A Kluyveromyces gene-modified cell

Abstract

The present invention relates to a genetically modified cell, and a method for producing fucosyllactose using the same, and in particular to a Kluyveromyces gene modified cell and its application, and belongs to the field of biological genetic engineering technology. The present invention provides a genetically modified cell, which comprises a recombinant nucleic acid sequence encoding a polypeptide having α-1,2-fucosyltransferase polypeptide (α-1,2-fucosyltransferase) activity. The α-1,2-fucosyltransferase polypeptide, including but not limited to a polypeptide having an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical amino acid sequence to SEQ ID NO: 1-25. The genetically modified cell improves the ability of the starting strain to synthesize fucosyllactose.

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 diverse mixture of oligosaccharides found in human milk. They are the third largest solid component of breast milk, after fat and lactose. Among them, 2'-fucosyllactose (2'-FL) and 3'-fucosyllactose (3'-FL) are key components of HMOs and have been shown to play an important role in the brain, intestinal tract, and growth and development of infants.

[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] Because chemical synthesis of fucosyllactose requires multiple protection and deprotection steps, the product yield is low and the production cost remains high. Therefore, fucosyllactose is often synthesized using enzymatic methods and microbial cell factories. Pure enzymatic methods generally require fucose as a substrate, which is costly. Cell factories, on the other hand, often allow for de novo synthesis, enabling low-cost, large-scale industrial production.

[0005] One feasible method for large-scale synthesis of fucosyllactose is the microbial cell factory method. Existing technologies mostly rely on Escherichia coli to biosynthesize fucosyllactose. Given a carbon source, the genetic pathway within E. coli is used to synthesize the precursor GDP-L-fucose and the acceptor lactose. Exogenous fucosyltransferase is then introduced into the engineered bacteria to combine the two precursors, resulting in industrial synthesis of fucosyllactose.

[0006] Saccharomyces cerevisiae strains are recognized as safe microorganisms, characterized by their non-pyrogenic properties, lack of toxic side effects, high food safety, and reduced subsequent isolation and purification costs. Furthermore, Saccharomyces cerevisiae is widely used in industry, but it cannot utilize lactose or transport it into cells. Although scientists have introduced a lactose transporter into Saccharomyces cerevisiae, the recombinant Saccharomyces cerevisiae produces only 0.5 g / L of 2'-fucosyllactose (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 utilize recombinant Saccharomyces cerevisiae to synthesize 2'-FL, which includes GDP-mannose-4,6-dehydratase, GDP-L-fucose synthase, and α-1,2-fucose transferase, achieving 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 an ABC transporter. Expressing heterologous ABC transporters in genetically modified yeast cells can increase the yield and purity of HMOs.

[0008] International publication WO2023122270 (A2) discloses a host cell capable of producing HMO, which contains one or more heterologous nucleic acids, each of which independently encodes: fucosyltransferase, GDP-fucosyl 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] The present invention aims to provide a genetically modified cell and its use in synthesizing fucosyllactose, which has the activity of synthesizing fucosyllactose de novo without undesirable side effects. The fucosyllactose includes 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 of the polypeptide recombinant nucleic acid sequence.

[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 typically guanosine diphosphate L-fucose (GDP-L-fucose). Suitable acceptor molecules for fucose residues include oligosaccharides, glycopeptides, glycoproteins, and glycolipids. Typically, 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 this application are Kluyveromyces gene-modified cells, which include Kluyveromyces cells that can produce fucosyllactose after being modified by the technical solution of 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, Thermococcus thermophilus, Escherichia coli, mammals, Caenorhabditis elegans, Schistosoma mansoni, Bacillus cereus, Pseudopedobater saltans, Helicobacter scutellariae, 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 low catalytic synthesis of 2'-FL activity, accompanied by the production of an undesirable side activity of synthesizing DFL (Difucosyllactose).

