Kluyveromyces cerevisiae gene modified cell and application thereof

Abstract

The invention relates to a Kluyveromyces cerevisiae gene modified cell containing GDP-mannose dehydratase polypeptide, which can be applied to synthesis of human milk oligosaccharide, and belongs to the field of synthetic biology. According to the technical scheme, the gene modified cell comprises a recombinant nucleic acid sequence for coding a polypeptide with GDP-mannose dehydratase polypeptide (GDP-mannose dehydratase polypeptide) activity. The polypeptide capable of catalyzing the conversion of GDP-mannose into GDP-4-keto-6-deoxymannose comprises a polypeptide of which the amino acid sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 and / or the amino acid sequence of polypeptides M1-M13, wherein the amino acid sequence of the polypeptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% identical to the amino acid sequence of the polypeptides M1-M13. The invention provides a kluyveromyces kluyveromyces genetically modified cell with fucosyllactose synthesis activity.

Description

Technical Field

[0001] The present invention relates to Kluyveromyces genetically modified cells containing GDP-mannose dehydratase polypeptide, which can be applied to the synthesis of human milk oligosaccharides (HMOs), and belongs to the technical field of biological genetic engineering. Background Art

[0002] Human milk oligosaccharides (HMOs) are a unique and rich variety of oligosaccharide mixtures present in human milk, and are the third largest solid component in breast milk after fat and lactose. Among them, 2'-fucosyllactose and 3-fucosyllactose in fucosyllactose are important components of human milk oligosaccharides, and it has been confirmed that they play an important role in the brain, intestine and growth and development of infants.

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

[0004] The synthesis of fucosyllactose mostly adopts chemical method, enzymatic method or microbial cell factory synthesis method. Although the chemical synthesis method has made great progress, during the synthesis process, it is necessary to repeat and protect and deprotect the fucosyllactose molecule many times, and the process is cumbersome, resulting in low product yield and high production cost. It is still very difficult to truly achieve the large-scale synthesis of 2'-FL and 3-FL by chemical synthesis method. Using microorganisms to synthesize fucosyllactose is a feasible method for large-scale production of fucosyllactose at present, and the fucosyllactose synthesized by microorganisms has the same function as natural fucosyllactose, and clinical trials will not cause adverse reactions. Compared with the chemical synthesis method, the microbial synthesis method is safer and faster. A large number of toxic reagents do not need to be introduced during the synthesis process, and food safety-level microorganisms can be used to produce 2'-FL and 3-FL, which greatly increases the safety of the product.

[0005] The biosynthesis of 2’-FL and 3-FL was first achieved through enzymatic catalysis. In 2000, Albermann et al. transformed GDP-D-mannose into GDP-fucose in vitro by heterologously 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. At the same time, they found that the activity of GDP-mannose-4,6-dehydratase could be inhibited by GDP-fucose. Therefore, GDP-mannose-4,6-dehydratase is a relatively key rate-limiting step enzyme. Therefore, it is necessary to study GDP-mannose-4,6-dehydratase in order to improve the synthesis efficiency and conversion rate of 2’FL and 3-FL. The research on the modification of key enzymes is crucial for improving the catalytic efficiency of enzymes.

[0006] Saccharomyces cerevisiae strains are recognized as safe microorganisms, characterized by not producing pyrogens, having no toxic side effects, having high food safety, and can reduce the subsequent separation and purification costs. Moreover, Saccharomyces cerevisiae is widely used in industry. However, Saccharomyces cerevisiae cannot utilize lactose or transport lactose into the cell. Although scientists have introduced lactose transporters into Saccharomyces cerevisiae, the yield of 2’-fucosyllactose synthesized by the recombinant Saccharomyces cerevisiae is only 0.5 g / L (Yue et al., Microb Cell Fact, 2018, 17:101. DOI: 10.1186 / s12934-018-0947-2), which is difficult to meet the needs of industrial production.

[0007] Chinese patents 202010187309.1 and 202010187632.9 use recombinant Saccharomyces cerevisiae to synthesize 2’-FL, which includes GDP-mannose-dehydratase, GDP-fucose synthase, and α-1,2-fucosyltransferase, with yields reaching 2.9 g / L and 3.8 g / L respectively.

[0008] As the importance of HMOs to the human body is gradually recognized, the social demand for them will gradually increase. To meet the social needs, it is necessary to optimize the key factors in the fucosyllactose synthesis pathway to improve the fucose synthesis efficiency and meet the social needs. Summary of the Invention

[0009] Object of the Invention: To provide a genetically modified cell and its application in the synthesis of fucosyllactose, which has the activity of de novo synthesizing fucosyllactose. The fucosyllactose includes but is not limited to 2’-fucosyllactose (2’-FL) and 3-fucosyllactose (3-FL).

[0010] Technical solution:

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

[0012] The GDP-mannose dehydratase polypeptide (GDP-mannose dehydratase) is also known as: GDP-D-mannose-4,6-dehydratase (GDP-mannose-4,6-dehydratase), or GDP-mannose-6-dehydrogenase (GDP-mannose-6-dehydrogenase). It 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 sources of the heterologous GDP-mannose dehydratase polypeptide gene include, but are not limited to, natural strains or genetically modified strains such as Escherichia coli, Caenorhabditis elegans, Homo sapien, Arabidopsis, Dictyostelium discoideum, Mus musculus, Drosophila melanogaster, Sinorhizobium fredii, Pandoraea vervacti, Caenorhabditis briggsae, Candidatus Curtissbacteria, Pseudomonas sp, Clostridium sp., Cricetulus griseus, Arthrobacter siccitolerans, Paraburkholderia piptadeniae, or Bacillus smithii.

[0014] (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 mined from nature, named BsGMD. Its amino acid sequence is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:7.

