesize 2'-FL.

[0225] The introduction of the marker gene does not affect the yield of 2'-FL or 3-FL synthesized by fermentation in cells modified with the Kluyvedo gene obtained in Example 2 or Example 3.

[0226] Example 5. Kluyveromyces KL-M1-J, KL-M13-J, KM-M1-J, KM-M13-J, KL-4-J, and KM-4-J, as well as KL-M1-1-J, KL-M13-1-J, KM-M1-1-J, KM-M13-1-J, and KM-4-1-J, were streaked onto solid media such as YDP and cultured at 30°C for 2-3 days. Single colonies were then picked and inoculated into 1.5 mL of LYPD liquid medium and cultured overnight at 30°C with shaking at 200 rpm. Subsequently, 2% of each colony was inoculated into 50 mL shake flasks of liquid medium and cultured at 30°C with shaking at 200 rpm until OD reached [value missing]. 600 =1, 2% of each yeast extract was inoculated into 1L of YPD medium (10g / L yeast extract, 20g / L peptone, 20g / L glucose) in a 3L fermenter. After 6-7 hours of cultivation, sucrose (50% mother liquor concentration) was added at a flow rate of 8mL / h, and lactose (40% mother liquor concentration) was added simultaneously to maintain a final lactose concentration of 15g / L. The total fermentation time was 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and the supernatant and precipitate were collected separately. The precipitate was homogenized using a high-pressure homogenizer, and the protein was removed by boiling and centrifugation. The supernatants were combined to obtain the final yeast fermentation product. The contents of 2'-FL and 3-FL in the supernatant were detected, and the results are recorded in Table 16.

[0227] Table 16

[0228]

[0229] While the invention has been described in considerable detail by way of illustration and example for purposes of clarity, it will be apparent to those skilled in the art that any equivalent aspects or modifications may be made. Therefore, this specification and the embodiments should not be construed as limiting the scope of the invention.

Claims

1. A genetically modified cell, characterized in that, The compound contains a recombinant nucleic acid sequence encoding a GDP-mannose dehydratase polypeptide, wherein the gene-modified cell is selected from yeast gene-modified cells, and the GDP-mannose dehydratase polypeptide includes, but is not limited to, amino acid sequences selected from polypeptides M1-M13, wherein polypeptides M1-M13 are obtained by modifying at least one site of the amino acid sequences shown in SEQ ID NO:1, namely positions 69-72, 118-119, 123-124, 176, 203-205, 213-217, 257-264, 273-276, 288, 304-312, 339-443, and 365, specifically, the modification is a mutation, and the mutation is selected from any of the following mutations: 1) In the amino acid sequence shown in SEQ ID NO:1, the amino acid fragment at positions 69-72 is replaced with ASIR by EKIK, thereby obtaining polypeptide M1; 2) In the amino acid sequence of polypeptide M1, the amino acid fragment at positions 118-119 is replaced by LD, thereby obtaining polypeptide M2; 3) In the amino acid sequence of polypeptide M2, the amino acid fragment at positions 123-124 is replaced by EK with QD, thereby obtaining polypeptide M3; 4) In the amino acid sequence of polypeptide M3, the amino acid fragment at position 176 is replaced with D instead of N, thereby obtaining polypeptide M4; 5) In the amino acid sequence of polypeptide M4, the amino acid fragment at positions 203-205 is replaced by IQL with RDI, thereby obtaining polypeptide M5; 6) In the amino acid sequence of polypeptide M5, the amino acid residues at positions 213-217 are replaced by LDTLY with KKVLK, thereby obtaining polypeptide M6; 7) In the amino acid sequence of polypeptide M6, the amino acid fragment at positions 257-264 is replaced by FTVREAVE with YTVKEIVK, thereby obtaining polypeptide M7; 8) In the amino acid sequence of polypeptide M7, the amino acid fragment at positions 273-276 is replaced by ELEF with ALRW, thereby obtaining polypeptide M8; 9) In the amino acid sequence of polypeptide M8, the amino acid fragment at position 288 is replaced by K with E, thereby obtaining polypeptide M9; 10) In the amino acid sequence of polypeptide M9, amino acid residues at positions 304-312 are replaced by ILEVDPSFL with VLKVDPAFR, thereby obtaining polypeptide M10. 11) In the amino acid sequence of polypeptide M10, the amino acid fragment at positions 339-443 is replaced by FEEMM with IEEMI, thereby obtaining polypeptide M11; 12) In the amino acid sequence of polypeptide M11, the amino acid fragment at positions 352-360 is replaced by KVAEEYAEK with AVAEKYAEL, thereby obtaining polypeptide M12. 13) In the amino acid sequence of polypeptide M12, the amino acid residue at position 365 is replaced by Y with E, thereby obtaining polypeptide M13.