[0019] The amino acid sequence of the α-1,2-fucosyltransferase polypeptide is shown in SEQ ID NO: 1. After comparison with the NCBI database, the amino acid sequence identity is the highest with Bacillus cereus 107, 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 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. The polypeptides shown in SEQ ID NOs: 2-25 are α-1,2-fucosyltransferase polypeptides formed by replacing one or more amino acid residues and / 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 the 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 the 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 the amino acid sequence shown in SEQ ID NO: 8 / or SEQ ID NO: 9, respectively;

[0027] 5) In the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13, respectively;

[0029] 7) In the amino acid sequence of 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 as shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively;

[0030] 8) In the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence shown in SEQ ID NO: 18;

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

[0033] 11) In the amino acid sequence of 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 the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, respectively;

[0034] 12) In the amino acid sequence of SEQ ID NO: 21, the amino acid residue at position 282 is replaced by E with D or Y, thereby obtaining a polypeptide with the amino acid sequence of 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 substituted from 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 amino acid sequence of the α-1,2-fucosyltransferase polypeptide includes, but is not limited to, an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 1-25, and comprises an amino acid substitution mutation that is functionally equivalent to the above schemes 1)-14). 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 above schemes 1)-14).

[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 set forth 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 that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 1 to 25, as well as a polypeptide comprising an amino acid sequence that is functionally equivalent to the amino acid sequence set forth 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 the 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] Can use methods known in the art, in the nutrient medium that is suitable for producing polypeptide, cultivate host cell.For example, can pass through shake flask culture, or in applicable substratum and under the condition of allowing polypeptide expression and / or separation, carry out small-scale or large-scale fermentation (comprising continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) in laboratory or industrial fermentor tank and cultivate cell.Cultivation is to use program known in the art, occurs in applicable nutrient medium, and described substratum comprises carbon and nitrogen source and inorganic salt.Suitable substratum can be purchased through commercial channels, or according to disclosed composition preparation.

[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 E. 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 NOs: 1-25 or to a polypeptide as shown in SEQ ID NOs: 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] Furthermore, in some embodiments, in addition to heterologous nucleic acids encoding one or more of the above-mentioned enzymes, the genetically modified yeast cell may also include one or more heterologous nucleic acids encoding GDP-mannose-4,6-dehydratase (e.g., from, but not limited to, E. coli) or GDP-L-fucose synthase (e.g., from, but not limited to, E. coli). The genetically modified yeast cell is capable of synthesizing 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 Curtiss bacteria, 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 are not limited to, Mus musculus, Homo sapiens, Marinobacter salarius, Sinorhizobium fischeri NGR234, Citrobacter rodentium, Pongoabelii, Caenorhabditis elegans, Candidatus Staskawiczbacteria bacterium, Azorhizobium caulinodans, or Candictus Nitrospiranitrificans.

[0052] In some embodiments, the Kluyveromyces genetically modified cells further comprise a deletion or disruption of the β-galactosidase gene (lac4) required for lactose metabolism in at least a portion of the original strain. Thus, the engineered Kluyveromyces strains described herein are capable of importing lactose without consuming it. Compared to the original strain, the expression of the β-galactosidase gene in the Kluyveromyces genetically modified cells is reduced, thereby reducing lactose consumption in the engineered Kluyveromyces strains.

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

[0054] In some embodiments, the Kluyveromyces genetically modified cell is a Kluyveromyces marxianus genetically modified cell, and the nucleic acid sequence of its β-galactosidase gene lac4 gene is Genebank No. XM_022818497.1, and the amino acid sequence is Genebank No. 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, a preferred technical solution is: the starting strain of the genetically modified Kluyveromyces lactis cells is K. lactis DSM70799; the starting strain of the genetically modified Kluyveromyces marxianus cells is K. marxianus DMKU3-1042. The starting strains of K. lactis and K. marxianus as described above can be easily purchased commercially or from culture collections.

[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 molecularly labeled gene-modified cell, comprising a nucleic acid sequence such as the one 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] More preferably, the starting strain of Kluyveromyces lactis is K. lactis DSM 70799, and the starting strain of Kluyveromyces marxianus is K. marxianus DMKU 3-1042.

[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 lactis cell K. lactis 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 the heterologous GDP-mannitol 4,6 dehydratase (gmd) gene into Kluyveromyces cells.

