Kluyveromyces cerevisiae gene modified cell

Abstract

The invention relates to a gene modified cell and a method for producing fucosyllactose by using the gene modified cell, in particular to a kluyveromyces kluyveromyces gene modified cell and application thereof, and belongs to the technical field of biological gene engineering. The present invention provides a genetically modified cell comprising a recombinant nucleic acid sequence encoding a polypeptide having an [alpha]-1, 2-fucosyltransferase polypeptide ([alpha]-1, 2-fucosyltransferase) activity. The alpha-1, 2-fucosyltransferase polypeptide includes, but is not limited to, polypeptides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% identical amino acid sequences with the amino acid sequences of SEQ ID NO: 1-25. According to the gene modified cell, the fucosyllactose synthesis capability of an original strain is improved.

Description

Technical Field

[0001] The present invention relates to a gene modified cell, in particular to a gene modified cell using Kluyveromyces as a host, and specifically to a Kluyveromyces gene modified cell and application thereof, belonging to the technical field of biological genetic engineering. Background Art

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

[0003] 2'-FL and 3'-FL have been approved as raw materials for infant formula by the U.S. FDA, the European Union, Australia, New Zealand, Canada and other countries and regions, and can be added to infant formula, regular food, dietary supplements and / or medical food.

[0004] Since the chemical synthesis of fucosyllactose requires multiple protection and deprotection, the product yield is low and the production cost remains high. Therefore, the synthesis of fucosyllactose mostly adopts enzymatic method and microbial cell factory synthesis method. Since the pure enzyme catalysis method generally requires fucose as a substrate, the cost is relatively high. Cell factories can often synthesize from scratch to achieve low-cost large-scale industrial production.

[0005] One of the feasible methods for large-scale synthesis of fucosyllactose is the microbial cell factory method. Existing technologies mostly use Escherichia coli to biosynthesize fucosyllactose. Under the premise of providing a carbon source, the gene pathway in Escherichia coli is used to synthesize the precursor of fucosyllactose, GDP-L-fucose, and the receptor lactose. The exogenous fucosyltransferase is transferred into the engineered bacteria to combine the two precursors, thereby industrially synthesizing fucosyllactose.

[0006] Saccharomyces cerevisiae strains are recognized as safe microorganisms, with the characteristics of not producing pyrogens, no toxic side effects, high food safety, and can reduce the subsequent separation and purification costs. Moreover, Saccharomyces cerevisiae is widely used in industry, but Saccharomyces cerevisiae cannot utilize lactose or transport lactose into cells. Although scientists have introduced lactose transporters into Saccharomyces cerevisiae, the yield of 2'-fucosyllactose synthesized by the recombinant Saccharomyces cerevisiae is only 0.5g / L (Yu 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-4,6-dehydratase, GDP-L-fucose synthase, and α-1,2-fucose transferase, with yields of 2.9 g / L and 3.8 g / L, respectively. Chinese patent 202080061688.8 discloses yeast cells genetically modified to produce one or more human milk oligosaccharides, wherein the yeast cells include GDP-mannose-4,6-dehydratase, GDP-L-fucose synthase, and ABC transporters, and expressing heterologous ABC transporters in genetically modified yeast cells can increase the yield and purity of HMO.

[0008] International publication WO2023122270 (A2) discloses a host cell capable of producing HMO, which comprises one or more heterologous nucleic acids, each of which independently encodes: fucosyltransferase, GDP-fucose synthase, GDP-mannodehydratase, and lactose permease. This patented technical solution reduces the DFL content in 2'-FL by using a recombinant strain.

[0009] With the gradual rise of HMOs in recent years, it is of great significance to optimize the key factors of the fucose synthesis pathway to improve the efficiency of fucose synthesis. Summary of the invention

[0010] Purpose of the invention: To provide a genetically modified cell and its application in synthesizing fucosyllactose, which has the activity of synthesizing fucosyllactose from scratch and has no undesirable side activities. The fucosyllactose includes 2'-fucosyllactose (2'-fucosyllactose, 2'-FL).

[0011] The technical solution of the present invention is:

[0012] A genetically modified cell comprising a polypeptide encoding an α-1,2-fucosyltransferase

[0013] -1,2-fucosyltransferase) activity.

[0014] The α-1,2-fucosyltransferase polypeptide refers to a polypeptide that can catalyze the transfer of a fucose residue from a donor substrate to an acceptor molecule. The donor substrate used to transfer the fucose residue to the acceptor molecule is usually guanosine diphosphate L-fucose (GDP-L-fucose). Suitable acceptor molecules for fucose residues include oligosaccharides, glycopeptides, glycoproteins and glycolipids. Generally, the fucose residue is transferred to, for example, an N-acetylglucosamine residue, an N-acetylgalactosamine residue, a galactose residue, a fucose residue, a sialic acid residue or a glucose residue of an oligosaccharide, or a sugar portion of a glycoprotein or glycolipid; further, the α-1,2-fucosyltransferase polypeptide has the activity of catalyzing the synthesis of 2'-FL using GDP-L-fucose and lactose as substrates.

[0015] The gene-modified cells described in the present application are Kluyveromyces gene-modified cells, which include Kluyveromyces cells that can produce fucosyllactose after being modified by the technical solution described in the present invention, and the starting strains thereof include but are not limited to Pichia kluyveri

[0016] Kluyveromyces lactis, Kluyveromyces polysporus, Kluyveromyces dobzhanskii, Kluyveromyces thermotolerans, Kluyveromyces yarrowii, or Kluyveromyces marxinus, etc.

[0017] The α-1,2-fucosyltransferase polypeptide is derived from Helicobacter pylori, Thermosynechococcus, Escherichia coli, mammals, Caenorhabditis elegans, Schistosoma mansoni, Bacillus cereus, Pseudopedobater saltans, Helicobacter muscardine, Bacillus fragilis, Bacteroides vulgaris, Bacteroides fragilis, or Bacillus smithii.

[0018] Preferably, the α-1,2-fucosyltransferase polypeptide is an α-1,2-fucosyltransferase (α-1,2-fucosyltransferase) excavated by the applicant from Bacillus smithii in nature, named BSFut-wt, whose amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 26. It has a lower catalytic synthesis of 2'-FL activity, accompanied by the production of undesirable DFL (Difucosyllactose) synthesis side activity.

[0019] The amino acid sequence of the α-1,2-fucosyltransferase polypeptide shown in SEQ ID NO:1 has the highest consistency with the amino acid sequence of Bacillus cereus107 (as of October 20, 2023) after comparison with the NCBI database, which is 69.31% (as of October 20, 2023); it has a similarity of 33.57% with the α-1,2-fucosyltransferase (α-1,2-fucosyltransferase,) derived from Helicobacter pylori, named HPFut-wt, the amino acid sequence is shown in SEQ ID NO:27, and the nucleotide sequence is shown in SEQ ID NO:28.

[0020] Further preferably, the α-1,2-fucosyltransferase polypeptide is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2-25, which is a derivative peptide of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, and these polypeptides have no side activities.

[0021] In one embodiment, the α-1,2-fucosyltransferase polypeptide is a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. The polypeptides shown in SEQ ID NO: 2-25 are α-1,2-fucosyltransferase polypeptides formed by replacing one or more amino acid residues / or fragments in the polypeptide shown in SEQ ID NO: 1. Specifically, the α-1,2-fucosyltransferase polypeptide is selected from the amino acid sequences obtained by any one of the following.