[0016] Compared with the NCBI database, as of November 15, 2023, the GDP-mannose dehydratase with the highest similarity to the amino acid sequence shown in SEQ ID NO:1 is the GDP-mannose dehydratase derived from Escherichia coli. The amino acid sequences of the two are only 69.03% identical, indicating that the similarity between SEQ ID NO:1 and the amino acid sequences of known enzymes is not high. The amino acid sequence of the GDP-mannose dehydratase derived from Escherichia coli is shown in SEQ ID NO:2.

[0017] In certain embodiments, the GDP-mannose dehydratase polypeptide comprises a polypeptide having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the polypeptide shown in SEQ ID NO:1, or SEQ ID NO:2, or polypeptide M1-M13. Among them, polypeptide M1-M13 is a derivative peptide of the polypeptide shown in SEQ ID NO:1.

[0018] The genetically modified cell includes, but is not limited to, a GDP-mannose dehydratase polypeptide having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the polypeptide shown in SEQ ID NO:1, or SEQ ID NO:2, or polypeptide M1-M13.

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

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

[0021] Preferably, the modification is a mutation selected from any one 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 by ASIR from 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 from IE, 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 from QD, thereby obtaining polypeptide M3;

[0025] 4) In the amino acid sequence of polypeptide M3, the amino acid fragment at position 176 is replaced by D from 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 from 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 from 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 YTVKEIVK from FTVREAVE, 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 from 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 from E, thereby obtaining polypeptide M9;

[0031] 10) In the amino acid sequence of polypeptide M9, the amino acid residues at positions 304-312 are replaced by VLKVDPAFR from ILEVDPSFL, 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 IEEMI from FEEMM, 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 AVAEKYAEL from KVAEEYAEK, 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 from E, thereby obtaining polypeptide M13.

[0035] Preferably, the GDP - mannose dehydratase polypeptide further comprises amino acid substitution mutations that are functionally equivalent to the above - described schemes 1) - 13). In certain embodiments, the substitution mutations include mutations to charged residues; in certain embodiments, the substitution mutations include mutations to basic residues. In certain embodiments, the substitution mutations include mutations homologous to the amino acid sequences of the above - described schemes 1) - 13).

[0036] The method for producing the GDP - mannose dehydratase polypeptide as described above comprises:

[0037] (1) Culturing the transformed host cell under conditions suitable for expressing the polypeptide;

[0038] (2) Recovering the polypeptide.

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

[0040] More 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.;

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

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

[0043] In a specific embodiment, step (1) includes: first, introducing a nucleic acid construct or a recombinant expression vector containing the nucleic acid encoding the GDP-mannose dehydratase polypeptide as described above into a host cell to construct an engineered host cell expressing the polypeptide; then, culturing the engineered host cell and inducing its expression of the polypeptide.

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

[0045] The host cells can be cultured in a nutrient medium known in the art suitable for producing the polypeptide. For example, the cells can be cultured by shake flask culture, or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, fed-batch fermentation, or solid-state fermentation) in a suitable medium and under conditions allowing polypeptide expression and / or isolation, in a laboratory or industrial fermentor. 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 published compositions.

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

[0049] Furthermore, in some embodiments, in addition to the heterologous nucleic acid encoding the above dehydratase, the genetically modified yeast gene cells may further 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 cells can de novo synthesize fucosyllactose using lactose and glucose, or glycerol, or sucrose as a carbon source.

[0050] The sources of the alpha-1,2-fucosyltransferase gene include, but are not limited to, natural strains or gene-modified strains such as Helicobacter pylori, Thermophilic Chlorella, Escherichia coli, Caenorhabditis elegans, Schistosoma mansoni, Bacucilius cereus, Pseudopedobater saltans, Helicobacter mustelae, Bacillus fragilis, Bacteroides vulgatus, Bacteroides fragilis, or Bacillus smithii.

[0051] The sources of the α-1,3-fucosyltransferase gene include, but are not limited to, natural strains or genetically modified strains such as European wild boar (Sus scrofa), green monkey (Chlorocebus sabaeus), chimpanzee (Pan troglodytes), Helicobacter pylori, Akkermansia muciniphila, Bacteroides fragilis, Zea mays, Escherichia coli, western lowland gorilla (Gorilla gorilla gorilla), rhesus macaque (Macaca mulatta), European rabbit (Oryctolagus cuniculus), Bornean orangutan (Pongo pygmaeus), brown rat (Rattus norvegicus), Akkermansia muciniphila, gibbon (Hylobates lar), domestic cattle (Bos taurus), agile gibbon (Hylobates agilis), brown lemur (Eulemur fulvus), Helicobacter hepaticus, Azospirillum brasilense, etc.

[0052] The sources of the heterologous GDP-fucose synthase gene include, but are not limited to, Escherichia coli, house mouse ( Mus musculus ), human (Homo sapiens), Marinobacter salarius, Sinorhizobium fredii, Citrobacter, Bornean orangutan (Pongo abelii), Caenorhabditis elegans, Candidatus Staskawiczbacteria, Azorhizobium caulinodans, or Candictus Nitrospira nitrificans and other natural strains or genetically modified strains.

[0053] In some embodiments, the Kluyveromyces genetically modified cell further comprises deletion or disruption of the β-galactosidase gene (lac4) required for lactose metabolism in at least a part of the parental strain. Therefore, the Kluyveromyces genetically modified cell described in the present application can import lactose without consuming lactose. Compared with the parental strain, the expression level of the β-galactosidase gene in the Kluyveromyces genetically modified cell is reduced. Therefore, the Kluyveromyces genetically modified cell of the present invention reduces lactose consumption. When the Kluyveromyces genetically modified cell is a Kluyveromyces lactis genetically modified cell, the nucleic acid sequence Genebank number of its β-galactosidase lac4 gene is M84410.1, and the amino acid sequence Genebank number is AAA35265.1; when the Kluyveromyces genetically modified cell is a Kluyveromyces marxianus genetically modified cell, the nucleic acid sequence Genebank number of its β-galactosidase gene lac4 gene is XM_022818497.1, and the amino acid sequence Genebank number is XP_022675157.1.

