Fucosyltransferase and use thereof

Abstract

The present invention relates to an α-1,2-fucosyltransferase and variants thereof, a polynucleotide encoding the α-1,2-fucosyltransferase or variants thereof, a vector comprising the polynucleotide, a host cell expressing the α-1,2-fucosyltransferase or variants thereof, and a method for producing fucosylated oligosaccharides using the α-1,2-fucosyltransferase or variants thereof, or using the host cell.

Description

Fucosyltransferases and their applications

[0001] Cross-citation of related applications

[0002] This application claims priority to Chinese patent application CN202411216820.4, filed on August 30, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biotechnology, and more particularly to α-1,2-fucosyltransferase and its applications. Background Technology

[0004] Human milk oligosaccharide fucosyltransferases belong to the glycosyltransferase family and are widely expressed in vertebrates, invertebrates, plants, and bacteria. They catalyze the transfer of fucose residues from donors (usually guanosine diphosphate fucose (GDP-L-fucose)) to receptors, which include oligosaccharides, (glyco)proteins, and (glyco)lipids. The resulting oligosaccharides, glycoproteins, glycolipids, and other glycoconjugates play important roles in numerous physiological and pathological processes, including cell adhesion, cell differentiation, immune responses, fertilization, viral and bacterial infections, and tumor development. Many anomalous glycan structures containing fucosyl groups are closely related to tumorigenesis and development. Fucosyl oligosaccharides and glycoconjugates (including glycoproteins and glycolipids) can serve as key molecular markers for tumor diagnosis and have been studied and developed into anti-tumor glycovaccines for cancer immunotherapy. Therefore, research on fucosyltransferases is increasingly attracting attention from scholars and pharmaceutical companies worldwide.

[0005] Human milk oligosaccharides (HMOs) are the third most important solid component of breast milk and play a crucial role in infant health and development. Studies have shown that infants who are not breastfed and are fed only formula or cow's milk have lower survival rates than breastfed infants. They are also more prone to various intestinal diseases and developmental delays. Research has found that formula or cow's milk differs from breast milk primarily in its very low or absent HMO content. Research on HMOs is increasingly important. There are many types of HMOs; to date, approximately 200 have been isolated and identified from human milk, with fucoidan being the most abundant and important. HMOs have physiological functions such as promoting the proliferation and colonization of bifidobacteria in the intestinal epithelium, inhibiting pathogen adhesion, maintaining the balance of the infant's intestinal flora, participating in the body's immunity, and promoting brain development, and have gradually become a focus of research.

[0006] Fucosyltransferases are crucial enzymes in the synthesis of fucosyllactose, and different types of fucosyltransferases directly determine the type of human milk oligosaccharide synthesized. Based on the fucosyl addition site, fucosyltransferases are classified into α-1,2-, α-1,3 / 4-, and O-fucosyltransferases, with α-1,2-fucosyltransferase being the most prevalent, present in 75% of women's breast milk. α-1,2-fucosyltransferases exhibit complete stereochemical and regiochemical control, good substrate specificity, and can almost quantitatively form glycosidic bonds, catalyzing the synthesis of 2'-fucosyllactose (2'-FL) by transferring glycosyl groups from an activated donor substrate to an acceptor substrate. Several α-1,2-fucosyltransferases have been identified, for example, in Helicobacter pylori and Escherichia coli, mammals, Caenorhabditis elegans and Schistosoma mansoni, and plants. However, most of these enzymes either cannot be expressed in active form in bacterial systems or cannot use lactose as a receptor. Several have been successfully applied to the synthesis of 2'-FL, namely: *Thermosynechococcus vestitus* Te2FT (UniProtKB:Q8DK72, GenBank:BAC08546.1), *H. pylori* FutC (UniProtKB:A4L7J1), *E. coli* O127:K63 WbiQ (UniProtKB:Q5J7C6), *E. coli* O86:K62:H2 WbnK (UniProtKB:Q58YV9), *E. coli* O128:B12 WbsJ (UniProtKB:Q6XQ53), and *E. coli* O126... WbgL (UniProtKB:A6M9C2), Bacteroides fragilis Wcfb (GenBank:CAH06753.1), Azospirillum lipoferum SAMT (GenBank:SMH41196.1), Helicobacter sp. 11S02629-2 BKHT (GenBank:PAF50342.1).Among them, FutC exhibits the highest activity and broad substrate specificity, but has low affinity for the receptor lactose; WbsJ also has broad substrate specificity, but shows the highest activity as a receptor for lactulose; WbwK has very strict substrate specificity, only utilizing the T antigen (Gal-β-1,3-GalNAc-α-O-Bn / Me) as a receptor substrate; WbiQ is a capsule protein, and its catalytic activity is severely affected by metal ions. It also exhibits strict substrate specificity, recognizing only receptors with the non-reducing terminal Gal-β1,3-GalNAc, and not those with other Gal-β receptors at the reducing terminal. Their yields of synthesized human milk oligosaccharides are all low, making it particularly important to obtain novel fucosyltransferases with high activity and high expression levels. Summary of the Invention