[0075] Preferably, the recombinant construction technology of the Kluyveromyces genetically engineered bacteria further 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 yeast.

[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 cell in synthesizing 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 medium capable of maintaining the growth and viability of the genetically modified cells that produce fucoidan. In some embodiments, the culture medium can also include appropriate salts, minerals, metals, or other nutrients. In some embodiments, a carbon source and nutrients necessary for cell growth are added to the culture medium in an incremental or continuous manner.

[0083] The application of the Kluyveromyces gene-modified cell 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 genetically modified yeast 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 the partial or complete modification of the coding region and / or control sequence of the reference gene (e.g., by deletion, insertion and / or substitution of one or more nucleotides), such 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 genetically modified Kluyveromyces cell, which includes but is not limited to the nucleotide sequence of the transferase polypeptide represented by the amino acid sequence of SEQ ID NO: 1-25, and has the following beneficial effects:

[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 Escherichia coli genetically modified cells, 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 food, health products, infant food and other fields.

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

[0095] Figure 1 .Lactose HPLC spectrum.

[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 an LC-MS analysis spectrum of a substance having an rt of approximately 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 embodiments 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 ideas and scope of use of the present invention fall within the scope of protection of the present invention.

[0104] Unless otherwise defined, all technical 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 and are not intended to limit the scope of protection of the present invention. In the following embodiments, if no specific experimental conditions are specified, the experimental methods are generally based on conventional molecular biology methods and conditions within the skill of the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual, or follow the conditions recommended by the manufacturer.

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

[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 a 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 producing fucosyllactose. The method includes providing a genetically modified yeast cell capable of producing fucosyllactose. The yeast cell includes a gene for an α-1,2-fucosyltransferase polypeptide as described herein. In some embodiments, the method further includes providing a culture medium and culturing the yeast cell in the culture medium under conditions suitable for the yeast cell to produce fucosyllactose.

[0110] Cultivation can be carried out in suitable container, includes but not limited to carrying out in suitable culture medium in cell culture plate, blisters or fermentation tank.Can use any suitable fermentation tank, includes but not limited to stirred fermentor, airlift fermentor, bubble fermentor or its any combination.In utilizing Kluyveromyces lactis as the particular embodiment of host cell, bacterial strain can be grown in fermentation tank.In addition, described method can be carried out with the fermentation of any scale known in the art, to support the industrial production of microbial products.Material and method for cell culture maintenance or growth are well known to those skilled in the art in microbiology or fermentation field.

[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. lactis 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 sequences of the α-1,2-fucosyltransferase polypeptide (futC) are shown in SEQ ID NOs: 1-25 or SEQ ID NO: 59, respectively.

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

[0117] The amino acid sequence of the α-1,2-fucosyltransferase polypeptide from Escherichia coli is shown in SEQ ID NO: 61, denoted as 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, the polypeptide shown in SEQ ID NO: 1 was synthesized and finally constructed into the PET32a vector to obtain the PET32a-wt plasmid. Yeast codon-optimized synthesis was performed based on the amino acid sequences HPFut-wt, ECFut-wt, BCFut-wt, and BFFut-wt, respectively, and finally constructed into the PET32a vector and named PET32a-HPFut-wt, PET32a-ECFut-wt, PET32a-BCFut-wt, and PET32a-BFFut-wt, respectively.

[0123] 2. Different mutation sites were obtained by computer-assisted rational design. According to the method of the 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 a recombinant plasmid series (pET32a-M1-M25) was constructed using a point mutagenesis kit (Fast Mutagenesis Kit).

[0124] 3. Transform the above recombinant plasmids 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 thaw, 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 45 seconds, then immediately cool them in an ice bath for 2 minutes, add 1 mL of fresh LB medium (LB medium: 1.0% peptone, 0.5% yeast extract, 1.0% NaCl, and 1.5% agar powder on the plate), and resuscitate and culture at 37°C and 100 rpm for 1 hour. Then, take 100 μL of the bacterial solution and spread it on an LB plate containing ampicillin (100 μg / mL). After culturing in a 37°C constant temperature incubator for 12 hours, pick a single colony for colony PCR to screen for positive transformants.