[0022] 1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid fragment at positions 5-7 is replaced by QVY to GIW, and the amino acid fragment at positions 28-41 is replaced by NDVYLDSSTSYEKY to DDVYLDIETYFEKN / or

[0023] KDVYLDTVTWYEKY, thereby obtaining a polypeptide with an amino acid sequence as shown in SEQ ID NO:2 / or SEQ ID NO:3;

[0024] 2) In the amino acid sequence shown in SEQ ID NO:2, the amino acid fragment at positions 55-62 is replaced by HIKPKHAS with HTKPKLAK / or PIKPKEAK, thereby obtaining a polypeptide with an amino acid sequence shown in SEQ ID NO:4 / or SEQ ID NO:5, respectively;

[0025] 3) In the amino acid sequence shown in SEQ ID NO:5, the amino acid fragment at positions 68-74 is replaced by NLSDLDE with KLSDIDE / or ELGSFDD, thereby obtaining a polypeptide with an amino acid sequence shown in SEQ ID NO:6 / or SEQ ID NO:7, respectively;

[0026] 4) In the amino acid sequence shown in SEQ ID NO:7, the amino acid fragment at positions 87-93 is replaced by KKSTYVE with KKDTVIV / or EKKTMIK, thereby obtaining a polypeptide with an amino acid sequence shown in SEQ ID NO:8 / or SEQ ID NO:9, respectively;

[0027] 5) In the amino acid sequence as shown in SEQ ID NO:9, the amino acid fragment at positions 110-117 is replaced by TYFKGYY with SFLYGYW / or AYLEGYW, thereby obtaining a polypeptide with an amino acid sequence as shown in SEQ ID NO:10 / or SEQ ID NO:11, respectively;

[0028] 6) In the amino acid sequence shown in SEQ ID NO: 10, the amino acid residue at position 122 is replaced by W to Y, and the amino acid fragment at positions 126-136 is replaced by EEDLLKDYQFT to KEDLLKDYQFL / or LEDLKKAFQFK, thereby obtaining a polypeptide with an amino acid sequence as shown in SEQ ID NO: 12 / or SEQ ID NO: 13, respectively;

[0029] 7) In the amino acid sequence shown in SEQ ID NO: 12, the amino acid fragment at positions 166-173 is replaced by WLNKEYRD with YLNKEYEE / or YLNKEYEE, thereby obtaining polypeptides with amino acid sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively;

[0030] 8) In the amino acid sequence as shown in SEQ ID NO:14, the amino acid fragment at positions 181-194 is replaced by LEWYLKAIAYVEKR with EEYYLKAIAYVEER / or VDYYLKAINYVLEK, thereby obtaining a polypeptide with an amino acid sequence as shown in SEQ ID NO:16 / or SEQ ID NO:17, respectively;

[0031] 9) In the amino acid sequence shown in SEQ ID NO: 16, the amino acid fragment at positions 201-205 is replaced by EIFSY with YIFSD, thereby obtaining a polypeptide with an amino acid sequence shown in SEQ ID NO: 18;

[0032] 10) In the amino acid sequence shown in SEQ ID NO: 18, the amino acid residue at position 211 is replaced by Q to K, and the amino acid residue at position 224 is replaced by D to S, thereby obtaining a polypeptide with the amino acid sequence shown in SEQ ID NO: 19;

[0033] 11) In the amino acid sequence shown in SEQ ID NO: 19, the amino acid fragment at positions 259-264 is replaced by NKWKDK with NKYKDK / or CEDEDA, thereby obtaining a polypeptide with an amino acid sequence shown in SEQ ID NO: 20 / or SEQ ID NO: 21, respectively;

[0034] 12) In the amino acid sequence shown in SEQ ID NO:21, the amino acid residue at position 282 is replaced by E with D / or Y, thereby obtaining a polypeptide with an amino acid sequence shown in SEQ ID NO:22 / or SEQ ID NO:23;

[0035] 13) In the amino acid sequence shown in SEQ ID NO:22, the amino acid residue at position 286 is replaced by K to D, thereby obtaining a polypeptide with the amino acid sequence shown in SEQ ID NO:24;

[0036] 14) In the amino acid sequence shown in SEQ ID NO:24, the amino acid fragment at positions 289-291 is replaced by IQL to LLLEKEEIEE, thereby obtaining the amino acid sequence shown in SEQ ID NO:25.

[0037] In certain embodiments, the α-1,2-fucosyltransferase polypeptide, its amino acid sequence includes but is not limited to an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical to SEQ ID NO: 1-25, and comprises an amino acid substitution mutation that is functionally equivalent to the schemes described in 1)-14) above. In certain embodiments, the substitution mutation comprises a mutation to a charged residue; in certain embodiments, the substitution mutation comprises a mutation to a basic residue. In certain embodiments, the substitution mutation comprises a mutation homologous to the amino acid sequence of the schemes described in 1)-14) above.

[0038] In certain embodiments, the α-1,2-fucosyltransferase polypeptide includes but is not limited to a polypeptide having an amino acid sequence and an amino acid sequence as described in any one of SEQ ID NOs: 1 to 25. In certain embodiments, the α-1,2-fucosyltransferase polypeptide includes but is not limited to a polypeptide having an amino acid sequence and an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical amino acid sequence to SEQ ID NOs: 1 to 25, and a polypeptide comprising an amino acid sequence functionally equivalent to that shown in SEQ ID NOs: 1 to 25.

[0039] The method for producing the α-1,2-fucosyltransferase polypeptide as described above comprises:

[0040] (1) culturing the transformed host cell under conditions suitable for expressing the polypeptide;

[0041] Preferably, the host cell is genetically engineered Escherichia coli BL21 (DE3); and

[0042] (2) Recovering the polypeptide.

[0043] In a specific embodiment, the step (1) comprises: firstly introducing a nucleic acid construct or a recombinant expression vector encoding the above-mentioned α-1,2-fucosyltransferase polypeptide 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 embodiment, the step (2) includes the steps of isolating and purifying the polypeptide from the culture.

[0045] Host cells can be cultivated in a nutrient medium suitable for producing polypeptides using methods known in the art. For example, cells can be cultured by shaking bottles, or in a suitable medium and under conditions that allow polypeptide expression and / or separation, small-scale or large-scale fermentations (including continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) can be carried out in a laboratory or industrial fermentor tank to cultivate cells. Cultivation is to use procedures known in the art, which occur in a suitable nutrient medium, and the medium comprises carbon and nitrogen sources and inorganic salts. Suitable medium can be purchased through commercial channels, or prepared according to disclosed compositions.

[0046] In some embodiments, the preferred host cell is Escherichia coli, or other transformable host cells. Further preferably, the host cell is a genetically modified Escherichia coli BL21 (DE3).

[0047] The genetically modified cells include, but are not limited to, polypeptides having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence such as SEQ ID NO: 1-25 or to a polypeptide as shown in SEQ ID NO: 1-25.

[0048] In some embodiments, preferably, the Kluyveromyces genetically modified cells include Kluyveromyces lactis genetically modified cells and Kluyveromyces marxianus genetically modified cells.

[0049] Further, in some embodiments, the genetically modified yeast cell may include one or more heterologous nucleic acids encoding GDP-mannose-4,6-dehydratase (e.g., from Escherichia coli, but not limited to Escherichia coli) and GDP-L-fucose synthase (e.g., from Escherichia coli, but not limited to Escherichia coli) in addition to heterologous nucleic acids encoding one or more of the above enzymes. The genetically modified yeast cell can synthesize 2'-fucosyllactose de novo using lactose and glucose, or glycerol, or sucrose as carbon sources.

[0050] The heterologous GDP-mannose-4,6-dehydratase (gmd) gene is from Escherichia coli. Other suitable sources of GDP-mannose-4,6-dehydratase include, but are not limited to, Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, Dictyostelium discoideum, Mus musculus, Drosophila melanogaster, Sinorhizobium fredii, Pandoraea vervacti, Caenorhabditis briggsae, Candidatus Curtissbacteria, Pseudomonas sp., Clostridium sp., Cricetulus griseus), Arthrobacter iccitolerans or Paraburkholderia piptadeniae, etc.

[0051] The heterologous GDP-L-fucose synthase (wcag) gene is from Escherichia coli. Other suitable sources of GDP-L-fucose synthase include, for example, but not limited to, Mus musculus, Homo sapiens, Marinobacter salarius, Sinorhizobium freundii NGR234, Citrobacter rodentium, Pongoabelii, Caenorhabditis elegans, Candidatus Staskawiczbacteria bacterium, Azorhizobium caulinodans, or Candictus Nitrospiranitrificans, etc.