2. The genetically modified cell of claim 1, wherein, The genetically modified cells were selected from Kluyveromyces lactis genetically modified cells and Kluyveromyces marxianus genetically modified cells.

3. The genetically modified cell of claim 1, wherein, The genetically modified cells were selected from Kluyveromyces sp. genetically modified cells.

4. The genetically modified cell of any one of claims 1-3, wherein, It also includes one or more heterologous nucleic acids encoding GDP-L-fucose synthase, alpha-1,2-fucosyltransferase, or alpha-1,3-fucosyltransferase.

5. The gene-modified cell as described in claim 4, characterized in that, The α-1,2-fucosyltransferase gene is derived from natural or genetically modified strains of Helicobacter pylori, Thermophilic Chlorella, Escherichia coli, Caenorhabditis elegans, Schistosoma mansoni, Bacillus cereus, Pseudopedobater saltans, Helicobacter mustelae, Bacillus fragilis, Bacteroides vulgatus, Bacteroides fragilis, or Bacillus smithii.

6. The gene-modified cell as described in claim 4, characterized in that, The α-1,3-fucosyltransferase gene is derived from, but is not limited to, European wild boar (Sus scrofa), green monkey (Chlorocebus sabaeus), chimpanzee (Pantroglodytes), Helicobacter pylori, Akkermansia muciniphila, Bacteroides fragilis, and maize (Zea mays), Escherichia coli, western lowland gorilla (Gorilla gorilla gorilla), rhesus monkey (Macacamulatta), rabbit (Oryctolagus cuniculus), Borneo orangutan (Pongo pygmaeus), brown rat (Rattus norvegicus), Akkermansia muciniphila, gibbon (Hylobates lar), domestic cattle (Bos taurus), and black-handed gibbon (Hylobates lar). Natural or genetically modified strains of *Agilis*, *Eulemur fulvus*, *Helicobacter hepaticus*, and *Azospirillum brasilense*.

7. The gene-modified cell as described in claim 4, characterized in that, The heterologous GDP-fucose synthase gene is derived from, but is not limited to, Escherichia coli and mice. Musmusculus Natural or genetically modified strains of Homo sapiens, Marinobacter salarius, Sinorhizobium fredii, Citrobacter, Pongo abelii, Caenorhabditis elegans, Candidatus Staskawiczbacteria, Azorhizobium caulinodans, or Candictus nitrospiranitrificans.

8. The gene-modified cell according to any one of claims 1-3, characterized in that, The genetically modified cells also include at least a portion of the β-galactosidase gene (lac4) of the starting strain that is missing or destroyed.

9. The method for preparing gene-modified cells according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Cultivate the starting strain; (2) An expression cassette was constructed and a heterologous GDP-mannose dehydrase gene was introduced into Kluyveromycetes cells; (3) Destroy the β-galactosidase gene lac4 in the strain obtained in step (1); (4) Construct an expression cassette and introduce a heterologous GDP-fucose synthase gene into Kluyveromycetes cells; (5) Construct an expression cassette and introduce a heterologous α-1,2-fucosyltransferase gene or α-1,3-fucosyltransferase gene into Kluyveromycetes cells.

10. The use of the gene-modified cells obtained in claim 9 in the synthesis of fucoidosyllactose.

11. A method for preparing fucoidosyl lactose, characterized in that, Obtained by fermentation of the gene-modified cells according to claim 9.

12. The application of claim 10, wherein the fucoidyl lactose is selected from 2'-fucosylvose or 3-fucosylvose.

13. The application as described in claim 10, wherein fucoidosyl lactose is synthesized de novo using lactose and glucose, or glycerol, or sucrose as carbon sources.

14. The preparation method according to claim 11, characterized in that, The fucoidyl lactose is selected from 2'-fucosyl lactose or 3-fucosyl lactose.

15. The preparation method according to claim 11, characterized in that, Fucosyl lactose can be synthesized de novo using lactose and glucose, or glycerol, or sucrose as carbon sources.

16. The method for preparing gene-modified cells as described in claim 9, characterized in that, Also includes: A molecular marker expression cassette was constructed, and the marker gene was introduced into Kluyveromycetes cells.

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