Fucosyltransferase polypeptide and genetically modified cell

Abstract

Provided is a polypeptide M1-M21 having an activity of synthesizing 3-FL, which polypeptide is obtained by means of modifying a polypeptide as set forth in SEQ ID NO: 1. Further provided is a genetically modified cell containing the gene of the polypeptide. The provided polypeptide has improved activity in catalyzing the synthesis of 3-FL and also has improved specificity in catalyzing a substrate compared to the polypeptide as set forth in SEQ ID NO: 1.

Description

Fucosyltransferase polypeptide and gene-modified cells

[0001] This application claims priority to the patent application filed on February 26, 2024, with application number CN202410206745.7, entitled "Fucosyltransferase polypeptide and its application", and applicant Shandong Henglu Biotechnology Co., Ltd. (the priority has been transferred to Hengrui (Qingdao) Biotechnology Co., Ltd.), the patent application filed on July 16, 2024, with application number CN202410947246.3, entitled "Genetically modified cells and their applications in synthesizing human milk oligosaccharides", and applicant Hengrui (Qingdao) Biotechnology Co., Ltd., and the patent application filed on July 17, 2024, with application number CN202410953965.6, entitled "A genetically modified cell and its application in synthesizing fucosyllactose", and applicant Hengrui (Qingdao) Biotechnology Co., Ltd., the entire contents of which are incorporated herein by reference and constitute a part of this application for all purposes. Technical Field

[0002] The present application relates to a series of fucosyltransferase polypeptides and genetically modified cells containing the polypeptide genes, which can be applied to the synthesis of human milk oligosaccharides (HMOs) and belong to the technical fields of enzyme engineering and biogenetic engineering. Background Art

[0003] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the application, and is not necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art known to a person skilled in the art.

[0004] Human milk oligosaccharides (HMOs) are a unique and rich mixture of oligosaccharides present in human milk and are the third largest solid component in breast milk after fat and lactose.

[0005] 3-Fucosyllactose (3-FL) is the only HMO whose concentration increases during lactation, suggesting a wider range of applications in areas such as follow-on formula. 3-FL has been approved for marketing by the US FDA, the EU, Australia, Singapore, and other countries and regions. Its intended uses include infant formula and follow-on formula, infant food, foods for special medical purposes, and dietary supplements.

[0006] In the microbial fermentation method for synthesizing 3-FL, α-1,3-fucosyltransferase is a key enzyme that catalyzes the conversion of GDP-L-fucose and lactose. Currently, α-1,3-fucosyltransferases are mostly derived from Helicobacter pylori and Escherichia coli. Furthermore, microbial methods for synthesizing 3-fucosyllactose often produce low yields and are accompanied by the undesirable production of lactose difucosyllactose (DFL), which increases the difficulty of separation.

[0007] Chinese Patent No. 202111404385.4 discloses a recombinant Escherichia coli expressing an α-1,3-fucosyltransferase from Bacteroides gallinaceum. Patent No. 202011223864.1 discloses a recombinant Bacillus subtilis strain for producing 3-FL, its construction method, and its use. The recombinant Bacillus subtilis 1643FL was obtained by integrating fusion gene fragments encoding fructose-6-phosphate isomerase, phosphomannose mutase, and mannose-1-phosphate guanylyltransferase into the manA locus of Bacillus subtilis. Chinese Patent No. 202310292697.3 discloses a mutant of Helicobacter pylori α-1,3-fucosyltransferase, which has mutations at amino acids 108 and / or 139 of the wild-type H. pylori α-1,3-fucosyltransferase.

[0008] Chinese patent 202110588386.2 discloses a method for synthesizing fucosylated lactose by in vitro multi-enzyme cascade catalysis. The method jointly utilizes GDP-L-fucose synthesis-related enzymes and L-fucosyltransferase to catalyze the reaction of substrate GDP-L-fucose and lactose to synthesize fucosylated lactose.

[0009] In summary, it is necessary to study the substrate specificity of α-1,3-fucosyltransferase in order to improve the synthesis efficiency and conversion rate of 3-FL and reduce or eliminate the production of impurity sugars. The modification of key enzymes is crucial to improving the catalytic efficiency of enzymes. Summary of the Invention

[0010] Purpose of the invention: To provide an α-1,3-fucosyltransferase polypeptide and its application in the synthesis of 3-fucosyllactose (3-FL).

[0011] The second object of the present application is to provide a genetically modified cell containing the α-1,3-fucosyltransferase polypeptide gene, which has the activity of synthesizing 3-fucosyllactose de novo.

[0012] The technical solution of this application:

[0013] The applicant discovered an α-1,3-fucosyltransferase polypeptide from Bacillus smithii in nature and named it BSFut3t-wt. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 8. The polypeptide shares 33.96% amino acid sequence identity with HPFutA-wt (amino acid sequence shown in SEQ ID NO: 2), an α-1,3-fucosyltransferase from Helicobacter pylori.

[0014] The fucosyltransferase polypeptide is a lactose-accepting fucosyltransferase polypeptide.

[0015] The inventors of this application have conducted a series of modifications to the polypeptide represented by SEQ ID NO:1, screening and obtaining a series of derivative peptides that catalyze the synthesis of fucosyllactose. These derivative peptides have improved catalytic ability for the synthesis of 3-FL and substrate specificity. These derivative peptides are transferase polypeptides formed by replacing one or more amino acid residues and / or fragments of the polypeptide represented by SEQ ID NO:1. Compared to the original polypeptide, these polypeptides have reduced enzymatic side activities and can reduce or avoid the formation of the undesirable byproduct lacto-N-difucotetraose (DFL) when catalyzing the synthesis of 3-fucosyllactose. This application describes several exemplary combinations of amino acid substitutions made to the polypeptide represented by SEQ ID NO:1.

[0016] Specifically, in a first aspect, the present application provides a series of α-1,3-fucosyltransferase polypeptides for synthesizing fucosylated lactose, wherein the α-1,3-fucosyltransferase polypeptides include a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 and its derivative peptides M1-M21, wherein the derivative peptides M1-M21 are as follows:

[0017] 1) Polypeptide M1, obtained by replacing the lysine (K) at position 2 of the amino acid sequence shown in SEQ ID NO: 1 with threonine (T);

[0018] 2) polypeptide M2, obtained by replacing lysine (K) at position 17 in the amino acid sequence of M1 with arginine (R);

[0019] 3) polypeptide M3, obtained by replacing the lysine-asparagine (KN) at positions 21-22 in the amino acid sequence of M2 with asparagine-serine (NS);

[0020] 4) Polypeptide M4, obtained by replacing the lysine (K) at position 27 in the amino acid sequence of M3 with asparagine (N);

[0021] 5) polypeptide M5, obtained by replacing phenylalanine-phenylalanine-serine-aspartic acid (FFSD) at positions 45-48 in the amino acid sequence of M4 with isoleucine-tyrosine-serine-cysteine ​​(IYSC);

[0022] 6) polypeptide M6, obtained by replacing glutamic acid-proline-aspartic acid-phenylalanine (EPDF) at positions 69-72 in the amino acid sequence of M5 with glutamine-proline-asparagine-leucine (QPNL);

[0023] 7) polypeptide M7, obtained by replacing tyrosine-lysine-tyrosine-lysine-glutamic acid-alanine-leucine (YKYKEAL) at positions 97-103 in the amino acid sequence of M6 with phenylalanine-leucine-tyrosine-threonine-glutamine-aspartic acid-tyrosine (FLYTQDY);

[0024] 8) polypeptide M8, obtained by replacing glutamic acid (E) at position 118 in the amino acid sequence of M7 with lysine (K);

[0025] 9) polypeptide M9, obtained by replacing the cysteine-phenylalanine-asparagine (CFN) at positions 125-127 in the amino acid sequence of M8 with asparagine-phenylalanine-isoleucine (NFI);

[0026] 10) polypeptide M10, obtained by replacing tyrosine-aspartic acid-glutamic acid (YDE) ​​at positions 133-135 in the amino acid sequence of M9 with alanine-histidine-arginine (AHR);