[0052] In certain embodiments, the Kluyveromyces genetically modified cells further comprise a β-galactosidase gene (lac4) required for lactose metabolism of at least a portion of the starting strain that is missing or destroyed. Therefore, the Kluyveromyces engineered bacteria described in the present application can import lactose without consuming lactose. Compared with the starting strain, the expression level of the β-galactosidase gene in the Kluyveromyces genetically modified cells is reduced, and therefore, the Kluyveromyces engineered bacteria reduces the consumption of lactose.

[0053] In some embodiments, the Kluyveromyces genetically modified cell is a Kluyveromyces lactis genetically modified cell, and the nucleic acid sequence of the β-galactosidase lac4 gene thereof has a Genebank number of M84410.1, and an amino acid sequence of Genebank number of AAA35265.1.

[0054] In some embodiments, the Kluyveromyces genetically modified cell is a Kluyveromyces marxianus genetically modified cell, and the nucleic acid sequence of the β-galactosidase gene lac4 gene has a Genebank number of XM_022818497.1, and an amino acid sequence of Genebank number of XP_022675157.1.

[0055] The Kluyveromyces gene-modified cells of the present invention can achieve a higher yield of synthetic fucosylated lactose without the need to introduce a heterologous transporter protein.

[0056] In one embodiment, the preferred technical solution is: the starting strain of the Kluyveromyces lactis gene-modified cells is Kluyveromyces lactis DSM70799; the starting strain of the Kluyveromyces marxianus gene-modified cells is Kluyveromyces marxianus DMKU3-1042. As mentioned above, the starting strains of Kluyveromyces lactis and Kluyveromyces marxianus can be easily purchased through commercial channels or culture collection centers.

[0057] The Kluyveromyces genetically modified cell has been genetically engineered to contain increased intracellular GDP-L-fucose and 2'-FL production capacity compared to the starting strain. Preferably, the genetically modified cell has been genetically engineered to:

[0058] 1) expressing a gene encoding a bifunctional fucokinase / L-fucose-1-phosphate-guanosyltransferase, which catalyzes the formation of GDP-fucose from L-fucose; or

[0059] 2) Overexpression of at least one of the genes encoding GDP-mannose-4,6-dehydratase and GDP-L-fucose synthase.

[0060] 3) expressing a gene encoding an α-1,2-fucosyltransferase polypeptide.

[0061] 4) Disruption of the galactosidase gene lac4.

[0062] Optionally, the GDP-mannose-4,6-dehydratase and GDP-L-fucose synthase may be genetically modified enzymes or wild-type enzymes.

[0063] The present invention also provides a gene-modified cell containing a molecular marker, wherein the gene-modified cell containing a molecular marker comprises a nucleic acid sequence as shown in SEQ ID NO:29.

[0064] The present invention also provides a method for preparing genetically modified cells. The method uses Kluyveromyces as a starting strain and integrates a heterologous α-1,2-fucosyltransferase gene, a heterologous GDP-L-fucose synthase gene (wcaG), and a heterologous GDP-mannitol-4,6-dehydratase gene (gmd) into the Kluyveromyces genome, respectively.

[0065] Preferably, the α-1,2-fucosyltransferase gene includes but is not limited to polypeptides having amino acid sequences as shown in SEQ ID NOs: 1-25.

[0066] Preferably, the genetically modified cells in the preparation method are genetically modified cells of Kluyveromyces; further preferably, they are genetically modified cells of Kluyveromyces lactis or Kluyveromyces marxianus.

[0067] Further preferably, the starting strain of Kluyveromyces lactis is K. lactic DSM70799, and the starting strain of Kluyveromyces marxianus is K. marxianus DMKU3-1042.

[0068] Preferably, the method for preparing the Kluyveromyces genetically modified cells specifically comprises the following steps:

[0069] (1) Cultivating Kluyveromyces cells;

[0070] Preferably, the Kluyveromyces cell is a Kluyveromyces lactic acid cell K.lactic / or a Kluyveromyces marxianus cell K.marxianus;

[0071] (2) constructing an expression cassette and introducing a heterologous α-1,2-fucosyltransferase gene into Kluyveromyces cells;

[0072] (3) destroying the lac4 gene in the strain obtained in step (1);

[0073] (4) constructing an expression cassette and introducing a heterologous GDP-L-fucose synthase gene (wcag) into Kluyveromyces cells; and / or

[0074] (5) Construct an expression cassette and introduce a heterologous GDP-mannitol 4,6 dehydratase (gmd) gene into Kluyveromyces cells.

[0075] Preferably, the recombinant construction technology of Kluyveromyces genetically engineered bacteria also includes:

[0076] (6) Construct a molecular marker expression cassette and introduce the marker gene into Kluyveromyces cells.

[0077] Preferably, the method further comprises the step of (7) recovering the genetically engineered Kluyveromyces bacteria.

[0078] Preferably, in the above method for preparing genetically modified Kluyveromyces cells, the steps are not ordered in any particular order.

[0079] In some embodiments of the present invention, the use of the genetically modified Kluyveromyces cells in the synthesis of 2'-fucosyllactose (2'-FL) is described.

[0080] Preferably, the Kluyveromyces genetically modified cells are Kluyveromyces lactis genetically modified cells or Kluyveromyces marxianus genetically modified cells.

[0081] In some embodiments, the carbon source in the culture medium comprises lactose and sucrose, or glycerol, or glucose.

[0082] In some embodiments, the culture medium can be any genetically modified cell that can maintain growth and vitality of the fucoidan-producing cell. In some embodiments, the culture medium can also include appropriate salts, minerals, metals or other nutrients. In some embodiments, the carbon source and nutrients necessary for each cell growth are added to the culture medium in an incremental or continuous manner.

[0083] The application of the Kluyveromyces gene-modified cells in synthesizing 2'-FL uses lactose and sucrose, or glycerol, or glucose as carbon sources.

[0084] In some embodiments, the culture medium does not contain fucose. In some embodiments, the method further comprises adjusting the mass ratio of the carbon source (such as sucrose) to lactose to adjust the yield of fucosyllactose.

[0085] Preferably, the fermentation product of the yeast genetically modified cells can be purified by centrifugation, filtration, decolorization, nanofiltration, chromatography, crystallization, recrystallization, etc. to obtain the product 2'-FL.

[0086] The term "heterologous": when used in reference to a polynucleotide, gene, nucleic acid, polypeptide or enzyme, refers to a polynucleotide, gene, nucleic acid, polypeptide or enzyme that is from or derived from a source other than the host organism's species.

[0087] The term "host cell" is defined as a cell that has been transformed or transfected or is capable of being transformed or transfected with an exogenous polynucleotide sequence, thereby comprising at least one non-naturally occurring sequence in the host cell.

[0088] The term "molecular marker" refers to a specific DNA fragment that can reflect certain differences in the genome between biological individuals or populations.

[0089] The term "deletion or disruption" refers to that the coding region and / or control sequence of the reference gene is partially or completely modified (e.g., by deletion, insertion and / or substitution of one or more nucleotides), so that the expression of the encoded polypeptide is absent (inactivated) or reduced, and / or the enzymatic activity of the encoded polypeptide is absent or reduced.

[0090] Beneficial Effects

[0091] The present invention provides a Kluyveromyces genetically modified cell, which includes but is not limited to the nucleotide sequence of the transferase polypeptide with an amino acid sequence such as SEQ ID NO: 1-25, and its beneficial effects are embodied in:

[0092] (1) Compared with the original strain, the Kluyveromyces gene-modified cells produced the activity of synthesizing 2'-FL and had no side activities (such as the side activity of producing DFL).

[0093] (2) Compared with 2'-FL produced by Escherichia coli or genetically modified cells of Escherichia coli, 2'-FL products synthesized by recombinant yeast genetically modified cells do not contain endotoxins and other allergens, are safe for consumption, and are more suitable for applications in the fields of food, health products, and infant food.