[0007] A first aspect of the invention provides α-1,2-fucosyltransferase derived from *Escherichia coli* O126, *Campylobacter hyointestinalis*, *Helicobacter muridarum*, *Patescibacteria group bacterium*, *Acetobacter sp.*, or *Corynebacterium urealyticum*, or variants thereof, said variant comprising, relative to wild-type *Corynebacterium urealyticum* α-1,2-fucosyltransferase, the amino acid substitution Q258S, and further comprising substitution of one or more amino acids at positions 56 to 66. In some embodiments, the substitution of one or more amino acids at positions 56 to 66 of said variant comprises substitution of amino acids at one, two, or three positions of positions 60, 62, and 64. In some embodiments, the amino acid substitution at position 60 of the variant is F60A or F60S, the amino acid substitution at position 62 is W62H or W62R, and the amino acid substitution at position 64 is N64D or N64E. In some embodiments, the variant further includes one or more amino acid substitutions selected from the group consisting of: V25I, Q203Y, H290W, M210I, and V304I.

[0008] In some embodiments, the amino acid sequence of the variant has at least 80% sequence identity with the amino acid sequence of wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase.

[0009] In some embodiments, the variant relative to wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase comprises any group of amino acid substitutions selected from Table 1.

[0010] A second aspect of the invention provides a polynucleotide encoding any of the aforementioned α-1,2-fucosyltransferases or variants thereof.

[0011] A third aspect of the invention provides a carrier comprising a polynucleotide encoding any of the aforementioned α-1,2-fucosyltransferases or variants thereof.

[0012] A fourth aspect of the invention provides a host cell that expresses any of the aforementioned α-1,2-fucosyltransferases or variants thereof, or contains the aforementioned polynucleotides or vectors.

[0013] A fifth aspect of the invention provides a genetically modified host cell expressing α-1,2-fucosyltransferase and comprising: (1) a gene for the synthesis pathway of the donor substrate of the α-1,2-fucosyltransferase; (2) a gene for a transport protein that transports the acceptor substrate of the α-1,2-fucosyltransferase into the host cell, or a gene for the synthesis pathway of the acceptor substrate of the α-1,2-fucosyltransferase; wherein the α-1,2-fucosyltransferase is any of the aforementioned α-1,2-fucosyltransferases or a variant thereof.

[0014] In some embodiments, the donor substrate is GDP-L-fucose and the acceptor substrate is lactose.

[0015] In some embodiments, the host cell is a microbial cell. In some embodiments, the host cell is a bacterial or yeast cell. In some embodiments, the host cell is a bacterium belonging to the genera *Escherichia*, *Corynebacterium*, *Bacillus*, *Lactobacillus*, *Bifidobacterium*, *Streptococcus*, *Lactococcus*, or *Pseudomonas*. In some embodiments, the host cell is *Escherichia coli*, *Corynebacterium glutamicum*, *Bacillus subtilis*, *Saccharomyces cerevisiae*, or *Yersinia lipolytica*.