[0027] 11) polypeptide M11, obtained by replacing the isoleucine-lysine (IK) at positions 138-139 in the amino acid sequence of M10 with phenylalanine-glutamic acid (FE);

[0028] 12) polypeptide M12, obtained by replacing tyrosine (Y) at position 141 in the amino acid sequence of M11 with isoleucine (I);

[0029] 13) Polypeptide M13, obtained by replacing the threonine (T) at position 156 with isoleucine (I) in the amino acid sequence of M12;

[0030] 14) polypeptide M14, obtained by replacing phenylalanine-leucine-isoleucine-glutamic acid (FLIE) at positions 162-165 in the amino acid sequence of M13 with tyrosine-arginine-valine-aspartic acid (YRVD);

[0031] 15) polypeptide M15, obtained by replacing the threonine (T) at position 223 in the amino acid sequence of M14 with cysteine ​​(C);

[0032] 16) polypeptide M16, obtained by replacing the serine-serine-tyrosine (SSY) at positions 227-229 in the amino acid sequence of M15 with lysine-serine-lysine (KSK);

[0033] 17) polypeptide M17, obtained by replacing lysine (K) at position 243 in the amino acid sequence of M16 with aspartic acid (D);

[0034] 18) polypeptide M18, obtained by replacing the threonine-glutamic acid (TE) at positions 247-248 in the amino acid sequence of M17 with serine-glutamine (SQ);

[0035] 19) polypeptide M19, obtained by replacing phenylalanine (F) at position 255 in the amino acid sequence of M18 with leucine (L);

[0036] 20) polypeptide M20, obtained by replacing asparagine-proline-glutamic acid-glutamic acid (NPEE) at positions 260-263 in the amino acid sequence of M19 with aspartic acid-proline-glutamic acid-lysine (DPEK);

[0037] 21) Polypeptide M21 is obtained by replacing alanine (A) at position 268 in the amino acid sequence of M20 with glutamic acid (E).

[0038] As described above, polypeptides M1-M21 reduce or avoid the production of the by-product DFL (Difucosyllactose).

[0039] In certain embodiments, the polypeptides described herein further include polypeptides having at least 60% identical amino acid sequence (sequence identity) to the amino acid sequence of the polypeptide shown in SEQ ID NO: 1 or polypeptides M1-M21, and having improved 3-FL catalytic synthesis activity.

[0040] In a second aspect, the present application provides a polynucleotide encoding the polypeptide as described in the first aspect above.

[0041] Polynucleotides encoding the polypeptide of SEQ ID NO: 1 and polypeptides M1-M21 can be prepared using recombinant DNA techniques known in the art. These methods include, but are not limited to, cloning, recombination, in vitro synthesis, and in vitro amplification. Polynucleotides encoding the polypeptides described herein can be obtained and expressed using a variety of methods. For example, the polynucleotides can be constructed into appropriate expression vectors and expressed using a variety of expression systems.

[0042] In a third aspect, the present application provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above.

[0043] The nucleic acid construct preferably further comprises one or more regulatory sequences operably linked to the polynucleotide, and the regulatory sequences can direct the production of the polypeptide in an appropriate expression host.

[0044] In a fourth aspect, the present application provides an expression vector comprising the polynucleotide as described in the second aspect above, or comprising the nucleic acid construct as described in the third aspect above.

[0045] The expression vector comprises the nucleic acid construct described in the third aspect of the present application or the polynucleotide described in the second aspect operably linked to a regulatory sequence capable of achieving expression of the polynucleotide sequence, such as a promoter sequence.

[0046] Plasmids and other related nucleic acids can be purified from cells using commercially available kits. The purified nucleic acids can be further used to produce other nucleic acids, used to transfect cells, incorporated into related vectors to infect organisms for expression, etc. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters that can be used to regulate the expression of specific target nucleic acids. The vectors optionally contain universal expression cassettes.

[0047] In a specific embodiment, the expression cassette comprises all elements for expressing the α-1,3-fucosyltransferase polypeptide, including elements necessary for transcription and translation in a host cell. For example, the expression cassette comprises a promoter and a terminator. The promoter and terminator are not particularly limited and may be promoters and terminators known in the art that can achieve expression of the α-1,3-fucosyltransferase polypeptide.

[0048] In a fifth aspect, the present application provides a transformed host cell, which is transformed with the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above.

[0049] In some embodiments, the host cell includes but is not limited to natural strains or genetically modified strains of bacteria, yeast, mold, etc.

[0050] Further preferably, the host cell includes but is not limited to natural strains or genetically modified strains of Escherichia sp., Bacillus sp., Kluyveromyces sp., etc.

[0051] Further preferably, the host cell includes but is not limited to natural strains or genetically modified strains such as Saccharomyces cerevisiae, Kluyveromyces marxianus, Kluyveromyces lactis, and Yarrowia lipolytica.

[0052] More preferably, the host cell is the genetically engineered Escherichia coli BL21 (DE3).

[0053] In a sixth aspect, the present application provides an enzyme agent or enzyme composition, which comprises the polypeptide as described in the first aspect above.

[0054] The enzyme or enzyme composition preferably comprises one or more of the polypeptide shown in SEQ ID NO: 1 and polypeptides M1-M21 described in the present application.

[0055] Depending on the reaction substrate and target product, the enzyme agent or composition may also contain other enzymes that are beneficial to the synthesis of fucosyllactose, such as: bifunctional enzyme L-fucose pyrophosphorylase (Lfucokinase / GDP-L-fucosepyrophosphorylase, FKP), glucokinase (glucokinase, Glk), mannose-phosphate mutase (mannose-phosphate mutase, ManB), mannose-1-phosphate guanylyltransferase (ManC), GDP-mannose dehydratase (GDP-mannose-4,6-dehydratase, GMD), etc.

[0056] In a seventh aspect, the present application provides a method for producing the polypeptide as described in the first aspect above, comprising:

[0057] (1) culturing a transformed host cell under conditions suitable for expressing the polypeptide; the transformed host cell is as described in the fifth aspect above; and

[0058] (2) Recovering the polypeptide.

[0059] In a specific embodiment, the step (1) comprises: firstly introducing a nucleic acid construct or a recombinant expression vector encoding the polypeptide as described in the first aspect above into a host cell to construct an engineered host cell expressing the polypeptide; then, culturing the engineered host cell and inducing it to express the polypeptide.

[0060] In a specific embodiment, the step (2) includes the steps of isolating and purifying the polypeptide from the culture.

[0061] 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 flasks, or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, fed-batch fermentation, or solid-state fermentation) in a laboratory or industrial fermentor tank in a suitable substratum and under conditions allowing polypeptide expression and / or separation.

[0062] The polypeptide can be recovered from the culture using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by a variety of conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0063] In an eighth aspect, the present application provides the use of the α-1,3-fucosylfucotransferase polypeptide described in the first aspect, the transformed host cell described in the fifth aspect, or the enzyme or enzyme composition described in the sixth aspect in synthesizing 3-fucosyllactose.

[0064] For such applications, suitable reaction conditions include: the presence of a suitable reaction substrate, a suitable polypeptide, or the presence of essential cofactors such as monovalent or divalent ions, a pH value within an appropriate range, a suitable temperature, etc. It is not necessary to meet the optimal value of each factor affecting the polypeptide described in this application, but the reaction conditions must enable the α-1,3-fucosyltransferase polypeptide described in this application to exert its enzymatic activity.

[0065] Preferably, the reaction substrates are GDP-L-fucose and lactose.

[0066] Preferably, the α-1,3-fucosyltransferase polypeptide is produced in a cell-free expression system such as, but not limited to, the PURExpress system (NEB) or in a host organism, after which the α-1,3-fucosyltransferase polypeptide listed above can be isolated and optionally further purified.

[0067] In one embodiment, at least one of the polypeptides described in the first aspect is mixed with GDP-L-fucose, a buffer solution (such as Tris-HCl or HEPES), and lactose, and incubated at a specific temperature (e.g., 35°C, 37°C, or 40°C) for a specific period of time (e.g., 24 hours). During this period, the lactose reacts with the GDP-L-fucose to convert it into 3-FL. 3-FL is then isolated and purified using methods known in the art. The concentration of 3-FL in the reaction solution is determined by HPLC chromatography.