[0094] (3) The process of fermenting and synthesizing 2'-FL by the Kluyveromyces gene-modified cells of the present invention is simple and easy, which improves production efficiency and reduces production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 .HPLC spectrum of lactose.

[0096] Figure 2 .HPLC spectrum of 2'-FL standard.

[0097] Figure 3 .HPLC spectrum of DFL standard.

[0098] Figure 4.HPLC profile of fermentation broth of Kluyveromyces lactis genetically modified cells KL-M20.

[0099] Figure 5 .HPLC profile of fermentation broth of Kluyveromyces marxianus genetically modified cells KM-M20.

[0100] Figure 6 .Mass spectrometry analysis of 2'-FL standard.

[0101] Figure 7 . Figure 5 The HPLC spectrum shown is the LC-MS analysis spectrum of the substance with rt of about 15.06 min. DETAILED DESCRIPTION

[0102] The experimental methods used in the following examples are conventional methods unless otherwise specified; all materials, reagents, etc., are commercially available unless otherwise specified.

[0103] The present invention is further described in detail below through examples. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention. Modifications or replacements to the details and forms of the technical solution without departing from the structural concept and scope of use of the present invention all fall within the protection scope of the present invention.

[0104] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art. In general, the nomenclature used in this specification and the experimental methods described below are well known and commonly used in the art.

[0105] It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. In the following specific embodiments, if the experimental methods of specific conditions are not indicated, they are generally in accordance with the conventional methods and conditions of molecular biology within the art, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: A Laboratory Manual", or the conditions recommended by the manufacturer.

[0106] In the following specific embodiments, if the experimental methods for specific conditions are not specified, they are usually based on conventional methods and conditions of molecular biology within the technology of the field, and such techniques and conditions are fully explained in the literature; all materials, reagents, etc., unless otherwise specified, can be obtained from commercial channels.

[0107] The fermentation product of the yeast gene modified cells of the present invention can be purified by centrifugation, filtration, decolorization, nanofiltration, chromatography purification and crystallization to obtain a reaction solution or solid with high purity; it can also be made into powder by spray drying or freeze drying technology.

[0108] Explanation of abbreviations in the examples: gmd: GDP-4,6-mannitol dehydratase; wcaG: GDP-L-fucose synthase; futC: α-1,2-fucosyltransferase polypeptide.

[0109] Also provided herein is a method for preparing fucoidan lactose. The method includes providing a genetically modified yeast cell capable of producing fucoidan lactose. The yeast cell includes the α-1,2-fucosyltransferase polypeptide gene described in the present application. In some embodiments, the method also includes providing a culture medium, and culturing the yeast cell in the culture medium under conditions suitable for the yeast cell to produce fucoidan lactose.

[0110] Cultivation can be carried out in suitable containers, including but not limited to carrying out in suitable culture medium in cell culture plates, culture bottles or fermentation tanks. Any suitable fermentation tank can be used, including but not limited to stirred fermentation tanks, airlift fermentation tanks, bubble fermentation tanks or any combination thereof. In the particular embodiment utilizing Kluyveromyces lactis as host cell, bacterial strain can be grown in a fermentation tank. In addition, the method can be carried out with the fermentation of any scale known in the art, to support the industrial production of microbial products. The materials and methods used to maintain or grow cell cultures are well known to those skilled in the art in microbiology or fermentation science fields.

[0111] In some embodiments, the culture medium comprises lactose and sucrose, or glucose. In some embodiments, the carbon source in the culture medium consists essentially of lactose and sucrose, or glucose. Preferably, in some embodiments, the carbon source in the culture medium consists of lactose and sucrose.

[0112] In the following examples, the starting strain of Kluyveromyces lactis is: K. lactic DSM70799; the starting strain of Kluyveromyces marxianus is: K. marxianus DMKU3-1042.

[0113] GDP-mannitol-4,6-dehydratase (Gmd) is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO:27.

[0114] GDP-L-fucose synthase (WcaG) is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO:28.

[0115] The amino acid sequence of the α-1,2-fucosyltransferase polypeptide (futC) is shown in SEQ ID NO: 1-25 or SEQ ID NO: 59, respectively.

[0116] The amino acid sequence of the α-1,2-fucosyltransferase polypeptide derived from Helicobacter pylori is shown in SEQ ID NO: 59, denoted by HPFut-wt.

[0117] The amino acid sequence of the α-1,2-fucosyltransferase polypeptide derived from Escherichia coli is shown in SEQ ID NO: 61, denoted by ECFut-wt.

[0118] The amino acid sequence of the α-1,2-fucosyltransferase polypeptide from Bacillus cereus is as follows

[0119] Shown in SEQ ID NO:63, represented by BCFut-wt.

[0120] The amino acid sequence of the α-1,2-fucosyltransferase polypeptide from Bacteroides fragilis is shown in SEQ ID NO:65, represented by BFFut-wt.

[0121] Example 1. Expression of polypeptides shown in SEQ ID NOs: 1-25 in Escherichia coli.

[0122] 1. Using the amino acid sequence of the polypeptide shown in SEQ ID NO: 1 (nucleotide sequence shown in SEQ ID NO: 26) as a template, synthesize the polypeptide shown in SEQ ID NO: 1, and finally construct it into the PET32a vector to obtain the PET32a-wt plasmid. Yeast codon optimization synthesis was performed according to the amino acid sequences HPFut-wt, ECFut-wt, BCFut-wt and BFFut-wt, and finally constructed into the PET32a vector, and named PET32a-HPFut-wt, PET32a-ECFut-wt, PET32a-BCFut-wt, PET32a-BFFut-wt, respectively.

[0123] 2. Different mutation sites were obtained by computer-assisted rational design. According to the method of Molecular Cloning Experiment Guide, the nucleotide sequence of PET32a-wt (SEQ ID NO: 26) was used to perform Primer-BLAST ( Primer designing tool(nih.gov) ) mutant primers were designed and the recombinant plasmid series (pET32a-M1-M25) were constructed using the Fast Mutagenesis Kit.

[0124] 3. The above recombinant plasmids were transformed into Escherichia coli BL21 (DE3) according to the following steps:

[0125] Take the prepared Escherichia coli BL21 (DE3) competent cells, place them on ice for 30 minutes to melt, take 100 μL of competent cells and 10 μL of pET32a-M1-M25 recombinant plasmid (concentration 50 ng / μL) respectively, mix them, place them in a 42°C water bath for heat shock for 45 seconds, then immediately cool them in an ice bath for 2 minutes, add 1 mL of fresh LB medium (LB medium: peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, and 1.5% agar powder on the plate), and resuscitate and culture at 37°C and 100 rpm for 1 hour. Then take 100 μL of the bacterial solution and spread it on an LB plate containing ampicillin (100 μg / mL). After culturing in a 37°C constant temperature incubator for 12 hours, pick a single colony for colony PCR to screen out positive transformants.

[0126] 4. Cultivate the positive transformants, extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the pET32a-M1-M25 recombinant plasmid has been successfully introduced into E. coli.

[0127] 5. The correct transformant was inoculated into LB liquid medium, placed on a shaker at 37°C and 200rpm for 12h to obtain seed liquid; then the seed liquid was inoculated into fresh LB medium at an inoculum amount of 1% (v / v), shaken and cultured at 37°C until OD600 was 0.8, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG), the final concentration of IPTG was 0.1mmol / L, and the induction was carried out at 16°C for 12h, the speed was 200rpm; after induction expression, the fermentation liquid was centrifuged at 5000r / min for 30min at 4°C to collect the bacteria; the bacteria were resuspended in 20mM pH7.4 PBS buffer, and ultrasonicated at a frequency of plus on 5s / off 5s for 30min to break the bacteria; the broken liquid was centrifuged at 13000×g and 4°C for 30min to remove cell debris and collect the supernatant.