[0054] The Kluyveromyces genetically modified cell described in the present invention can achieve a relatively high yield of fucosylated lactose without importing a heterologous transporter. The Kluyveromyces, preferably Kluyveromyces lactis and Kluyveromyces marxianus.

[0055] In one embodiment, the preferred technical solution is: the parental strain of the Kluyveromyces lactis genetically modified cell is Kluyveromyces lactis DSM70799; the parental strain of the Kluyveromyces marxianus genetically modified cell is Kluyveromyces marxianus DMKU3-1042. As described above, the parental strains of Kluyveromyces lactis and Kluyveromyces marxianus can be easily purchased through commercial channels or from a culture collection center.

[0056] The Kluyveromyces genetically modified cell, compared with the parental strain, has been genetically engineered to contain increased intracellular GDP-fucose and the production capacity of fucosyllactose.

[0057] Preferably, the Kluyveromyces genetically modified cell has been genetically engineered and has the following characteristics:

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

[0059] 2) Express a gene encoding an α-1,2-fucosyltransferase polypeptide or an α-1,3-fucosyltransferase polypeptide.

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

[0061] Optionally, the amino acid sequence of the GDP-mannose dehydratase has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical amino acid sequence to 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 gene-modified enzymes or wild-type.

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

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

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

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

[0067] Preferably, the gene-modified cell in the preparation method is a Kluyveromyces gene-modified cell; more preferably, it is a Kluyveromyces lactis gene-modified cell or a Kluyveromyces marxianus gene-modified cell.

[0068] More preferably, the starting strain of Kluyveromyces lactis is K. lactis DSM70799, and the starting strain of Kluyveromyces marxianus is K. marxianus DMKU3-1042.

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

[0070] (1) Culturing the starting strain;

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

[0072] Preferably, the Kluyveromyces cell is a natural strain or a genetically modified strain of Kluyveromyces lactis cell K.lactic / or Kluyveromyces marxianus cell K.marxianus.

[0073] Preferably, the starting strain is selected from Kluyveromyces lactis cell K.lactic DSM70799;

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

[0075] (2) Construct an expression cassette and introduce a heterologous GDP-mannose dehydratase gene into the Kluyveromyces cell;

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

[0077] (3) Disrupt 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 the Kluyveromyces cell;

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

[0080] Optionally, the recombinant construction technique of the genetically modified Kluyveromyces cell further includes:

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

[0082] Preferably, it further includes the step of (7) recovering the genetically modified Kluyveromyces cell.

[0083] Optionally, in the preparation method of the genetically modified Kluyveromyces cell as described above, the steps are not in a specific order.

[0084] In some embodiments of the present invention, a method for preparing fucosyllactose is provided, and the preparation method synthesizes fucosyllactose by fermentation of the genetically modified cell described in the present application.

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

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

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

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

[0089] In some embodiments, the fermentation in the preparation method requires the use of a culture medium, and the culture medium can be any medium that can maintain the growth and viability of the genetically modified cells producing fucosyllactose. In some embodiments, the culture medium may further 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 increasing or continuous manner.

[0090] The preparation method de novo synthesizes fucosyllactose using lactose and sucrose, or glycerol, or glucose as a carbon source.

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

[0092] In some embodiments, it further includes adjusting the mass ratio of the carbon source (such as sucrose) to lactose to adjust the yield of fucosyllactose.

[0093] Preferably, the fermentation product of the yeast genetically modified cells can be purified by means such as centrifugation, filtration, decolorization, nanofiltration, chromatographic purification, crystallization, and recrystallization to obtain pure fucosyllactose.

[0094] This application also provides a preparation of fucosyllactose. The preparation method uses the genetically modified cells of this application and uses lactose and sucrose, or glycerol, or glucose as a carbon source to ferment and synthesize fucosyllactose.

[0095] The term "heterologous": when referring to a polynucleotide, gene, nucleic acid, polypeptide or enzyme, it refers to a polynucleotide, gene, nucleic acid, polypeptide or enzyme from a source other than the host organism species or derived from a source other than the host organism species.

[0096] The term "molecular marker": refers to a specific DNA fragment that can reflect a certain difference in the genome between biological individuals or populations.

[0097] The term "deletion or disruption": refers to the coding region and / or control sequence of a reference gene being partially or completely modified (for example, by deleting, inserting and / or substituting one or more nucleotides), so that the expression of the encoded polypeptide does not exist (inactivated) or is reduced, and / or the enzymatic activity of the encoded polypeptide does not exist or is reduced.

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

[0099] Advantages:

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

[0101] (1) Compared with the starting strain, the Kluyveromyces genetically modified cell has the activity of synthesizing fucosyllactose.

[0102] (2) Compared with the fucosyllactose produced by Escherichia coli or genetically modified Escherichia coli cells, the fucosyllactose product synthesized by the recombinant yeast genetically modified cell containing the nucleotide sequence of SEQ ID NO:1 or the polypeptides M1-M13 does not contain endotoxins and other sensitizing substances, has the characteristic of food safety, and is more suitable for applications in the fields of food, health products, infant food, etc.

[0103] (3) The Kluyveromyces genetically modified cell described in the present invention has a simple and feasible process for fermentatively synthesizing fucosyllactose, improves production efficiency, and reduces production costs.

[0104] The technology of the present invention provides excellent microbial resources for the biological preparation of GDP-fucose and fucosyllactose.

[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, is conducive to the application of large-scale industrial production, and has important practical value. Brief Description of the Drawings

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

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

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

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

[0110] Figure 5 . Mass spectrum of 2’-FL standard.

[0111] Figure 6 . Mass spectrum of 3-FL standard.