[0068] Preferably, substantially all proteins, as well as amino acids, RNA, and DNA, are removed from the mixture containing 3-FL (preferably after clarification). In this step, proteins and related impurities can be removed from the mixture containing 3-FL in a conventional manner. Separation and purification can be performed using existing separation and purification techniques.

[0069] In a ninth aspect, the present application provides a genetically modified cell comprising a heterologous nucleic acid sequence encoding the polypeptide as described in the first aspect, preferably the polynucleotide described in the second aspect.

[0070] Preferably, the genetically modified cell further comprises a heterologous nucleic acid sequence encoding a GDP-L-fucose synthase polypeptide (GMER) and / or a GDP-mannose dehydratase polypeptide (GMD).

[0071] Preferably, the genetically modified cells include, but are not limited to, genetically modified cells of yeast sp., Bacillus sp., and Escherichia sp.

[0072] Preferably, the genetically modified cells include but are not limited to genetically modified cells of Escherichia coli, Bacucilius cereus, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Saccharomyces paradoxus, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces cariocas, Saccharomyces kudriavzevii and Saccharomyces cerevisiae.

[0073] Preferably, the genetically modified cell further comprises a nucleic acid sequence of a marker gene.

[0074] Preferably, the GDP-fucose synthase polypeptide gene can be derived from existing publicly available GDP-fucose synthase polypeptides, such as natural strains of Escherichia coli, Mus musculus, Homo sapiens, Marinobacter salarius, Sinorhizobium fredii, Caenorhabditis elegans, or genetically modified strains thereof.

[0075] Preferably, the source of the GDP-mannose dehydratase polypeptide gene can be selected from existing disclosed GDP-mannose dehydratase polypeptides, such as natural strains of Escherichia coli, Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, Dictyostelium discoideum, Mus musculus, Caenorhabditis briggsae, Bacillus smithii, and Bacillus smithii, or genetically modified strains thereof.

[0076] Preferably, the yeast genetically modified cells include, but are not limited to, Kluyveromyces lactis genetically modified cells, Kluyveromyces marxianus genetically modified cells and Saccharomyces cerevisiae genetically modified cells.

[0077] Preferably, the amino acid sequence of the molecular marker of the genetically modified cell containing the molecular marker is shown in SEQ ID NO: 7. Preferably, the nucleotide sequence of the molecular marker of the genetically modified cell containing the molecular marker is shown in SEQ ID NO: 9.

[0078] The present application also provides a method for preparing the genetically modified cells, wherein yeast cells are used as a starting strain, and a heterologous gene encoding the polypeptide described in the first aspect, a heterologous GDP-fucose synthase gene, and a heterologous GDP-mannose dehydratase gene are respectively integrated into the Kluyveromyces genome.

[0079] Preferably, the Kluyveromycin gene-modified cell further comprises a disrupted β-galactosidase gene (lac4).

[0080] Preferably, the genetically modified Saccharomyces cerevisiae cell further comprises a lactose permease (LAC12) gene and a fucosyllactose export transporter (CDT2) gene.

[0081] Preferably, the lactose permease is a lactose transporter responsible for transferring lactose from the extracellular space to the intracellular space. In some embodiments, the source of the lactose permease (LAC12) includes but is not limited to natural strains such as Neurospora crassa, Neofusicoccum parvum, Scheffersomyces stipitis, Aspergillus lentulus, Beauveria bassiana, Kluyveromyces lactis, Kluyveromyces marxianus, Helicobacter pylori, Zymomonas mobilis, or Escherichia coli, or genetically modified strains thereof.

[0082] In some embodiments, the transporter polypeptide that exports fucosyllactose is a transporter polypeptide that exports 3-fucosyllactose, and its sources include but are not limited to natural strains such as Escherichia coli, Kluyveromyces marxianus, Kluyveromyces lactis, Neurospora crassa, or genetically modified strains thereof.

[0083] Preferably, the starting strain of Kluyveromyces lactis is K. lactis DSM70799, the starting strain of Kluyveromyces marxianus is K. marxianus DMKU3-1042, and the starting strain of Saccharomyces cerevisiae is CCTCC NO: M20231127 (Saccharomyces cerevisiae SctgtP8).

[0084] Preferably, the method for preparing the genetically modified yeast cells comprises the following steps:

[0085] (1) Cultivate the starting strain;

[0086] Preferably, the starting strain includes but is not limited to natural Kluyveromyces cells or genetically modified cells thereof;

[0087] Preferably, the Kluyveromyces cell is selected from natural cells of Kluyveromyces lactis K. lactis or Kluyveromyces marxianus K. marxianus or genetically modified cells thereof.

[0088] Preferably, the starting strain is selected from Kluyveromyces lactis DSM70799;

[0089] Preferably, the starting strain is selected from Kluyveromyces marxianus DMKU3-1042.

[0090] Preferably, the starting strain is selected from Saccharomyces cerevisiae SctgtP8.

[0091] (2) constructing an expression cassette and introducing heterologous GDP-mannose dehydratase gene and heterologous GDP-fucose synthase gene into yeast cells;

[0092] (3) Constructing an expression cassette and introducing the gene encoding the α-1,3-fucosyltransferase polypeptide described in the present application into yeast cells.

[0093] Optionally, the method for preparing the genetically modified cells further comprises:

[0094] (4) Construct a molecular marker expression cassette and introduce the marker gene into Kluyveromyces cells.

[0095] If the starting strain is Kluyveromyces yeast cells, it also includes:

[0096] (5) destroying the β-galactosidase gene (lac4) in the strain obtained in step (1);

[0097] If the starting strain is a Saccharomyces cerevisiae cell, it also includes:

[0098] (6) Introduce the lactose permease (LAC12) gene and the fucosyllactose export transporter (CDT2) gene.

[0099] Optionally, the above steps may be performed in any order.

[0100] The gene cell described in the present application can synthesize fucosyllactose de novo using lactose as a substrate and glucose, glycerol or sucrose as a carbon source.

[0101] The term "identity" refers to amino acid sequence identity and refers to the percentage of two or more sequences or subsequences that are identical, or that have identical amino acid residues or nucleotides, when compared and aligned for maximum correspondence. An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0102] The term "polypeptide" refers to any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds. Beneficial effects:

[0103] This application provides polypeptides represented by SEQ ID NO: 1 and polypeptides M1-M21, which enhance the ability of the polypeptide represented by SEQ ID NO: 1 to catalyze the synthesis of 3-FL and improve its substrate specificity. The catalytic reaction of polypeptides M1-M21 reduces the DFL-producing side effect of the polypeptide represented by SEQ ID NO: 1. Furthermore, this application provides polynucleotides encoding the polypeptides and related nucleic acid products, transformed host cells, enzymes containing the polypeptides, and methods for catalytically synthesizing 3-FL using the polypeptides as catalysts.

[0104] The technical solution of the present application has positive significance for the industrial production of human milk oligosaccharides. The method is green, efficient, and sustainable, is conducive to the application of industrial large-scale production, and has important practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] The drawings constituting part of the present application are provided to provide a further understanding of the present application. The illustrative embodiments and their descriptions of the present application are provided to explain the present application and do not constitute an undue limitation on the present application. The following describes the implementation scheme of the present application in detail in conjunction with the drawings, wherein:

[0106] Figure 1. HPLC chromatograms of lactose, 3-FL standard, DFL standard, reaction solutions of the peptide shown in SEQ ID NO: 1, and peptide M18. The RT of lactose is 9.98 min, the RT of the 3-FL standard is 11.43 min, and the RT of the DFL standard is 13.00 min.

[0107] Figure 2. Mass spectrometry analysis of the 3-FL standard. The results show that the sodium addition mode of the 3-FL standard is 511.17 and the molecular weight of 3-FL is 488.2, which is within the allowable error range with the theoretical molecular weight of 488.4.