[0128] 6. The soluble polypeptide sequence was purified by nickel column affinity chromatography. The process was as follows: deionized water was added to the top of the nickel column, and after natural elution, it was eluted with 5 volumes of Binding buffer; the crude enzyme solution filtered through a 0.45 μm filter membrane was then loaded onto the column, and the sample was fully combined with the nickel column at a flow rate of 1.5 mL / min. After the sample was dried, it was continuously eluted with 5 column volumes of Washing buffer to remove impurities, and finally the target protein was eluted with 5 times the Elution buffer, and the eluate was collected; then, the expression of the target protein was analyzed by SDS-PAGE.

[0129] The SDS-PAGE results showed that the genetically engineered bacteria had obvious specific expression bands after induction, and the molecular weight of the bands was basically consistent with the expected molecular weight of 35.3 kDa. It can be seen that the polypeptides shown in SEQ ID NO: 1-25 were obtained. For detailed information, see Table 1.

[0130] Table 1. Correspondence between the transferase polypeptides and their amino acid sequence numbers

[0131] Plasmids Peptide naming Transferase polypeptide amino acid sequence PET32a-WT BSFut-wt SEQ ID NO:1 PET32a-M1 M1 SEQ ID NO:2 PET32a-M2 M2 SEQ ID NO:3 PET32a-M3 M3 SEQ ID NO:4 PET32a-M4 M4 SEQ ID NO:5 PET32a-M5 M5 SEQ ID NO:6 PET32a-M6 M6 SEQ ID NO:7 PET32a-M7 M7 SEQ ID NO:8 PET32a-M8 M8 SEQ ID NO:9 PET32a-M9 M9 SEQ ID NO:10 PET32a-M10 M10 SEQ ID NO:11 PET32a-M11 M11 SEQ ID NO:12 PET32a-M12 M12 SEQ ID NO:13 PET32a-M13 M13 SEQ ID NO:14 PET32a-M14 M14 SEQ ID NO:15 PET32a-M15 M15 SEQ ID NO:16 PET32a-M16 M16 SEQ ID NO:17 PET32a-M17 M17 SEQ ID NO:18 PET32a-M18 M18 SEQ ID NO:19 PET32a-M19 M19 SEQ ID NO:20 PET32a-M20 M20 SEQ ID NO:21 PET32a-M21 M21 SEQ ID NO:22 PET32a-M22 M22 SEQ ID NO:23 PET32a-M23 M23 SEQ ID NO:24 PET32a-M24 M24 SEQ ID NO:25 PET32a-HPFut-wt HPFut-wt N / A PET32a-ECFut-wt ECFut-wt N / A PET32a-BCFut-wt BCFut-wt N / A PET32a-BFFut-wt BFFut-wt N / A

[0132] Example 2. Construction of genetically modified Kluyveromyces lactis cells.

[0133] (1) Construct an expression cassette for Kluyveromyces lactis-modified cells.

[0134] 1) Construction of the KL-ΔLAC4 knockout cassette.

[0135] Referring to the applicant's prior patent: 202211453172.5, invention name: Recombinant yeast and its application, a KL-ΔLAC4 knockout box was constructed.

[0136] 2) Construction of KL-ΔLAC4::GMD expression cassette.

[0137] Using the Kluyveromyces lactis genome as a template, the upstream and downstream homology arm sequences lac4 up and lac4-do, the promoter sequence tef1 and the terminator sequence adh1 were amplified; using the synthesized gmd sequence as a template, the GMD target gene sequence was amplified; using the expression cassette KL-ΔLAC4 as a template, the G418+loxp sequence was amplified, and lac4 up, tef1, gmd, adh1, G418+loxp and lac4-do were fused by successive fusion PCR of two fragments. Finally, using primers lac4-upF and lac4-doR as primers (Table 2), the fusion PCR system was used as a template, and the gmd expression cassette KL-ΔLAC4::GMD was amplified by PCR.

[0138] Table 2

[0139]

[0140] 3) Construction of KL-ΔXK::GMER expression cassette.

[0141] Using the Kluyveromyces lactis genome as a template, the upstream and downstream homology arm sequences xk-up and xk-do, the promoter sequence pgk1 and the terminator sequence tdh3 of the xylulose kinase XKb coding region were amplified; using the synthetic gmer sequence as a template, the GMER target gene sequence was amplified; using the existing plasmid in the laboratory as a template, the G418+loxp sequence was amplified, and xyl1 up, pgk1, gmer, tdh3, G418+loxp and xyl1-do were fused by successive fusion PCR of two fragments. Finally, primers xK-upF and xK-doR were used as primers (Table 3) and the fusion PCR system was used as a template to obtain the gmer expression cassette KL-ΔXK::GMER by PCR amplification.

[0142] Table 3

[0143]

[0144] 4) Construction of KL-ΔGK::BSfutC-wt, KL-ΔGK::BSfutC-M1, KL-ΔGK::BSfutC-M24, KL-ΔGK::HPFut-wt, KL-ΔGK::ECFut-wt, KL-ΔGK::BCFut-wt, and KL-ΔGK::BFFut-wt expression cassettes.

[0145] 2. Using Kluyveromyces lacticus DSM70799 genomic DNA as template, primers galk up-F and galk up-R and galk down-F and galk down-R were used for PCR amplification to obtain the upstream and downstream homology arms respectively; loxp-F and loxp-R were used as primers and the G418 resistance plasmid was used as a template to amplify the resistance screening marker containing G418 resistance and loxp sites; futC-F and futC-R were used as primers and PET32a-WT, PET32a-M1 to PET32a-M24 were used as templates respectively to amplify the original sequence and mutant sequence of the fucosyltransferase gene; tdh3-F and tdh3-R, tef-F and tef-R were used as primers and the lactic acid Kluyveromyces genome was used as a template to amplify the promoter tdh3 and terminator sequence tef of the transferase respectively; KL-PF and KL-PR were used as primers and the PUC19 plasmid was used as a template to amplify the plasmid vector sequence by PCR. Fusion PCR was used to fuse the upstream homology arm, promoter, target gene (original sequence and mutant sequence), terminator, G418+loxp and downstream homology arm in two successive fusions to obtain ΔGK::bs-wt, ΔGK::bs-M1 to ΔGK::bs-M24 expression cassettes; the expression cassette was connected to the plasmid vector sequence by ABclonal to obtain plasmids PUC-KL-WT, PUC-KL-M1 to PUC-KL-M24 carrying ΔGK::BSfut-wt, ΔGK::BSfut-M1 to ΔGK::BSfut-M24 expression cassettes, respectively. Using PUC-KL-WT as a template, the plasmid backbone, G418 resistance and promoter terminator parts were amplified. Using PET32a-HPFut-wt, PET32a-ECFut-wt, PET32a-BCFut-wt and PET32a-BFFut-wt as templates, PCR amplification was performed to obtain HPFut-wt, ECFut-wt, BCFut-wt and BFFut-wt sequences, respectively. By connecting through ABclonal, plasmids PUC-KL-HPFut-wt, PUC-KL-ECFut-wt, PUC-KL-BCFut-wt and PUC-KL-BFFut-wt carrying ΔGK::HPFut-wt, ΔGK::ECFut-wt, ΔGK::BCFut-wt and ΔGK::BFFut-wt expression cassettes were obtained, respectively.

[0146] Using primers galk up-F and galk down-R as templates, and 25 plasmids PUC-KL-WT, PUC-KL-M1 to PUC-KL-M24, as well as PUC-KL-HPFut-wt, PUC-KL-ECFut-wt, PUC-KL-BCFut-wt and PUC-KL-BFFut-wt as templates, KL-ΔGK::BSfut-wt, KL-ΔGK::BSfut-M1 to KL-ΔGK::BSfut-M24 expression cassettes, as well as KL-ΔGK::HPFut-wt, KL-ΔGK::ECFut-wt, KL-ΔGK::BCFut-wt, KL-ΔGK::BFFut-wt were amplified for the next step of constructing recombinant strains.

[0147] Table 4. Primers for constructing KL-ΔGK::BSfutC-wt, KL-ΔGK::BSfutC-M1, KL-ΔGK::BSfutC-M24, KL-ΔGK::HPFut-wt, KL-ΔGK::ECFut-wt, KL-ΔGK::BCFut-wt, KL-ΔGK::BFFut-wt expression cassettes

[0148]

[0149] (2) Transformation of the recombinant expression cassette and verification of the recombinant strain.