[0126] 4. Culture 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 transformants were inoculated into LB liquid medium and cultured on a shaker at 37°C at 200 rpm for 12 h to obtain seed liquid. The seed liquid was then inoculated into fresh LB medium at a 1% (v / v) inoculum and cultured at 37°C with shaking to an OD600 of 0.8. The cells were then induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mmol / L and incubated at 16°C at 200 rpm for 12 h. After induction, the fermentation broth was centrifuged at 5000 rpm for 30 min at 4°C to collect the cells. The cells were resuspended in 20 mM PBS buffer, pH 7.4, and disrupted by sonication at a rate of plus on 5s / off 5s for 30 min. The disrupted liquid was centrifuged at 13,000 × g at 4°C for 30 min to remove cell debris, and the supernatant was collected.

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

[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. Therefore, it can be seen that the polypeptides shown in SEQ ID NO: 1-25 were obtained. For detailed information, please see Table 1.

[0130] Table 1. Corresponding relationship between 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 a Kluyveromycin-modified cell expression cassette.

[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 cassette 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; the synthesized gmd sequence was used as a template to amplify the GMD target gene sequence; 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 gmd expression cassette KL-ΔLAC4::GMD was amplified by PCR using the fusion PCR system as a template.

[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 of the xylulose kinase XKb coding region, the promoter sequence pgk1, and the terminator sequence tdh3 were amplified; using the synthesized gmer sequence as a template, the GMER target gene sequence was amplified; using the existing plasmid in the laboratory as a template, the G418+loxp sequence was amplified, and by sequential fusion PCR of two fragments, xyl1 up, pgk1, gmer, tdh3, G418+loxp, and xyl1-do were fused. Finally, using primers xK-upF and xK-doR as primers (Table 3), the fusion PCR system was used as a template to amplify the gmer expression cassette KL-ΔXK::GMER.

[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 lactis DSM70799 genomic DNA as template, primers galk up-F and galk up-R and galk down-F and galk down-R was 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. 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 cassettes were 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 lactis DSM70799 cells. The specific method is:

[0151] 1) Prepare competent yeast cells: Take a small amount of frozen yeast strain and streak it on a solid culture medium plate, invert and culture at 30℃ for 2 days. Pick a single yeast colony in 50mL liquid culture medium and culture at 30℃, 220rpm until OD 600 The nitric oxide concentration (DNA saturation) should be between 0.8 and 1.5. Collect the cells, wash with 25 mL of sterile water, centrifuge at 1500 × g for 10 min at room temperature, and discard the supernatant. Add 1 mL of 100 mM lithium chloride buffer, resuspend the pellet, centrifuge at 12,000 rpm for 30 s, and discard the supernatant. Add 400 μL of 100 mM lithium chloride buffer again, resuspend the pellet, and obtain competent yeast cells. Aliquot 50 μL / tube for transformation.

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

[0153] 2) Transformation: Centrifuge the competent yeast prepared above and remove any residual lithium chloride solution with a tip. For each transformation, add the following solution in the following order: 50% PEG3350 (240 μL); 1 M LiCl (36 μL); 2 mg / mL single-stranded Salmon sperm DNA (25 μL); and 50 μL of 5-10 μg / 50 μL plasmid DNA in water. Vortex vigorously until the precipitated yeast cells are completely distributed. Incubate in a 30°C waterbath for 30 min. Heat shock the cells in a 42°C waterbath for 20-25 min. Centrifuge at 8000 rpm for 10 min and harvest the yeast cells. Resuspend the yeast in 500 μL of liquid culture medium and incubate on a shaker at 30°C. After 1-4 h, spread 25-100 μL of the culture medium onto selective culture plates and incubate them upside down at 30°C.

[0154] 3) Verification: The correspondence between the recombinant strains and their genotypes is shown in Table 5. To verify the correctness of the above strains, we extracted the genomes of the transformants and the original strains and performed PCR amplification using primers corresponding to the knockout or expression cassette. If a single band was obtained after PCR amplification and the size was consistent with the knockout or expression cassette, the strain was considered correct; otherwise, the strain was considered a false positive.