[0108] Figure 3. LC-MS analysis of the material with an RT of approximately 11.43 min in the HPLC spectrum of the peptide M18 reaction solution in Figure 1. The results show that it is consistent with the mass spectrum of the 3-FL standard, thus proving the successful synthesis of 3-FL. DETAILED DESCRIPTION

[0109] The present application is further described in detail below through examples. It should be understood that the specific implementation methods described here are only used to explain the present application and are not used to limit the present application. 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 application fall within the scope of protection of the present application.

[0110] 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.

[0111] In the following examples, the alpha-1,3-fucosyltransferase (BSFUT3t-wt) involved is derived from Bacillus smithii, with an amino acid sequence as shown in SEQ ID NO: 1 and a nucleotide sequence as shown in SEQ ID NO: 8; the alpha-1,3-fucosyltransferase (HPFutA-wt) involved is derived from Helicobacter pylori, with an amino acid sequence as shown in SEQ ID NO: 2. The GDP-fucose synthase polypeptide (GDP-L-fucose synthase, GMER) is derived from Escherichia coli, with an amino acid sequence as shown in SEQ ID NO: 3. The GDP-mannose dehydratase polypeptide (GDP-mannose dehydratase, GMD) is derived from Escherichia coli, with an amino acid sequence as shown in SEQ ID NO: 4. The lactose permease is derived from Kluyveromyces lactis and is abbreviated as LAC12, with an amino acid sequence shown in SEQ ID NO: 5. The fucosyllactose export transporter (CDT2) is derived from Neurospora crassa and has an amino acid sequence shown in SEQ ID NO: 6. The amino acid sequence of the molecular marker is shown in SEQ ID NO: 7, and the nucleotide sequence is shown in SEQ ID NO: 9.

[0112] 3-FL and DFL standards are produced by ELICITYL, France.

[0113] Example 1. Expression of the polypeptide shown in SEQ ID NO: 1 and polypeptides M1-M21 in Escherichia coli.

[0114] 1. Using the amino acid sequences of the polypeptides shown in SEQ ID NO: 1 and SEQ ID NO: 2 as templates, their nucleotide sequences were synthesized respectively and finally constructed into the PET32a vector to obtain PET32a-WT and PET32a-HP-WT plasmids.

[0115] 2. Referring to the methods in the Molecular Cloning Guide, mutant primers were designed based on the sequence shown in SEQ ID NO:8 of pET32a-wt using Primer-BLAST (Primer designing tool (nih.gov, website: https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). Recombinant plasmid series (pET32a-M1 to pET32a-M21) were constructed using the Fast Mutagenesis Kit.

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

[0117] Take the prepared Escherichia coli BL21 (DE3) competent cells, place them on ice for 30 minutes to thaw, take 100 μL of competent cells and 10 μL of pET32a-M1 to pET32a-M21 recombinant plasmids (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 positive transformants.

[0118] 4. Culture positive transformants, extract their plasmids, and use double enzyme digestion and gene sequencing to verify whether the recombinant plasmids pET32a-M1 to pET32a-M21 are successfully introduced into E. coli.

[0119] 5. Inoculate the correct transformant into LB liquid medium and culture it on a shaker at 37°C and 200 rpm for 12 hours to obtain seed solution. Then, inoculate the seed solution into fresh LB medium at a 1% (v / v) inoculum and culture it at 37°C with shaking until the OD 600 The expression of the culture medium was 0.8, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG) with a final IPTG concentration of 0.1 mmol / L, and induced at 16°C for 12 h at a speed of 200 rpm; after induction of expression, the fermentation broth was centrifuged at 5000 r / min for 30 min at 4°C to collect the bacteria; the bacteria were resuspended in 20 mM pH 7.4 PBS buffer and ultrasonicated at a frequency of plus on 5s / off 5s for 30 min to break the bacteria; the broken liquid was centrifuged at 13000×g and 4°C for 30 min to remove cell debris and collect the supernatant.

[0120] 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.

[0121] 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 and SEQ ID NO: 2, as well as M1-M21 polypeptides were obtained. The relevant information is shown in Table 1.

[0122] Table 1. Corresponding numbers of α-1,3-fucosyltransferase polypeptides and their amino acid sequences

[0123] Example 2. Determination of the ability of the polypeptides obtained in Example 1 (polypeptides represented by SEQ ID NO: 1 and SEQ ID NO: 2, and polypeptides M1-M21) to serve as catalysts for the synthesis of 3-FL.

[0124] 3-FL was synthesized using one of the following purified polypeptides obtained in Example 1 as a catalyst: the polypeptide represented by SEQ ID NO: 1, the polypeptide represented by SEQ ID NO: 2, and the M1-M21 polypeptides, respectively, with lactose and GDP-L-fucose as reaction substrates. The specific procedures are as follows:

[0125] 40 mM lactose, 15 mM GDP-L-fucose, 10 mM MnCl2, and 25 mM Tris-HCl, pH 7.5, were each added with 0.2 mg / mL of one of the following purified polypeptides obtained in Example 1: the polypeptide shown in SEQ ID NO: 1, the polypeptide shown in SEQ ID NO: 2, and the M1-M21 polypeptides, mixed well, reacted at 37°C for 6 hours, terminated the reaction, and purified by gel column method.

[0126] Identification of catalytic synthesis products:

[0127] HPLC detection method: Detection conditions: chromatographic column model: Shodex Asahipak NH2P-504E, 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.

[0128] LC-MS analysis conditions are as follows:

[0129] Chromatographic column model: Shodex Asahipak NH2P-504E, detector: UV detector (Hitachi Chromaster), detection wavelength: 210 nm, injection volume: 10 μL, flow rate: 0.5 mL / min, column temperature: 35°C, mobile phase: acetonitrile:water = 65:35; ESI-MS mode, molecular weight scan range: 100-800.

[0130] The standard and reaction solution were tested using the aforementioned analytical method. HPLC analysis revealed that the peak elution time (RT) of the 3-FL standard was 11.43 min. The reaction solutions of the polypeptide described in Example 1 all exhibited a strong absorption peak around 11.43 min, consistent with the peak elution time of the 3-FL standard, indicating that the polypeptide-catalyzed reaction in Example 1 produced 3-FL.

[0131] In addition: In the HPLC analysis spectrum shown in Figure 1, the rt of the DFL standard is 13.00 min, and the reaction solutions of the polypeptide represented by SEQ ID NO: 1 and polypeptides M1 and M3 have a weak peak at rt of 13.00 min; the reaction solutions of polypeptide M2 ​​and polypeptides M4-M21 have no peak at this position, wherein Figure 1 also exemplarily shows the HPLC spectrum of the reaction solution of the polypeptide described in M18.

[0132] HPLC analysis results showed that the reaction solution of the polypeptide represented by SEQ ID NO: 2 also had a weak peak at rt of 13.00 min, indicating that the reaction products of the polypeptide represented by SEQ ID NO: 1 and SEQ ID NO: 2 both contained undesirable DFL.

[0133] The LC-MS analysis conditions are shown above, and the analysis results show:

[0134] The product of the HPLC chromatogram peak near rt = 11.43 min in the reaction solution was analyzed by LC-MS. The spectrum is shown in Figure 2. The M+Na value in the fermentation broth was 511.17, consistent with the mass spectrum of the 3-FL standard shown in Figure 2 and within the allowable error range of the theoretical molecular weight of 3-FL of 488.4. This demonstrates that the polypeptides represented by SEQ ID NO: 1 and M1-M21 from Bacillus smithii can replace the previously reported polypeptide represented by SEQ ID NO: 2 from Helicobacter pylori for the synthesis of 3-FL.

[0135] The yields of 3-FL and DFL in the fermentation broth were detected by the above-mentioned HPLC analysis method, and the results are recorded in Table 2.

[0136] Table 2. Studies on the synthesis of 3-FL catalyzed by various peptides

[0137] Table 2 Data Description:

[0138] (1) The catalytic reaction liquid of α-1,3-fucosyltransferase from Helicobacter pylori showed a 3-FL yield of 0.2 mg / mL and a DFL yield of 0.1 mg / mL, which proved that the α-1,3-fucosyltransferase from Bacillus smithii had a higher yield of 3-FL (0.5 mg / mL) than that from Helicobacter pylori.