[0150] The expression cassettes constructed in (1) above were respectively transferred into the starting strain Kluyveromyces lacticus DSM70799 cells. The specific method is:

[0151] 1) Prepare yeast competent cells: Take a small amount of frozen yeast strains and streak them on a plate solid culture medium, invert and culture at 30°C for 2 days. Pick a single yeast colony in 50mL liquid culture medium and culture at 30°C, 220rpm until OD 600 Between 0.8 and 1.5. Collect the cells and 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 the competent yeast cells, and dispense them into 50 μL / tube for transformation.

[0152] Meanwhile, boil 1 mL of salmon sperm DNA for 5 min and quickly put it on ice to prepare single-stranded DNA.

[0153] 2) Transformation: Centrifuge the competent yeast prepared above and remove the residual lithium chloride solution with Tips. For each transformation, add the following order: 50% PEG3350 (240 μL); 1M LiCl (36 μL); 2mg / mL single-stranded Salmon sperm DNA (25 μL); 5-10 μg / 50 μL plasmid DNA aqueous solution (50 μL), vortex vigorously until the precipitated bacteria are completely distributed; incubate in a 30℃ water bath for 30 minutes; heat shock in a 42℃ water bath for 20-25 minutes; centrifuge at 8000rpm for 10 minutes, and collect the yeast cells; then, resuspend the yeast in 500 μL liquid culture medium and incubate in a shaking table at 30℃; after 1-4 hours, take 25-100 μL of bacterial solution and spread it on a selective culture medium plate, and incubate it upside down at 30℃.

[0154] 3) Verification: The correspondence between the recombinant strain and its genotype is shown in Table 5. To verify the correctness of the above strains, we extracted the genomes of the above transformants and the original strains, and amplified the genomes by PCR using the corresponding knockout cassette or expression cassette amplification primers. If the size of the only band obtained after PCR amplification is consistent with the knockout cassette or expression cassette, the strain is judged to be correct, otherwise the strain is judged to be a false positive error.

[0155] Table 5. Kluyveromyces lactis engineered strains and their genotypes (α-1,2-fucosyltransferase gene)

[0156]

[0157]

[0158] Example 3. Construction of genetically modified cells of Kluyveromyces marxianus.

[0159] (1) Construction of Max-Kluyveromycin-modified cell expression cassette.

[0160] 1) Construction of KM-ΔLAC4 knockout cassette.

[0161] Refer to the applicant's prior patent: 202211453172.5, invention name: Recombinant yeast and its application to construct KM-ΔLAC4 knockout box.

[0162] 2) Construction of KM-ΔLAC4::GMD expression cassette.

[0163] Using the Kluyveromyces marxianus genome as a template, the upstream and downstream homology arm sequences km-lac4 up and km-lac4-do were amplified. Using the KL-ΔLAC4::GMD expression cassette as a template, the GMD expression cassette and G418+loxp site were amplified. By means of three-fragment fusion PCR, km-lac4 up, GMD expression cassette+G418+loxp and km-lac4-do were fused. Finally, primers lac4-upF1 and lac4-doR1 were used as primers (Table 6) and the fusion PCR system was used as a template to obtain the gmd expression cassette KM-ΔLAC4::GMD by PCR amplification.

[0164] Table 6

[0165]

[0166] 3) Construction of KM-ΔXK::GMER expression cassette.

[0167] Using the Kluyveromyces marxianus genome as a template, the upstream and downstream homology arm sequences km-xk up and km-xk-do of the Kluyveromyces marxianus xylulose kinase were amplified. Using the 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, primers xk-upF1 and xk-doR1 were used as primers (Table 7) and the fusion PCR system was used as a template to obtain the gmer expression cassette KM-ΔXK::GMER by PCR amplification.

[0168] Table 7

[0169]

[0170] 4) Construction of KM-ΔGK::BSfutC-wt, KM-ΔGK::BSfutC-M1, KM-ΔGK::BSfutC-M24, KM-ΔGK::HPFut-wt, KM-ΔGK::ECFut-wt, KM-ΔGK::BCFut-wt, and KM-ΔGK::BFFut-wt expression cassettes.

[0171] Using Kluyveromyces marxianus DMKU3-1042 genomic DNA as template, primers glk up-F1 and glk up-R1 and glk down-F1 and glk down-R1 were PCR amplified to obtain the upstream and downstream homology arms, respectively; the expression cassettes of KL-ΔGK::BSfut-wt, KL-ΔGK::BSfut-M1 to KL-ΔGK::BSfut-M24, and KL-ΔGK::HPFut-wt, KL-ΔGK::ECFut-wt, KL-ΔGK::BCFut-wt, and KL-ΔGK::BFFut-wt were used as templates to PCR amplify the fut-wt / fut mutant expression cassette + G418 + loxp sequence, and the upstream and downstream homology arms and the fut-wt / fut mutant expression cassette + G418 + loxp sequence were fused by fusion PCR, respectively, using the fusion system as templates with primers glk up-F1 and glk down-R1. Using down-R1 as a template, the expression cassettes of KM-ΔGK::BSfut-wt, KM-ΔGK::BSfut-M1 to KM-ΔGK::BSfut-M24, as well as KM-ΔGK::HPFut-wt, KM-ΔGK::ECFut-wt, KM-ΔGK::BCFut-wt, and KM-ΔGK::BFFut-wt were amplified for the next step of constructing recombinant strains.

[0172] Table 8. Primers for constructing KM-ΔGK::BSfut-wt, KM-ΔGK::BSfut-M1 to KM-ΔGK::BSfut-M24 expression cassettes, and KM-ΔGK::HPFut-wt, KM-ΔGK::ECFut-wt, KM-ΔGK::BCFut-wt, KM-ΔGK::BFFut-wt expression cassettes

[0173]

[0174] (2) Transformation of recombinant plasmid and verification of recombinant strain:

[0175] The expression cassette constructed in (1) above was transformed into the starting strain K. marxianus DMKU3-1042 cells. The specific method is as described in part (2) of Example 2, transformation of the recombinant expression cassette and verification of the recombinant strain.

[0176] The correspondence between the recombinant strains and their genotypes is shown in Table 9. To verify the correctness of the above strains, we extracted the genomes of the above transformants and the original strains, and amplified the genomes by PCR using the corresponding knockout cassette or expression cassette amplification primers. If the size of the only band obtained after PCR amplification is consistent with the knockout cassette or expression cassette, the strain is judged to be correct, otherwise the strain is judged to be a false positive error.

[0177] Table 9. Kluyveromyces marxianus engineered bacteria and their genotypes (α-1,2-fucosyltransferase gene)

[0178] strains genotype K.marxianusDMKU3-1042 wt KM-1 K.marxianusDMKU3-1042;ΔLAC4::GMD KM-2 KM-1;KM-ΔXK::GMER KM-3 KM-2;KM-ΔGK::BSFut-wt KM-M1 KM-2;KM-ΔGK::BSFut-M1 KM-M2 KM-2;KM-ΔGK::BSFut-M2 KM-M3 KM-2;KM-ΔGK::BSFut-M3 KM-M4 KM-2;KM-ΔGK::BSFut-M4 KM-M5 KM-2;KM-ΔGK::BSFut-M5 KM-M6 KM-2;KM-ΔGK::BSFut-M6 KM-M7 KM-2;KM-ΔGK::BSFut-M7 KM-M8 KM-2;KM-ΔGK::BSFut-M8 KM-M9 KM-2;KM-ΔGK::BSFut-M9 KM-M10 KM-2;KM-ΔGK::BSFut-M10 KM-M11 KM-2;KM-ΔGK::BSFut-M11 KM-M12 KM-2;KM-ΔGK::BSFut-M12 KM-M13 KM-2;KM-ΔGK::BSFut-M13 KM-M14 KM-2;KM-ΔGK::BSFut-M14 KM-M15 KM-2;KM-ΔGK::BSFut-M15 KM-M16 KM-2;KM-ΔGK::BSFut-M16 KM-M17 KM-2;KM-ΔGK::BSFut-M17 KM-M18 KM-2;KM-ΔGK::BSFut-M18 KM-M19 KM-2;KM-ΔGK::BSFut-M19 KM-M20 KM-2;KM-ΔGK::BSFut-M20 KM-M21 KM-2;KM-ΔGK::BSFut-M21 KM-M22 KM-2;KM-ΔGK::BSFut-M22 KM-M23 KM-2;KM-ΔGK::BSFut-M23 KM-M24 KM-2;KM-ΔGK::BSFut-M24 KM-4 KM-2;KM-ΔGK::HPFut-wt KM-5 KM-2;KM-ΔGK::ECFut-wt KM-6 KM-2;KM-ΔGK::BCFut-wt KM-7 KM-2;KM-ΔGK::BFFut-wt

[0179] Example 4. Construction of genetically modified Kluyveromyces lactis cells carrying marker genes and identification of marker genes.