[0016] In some embodiments, the GDP-L-fucose synthesis pathway gene includes a GDP-D-mannose synthesis pathway gene, a gene encoding GDP-mannose-4,6-dehydratase (GMD), and a gene encoding GDP-L-fucose synthase (GFS); preferably, the GDP-mannose-4,6-dehydratase (GMD) is GDP-mannose-4,6-dehydratase (EcGMD) derived from Escherichia coli or a functional variant thereof, and / or the GDP-L-fucose synthase (GFS) is GDP-L-fucose synthase (WcaG) derived from Escherichia coli or a functional variant thereof.

[0017] In some embodiments, the GDP-L-fucose synthesis pathway gene includes a gene encoding a bifunctional enzyme of fucokinase / GDP-L-fucose pyrophosphorylase; preferably, the bifunctional enzyme of fucokinase / GDP-L-fucose pyrophosphorylase is a bifunctional enzyme of fucokinase / GDP-L-fucose pyrophosphorylase derived from Bacteroides fragilis or a functional variant thereof.

[0018] In some embodiments, the GDP-fucose synthesis pathway gene includes a GDP-D-mannose synthesis pathway gene, a gene encoding GDP-D-mannose-4,6-dehydratase, a gene encoding a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose, and a gene encoding GDP-D-rhamnose-3,5-epimerase; preferably, the GDP-mannose-4,6-dehydratase (GMD) is derived from *Escherichia coli* GMD (EcGMD) or a functional variant thereof, the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose is derived from *Campylobacter jejuni* DdahC or a functional variant thereof, and / or the GDP-D-rhamnose-3,5-epimerase is derived from rice (*Oryza sativa*). GDP-mannose-3,5-epimerase (OsGME) or its functional variants thereof.

[0019] In some embodiments, the host cell contains a gene encoding a lactose transporter; preferably, the lactose transporter is a lactose permease; more preferably, the lactose permease is a lactose permease derived from Escherichia coli or Kluyveromyces lactis, or a functional variant thereof.

[0020] In some embodiments, the host cell further contains the gene for α-1,3-fucosyltransferase or α-1,3 / 4-fucosyltransferase.

[0021] A sixth aspect of the invention provides a method for producing fucosyl oligosaccharides, comprising contacting one or more glycosyltransferases with their donor and acceptor substrates to synthesize the fucosyl oligosaccharides, wherein the one or more glycosyltransferases include at least α-1,2-fucosyltransferase, said α-1,2-fucosyltransferase being any of the aforementioned α-1,2-fucosyltransferases or a variant thereof.

[0022] In some embodiments, the fucoidosyl oligosaccharide includes 2'-fucosyllactose (2'-FL) and / or difucosyllactose (DFL).

[0023] In some embodiments, the donor substrate includes GDP-L-fucose, and the acceptor substrate includes lactose.

[0024] In some embodiments, the synthesis is carried out under intracellular, extracellular, or cell-free conditions.

[0025] A seventh aspect of the invention provides a method for producing fucoidan oligosaccharides, comprising culturing any of the aforementioned host cells under conditions suitable for producing the fucoidan oligosaccharides to synthesize the fucoidan oligosaccharides.

[0026] In some embodiments, the fucoidosyl oligosaccharide includes 2'-fucosyllactose (2'-FL) and / or difucosyllactose (DFL).

[0027] In some embodiments, the culture medium used to culture the host cells contains at least one carbon source.

[0028] In some embodiments, the culture medium used to culture the host cells contains lactose or a precursor substance capable of synthesizing lactose through intracellular synthetic pathways.

[0029] In some embodiments, the method further includes recovering the fucosyl oligosaccharide from the culture medium and / or host cells.

[0030] An eighth aspect of the invention provides the use of any of the aforementioned host cells in the production of fucosyl oligosaccharides. In some embodiments, the fucosyl oligosaccharides comprise 2'-fucosyllactose (2'-FL) and / or difucosyllactose (DFL). Detailed Implementation

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples described herein are illustrative only and not intended to be restrictive.

[0033] When the terms “about” and “approximately” are used with numerical variables, they generally mean that the value of the variable and all values ​​of the variable are within the measurement or experimental error (e.g., the 95% confidence interval of the mean) or within a wider range of specified values ​​(e.g., ±5% or ±10%).