[0139] (2) In the catalytic reaction liquids of the α-1,3-fucosyltransferase polypeptide having the amino acid sequence shown in SEQ ID NO: 1, DFL was detected as a byproduct in addition to 3-FL. In the catalytic reaction liquids of M2, M4-M21, only 3-FL was detected, and no byproduct DFL was detected.

[0140] The above results demonstrate that the peptides M2, M4-M21 derived from α-1,3-fucosyltransferase of Bacillus smithii have improved specificity in catalyzing the synthesis of 3-FL and eliminated the side activity of synthesizing DFL.

[0141] (3) Peptides M2-M5 and M7-M21 increased the activity of the polypeptide shown in SEQ ID NO:1 in synthesizing 3-FL.

[0142] Example 3. Synthesis of 3-fucosyllactose using Escherichia coli as a substrate.

[0143] Referring to Huang et al. (Huang, D., Yang, K., Liu, J., Xu, Y., Wang, Y., Wang, R., Liu, B., & Feng, L. (2017). Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement. Metabolic engineering, 41, 23–38.), Escherichia coli BL21 was used as the starting strain to construct the strain BL21ΔlacZΔlonΔwcaJ, and the above-mentioned constructs PET32a-M1 and PET32a-M21 were transformed into the Escherichia coli chassis cells BL21ΔlacZΔlonΔwcaJ by electroporation to construct EC-M1 and EC-M21.

[0144] Method for culturing Escherichia coli to produce 3-FL:

[0145] E. coli strains EC-M1 and EC-M21 were cultured on LB solid medium at 37°C for 10-12 hours, and a single colony was inoculated into 20 mL of liquid LB and cultured in a 250 mL shake flask at 37°C and 220 rpm for 10-12 hours. Each strain was inoculated into 5 mL of LB, and when the strain reached stationary phase, 1 mL of culture was inoculated into 100 mL of LB medium containing 36 g / L glucose (or glycerol) as a carbon source for growth in a 500 mL shake flask. When the OD 600 When the pH reached approximately 0.6, 0.1 mM IPTG was added at 25°C for induction. After 2 hours and 10 hours of additional culture, 5 g / L lactose was added to supplement 3-FL production. At the same time, the culture medium was supplemented with ampicillin to a final concentration of 100 μg / mL. After 10 minutes, the culture was boiled and centrifuged, and the supernatant was collected for analysis of 3-FL and DFL production. The results are reported in Table 3.

[0146] Table 3

[0147] Example 4. Synthesis of 3-fucosyllactose using Bacillus as the chassis.

[0148] To construct Bacillus subtilis chassis cells, refer to Zhang et al. (Zhang, Q., Liu, Z., Xia, H., Huang, Z., Zhu, Y., Xu, L., Liu, Y., Li, J., Du, G., Lv, X., & Liu, L. (2022). Engineered Bacillus subtilis for the de novo production of 2'-fucosyllactose. Microbial cell factories, 21(1), 110.), and use Bacillus subtilis with the deposit number of CCTCC NO: M20231126 (Bacillus subtilis BStgtP8) as the starting strain to construct Bacillus subtilis chassis cells BSP43-manB-P43-manC-P43-gmd-P43-wcaG. At the same time, P43-M1 and P43-M21 expression cassettes were constructed, and the expression cassettes were transferred into BSP43-manB-P43-manC-P43-gmd-P43-wcaG and inserted into the manP gene, thereby constructing BS-M1 and BS-M21.

[0149] Bacillus subtilis strains BS-M1 and BS-M21 were grown on LB solid medium at 37°C for 10-12 hours. A single colony was inoculated into 20 mL of liquid LB in a 250 mL shake flask and incubated at 37°C and 220 rpm for 10-12 hours. The Te seed culture was further inoculated into 30 mL of fermentation medium at a rate of 10% and incubated in a 250 mL shake flask at 37°C and 220 rpm for 72 hours. During shake flask fermentation, the following culture medium was used: 6 g / L trypsin, 12 g / L yeast extract, 12.5 g / L K2HPO4·3H2O, 2.5 g / L KH2PO4, and 10 mL / L of a trace metal solution (composed of 4 g / L FeSO4·7H2O, 4 g / L CaCl2, 1 g / L MnSO4·H2O, 0.2 g / L NaMoO4·2H2O, 0.2 g / L ZnSO4·7H2O, 0.1 g / L AlCl3·6H2O, 0.1 g / L CuCl2·2H2O, and 0.05 g / L H3BO4). Sterilized sucrose and lactose were added to the sterilized shake flasks to final concentrations of 20 and 10 g / L, respectively. Following fermentation, the culture was boiled for 10 minutes, and the supernatant was centrifuged and analyzed for 3-FL and DFL production. The results are reported in Table 4.

[0150] Table 4

[0151] The above process was scaled up to a 1L fermenter using continuous fed-batch fermentation. Sucrose concentrations in the fermentation broth were maintained between 17g / L and 22g / L, and lactose concentrations were maintained between 10g / L and 20g / L. Fermentation was terminated after 30 hours of continuous feeding. The culture broth was boiled for 10 minutes, and the supernatant was centrifuged and analyzed for 3-FL content. The results were: 10.8g / L in the BS-M1 fermentation broth, and 29.7g / L in the BS-M24 fermentation broth.

[0152] Example 5. Synthesis of 3-fucosyllactose using Saccharomyces cerevisiae as a substrate.

[0153] The Saccharomyces cerevisiae chassis cells were constructed according to Xu et al. (Xu, M., Meng, X., Zhang, W., Shen, Y., & Liu, W. (2021). Improved production of 2'-fucosyllactose in engineered Saccharomyces cerevisiae expressing a putative α-1,2-fucosyltransferase from Bacillus cereus. Microbial cell factories, 20(1), 165.), with the accession number CCTCC The Saccharomyces cerevisiae strain NO:M20231127 (SctgtP8) was used as the starting strain, and SC-Δgal80-Pgal1-lac12-Pgal1-gmd-wcaG was constructed. The pRS305-Pgal-M1 and pRS305-Pgal-M21 plasmids were constructed and transformed with SC-Δgal80-Pgal1-lac12-Pgal1-gmd-wcaG to obtain strains SC-M1 and SC-M21.

[0154] Take Saccharomyces cerevisiae SC-M1 and SC-M21 respectively and let them grow rapidly until they enter the late logarithmic phase or the stationary phase. Streak the strains on solid culture medium such as YPD, culture at 30℃ for 2-3 days, pick a single colony and inoculate it into 1.5mL YPD liquid culture medium, and culture it at 30℃ and 200rpm overnight. Then, inoculate 2% of the inoculum into 50mL liquid culture medium shake flasks and culture at 30℃ and 200rpm until the OD 600=1, and were inoculated at a 2% inoculum into 1.5 L of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) (3 L fermentor). After 5-6 h 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 h. After 72 h of fermentation, the fermentation broth was centrifuged, and the supernatant and precipitate were collected. The precipitate was disrupted using a high-pressure homogenizer, boiled, and centrifuged to remove protein. The supernatants were combined to obtain the final fermentation product of the yeast. The supernatants were assayed for 3-FL and DFL production, and the results are reported in Table 5.

[0155] Table 5

[0156] Example 6. Synthesis of 3-fucosyllactose using Kluyveromyces as a substrate.

[0157] Kluyveromyces lactis chassis cells were constructed according to the construction and transformation method of reference Li et al. (Li, F., Ma, W., Liu, L., Niu, K., Liu, D., Yin, W., Zhang, X., Han, L., & Fang, X. (2023). Reprogramming the Metabolic Network in Kluyveromyces lactis with a Transcriptional Switch for De Novo Lacto-N-biose Synthesis. Journal of agricultural and food chemistry, 71(23), 9031–9039. https: / / doi.org / 10.1021 / acs.jafc.3c01779) and the deposit number was CCTCC The lactic acid Kluyveromyces strain of NO: M2022118 (Kluyveromyces HLLWF3) was used as the starting strain to obtain the strain KL-ΔLAC4::GMD-ΔXK::GMER. At the same time, the expression cassettes ΔGK::Ptef1-M1 and ΔGK::Ptef1-M21 were constructed, and the expression cassettes were respectively transferred into the strain KL-ΔLAC4::GMD-ΔXK::GMER to obtain the KL-M1 and KL-M21 strains.