[0180] (1) The artificial amino acid sequence containing the encryption tag was sent for synthesis (Shanghai Shenggong Biotechnology Co., Ltd.), and the artificial sequence containing the encryption tag was randomly inserted into the genome of lactic acid yeast or Kluyveromyces marxii in the same manner as in Example 2.

[0181] The artificial amino acid sequence containing the encrypted tag is as follows:

[0182] The correspondence between the labeled recombinant strains and their genotypes is shown in Table 11.

[0183] Table 11

[0184]

[0185]

[0186] (3) Verification.

[0187] To verify the correctness of the above strains, we extracted the genomes of the above transformants and original strains, and amplified the genomes by PCR using the corresponding knockout box or expression box amplification primers. If the size of the only band obtained after PCR amplification is consistent with the knockout box or expression box, the strain is judged to be correct, otherwise the strain is judged to be a false positive error. For the verification of the label, PCR amplification is performed using primers, and further verification is performed by sequencing, otherwise the strain is judged to be a false positive error.

[0188] Example 5. Construction of Kluyveromyces marxii genetically modified cells with marker genes and identification of marker genes

[0189] The artificial amino acid sequence containing the encryption tag was sent for synthesis (Shanghai Shenggong Biotechnology Co., Ltd.), and the artificial sequence containing the encryption tag was randomly inserted into the lactic acid yeast or Kluyveromyces marxiensis genome in the same manner as in Example 2.

[0190] The artificial amino acid sequence containing the encrypted tag is as follows:

[0191] TFLSHIHLNGIINGYANVAPTANGFIHMAHWGSDEDIANSINGDAIQIATCNYYTIADEIECHANGAKRHNQANDIKANGLINGYIVANPFNDENIANFMMIANYITIAWGIEYISICQTINSHENCINNITYNGFAYDHEEIKFRDEDHAKLHIHSLFTNGGINGNENGHVPFING

[0192] Table 12. Correspondence of genotypes as shown in Table

[0193]

[0194]

[0195] (2) Verification.

[0196] To verify the correctness of the above strains, we extracted the genomes of the above transformants and original strains, and amplified the genomes by PCR using the corresponding knockout box or expression box amplification primers. If the size of the only band obtained after PCR amplification is consistent with the knockout box or expression box, the strain is judged to be correct, otherwise the strain is judged to be a false positive error. For the verification of the label, PCR amplification is performed using primers, and further verification is performed by sequencing, otherwise the strain is judged to be a false positive error.

[0197] Example 6. Fermentation synthesis of 2'-FL by genetically modified Kluyveromyces yeast cells

[0198] Yeast was cultured with glucose as the carbon source. The genetically modified cells obtained in Examples 2-5 were taken respectively, and the genetically modified cells of Kluyveromyces were grown rapidly until the growth entered the late logarithmic phase or the stable phase. The strains were streaked and cultured in solid culture media such as YDP, and after culturing at 30°C for 2-3 days, single colonies were picked and inoculated into 1.5mL YPD liquid culture medium, and cultured at 30°C and 200rpm overnight. Subsequently, they were inoculated into 50mL liquid culture medium shake flasks at a 2% inoculation rate, and cultured at 30°C and 200rpm until OD 600 =1, lactose and sucrose were added to a final concentration of 10 g / L, and 3% (w / v) sucrose were added, and the total fermentation time was 72 h at 30°C and 200 rpm. After 72 h of fermentation, samples were taken and boiled for 10 min, and the final fermentation product of yeast was obtained after centrifugation, and the 2'-FL content in the supernatant was detected.

[0199] HPLC detection method: Detection conditions: Chromatographic column model: Shodex Asahipak NH2P-50 4E, mobile phase: 65% acetonitrile aqueous solution, flow rate: 0.5 ml / min, column temperature: 35°C, injection volume: 10 μL, evaporative light detector, evaporation temperature 75°C, nebulization temperature 45°C.

[0200] The LC-MS analysis conditions are as follows:

[0201] Chromatographic column model: Shodex Asahipak NH2P-50 4E, detector: UV detector (Hitachi Chromaster), detection wavelength: 210nm, injection volume: 10μL, flow rate: 0.5mL / min, column temperature: 35℃, mobile phase: acetonitrile: water

[0202] =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 peak time rt of 2'-FL standard is 15.06min ( Figure 2 ); HPLC analysis of DFL standard ( Figure 3 )rt is 17.7min.

[0205] (2) The fermentation broths of the genetically modified cells KL-M1 to KL-M24, KM-M1 to KM-24, KL-M1-1 to KL-M24-1, and KM-M1-1 to KM-24-1 obtained in Example 2-5 all showed a strong absorption peak at around 15.06 min (see Appendix Figure 4 and attached Figure 5), which is consistent with the peak time of the 2'-FL standard, indicating that 2'-FL is produced in the fermentation broth of the genetically modified cells obtained in Example 2-5.

[0206] (3) The fermentation liquid of the genetically modified cells obtained in Example 2-5 had no absorption peak near 17.7 min, indicating that no DFL was generated in the fermentation liquid of the genetically modified cells obtained in Example 2-5.

[0207] (4) No 2'-FL and DFL were produced in the fermentation broth of the starting strain.

[0208] The LC-MS analysis conditions are shown above, and the analysis results show:

[0209] The product of the HPLC chromatogram peak of the reaction solution near rt = 15.06min was analyzed by LC-MS, and its spectrum is shown in the attached Figure 7 The MH value of the fermentation broth was 487.17, which was consistent with the Figure 6 The mass spectrum of the 2'-FL standard is consistent with the theoretical molecular weight of 2'-FL, which is 488.44, and is within the allowable error range. This proves that the fermentation process of the genetically modified cells obtained in Example 2-5 successfully synthesized 2'-FL, and there was no byproduct DFL in the product.

[0210] The yields of 2'-FL and DFL in the fermentation broth were detected by the above-mentioned HPLC analysis method, and the results are recorded in Tables 13-16.