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

[0113] Figure 8 . Mass spectrum of the substance at rt = 14.16 min in the HPLC chromatogram of the fermentation broth of KM-M10-1 strain in Example 4. Detailed implementation manners

[0114] The present invention will be further described in detail below through examples. It should be understood that the specific implementation manners described herein are only used to explain the present invention and are not used to limit the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several modifications or substitutions can also be made to the details and forms of the technical solutions 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 terms used are only for describing specific implementation manners 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, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments and not for limiting the protection scope of the present invention. For the experimental methods without specific conditions indicated in the following specific implementation manners, they are usually carried out according to the conventional methods and conditions of molecular biology in this technical field, 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] For the experimental methods without specific conditions indicated in the following specific implementation manners, they are usually carried out according to the conventional methods and conditions of molecular biology in this technical field, and such techniques and conditions are fully explained in the literature; all materials, reagents, etc., unless otherwise specified, can be obtained from commercial sources.

[0117] The present application 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 the present 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 the yeast cells to produce fucosyllactose.

[0118] Cultivation can be carried out in a suitable container, including but not limited to a cell culture plate, a culture flask or a fermenter in a suitable culture medium. Any suitable fermenter can be used, including but not limited to a stirred fermenter, an air-lift fermenter, a bubble fermenter or any combination thereof. In a specific embodiment using Kluyveromyces lactis as the host cell, the strain can grow in a fermenter. In addition, the method can be carried out by fermentation on any scale known in the art to support the industrial production of microbial products. The materials and methods for maintaining or growing cell cultures are well known to those skilled in the fields 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 consists essentially of lactose and sucrose, or glucose. Preferably, in some embodiments, the carbon source in the culture medium consists of lactose and sucrose.

[0120] The following is an explanation of the abbreviated symbols in the examples below:

[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 examples:

[0123] The starting strains of Kluyveromyces lactis include: K. lactis DSM70799; the starting strain of Kluyveromyces marxianus: K. marxianus DMKU3-1042.

[0124] GDP-mannose dehydratase (GMD): The amino acid sequence is as shown in SEQ ID NO:1, SEQ ID NO:2, or polypeptide M1-M13;

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

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

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

[0128] The 2’-FL and 3-FL standards are produced by the French company ELICITYL.

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

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

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

[0132] 2. By using computer-aided rational design, different mutation sites are obtained. Referring to the method in "Molecular Cloning: A Laboratory Manual", based on the sequence of SEQ ID NO:7 of PET32a-wt, mutant primers are designed using Primer-BLAST ( Primer designing tool(nih.gov) ), and a series of recombinant plasmids (pET32a-M1-M13) are constructed using a site-directed mutagenesis kit (Fast Mutagenesis Kit).

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

[0134] Take the prepared Escherichia coli BL21(DE3) competent cells, place them on ice for 30 min to melt. Take 100 μL of the competent cells and mix them with 10 μL of the pET32a-M1-M13 recombinant plasmid (concentration 50 ng / μL) respectively, place them in a 42 °C water bath for heat shock for 45 s, then immediately cool them in an ice bath for 2 min, add 1 mL of fresh LB medium (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, 1.5% agar powder is added to the plate), resuscitate and culture them at 37 °C and 100 rpm for 1 h. Then take 100 μL of the bacterial solution and spread it on an LB plate containing ampicillin (100 μg / mL), and culture it in a 37 °C constant temperature incubator for 12 h. Then pick single colonies for colony PCR to screen out positive transformants.

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

[0136] 5. Inoculate the correct transformants into LB liquid medium, place them on a shaker at 37 °C and 200 rpm for 12 h to obtain a seed solution; then, inoculate the seed solution into fresh LB medium at an inoculation amount of 1% (v / v), and culture it with shaking at 37 °C until the OD 600 reaches 0.8, and then induce it with isopropyl-β-D-thiogalactopyranoside (IPTG). The final concentration of IPTG is 0.1 mmol / L, and it is induced at 16 °C for 12 h with a rotation speed of 200 rpm; after induction expression, centrifuge the fermentation broth at 5000 r / min for 30 min at 4 °C to collect the cells; resuspend the cells with 20 mM pH 7.4 PBS buffer, and ultrasonicate them at a frequency of plus on 5 s / off 5 s for 30 min to break the cells; centrifuge the broken liquid at 13000×g and 4 °C for 30 min to remove cell debris, and then collect the supernatant.

[0137] 6. Purify the soluble polypeptide sequence by nickel column affinity chromatography. The process is as follows: Add deionized water to the nickel column until it reaches the top of the column, and after natural elution, elute it with 5 times the volume of Binding buffer; then load the crude enzyme solution filtered through a 0.45 μm filter membrane onto the column, and allow the sample to fully bind to the nickel column at a flow rate of 1.5 mL / min. After the sample has drained, continuously elute with 5 times the column volume of Washing buffer in a gradient to remove impurities, and finally elute the target protein with 5 times the volume of Elution buffer, and collect the eluate; then, analyze the expression of the target protein by SDS-PAGE.

[0138] The SDS-PAGE results show that there are obvious specific expression bands after induction in the gene-modified cells, and the molecular weight of the bands is basically consistent with the expected molecular weight of 35.3 kDa. Therefore, it can be known that the polypeptides shown in SEQ ID NO:1 and SEQ ID NO:2 and polypeptide M1-M13 are obtained. For relevant information, see Table 1.

[0139] Table 1. Corresponding relationship between GDP-mannose dehydratase polypeptides and their amino acid sequence numbers or names

[0140] Recombinant plasmid GDP - mannose dehydratase polypeptide naming / 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 Kluyveromyces lactis gene-modified cells.

[0142] (1) Construct a Kluyveromyces lactis modified cell expression cassette.

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

[0144] Refer to the applicant's prior patent: 202211453172.5, invention title: Recombinant yeast and its application, construct the KL-ΔLAC4 knockout cassette.