[0158] Take Kluyveromyces KL-M1 and KL-M21 respectively and let them grow rapidly until they enter the late logarithmic phase or the stationary phase. Streak the strains on solid culture medium such as YPD, culture at 30℃ for 2-3 days, pick a single colony and inoculate it into 1.5mL YPD liquid culture medium, and culture it at 30℃ and 200rpm overnight. Then inoculate it into 50mL liquid culture medium shake flasks at a 2% inoculum volume and culture it at 30℃ and 200rpm until the OD 600 =1, and were inoculated at a 2% inoculum 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 h of culture, sucrose (mother liquor concentration of 50%) was added at a rate of 8 mL / h. Simultaneously, lactose (mother liquor concentration of 40%) was added to maintain a final lactose concentration of 15 g / L. The total fermentation time was 72 h. After 72 h 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 3-FL and DFL contents in the supernatants were measured, and the results are recorded in Table 6.

[0159] Table 6

[0160] Example 7. Preparation of genetically modified Kluyveromyces lactis cells.

[0161] (1) Construct a Kluyveromycin-modified cell expression cassette.

[0162] 1) Construction of the KL-ΔLAC4 knockout cassette.

[0163] Referring to the applicant's prior patent: CN202211453172.5, invention name: Recombinant yeast and its application, a KL-ΔLAC4 knockout cassette was constructed.

[0164] 2) Construction of KL-ΔLAC4::GMD expression cassette.

[0165] 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 lac4up, 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 7), the fusion PCR system was used as a template to obtain the expression cassette KL-ΔLAC4::GMD by PCR amplification.

[0166] Table 7

[0167] 3) Construction of KL-ΔXK::GMER expression cassette.

[0168] 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 8), the fusion PCR system was used as a template to amplify the gmer expression cassette KL-ΔXK::GMER.

[0169] Table 8

[0170] 4) Construction of KL-ΔGK::BSFut3t-WT, KL-ΔGK::BSFut3t-M1 to KL-ΔGK::BSFut3t-M21, KL-ΔGK::HPFutA-WT expression cassettes.

[0171] Using the genomic DNA of Kluyveromyces lactis DSM70799 as a template, PCR amplification was performed with primers galk up-F and galk up-R as well as galk down-F and galk down-R to obtain the upstream and downstream homology arms, respectively; using loxp-F and loxp-R as primers and the G418 resistance plasmid as a template, a resistance screening marker containing G418 resistance and loxp sites was amplified; using PET32a-BSFut3t-WT, PET32a-M1 to PET32a-M21 and PET32a-HPFutA-WT as templates, the original sequence and mutant sequence of the Fut3t gene were amplified by PCR; using the genome of Kluyveromyces lactis as a template, the promoter tdh3 and terminator sequence tef of the transferase were amplified by PCR; using KL-PF and KL-PR as primers and the PUC19 plasmid as a template, the plasmid vector sequence was amplified by PCR. The upstream homology arm, promoter, target gene (original sequence and mutant sequence), terminator, G418+loxp and downstream homology arm were fused PCR to obtain ΔGK::BSFut3t-WT, ΔGK::BSFut3t-M1 to ΔGK::BSFut3t-M21 expression cassettes, and ΔGK::HPFutA-WT; the expression cassettes were connected to the plasmid vector sequence by ABclonal to obtain plasmids PUC-KL-WT, PUC-KL-M1 to PUC-KL-M21, and PUC-KL-HPFutA-WT carrying the ΔGK::BsFut3t-WT, ΔGK::HPFutA-WT, ΔGK::BSFut3t-M1 to ΔGK::BSFut3t-M21 expression cassettes, respectively.

[0172] Using primers galk up-F and galk down-R as templates, and PUC-KL-WT, PUC-KL-M1 to PUC-KL-M21, and PUC-KL-HPFutA-WT as templates, KL-ΔGK::BSFut3t-WT, KL-ΔGK::BSFut3t-M1 to KL-ΔGK::BSFut3t-M21 expression cassettes, and KL-ΔGK::HPFutA-WT were amplified for the next step of constructing recombinant strains.

[0173] Table 9. Primers for constructing KL-ΔGK::BSFut3t-WT, KL-ΔGK::BSFut3t-M1 to KL-ΔGK::BSFut3t-M21 expression cassettes, and KL-ΔGK::HPFutA-WT expression cassettes

[0174] (2) Transformation of the recombinant expression cassette and verification of the recombinant strain.

[0175] The expression cassettes constructed in (1) above were respectively transferred into the starting strain Kluyveromyces lactis DSM70799 cells. The specific method is:

[0176] 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.

[0177] Meanwhile, boil 1 mL of salmon sperm DNA for 5 min and quickly place on ice to prepare single-stranded DNA.

[0178] 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 5-10 μg / 50 μL plasmid DNA in water (50 μL). 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.

[0179] 3) Verification: The correspondence between the recombinant strains and their genotypes is shown in Table 10. 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.

[0180] 4) Resistance Removal: Using the same transformation method, a plasmid carrying Cre recombinase was transferred into the strain and plated on a YPD plate containing 0.4 mg / mL culture medium. After culturing for 48 h, a single colony was picked and added to 2 mL of YPD liquid medium. After overnight culture, the collected bacteria were centrifuged and transferred to 2 mL of YPD liquid medium. 2% galactose was added and induced for 4-6 hours. The bacteria were streaked onto a YPD plate. After 2 days, the strain was picked for verification. A single colony that did not grow on plates containing G418 or hygromycin but grew on YPD plates was selected as the target strain for resistance removal.

[0181] Table 10. Genetically modified Kluyveromyces lactis cells and their genotypes (Note: The starting strain is K. lactis DSM 70799)

[0182] The recombinant strains described in Table 10 were respectively introduced with encryption tags (amino acid sequence as SEQ ID NO: 7, nucleotide sequence as SEQ ID NO: 9), and strains KL-1-J to KL-4-J, and KL-M1-J to KL-M21-J were obtained. Among them, strain KL-M17-J was deposited with the China Center for Type Culture Collection (Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on June 26, 2024. The strain name is Kluyveromyces lactis KL-3FL-6 (Kluyveromyces lactis KL-3FL-6), and the deposit number is CCTCC NO: M 20241375.

[0183] Verification of marker genes: To verify the correctness of the above strains, extract the genomes of the transformants and the original strain and perform 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 is obtained after PCR amplification, the strain is considered correct; otherwise, the strain is considered a false positive. For tag verification, perform PCR amplification using primers and further verify by sequencing; otherwise, the strain is considered a false positive.

[0184] Example 8. Construction of genetically modified Kluyveromyces marxianus cells.

[0185] (1) Construct a Max-Kluyveromycin-modified cell expression cassette.

[0186] 1) Construction of the KM-ΔLAC4 knockout cassette.

[0187] Refer to the applicant's prior patent: CN202211453172.5, invention name: Recombinant yeast and its application to construct KM-ΔLAC4 knockout cassette.

[0188] 2) Construction of KM-ΔLAC4::GMD expression cassette.

[0189] 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 11) and the fusion PCR system was used as a template to obtain the gmd expression cassette KM-ΔLAC4::GMD by PCR amplification.

[0190] Table 11

[0191] 3) Construction of KM-ΔXK::GMER expression cassette.

[0192] 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 12) and the fusion PCR system was used as a template to obtain the gmer expression cassette KM-ΔXK::GMER.

[0193] Table 12

[0194] 4) Construction of KM-ΔGK::BSFut3t-WT, KM-ΔGK::BSFut3t-M1 to KM-ΔGK::BSFut3t-M21, KM-ΔGK::HPFutA-WT expression cassettes.