[0211] Table 13. Study on the synthesis of 2'-FL by Kluyveromyces lactis gene-modified cells obtained in Example 2

[0212]

[0213]

[0214] Table 14. Study on the synthesis of 2'-FL by Kluyveromyces maximus gene-modified cells obtained in Example 3

[0215] strains 2'-FL, mg / mL DFL, mg / mL K.marxianusDMKU3-1042 0 Not detected KM-1 0 Not detected KM-2 0 Not detected KM-3 1.8 0.07 KM-M1 3.6 Not detected KM-M2 4.2 Not detected KM-M3 4.8 Not detected KM-M4 5.2 Not detected KM-M5 6.7 Not detected KM-M6 5.6 Not detected KM-M7 6.3 Not detected KM-M8 6.5 Not detected KM-M9 6.3 Not detected KM-M10 7.5 Not detected KM-M11 6.4 Not detected KM-M12 6.8 Not detected KM-M13 7.5 Not detected KM-M14 8.3 Not detected KM-M15 7.9 Not detected KM-M16 8.3 Not detected KM-M17 8.6 Not detected KM-M18 10.2 Not detected KM-M19 11.6 Not detected KM-M20 12.5 Not detected KM-M21 13.3 Not detected KM-M22 33.6 Not detected KM-M23 36.5 Not detected KM-M24 34.1 Not detected KM-4 1.3 0.23 KM-5 0.8 Not detected KM-6 1.0 Not detected KM-7 0.6 Not detected

[0216] Table 15. Study on the synthesis of 2'-FL by Kluyveromyces lactis gene-modified cells obtained in Example 4

[0217]

[0218]

[0219] Table 16. Study on the synthesis of 2'-FL by Kluyveromyces maximus gene-modified cells obtained in Example 5

[0220]

[0221]

[0222] Table 13-16 Data Description:

[0223] (1) The starting strains of K. lactis and K. max axon and the genetically modified cells KL-1, KL-2, KM-1 and KM-2 were all unable to synthesize 2'-FL; however, KL-3 and KM-3 were able to produce 2'-FL, indicating that K. lactis / K. max axon cells themselves did not have the ability to synthesize 2'-FL, and that even after the introduction of Gmd (GDP-mannose-4,6-dehydratase) and WcaG (GDP-L-fucose synthase), 2'-FL synthesis could not be achieved. The introduction of BSFut-wt successfully detected the production of 2'-FL, thus proving that BSFut-wt had α-1,2-fucosyltransferase activity, but the activity was low.

[0224] (2) The Kluyveromyces gene-modified cells of the present invention have the activity of synthesizing 2'-FL, and no DFL is detected in the fermentation broth.

[0225] (3) The introduction of the marker gene in Example 4 and Example 5 does not affect the yield of 2'-FL synthesized by the Kluyveromycin gene-modified cells obtained in Example 2 or Example 3.

[0226] Example 7. Take Kluyveromyces KL-M1-1, KL-M24-1, KM-M1-1, KM-M24-1, KL-6-1 and KM-6-1 respectively, 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 a 2% inoculum, 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 of 2%, and after culturing for 6-7h, began to flow sucrose (mother liquor concentration 50%), and the sucrose addition flow rate was controlled at 8mL / h, and lactose (mother liquor concentration was 40%) was added at the same time to maintain the final lactose concentration of 15g / L, and the total fermentation time was 72h. After 72h of fermentation, the fermentation liquid was centrifuged, and the supernatant and precipitate were collected respectively. The precipitate was crushed by a high-pressure homogenizer, boiled and centrifuged to remove the protein, and the supernatant was combined to obtain the final fermentation product of the yeast, and the 2'-FL and DFL contents in the supernatant were detected. The results are recorded in Table 6.

[0227] Table 6

[0228] Genetically modified cells 2'-FL yield, g / L DFL yield, g / L KL-M1-1 25 Not detected KL-M24-1 35 Not detected KM-M1-1 23 Not detected KM-M24-1 30 Not detected KL-6-1 12 Not detected KM-6-1 8 Not detected

[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 The invention comprises a recombinant nucleic acid sequence encoding an α-1,2-fucosyltransferase polypeptide.

2. The genetically modified cell according to claim 1, characterized in that for Kluyveromyces gene-modified cells; Preferably, the Kluyveromyces genetically modified cells include but are not limited to Pichia kluyveri, Kluyveromyces lactis, Kluyveromyces polysporus, Kluyveromyces dobzhanskii, Kluyveromyces thermotolerans, Kluyveromyces yarrowii, or Kluyveromyces marxinus genetically modified cells; More preferably, the Kluyveromyces genetically modified cell is a Kluyveromyces lactis genetically modified cell or a Kluyveromyces marxinus genetically modified cell.

3. The genetically modified cell according to any one of claims 1 or 2, characterized in that The α-1,2-fucosyltransferase polypeptide is derived from Helicobacter pylori, Thermosynechococcus, Escherichia coli, mammals, Caenorhabditis elegans, Schistosoma mansoni, Bacillus cereus, Pseudopedobater saltans, Helicobacter muscardineus, Bacillus fragilis, Bacteroides vulgaris, Bacteroides fragilis, or Bacillus smithii; Preferably, the α-1,2-fucosyltransferase polypeptide includes, but is not limited to, a polypeptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical to an amino acid sequence of SEQ ID NOs: 1-25; Further preferably, the α-1,2-fucosyltransferase polypeptide is a polypeptide having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:

25.

4. The genetically modified cell according to any one of claims 1 to 3, characterized in that The genetically modified yeast gene cell further comprises one or more heterologous nucleic acids encoding GDP-mannose-4,6-dehydratase and GDP-L-fucose synthase; Preferably, the GDP-mannose-4,6-dehydratase and GDP-L-fucose synthase are genetically modified enzymes or wild-type enzymes; Preferably, the heterologous GDP-mannose-4,6-dehydratase (gmd) gene is from Escherichia coli. Other suitable sources of GDP-mannose-4,6-dehydratase include, but are not limited to, Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, Dictyostelium discoideum, Mus musculus, Drosophila melanogaster, Sinorhizobium fredii, Pandoraea vervacti, Caenorhabditis briggsae, Candidatus Curtissbacteria, Pseudomonas sp., Clostridium sp., Cricetulus griseus), Arthrobacter iccitolerans, or Paraburkholderia piptadeniae; Preferably, the heterologous GDP-L-fucosesynthase (wcag) gene is from Escherichia coli. Other suitable sources of GDP-L-fucose synthase include, for example, but not limited to, Mus musculus, Homo sapiens, Marinobacter salarius, Sinorhizobium freundii NGR234, Citrobacter rodentium, Pongoabelii, Caenorhabditis elegans, Candidatus Staskawiczbacteria bacterium, Azorhizobium caulinodans or Candictus Nitrospiranitrificans.

5. The genetically modified cell according to any one of claims 1 to 4, characterized in that At least a portion of the β-galactosidase gene (lac4) of the starting strain is deleted or disrupted.

6. The genetically modified cell according to any one of claims 1 to 5, characterized in that It also includes molecular marker genes, Preferably, the nucleotide sequence of the marker gene is as shown in SEQ ID NO:

29.

7. The genetically modified cell according to any one of claims 1 to 6, characterized in that Ability to import lactose without consuming it.

8. The genetically modified cell according to any one of claims 1 to 6, characterized in that The production method of the α-1,2-fucosyltransferase polypeptide comprises the following steps: (1) culturing the transformed host cell under conditions suitable for expressing the polypeptide; and (2) Recovering the polypeptide.

9. The method for preparing a genetically modified cell according to any one of claims 1 to 6, characterized in that: Include one or more of the following steps: (1) Cultivating the starting strain; (2) destroying the lac4 gene in the strain obtained in step (1); (3) constructing an expression cassette and introducing the α-1,2-fucosyltransferase gene shown in SEQ ID NO: 1-25 into the starting strain; (4) constructing an expression cassette and introducing a heterologous GDP-L-fucose synthase gene (wcag) into the starting strain; (5) constructing an expression cassette and introducing a heterologous GDP-mannitol 4,6 dehydratase (gmd) gene into Kluyveromyces cells; Optionally, the method for preparing the genetically modified cell further comprises: (6) constructing a molecular marker expression cassette and introducing the marker gene into the starting strain; Optionally, the method for preparing the genetically modified cell further comprises: (7) A step of recovering the genetically modified cells. Preferably, the starting strain as described in (1) above is a Kluyveromyces cell; Preferably, the Kluyveromyces yeast cell is a Kluyveromyces lactis strain and / or a Kluyveromyces marxianus strain. Preferably, in the above method for preparing genetically modified Kluyveromyces cells, the steps are not ordered in any particular order.

10. Use of the genetically modified cell according to any one of claims 1 to 6 in the synthesis of 2'-fucosyllactose (2'-FL); In the application, preferably, the carbon source in the culture medium comprises lactose and sucrose, or glycerol, or glucose. In the application, preferably, the culture medium also includes metal salts, minerals or other nutrients. In the application, preferably, the culture medium does not contain fucose.

Images