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

[0146] Using the Kluyveromyces lactis genome as a template, amplify the upstream and downstream homologous arm sequences lac4 up and lac4-do, the promoter sequence tef1 and the terminator sequence adh1; using the synthesized FutC and FutA sequences as templates, amplify the target gene sequences of FutC and FutA; using the expression cassette KL-ΔLAC4 as a template, amplify the G418+loxp sequence. By successive fusion PCR of two fragments, fuse lac4 up, tef1, FutC / FutA, adh1, G418+loxp and lac4-do. Finally, use the primers lac4-upF and lac4-doR (Table 2) and the fusion PCR system as a template, and perform PCR amplification to obtain the expression cassette KL-ΔLAC4::FutC / KL-ΔLAC4::FutA.

[0147] Table 2

[0148]

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

[0150] Using the Kluyveromyces lactis genome as a template, amplify the upstream and downstream homologous arm sequences xk-up and xk-do of the coding region of xylulokinase XKb, the promoter sequence pgk1 and the terminator sequence tdh3; using the synthesized gmer sequence as a template, amplify the target gene sequence of GMER; using the existing plasmid in the laboratory as a template, amplify the G418+loxp sequence. By successive fusion PCR of two fragments, fuse xyl1 up, pgk1, gmer, tdh3, G418+loxp and xyl1-do. Finally, use the primers xK-upF and xK-doR (Table 3) and the fusion PCR system as a template, and perform PCR amplification to obtain the gmer expression cassette KL-ΔXK::GMER.

[0151] Table 3

[0152]

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

[0154] 2. Using the genomic DNA of Kluyveromyces lactis K. lactic DSM70799 as a template, PCR amplification was performed with primers galk up-F and galk up-R as well as galk down-F and galk down-R to obtain the upstream and downstream homology arms respectively; using loxp-F and loxp-R as primers and the G418 resistance plasmid as a template, amplification was performed to obtain the resistance screening marker containing G418 resistance and loxp sites; using PET32a-BSGMD-WT, PET32a-M1 to PET32a-M13 and PET32a-ECGMD-WT as templates, PCR amplification was performed to obtain the original sequence and mutant sequence of the GMD gene; using the genome of Kluyveromyces lactis as a template, PCR amplification was performed to obtain the promoter tdh3 and terminator sequence tef of the transferase respectively; using KL-PF and KL-PR as primers and the PUC19 plasmid as a template, PCR amplification was performed to obtain the plasmid vector sequence. The upstream homology arm, promoter, target gene (original sequence and mutant sequence), terminator, G418+loxp and downstream homology arm were fused PCR to obtain ΔGK::BSGMD-WT, ΔGK::BSGMD-M1 to ΔGK::BSGMD-M13 expression cassettes, and ΔGK::ECGMD-WT; the expression cassette was connected to the plasmid vector sequence by ABclonal to obtain plasmids PUC-KL-WT, PUC-KL-M1 to PUC-KL-M13, and PUC-KL-ECGMD-WT carrying ΔGK::BsGMD-WT, ΔGK::ECGMD-WT, ΔGK::BSGMD-M1 to ΔGK::BSGMD-M13 expression cassettes, respectively.

[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, KL-ΔGK::BSGMD-WT, KL-ΔGK::BSGMD-M1 to KL-ΔGK::BSGMD-M13 expression cassettes, and KL-ΔGK::ECGMD-WT were amplified for the next step of constructing recombinant strains.

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

[0157]

[0158] (2) Transformation of the recombinant expression cassette and verification of the recombinant strain.

[0159] The expression cassettes constructed in (1) above were respectively transferred into the cells of the starting strain Kluyveromyces lactis DSM70799. The specific method is as follows:

[0160] 1) First, prepare yeast competent cells: streak a small amount of the yeast strain cryopreservation on a solid plate medium and incubate it upside down at 30 °C for 2 days. Pick a yeast single colony into 50 mL of liquid medium and culture it at 30 °C and 220 rpm until the OD 600 is between 0.8 and 1.5. Collect the cells and wash them 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 to resuspend the precipitate, centrifuge at 12000 rpm for 30 s, and discard the supernatant. Add 400 μL of 100 mM lithium chloride buffer again to resuspend the precipitate to obtain yeast cell competent cells, aliquot them at 50 μL / tube for transformation.

[0161] Meanwhile, boil 1 mL of salmon sperm DNA for 5 min and quickly ice-bath it to prepare single-stranded DNA.

[0162] 2) Transformation: Centrifuge the prepared competent yeast cells and remove the residual lithium chloride solution with a Tips. For each transformation, add the following in sequence: 50% PEG3350 (240 μL); 1 M 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 evenly distributed; incubate in a 30 °C water bath for 30 min; heat shock in a 42 °C water bath 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 it on a shaker at 30 °C; after 1 - 4 h, take 25 - 100 μL of the bacterial solution and spread it on a selective medium plate and incubate it upside down at 30 °C.

[0163] 3) Verification: The correspondence between the 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 above transformants and the original strains, and performed PCR amplification on the genomes using the corresponding knockout cassette or expression cassette amplification primers. If there is exactly one band after PCR amplification and the size of the band is consistent with that of the knockout cassette or expression cassette, the strain is determined to be correct; otherwise, the strain is determined to be a false positive error.

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

[0165] Microbial cell Genotype K.lactic DSM70799 wt KL - 1 K.lactic DSM70799; 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 respectively introduced into the recombinant strains described in Table 5 to obtain a total of 17 strains such as KL-X-J and KL-Y-J. Among them: X represents values 1-4; Y represents polypeptides M1-M13.

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

[0168] Microbial cell Genotype K.lactic DSM70799 WT KL - 1 - 1 K.lactic DSM70799; 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 respectively introduced into the recombinant strains described in Table 6 to obtain a total of 17 strains such as KL-X-1-J and KL-Y-1-J. Among them: X represents values 1-4; Y represents polypeptides M1-M13.

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

[0171] (1) Construction of the Kluyveromyces marxianus modification cell expression cassette.