[0195] 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 (Table 13) were PCR amplified to obtain the upstream and downstream homology arms, respectively; KL-ΔGK::BSFut3t-wt, KL-ΔGK::BSFut3t-M1 to KL-ΔGK::BSFut3t-M21 expression cassettes, and KL-ΔGK::HPFutA-wt expression cassette were used as templates to PCR amplify the BSFut3t-wt / BSFut3t mutant expression cassette / HPFutA-wt expression cassette+G418+loxp sequence, and the upstream and downstream homology arms and BSFut3t-wt / BSFut3t mutant expression cassette / HPFutA-wt expression cassette+G418+loxp sequence were fused using the fusion PCR method, respectively, using the fusion system as a template with primers glk up-F1 and glk down-R1. Down-R1 was used as a template to amplify the KM-ΔGK::BSFut3t-WT, KM-ΔGK::BSFut3t-M1 to KM-ΔGK::BSFut3t-M21, and KM-ΔGK::HPFutA-wt expression cassettes for the next step of constructing recombinant strains.

[0196] Table 13. Primers for constructing KM-ΔGK::BSFut3t-WT, KM-ΔGK::BSFut3t-M1 to KM-ΔGK::BSFut3t-M21, KM-ΔGK::HPFutA-wt expression cassettes

[0197] (2) Transformation of recombinant plasmid and verification of recombinant strain:

[0198] 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.

[0199] The correspondence between the recombinant strains and their genotypes is shown in Table 14. To verify the correctness of the above strains, we extracted the genomes of the above transformants and the original strain and performed PCR amplification on the genome using primers amplifying the corresponding knockout cassette or expression cassette. If the PCR amplification yielded a single band with a size consistent with the knockout cassette or expression cassette, the strain was considered correct. Otherwise, the strain was considered a false positive. Further, the resistance was removed according to Example 3 to obtain the target strain.

[0200] Table 14. Genetically modified Kluyveromyces marxianus cells and their genotypes (Note: The starting strain is K. marxianus DMKU3-1042)

[0201] The encrypted markers (amino acid sequence such as SEQ ID NO: 7, nucleotide sequence such as SEQ ID NO: 9) were introduced into the recombinant strains described in Table 14 to obtain strains KM-1-J to KM-4-J, and KM-M1-J to KM-M21-J, respectively.

[0202] Verification of marker genes: Verification was performed according to the method described in Example 7.

[0203] Example 9. Construction of genetically modified cells of Saccharomyces cerevisiae.

[0204] (1) Construction of SC-ΔARO3::GMD-GMER / SC-ΔARO3::GMD-GMER expression cassette

[0205] The upstream and downstream homology arm sequences aro3-up and aro3-do, the promoter sequence TDH3p and the terminator sequence PGK1t, the promoter sequence TFF1p and the terminator sequence GPMt were amplified using the Saccharomyces cerevisiae genome as a template; the GMD target gene sequence was amplified using the synthesized GMD sequence as a template; the GMER target gene sequence was amplified using the synthesized GMER as a template; referring to the applicant's prior patent: 202211453172.5, invention name: Recombinant yeast and its application, KL-Δ The LAC4 knockout cassette was used as a template to amplify the G418+loxp sequence. Aro3-up, TDH3p, GMD, PGK1t, TEF1p, GMER, ADH1t, G418+loxp and aro3-do were fused by successive fusion PCR of two fragments. Finally, primers ARO3-F and ARO3-R were used as primers and the fusion PCR system was used as a template to obtain the SC-ΔARO3::GMD-GMER / SC-ΔARO3::GMD-GMER expression cassette by PCR amplification.

[0206] Table 15. Plasmids and primers

[0207] (2) Construction of SC-ΔTRP3::CDT2-LAC12 expression cassette

[0208] Using the Saccharomyces cerevisiae genome as a template, the upstream and downstream homology arm sequences trp3-up and trp3-do of the TRP3 coding region, the promoter sequence TFF1p and the terminator sequence CYC1t, the terminator sequence GPMt and the promoter sequence TPILp were amplified respectively; using the synthesized CDT2 sequence as a template, the CDT2 target gene sequence was amplified; using the Kluyveromyces lactis genome as a template, the LAC12 gene was amplified; using SC-ΔARO3::GMD-GMER as a template, the G418+loxp sequence was amplified, and trp3-up, TFF1p, CDT2, CYC1t, GPMt, LAC12, TPILp, G418+loxp and trp3-do were fused by successive fusion PCR of two fragments. Finally, using primers TRP3-F and TRP3-R as primers and the fusion PCR system as a template, the expression cassette SC-ΔTRP3::CDT2-LAC12 was amplified by PCR.

[0209] Table 16. Plasmids and primers

[0210] (3) Construction of SC-ΔGK::BSFut3t-WT, SC-ΔGK::BSFut3t-M1 to SC-ΔGK::BSFut3t-M13, and SC-ΔGK::HPFutA-WT expression cassettes.

[0211] Using Saccharomyces cerevisiae genomic DNA as a template, PCR amplification was performed to obtain the upstream and downstream homology arms gk-up and gk-down, the promoter sequence CUP1p and the terminator sequence GPMt; using the SC-ΔTRP3::CDT2-LAC12 expression cassette as a template, a resistance screening marker containing G418 resistance and loxp sites was amplified; using PET32a-BSFut3t-WT, PET32a-BSFut3t-M1 to PET32a-BSFut3t-M13, and PET32a-HPFutA-WT as templates, PCR amplification was performed to obtain BSFut3t-WT and mutant sequences, as well as HPFu tA-WT gene sequence; by using two fragments in successive fusion PCR, gk-up, CUP1p, BSFut3t-WT and mutant sequence / HPFutA-WT gene sequence, GPMt, G418+loxp and gk-do were fused, and finally primers GK-F and GK-R were used as primers, and the fusion PCR system was used as a template, and PCR amplification was performed to obtain expression cassettes SC-ΔGK::BSFut3t-WT, SC-ΔGK::BSFut3t-M1 to SC-ΔGK::BSFut3t-M13 and SC-ΔGK::HPFutA-WT expression cassettes for the next step of constructing recombinant strains.

[0212] Table 17. Primers for constructing SC-ΔGK::BSFut3t-WT, SC-ΔGK::BSFut3t-M1 to SC-ΔGK::BSFut3t-M13, and SC-ΔGK::HPFutA-WT expression cassettes

[0213] (2) Transformation of the recombinant expression cassette and verification of the recombinant strain.

[0214] The expression cassettes constructed in (1) were respectively transferred into the starting strain Saccharomyces cerevisiae SctgtP8 cells. The specific method is:

[0215] 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.

[0216] Meanwhile, boil 1 mL of salmon sperm DNA for 5 min and quickly place on ice to prepare single-stranded DNA.

[0217] 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 5-10 μg / 50 μL plasmid DNA in water (50 μL). 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.

[0218] 3) Verification: Verification of marker genes: Verification was performed according to the method described in Example 7. The corresponding relationship between the recombinant strains and their genotypes is shown in Table 18.

[0219] 4) Removal of resistance

[0220] Using the same transformation method, the plasmid carrying Cre recombinase was transferred into the strain and spread on a YPD plate containing 0.4 mg / mL and cultured for 48 hours. The grown single colonies were picked and placed in 2 mL of YPD liquid medium. After overnight culture, the bacteria were collected by centrifugation and transferred to 2 mL of YP liquid medium. 2% galactose was added and induced for 4-6 hours. The bacteria were streaked on a YPD plate. After 2 days, the strains were picked for verification. The single colonies that did not grow on plates containing G418 or hygromycin but grew on YPD plates were selected as the target strains for resistance removal.

[0221] Table 18. Saccharomyces cerevisiae engineered bacteria and their genotypes

[0222] (Note: The starting strain is: Saccharomyces cerevisiae CCTCC NO: M20231127)

[0223] The encryption markers (amino acid sequence as shown in SEQ ID NO: 7, nucleotide sequence as shown in SEQ ID NO: 9) were introduced into the recombinant strains described in Table 18 to obtain SC-1-J to SC-4-J and SC-M1-J to SC-M21-J strains, respectively.

[0224] Verification of marker genes: 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.