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

[0156]

[0157]

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

[0159] (1) Construct a Max-Kluyveromycin-modified cell expression cassette.

[0160] 1) Construction of the 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 cassette.

[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 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 amplify the gmd expression cassette KM-ΔLAC4::GMD.

[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 G418+loxp site were amplified. By three-fragment fusion PCR, km-xk up, GMD expression cassette+G418+loxp and km-xk-do were fused. Finally, primers xk-upF1 and xk-doR1 were used as primers (Table 7) and the fusion PCR system was used as a template to obtain the gmer expression cassette KM-ΔXK::GMER.

[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] The genomic DNA of K. marxianus DMKU3-1042 was used as template and primers galk up-F1 and galk up-R1 and galk down-F1 and galk down-R1 was amplified by PCR to obtain the upstream and downstream homology arms, respectively; the KL-ΔGK::BSfut-wt, KL-ΔGK::BSfut-M1 to KL-ΔGK::BSfut-M24 expression cassettes, and KL-ΔGK::HPFut-wt, KL-ΔGK::ECFut-wt, KL-ΔGK::BCFut-wt, KL-ΔGK::BFFut-wt expression cassettes 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 a template with primers glk up-F1 and glk 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 and used for the construction of the recombinant strain in the next step.

[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 Example 2, Part (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 transformants and the original strains and performed PCR amplification using primers corresponding to the knockout or expression cassette. If a single band was obtained after PCR amplification and the size was consistent with the knockout or expression cassette, the strain was considered correct; otherwise, the strain was considered a false positive.

[0177] Table 9. Kluyveromyces marxianus engineered strains 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 Sangon Biotechnology Co., Ltd.), and the artificial sequence containing the encryption tag was randomly inserted into the genome of lactic acid yeast or Kluyveromyces marxianus 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 tagged 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 transformants and the original strain and performed PCR amplification using primers corresponding to the knockout or expression cassette. If a single band with the same size as the knockout or expression cassette was obtained after PCR amplification, the strain was considered correct; otherwise, the strain was considered a false positive. For tag verification, PCR amplification using primers was performed and further verified by sequencing; otherwise, the strain was considered a false positive.

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

[0189] The artificial amino acid sequence containing the encryption tag was sent for synthesis (Shanghai Sangon Biotechnology Co., Ltd.), and the artificial sequence containing the encryption tag was randomly inserted into the genome of lactic acid yeast or Kluyveromyces marxianus 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 transformants and the original strain and performed PCR amplification using primers corresponding to the knockout or expression cassette. If a single band with the same size as the knockout or expression cassette was obtained after PCR amplification, the strain was considered correct; otherwise, the strain was considered a false positive. For tag verification, PCR amplification using primers was performed and further verified by sequencing; otherwise, the strain was considered a false positive.

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

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

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

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

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

[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 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 was 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 reaction solution HPLC chromatogram peak 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 Kluyveromycin gene-modified cells obtained in Example 5

[0220]

[0221]

[0222] Table 13-16 Data Description:

[0223] (1) The starting strains of Kluyveromyces lactis and Kluyveromyces marxianus and the genetically modified cells KL-1, KL-2, KM-1 and KM-2 were all unable to synthesize 2'-FL; KL-3 and KM-3 were able to produce 2'-FL, indicating that Kluyveromyces lactis / Kluyveromyces marxianus cells themselves do not have the ability to synthesize 2'-FL, and 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 has α-1,2 fucosyltransferase activity, but the activity is relatively 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 Examples 4 and 5 did not affect the yield of 2'-FL produced by fermentation of the Kluyveromyces gene-modified cells obtained in Examples 2 or 3.