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

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

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

[0175] Using the Kluyveromyces marxianus 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. By means of three-fragment fusion PCR, km-lac4 up, the FutC or FutA expression cassette + G418 + loxp, and km-lac4-do were fused. Finally, using the primers lac4-upF1 and lac4-doR1 (Table 7), and using the fusion PCR system as a template, PCR amplification was performed to obtain the FutC and FutA expression cassettes KM-ΔLAC4::FutC and KM-ΔLAC4::FutA.

[0176] Table 7

[0177]

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

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

[0180] Table 8

[0181]

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

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

[0184] / ECGMD+G418+loxp sequence. By means of fusion PCR, the upstream and downstream homologous arms and the BSGMD-wt / GMD mutant expression cassette / ECGMD expression cassette+G418+loxp sequence were fused respectively. 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 respectively for the construction of the next recombinant strains.

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

[0186]

[0187]

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

[0189] The expression cassettes constructed in (1) above were transferred into the cells of the starting strain Kluyveromyces marxianus DMKU3-1042. The specific method was to refer to Part (2) of Example 2 for the transformation of the recombinant expression cassette and the verification of the recombinant strain.

[0190] The corresponding relationship between the recombinant strains and their genotypes is shown in Tables 10-11. To verify the correctness of the above strains, we extracted the genomes of the above transformants and the original strain, and performed PCR amplification of the genome with the corresponding knockout cassette or expression cassette amplification primers. If there is exactly one band with a size consistent with the knockout cassette or expression cassette after PCR amplification, the strain is determined to be correct; otherwise, the strain is determined to be a false positive error.

[0191] Table 10. Kluyveromyces marxianus genetically modified cells 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] The recombinant strains described in Table 10 were respectively introduced with encrypted tags to obtain a total of 17 recombinant strains such as KM-X-J and KM-Y-J. Among them: X represents the values 1-4; Y represents polypeptides M1-M13.

[0194] Table 11. Kluyveromyces marxianus genetically modified cells 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-M10 KM-M11-1 KM-2; KM-ΔGK::BSGMD-M11 KM-M12-1 KM-2; KM-ΔGK::BSGMD-M12 KM-M13-1 KM-2; KM-ΔGK::BSGMD-M13

[0196] The recombinant strains described in Table 11 were respectively introduced with encrypted tags to obtain a total of 17 strains such as KM-X-1-J and KM-Y-1-J. Among them: X represents the values 1-4; Y represents polypeptides M1-M13.

[0197] Verification of marker genes: To verify the correctness of the above strains, we extracted the genomes of the above transformants and the original strains, and performed PCR amplification on the genomes using the corresponding knockout cassette or expression cassette amplification primers. If there is and only one band obtained after PCR amplification and its size is consistent with that of the knockout cassette or expression cassette, the strain is determined to be correct; otherwise, the strain is determined to be a false positive error. For the verification of the tags, PCR amplification is carried out using primers, and further verification is carried out by sequencing; otherwise, the strain is determined to be a false positive error.

[0198] Example 4. Study on the fermentative synthesis of fucosyllactose by Kluyveromyces genetically modified cells.

[0199] Yeast was cultured with glucose as the carbon source. The genetically modified cells obtained in Examples 2-3 were respectively taken, and the Kluyveromyces genetically modified cells were allowed to grow rapidly until the growth entered the late logarithmic phase or the stationary phase. The strains were respectively streaked on solid media such as YDP, and after culturing at 30 °C for 2-3 days, single colonies were picked and inoculated into 1.5 mL YPD liquid medium, and cultured overnight at 30 °C and 200 rpm on a shaker. Subsequently, they were respectively inoculated into 50 mL liquid medium shake flasks at an inoculation amount of 2%, and cultured at 30 °C and 200 rpm until OD 600 = 1, and lactose with a final concentration of 10 g / L and sucrose at 3% (w / v) were supplemented, and cultured at 30 °C and 200 rpm on a shaker for a total fermentation duration of 72 h. After 72 h of fermentation, samples were taken and boiled for 10 min, and after centrifugation, the final fermentation products of the yeast were obtained, and the 2'-FL content in the supernatant was detected.

[0200] HPLC detection method: Detection conditions: Chromatographic column model: Shodex Asahipak NH 2 P-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.

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

[0202] Chromatographic column model: Shodex Asahipak NH 2 P-50 4E, detector: UV detector (Hitachi Chromaster), detection wavelength: 210 nm, injection volume: 10 μL, flow rate: 0.5 mL / min, column temperature: 35°C, mobile phase: acetonitrile: water = 65:35; ESI-MS mode, molecular weight scanning range 100-800.

[0203] The standard product and the reaction solution were tested according to the above analytical method. The HPLC analysis results showed:

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

[0205] (2) The Kluyveromyces genetically modified cells (KL-3, KL-4, KM-3, KM-4 and strains introduced with M1-M13 genes, as well as the above strains introduced with marker genes) introduced with the α-1,2-fucosyltransferase gene obtained in Example 2-3 showed a strong absorption peak at around 13.43 min, which was consistent with the peak time of the 2'-FL standard, indicating that 2'-FL was produced in the fermentation broth of the genetically modified cells obtained in Example 2-3.

[0206] (3) The Kluyveromyces genetically modified cells (KL-3-1, KL-4-1, KM-3-1, KM-4-1 and strains introduced with M1-M13 genes, as well as the above strains introduced with marker genes) introduced with the α-1,3-fucosyltransferase gene obtained in Example 2-3 showed a strong absorption peak at around 14.16 min, which was consistent with the peak time of the 3-FL standard, indicating that 3-FL was produced in the fermentation broth of the genetically modified cells obtained in Example 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 of the reaction solution HPLC chromatographic peaks near rt=13.43min and rt=14.16min were analyzed by LC-MS, and the MH value was 487.17, which was consistent with the standard mass spectrum results of 2'-FL and 3-FL shown, and within the allowable error range of the theoretical molecular weight of 2'-FL and 3-FL of 488.44.