[0225] Example 10. Genetically modified cells from Examples 7-9 synthesize fucosyllactose

[0226] Yeast was cultured with glucose as the carbon source. Genetically modified cells obtained in Examples 7-9 were taken respectively, and the genetically modified cells were allowed to grow rapidly until the growth entered the late logarithmic phase or the stable phase. The strains were streaked and cultured in YPD solid culture medium respectively. 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 inoculum 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 hours. After 72 hours of fermentation, samples were taken and boiled for 10 minutes. The final yeast fermentation product was obtained by centrifugation, and the 3-FL content in the supernatant was determined by HPLC. The results are recorded in Tables 19 to 21.

[0227] Note: For the genetically modified Saccharomyces cerevisiae cells of Example 9, the fermentation broth also contained CuCl2 at a final concentration of 100 μM. Other procedures were the same as those for the Kluyveromyces fermentation experiments of Examples 7 and 8.

[0228] Table 19. Study on the synthesis of 3-FL by Kluyveromycin gene-modified cells

[0229] Table 20. Study on the synthesis of 3-FL by Kluyveromycin gene-modified cells

[0230] Table 21. Study on 3-FL synthesis by genetically modified cells of Saccharomyces cerevisiae

[0231] (Starting strain: Saccharomyces cerevisiae CCTCC NO: M20231127)

[0232] Example 11. Kluyveromyces KL-M9, KL-M17, SC-M9-J, SC-M17-J, KM-M9-J, and KM-M17-J were streaked onto solid media such as YPD. After culturing at 30°C for 2-3 days, single colonies were picked and inoculated into 1.5 mL YPD liquid medium and cultured overnight at 30°C and 200 rpm. Subsequently, 2% of the inoculum was inoculated into 50 mL liquid medium shake flasks and cultured at 30°C and 200 rpm until the OD 600 =1, and were inoculated at a 2% inoculum 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 h of culture, sucrose (mother liquor concentration of 50%) was added at a rate of 8 mL / h. Simultaneously, lactose (mother liquor concentration of 40%) was added to maintain a final lactose concentration of 15 g / L. The total fermentation time was 72 h. After 72 h 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, and the supernatants were combined to obtain the final yeast fermentation product. The 3-FL and DFL contents in the supernatant were determined by HPLC. The results are recorded in Table 22.

[0233] Table 22

[0234] Note: For the genetically modified cells SC-M9-J and SC-M17-J, the fermentation broth also contained CuCl2 at a final concentration of 100 μM. Other procedures were the same as those for the Kluyveromyces fermentation experiments in Examples 7 and 8.

[0235] The present application discloses a series of α-1,3-fucosyltransferase polypeptides, their preparation methods and applications, DNA molecules encoding the polypeptides, vectors, and host cells. Those skilled in the art can refer to the content of this application and appropriately improve the process parameters for implementation. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0236] Although the present application has been described in considerable detail by way of illustration and example for the purpose of clear 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 interpreted as limiting the scope of the present application.

[0237]

[0238]

Claims

1. An α-1,3-fucosyltransferase polypeptide, which is obtained by modifying at least one of positions 2, 17, 21-22, 27, 45-48, 69-72, 97-103, 118, 125-127, 133-135, 138-139, 141, 156, 162-165, 223, 227-229, 243, 247-248, 255, 260-263, and 268 of the amino acid sequence of SEQ ID NO: 1; Preferably, the modification is a mutation, and the α-1,3-fucosyltransferase polypeptide is selected from any one of the following mutations: 1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid at position 2 is substituted from K to T, thereby obtaining polypeptide M1; 2) In the amino acid sequence of M1, the amino acid at position 17 is replaced by K to R, thereby obtaining polypeptide M2; 3) In the amino acid sequence of M2, the amino acid segment at positions 21-22 is replaced by KN to NS, thereby obtaining polypeptide M3; 4) In the amino acid sequence of M3, the amino acid at position 27 is replaced by K to N, thereby obtaining polypeptide M4; 5) In the amino acid sequence of M4, the amino acid fragment at positions 45-48 is replaced by FFSD to IYSC, thereby obtaining polypeptide M5; 6) In the amino acid sequence of M5, amino acid residues 69-72 are replaced by EPDF with QPNL, thereby obtaining polypeptide M6; 7) In the amino acid sequence of M6, the amino acid segment at positions 97-103 is replaced by YKYKEAL to FLYTQDY, thereby obtaining polypeptide M7; 8) In the amino acid sequence of M7, the amino acid at position 118 is replaced by E to K, thereby obtaining polypeptide M8; 9) In the amino acid sequence of M8, the amino acid fragment at positions 125-127 is replaced by CFN to NFI, thereby obtaining polypeptide M9; 10) In the amino acid sequence of M9, the amino acid fragment at positions 133-135 is replaced by YDE to AHR, thereby obtaining polypeptide M10; 11) In the amino acid sequence of M10, the amino acid fragment at positions 138-139 is replaced by IK to FE, thereby obtaining polypeptide M11; 12) In the amino acid sequence of M11, the amino acid at position 141 is substituted from Y to I, thereby obtaining polypeptide M12; 13) In the amino acid sequence of M12, the amino acid at position 156 is replaced from T to I, thereby obtaining polypeptide M13; 14) In the amino acid sequence of M13, the amino acid fragment at positions 162-165 is replaced by FLIE with YRVD, thereby obtaining polypeptide M14; 15) In the amino acid sequence of M14, the amino acid at position 223 is replaced by T to C, thereby obtaining polypeptide M15; 16) In the amino acid sequence of M15, the amino acid segment at positions 227-229 is replaced by SSY to KSK, thereby obtaining polypeptide M16; 17) In the amino acid sequence of M16, the amino acid at position 243 is substituted from K to D, thereby obtaining polypeptide M17; 18) In the amino acid sequence of M17, the amino acid segment at positions 247-248 is replaced by TE to SQ, thereby obtaining polypeptide M18; 19) In the amino acid sequence of M18, the amino acid at position 255 is substituted from F to L, thereby obtaining polypeptide M19; 20) In the amino acid sequence of M19, the amino acid fragment at positions 260-263 is replaced by NPEE with DPEK, thereby obtaining polypeptide M20; 21) In the amino acid sequence of M20, the amino acid at position 268 is substituted from A to E, thereby obtaining polypeptide M21. 2 . A polynucleotide encoding the α-1,3-fucosyltransferase polypeptide according to claim 1 .

3. A nucleic acid construct comprising the polynucleotide according to claim 2; The nucleic acid construct preferably further comprises one or more regulatory sequences operably linked to the polynucleotide, and the regulatory sequences can direct the production of the polypeptide in an appropriate expression host cell. 4 . An expression vector comprising the polynucleotide according to claim 2 , or comprising the nucleic acid construct according to claim 3 .

5. A transformed host cell transformed with the polynucleotide according to claim 2, the nucleic acid construct according to claim 3, or the expression vector according to claim 4. 6 . An enzyme agent or enzyme composition comprising one or more of the α-1,3-fucosyltransferase polypeptides of claim 1 .

7. A genetically modified cell comprising a heterologous nucleic acid sequence encoding the α-1,3-fucosyltransferase polypeptide of claim 1; Preferably, the genetically modified cells are selected from genetically modified cells of Bacillus cereus, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Saccharomyces paradoxus, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces rioyo, Saccharomyces kuyveri and Escherichia coli.

8. The genetically modified cell according to claim 7, wherein The genetically modified cell further comprises a heterologous nucleic acid sequence encoding a GDP-L-fucose synthase polypeptide (GMER) and / or a GDP-mannose dehydratase polypeptide (GMD).

9. A method for preparing 3-fucosyllactose, characterized in that: Utilizing the genetically modified cell according to any one of claims 7 to 8, fucosyllactose is synthesized de novo using lactose as a substrate and glucose, glycerol or sucrose as a carbon source.

10. A method for preparing 3-fucosyllactose, characterized in that: The steps include: a) catalyzing the synthesis of 3-fucosyllactose using one or more of the α-1,3-fucosyltransferase polypeptides of claim 1, the transformed host cell of claim 5, or the enzyme agent or enzyme composition of claim 6 as catalysts and GDP-L-fucose and lactose as substrates; Further preferably, the preparation method further comprises the step b) purifying and / or recovering 3-fucosyllactose.

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