[0034] The term "comprising," or its variations such as "containing," "having," or "including," means to include the stated steps or elements, but does not exclude any other steps or elements. "Constitutes of," means to exclude steps or elements not listed. "Substantially constitutes of," means to include steps or elements that do not substantially affect the fundamental and novel features of the protected invention. The term "comprising" and its variations also include the cases of "consisting of specific steps or elements" and "substantially constitutes specific steps or elements."

[0035] When referring to a numerical range, it should be understood that the specific values ​​of its upper and lower limits are disclosed, as well as all intermediate ranges included therein, such as the intermediate range between its upper or lower limit and any intermediate value, or the intermediate range between any two intermediate values. Furthermore, any intermediate ranges, subranges, and all individual numerical values ​​described in the numerical range can be excluded from the numerical range.

[0036] The term “and / or” should be understood as any one of the multiple elements connected by the term, or a combination of any number of elements.

[0037] This invention discovers novel α-1,2-fucosyltransferases and their variants that catalyze the transfer of fucose from the donor substrate to the acceptor substrate, using GDP-L-fucose as the donor substrate and lactose as the acceptor substrate, to synthesize 2'-fucosyllactose (2'-FL). The α-1,2-fucosyltransferases and their variants of this invention can be used to produce human milk oligosaccharides (HMOs), particularly fucosyl oligosaccharides, especially 2'-fucosyllactose (2'-FL). The α-1,2-fucosyltransferases and their variants of this invention can improve the production efficiency and yield of fucosyl oligosaccharides (e.g., 2'-FL), especially compared to most naturally occurring α-1,2-fucosyltransferases. The α-1,2-fucosyltransferase and its variants of the present invention can be used to produce fucosyl oligosaccharides (HMOs), particularly 2'-fucosyllactose (2'-FL), in vitro or in vivo. These HMOs can be introduced into host cells (e.g., microbial host cells) via gene recombination technology for expression within the host cells and for the synthesis of HMOs in the cells. The α-1,2-fucosyltransferase and its variants of the present invention can also be combined with other glycosyltransferases (e.g., fucosyltransferases, galactosyltransferases, acetylglucosamine transferases, and / or sialic acid transferases) to synthesize more complex oligosaccharides.

[0038] It is speculated that the novel α-1,2-fucosyltransferase and its variants of the present invention may also be able to catalyze the transfer of fucose from the donor substrate to the acceptor substrate, using GDP-L-fucose as the donor substrate and lact-N-tetrasaccharide (LNT) as the acceptor substrate, to synthesize lact-N-fucopentose I (LNFP-I).

[0039] In some embodiments, the α-1,2-fucosyltransferase is a wild-type α-1,2-fucosyltransferase. In some embodiments, the α-1,2-fucosyltransferase is derived from *Helicobacter pylori* 26695-1MET, *Campylobacter sp. MIT 12-5580*, *Bacteroides fragilis*, *Escherichia coli* O126, *Helicobacter japonicus*, *Helicobacter trogontum*, *Poseidonibacter sp. SJOD-M-5*, *Pedobacter sp. BS3*, *Christiangramia sabulilitoris*, *Campylobacter hyointestinalis*, *Cupriavidus plantarum*, *Helicobacter himalayensis*, *Herbaspirillum rubrisubalbicans*, and *Bacteroides*. The following bacteria are listed: *Caccae*, *Campylobacter lari*, *Pseudomonas fluorescens*, *Helicobacter muridarum*, *Helicobacter mustelae*, *Candidatus melainabacteria bacterium*, *Neocallimastix californiae*, *Candidatus falkowbacteria bacterium RIFOXYA2_FULL_47_19*, *Patescibacteria group bacterium*, *Alphaproteobacteria bacterium*, and *Acetobacter sp.*α-1,2-fucosyltransferases of Candidatus Gastranaerophilales bacterium, Parcubacteria group bacterium, Desulfuromonadales bacterium, Lachnospiraceae bacterium, and artificially constructed or Corynebacterium urealyticum. In a preferred embodiment, the α-1,2-fucosyltransferase is a polypeptide sequence derived from *Escherichia coli* O126 (SEQ ID NO:7), a polypeptide sequence derived from *Campylobacter hyointestinalis* (SEQ ID NO:19), a polypeptide sequence derived from *Helicobacter muridarum* (SEQ ID NO:33), a polypeptide sequence derived from *Patescibacteria group bacterium* (SEQ ID NO:43), or a polypeptide sequence derived from *Acetobacters p.* (SEQ ID NO:47).