[0210] The yields of 2'-FL and 3-FL in the fermentation broth were detected by the above-mentioned HPLC analysis method, and the results are recorded in Tables 12-15.

[0211] Table 12. Study on the synthesis of fucosyllactose by Kluyveromyces lactis gene-modified cells obtained in Example 2

[0212]

[0213]

[0214] Table 13. Study on the synthesis of fucosyllactose by Kluyveromyces cerevisiae gene modified cells obtained in Example 3

[0215]

[0216] Table 14. Study on the synthesis of fucosyllactose by Kluyveromyces lactis gene-modified cells obtained in Example 2

[0217]

[0218]

[0219] Table 15. Study on the synthesis of fucosyllactose by Kluyveromyces cerevisiae gene-modified cells obtained in Example 3

[0220]

[0221] Table 12-15 Data Description:

[0222] (1) The starting strains and genetically modified cells of K. lactis and K. marxianus, KL-1, KL-2, KM-1 and KM-2, could not synthesize 2'-FL; KL-3 and KM-3 could produce 2'-FL; KL-1-1, KL-2-1, KM-1-1 and KM-2-1 could not synthesize 3-FL; KL-3-1 and KM-3-1 could produce 2'-FL.

[0223] Description: Kluyveromyces lactis / Kluyveromyces max cells do not have the ability to synthesize 2'-FL by themselves, and even after the introduction of GMD and GMER genes, they are unable to synthesize 2'-FL or 3-FL. The production of 2'-FL or 3-FL was detected in the gene-modified cells described in the present application.

[0224] (2) The Kluyveromycin gene-modified cells of the present invention have the activity of synthesizing 2'-FL.

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

[0226] Example 5. Take 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, respectively, and streak the strains in solid culture medium such as YDP, culture at 30°C for 2-3 days, pick a single colony and inoculate it in 1.5mL YPD liquid culture medium, and culture it at 30°C and 200rpm overnight. Then, inoculate them in 50mL liquid culture medium shake flasks at an inoculum size of 2%, and culture them at 30°C and 200rpm until OD 600 =1, and inoculated into 1L YPD medium (yeast extract powder 10g / L, peptone 20g / L, glucose 20g / L) (3L volume fermenter) at an inoculum size 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. At the same time, lactose (mother liquor concentration was 40%) was added 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. The 2'-FL and 3-FL contents in the supernatant were detected, and the results are recorded in Table 16.

[0227] Table 16

[0228]

[0229] 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 genetically modified cell, characterized in that, it comprises a recombinant nucleic acid sequence encoding a GDP-mannose dehydratase polypeptide; Preferably, the genetically modified cell includes, but is not limited to, a genetically modified yeast cell; More preferably, the genetically modified cell includes, but is not limited to, a genetically modified Kluyveromyces sp. cell; More preferably, the genetically modified cell includes, but is not limited to, a genetically modified Kluyveromyces lactis cell and a genetically modified Kluyveromyces marxianus cell.

2. The genetically modified cell according to claim 1, characterized in that, the GDP-mannose dehydratase polypeptide includes, but is not limited to, a polypeptide having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical to the polypeptide shown in SEQ ID NO:1, or SEQ ID NO:2, or polypeptide M1-M13; Preferably, the GDP-mannose dehydratase polypeptide includes, but is not limited to, the polypeptide shown in SEQ ID NO:1, SEQ ID NO:2, or polypeptide M1-M13.

3. The genetically modified cell according to claim 2, characterized in that, the polypeptide M1-M13 is obtained by modifying at least one site among positions 69-72, 118-119, 123-124, 176, 203-205, 213-217, 257-264, 273-276, 288, 304-312, 339-443, 365 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 69-72 is replaced by ASIR from 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 from IE, 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 from QD, thereby obtaining polypeptide M3; 4) In the amino acid sequence of polypeptide M3, the amino acid fragment at position 176 is replaced by D from 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 from 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 from 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 YTVKEIVK from FTVREAVE, 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 from 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 from E, thereby obtaining polypeptide M9; 10) In the amino acid sequence of polypeptide M9, the amino acid residues at positions 304 - 312 are replaced by VLKVDPAFR from ILEVDPSFL, thereby obtaining polypeptide M10; 11) In the amino acid sequence of polypeptide M10, the amino acid fragment at positions 339 - 443 is replaced by IEEMI from FEEMM, thereby obtaining polypeptide M11; 12) In the amino acid sequence of polypeptide M11, the amino acid fragment at positions 352 - 360 is replaced by AVAEKYAEL from KVAEEYAEK, thereby obtaining polypeptide M12; 13) In the amino acid sequence of polypeptide M12, the amino acid residue at position 365 is replaced by Y from E, thereby obtaining polypeptide M13.

4. The genetically modified cell according to any one of claims 1 - 3, wherein, the method for producing the GDP - mannose dehydratase polypeptide comprises: (1) culturing the transformed host cell under conditions suitable for expressing the polypeptide; and (2) recovering the polypeptide. Preferably, the host cell includes, but is not limited to, natural strains or genetically modified strains such as bacteria, yeast, molds, etc.; More 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.; More preferably, the host cell, the yeast cell includes, but is not limited to, natural strains or genetically modified strains such as Saccharomyces cerevisiae, Cloveromyces cerevisiae, Yarrowia lipolytica, Saccharomyces paradoxus, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces cariocas, Saccharomyces mikatae, and Saccharomyces kudriavzevii, or other nucleic acid constructs capable of transforming the polynucleotides of the polypeptides shown in SEQ ID NO:1, SEQ ID NO:2 and / or the derivative peptides M1 - M13; Preferably, the yeast cell is selected from Kluyveromyces yeast cells; More preferably, it is selected from Kluyveromyces lactis cells and Kluyveromyces marxianus cells; More preferably, the host cell is the genetically engineered Escherichia coli strain E. coli BL21(DE3); Preferably, the yeast cells include but are not limited to Kluyveromyces cells; More preferably, the Kluyveromyces cells include but are not limited to natural strains or genetically modified strains of Kluyveromyces lactis cells and Kluyveromyces marxianus cells.