[0226] Example 7. Kluyveromyces KL-M1-1, KL-M24-1, KM-M1-1, KM-M24-1, KL-6-1, and KM-6-1 were streaked onto solid media such as YDP. After culturing at 30°C for 2-3 days, a single colony was picked and inoculated into 1.5 mL YPD liquid medium and incubated at 30°C and 200 rpm overnight. Subsequently, each colony was inoculated into a 50 mL liquid medium shake flask at a 2% inoculum size and incubated at 30°C and 200 rpm until the OD 600 =1, and 2% inoculum was inoculated into 1 L of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) (3 L fermentor). After 6-7 hours of culture, sucrose (50% mother liquor concentration) was added at a rate of 8 mL / h. Simultaneously, lactose (40% mother liquor concentration) was added to maintain a final lactose concentration of 15 g / L. The total fermentation time was 72 hours. After 72 hours of fermentation, the fermentation broth was centrifuged, and the supernatant and precipitate were collected separately. The precipitate was disrupted using a high-pressure homogenizer, boiled, and centrifuged to remove protein. The supernatants were combined to obtain the final yeast fermentation product. The 2'-FL and DFL contents in the supernatants were measured, and 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 clarity of understanding, it will be apparent to those skilled 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 invention.

Claims

1. A genetically modified cell, characterized in that Contains a polypeptide encoding an α-1,2-fucosyltransferase The invention relates to a recombinant nucleic acid sequence of an α-1,2-fucosyltransferase, wherein the α-1,2-fucosyltransferase polypeptide is selected from one of the polypeptides represented by an amino acid sequence such as SEQ ID NO: 2, 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 or SEQ ID NO: 25, and the genetically modified cell is selected from a Kluyveromyces genetically modified cell.

2. The genetically modified cell according to claim 1, wherein The Kluyveromyces genetically modified cell is selected from the group consisting of Pichia kluyveri, Kluyveromyces lactis, Kluyveromyces polysporus, Kluyveromyces dobzhanskii, Kluyveromyces thermotolerans, Kluyveromyces yarrowii, and Kluyveromyces marxinus genetically modified cells.

3. The genetically modified cell according to claim 1, wherein The Kluyveromyces genetically modified cells are selected from Kluyveromyces lactis genetically modified cells or Kluyveromyces marxinus genetically modified cells.

4. The genetically modified cell according to any one of claims 1 to 3, wherein The genetically modified cell further comprises a heterologous nucleic acid encoding one or more of GDP-mannose-4,6-dehydratase and GDP-L-fucose synthase.

5. The genetically modified cell according to any one of claims 1 to 3, wherein 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 3, wherein It also includes a molecular marker gene, the nucleotide sequence of which is shown in SEQ ID NO:

29.

7. The genetically modified cell according to any one of claims 1 to 3, wherein Ability to import lactose without consuming it.

8. The genetically modified cell according to any one of claims 1 to 3, wherein 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 3, wherein: Include one or more of the following steps: (1) Cultivate the starting strain; (2) destroying the lac4 gene in the strain obtained in step (1); (3) constructing an expression cassette and introducing an α-1,2-fucosyltransferase gene into the starting strain, wherein the α-1,2-fucosyltransferase polypeptide is one of the polypeptides represented by the amino acid sequence of SEQ ID NO:2, 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 or SEQ ID NO:25; (4) constructing an expression cassette and introducing a heterologous GDP-L-fucose synthase gene (wcag) into the starting strain; (5) Construct an expression cassette and introduce the heterologous GDP-mannitol 4,6 dehydratase (gmd) gene into Kluyveromyces cells.

10. The method for preparing genetically modified cells according to claim 9, wherein: Also includes: (6) Construct a molecular marker expression cassette and introduce the marker gene into the starting strain.

11. The method for preparing genetically modified cells according to claim 9, wherein: Also includes: (7) A step of recovering the genetically modified cells.

12. The method for preparing genetically modified cells according to claim 9, wherein: The starting strain is selected from Kluyveromyces cells.

13. The method for preparing genetically modified cells according to claim 9, wherein: The Kluyveromyces yeast cell is selected from the group consisting of Kluyveromyces lactis and Kluyveromyces marxianus.

14. Use of the genetically modified cell according to any one of claims 1 to 3 in synthesizing 2'-fucoylactose.

15. The use according to claim 14, characterized in that The carbon source in the culture medium comprises lactose and one of sucrose, glycerol or glucose.

16. The use according to claim 14, characterized in that The culture medium may also contain metal salts, minerals or other nutrients.

17. The use according to claim 14, characterized in that The culture medium does not contain fucose.

Images