5. The genetically modified cell according to any one of claims 1-4, characterized in that it further comprises one or more heterologous nucleic acids encoding GDP-L-fucose synthase, alpha-1,2-fucosyltransferase or alpha-1,3-fucosyltransferase nucleic acids; Preferably, the source of the alpha-1,2-fucosyltransferase gene includes but is not limited to natural strains or genetically modified strains such as Helicobacter pylori, Thermophilic Chlorella, Escherichia coli, Caenorhabditis elegans, Schistosoma mansoni, Bacucilius cereus, Pseudopedobater saltans, Helicobacter mustelae, Bacillus fragilis, Bacteroides vulgatus, Bacteroides fragilis, or Bacillus smithii, etc.; Preferably, the source of the α-1,3-fucosyltransferase gene includes, but is not limited to, natural strains or genetically modified strains such as European wild boar (Sus scrofa), green monkey (Chlorocebus sabaeus), chimpanzee (Pan troglodytes), Helicobacter pylori, Akkermansia muciniphila, Bacteroides fragilis, Zea mays, Escherichia coli, western lowland gorilla (Gorilla gorilla gorilla), rhesus macaque (Macaca mulatta), European rabbit (Oryctolagus cuniculus), Bornean orangutan (Pongo pygmaeus), brown rat (Rattus norvegicus), Akkermansia muciniphila, gibbon (Hylobates lar), domestic cattle (Bos taurus), agile gibbon (Hylobates agilis), brown lemur (Eulemur fulvus), Helicobacter hepaticus, Azospirillum brasilense, etc.; Preferably, the heterologous GDP-L-fucose synthase (WcaG) gene is derived from, including but not limited to, Escherichia coli, Mus musculus Mus musculus ), Homo sapiens, Marinobacter salarius, Sinorhizobium fredii, Citrobacter (citrobacter), Bornean orangutan (Pongo abelii), Caenorhabditis elegans, Candidatus Staskawiczbacteria, Azorhizobium caulinodans or Candictus Nitrospira nitrificans and other natural strains or genetically modified strains.

6. The genetically modified cell according to any one of claims 1-5, characterized in that the Kluyveromyces genetically modified cell can import lactose without consuming or with low consumption of lactose; Preferably, the genetically modified cell further comprises deletion or disruption of at least a part of the β-galactosidase gene (lac4) of the starting strain; Preferably, when the Kluyveromyces gene-modified cell is a Kluyveromyces lactis gene-modified cell, the nucleic acid sequence Genebank number of its β-galactosidase lac4 gene is M84410.1, and the amino acid sequence Genebank number is AAA35265.1; preferably, when the Kluyveromyces gene-modified cell is a Kluyveromyces marxianus gene-modified cell, the nucleic acid sequence Genebank number of its β-galactosidase gene lac4 gene is XM_022818497.1, and the amino acid sequence Genebank number is XP_022675157.

1.

7. The method for preparing the gene-modified cell according to any one of claims 1-6, characterized in that it comprises the following steps: (1) Culturing the starting strain; (2) Constructing an expression cassette and introducing a heterologous GDP-mannose dehydratase gene into Kluyveromyces cells; (3) Disrupting the β-galactosidase gene lac4 in the strain obtained in step (1); (4) Constructing an expression cassette and introducing a heterologous GDP-fucose synthase gene into Kluyveromyces cells; (5) Constructing an expression cassette and introducing a heterologous α-1,2-fucosyltransferase gene or α-1,3-fucosyltransferase gene into Kluyveromyces cells; Optionally, the recombinant construction technique of the Kluyveromyces gene-modified cell further comprises: (6) Constructing a molecular marker expression cassette and introducing a marker gene into Kluyveromyces cells; Preferably, it further comprises the step of (7) recovering the Kluyveromyces gene-modified cell; Optionally, in the method for preparing the Kluyveromyces gene-modified cell as described above, the steps are not in a specific order; Preferably, (1) the starting strain includes but is not limited to a natural strain or a gene-modified strain of Kluyveromyces cells; Preferably, the Kluyveromyces cells include but are not limited to natural strains or gene-modified strains of Kluyveromyces lactis cells K.lactic / or Kluyveromyces marxianus cells K.marxianus; Preferably, the starting strain is selected from Kluyveromyces lactis cells K.lactic DSM70799; Preferably, the starting strain is selected from Kluyveromyces marxianus cells K.marxianus DMKU3-1042; Preferably, as described in (2) above, the GDP-mannose dehydratase gene includes the gene of the polypeptide with the amino acid sequence shown in SEQ ID NO:1, or the gene of any one of the polypeptides M1-M13.

8. The application of the gene-modified cell according to any one of claims 1-6 in the synthesis of fucosyllactose; Preferably, the fucosyllactose includes but is not limited to 2'-fucosyllactose or 3-fucosyllactose; Preferably, the gene-modified cell includes but is not limited to Kluyveromyces gene-modified cells; Preferably, the Kluyveromyces gene-modified cell includes but is not limited to Kluyveromyces lactis gene-modified cells or Kluyveromyces marxianus gene-modified cells; Preferably, in the above application, lactose and glucose, or glycerol, or sucrose are used as carbon sources to de novo synthesize fucosyllactose.

9. A method for preparing fucosyllactose, characterized in that, it is obtained by fermentation of one or more of the gene-modified cells described in any one of claims 1-6; Preferably, the fucosyllactose includes but is not limited to 2'-fucosyllactose or 3-fucosyllactose; Preferably, in the preparation method, lactose and glucose, or glycerol, or sucrose are used as carbon sources to de novo synthesize fucosyllactose.

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