[0040] In some embodiments, the α-1,2-fucosyltransferase comprises an amino acid sequence selected from SEQ ID NO:1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57, and 59. In a preferred embodiment, the α-1,2-fucosyltransferase comprises an amino acid sequence selected from SEQ ID NO:7,19,33,43, and 47.

[0041] In some embodiments, the α-1,2-fucosyltransferase is an α-1,2-fucosyltransferase derived from *Corynebacterium urealyticum*, which exhibits higher production efficiency and yield of fucosyl oligosaccharides (e.g., 2'-FL) compared to most naturally occurring α-1,2-fucosyltransferases. In some embodiments, the α-1,2-fucosyltransferase derived from *Corynebacterium urealyticum* comprises the amino acid sequence shown in SEQ ID NO:59.

[0042] In some embodiments, the invention also relates to variants of α-1,2-fucosyltransferase having activity comparable to or higher than that of wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase, and having an amino acid sequence that is at least 80% sequence identical to that of wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identical.

[0043] In some embodiments, the present invention also relates to a variant of wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase, which has increased α-1,2-fucosyltransferase activity compared to wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase, thereby further improving the production efficiency and yield of fucosyl oligosaccharides (e.g., 2'-FL). In some embodiments, compared to wild-type Corynebacterium urealyticum... Compared to α-1,2-fucosyltransferase (urealyticum), the variant is able to increase the yield of fucosyl oligosaccharides (e.g., 2'-FL) by at least 40%, at least 45%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, and to [a certain percentage]. The yield of fucoidan oligosaccharides (e.g., 2'-FL) is at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%. The yield of fucoidan oligosaccharides (e.g., 2'-FL) can be determined by methods well known to those skilled in the art, such as by HPLC. When host cells expressing the α-1,2-fucosyltransferase or a variant thereof are used to express fucosyl oligosaccharides (e.g., 2'-FL), the yield of fucosyl oligosaccharides (e.g., 2'-FL) in the host cells can be determined by methods well known to those skilled in the art, such as culturing the host cells under conditions suitable for fucosyl oligosaccharide (e.g., 2'-FL) synthesis and determining the yield by HPLC. In some embodiments, conditions suitable for fucosyl oligosaccharide (e.g., 2'-FL) synthesis may, for example, involve culturing the host cells for 48 hours in a medium containing a carbon source (e.g., glucose) and an acceptor substrate of α-1,2-fucosyltransferase (e.g., lactose).

[0044] In some embodiments, the amino acid alterations of the variant relative to wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase include the substitution of amino acid Q258S, and further include the substitution of one or more amino acids at positions 56 to 66. In some embodiments, the substitution of one or more amino acids at positions 56 to 66 includes the substitution of amino acids at one, two, or three positions of positions 60, 62, and 64. In some embodiments, the amino acid substitution at position 60 includes F60A or F60S. In some embodiments, the amino acid substitution at position 62 includes W62H or W62R. In some embodiments, the amino acid substitution at position 64 includes N64D or N64E. In some embodiments, the variant further includes one or more amino acid substitutions selected from the group consisting of V25I, Q203Y, H290W, M210I, and V304I.

[0045] In some embodiments, the variant comprises an amino acid substitution Q258S, and also comprises an amino acid substitution selected from N64 and N64E. In some embodiments, the variant further comprises one or more amino acid substitutions selected from the group consisting of: V25I, amino acid substitutions selected from F60A or F60S, amino acid substitutions selected from W62H or W62R, Q203Y, H290W, M210I, and V304I.

[0046] In some embodiments, the parent polypeptide of the variant is the amino acid sequence of wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase. In some embodiments, the positions of the amino acid substitutions mentioned herein correspond to the amino acid positions of the wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase amino acid sequence (i.e., SEQ ID NO:59).

[0047] In some embodiments, the α-1,2-fucosyltransferase variant has at least 80% sequence identity with the amino acid sequence of the parent polypeptide, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity.

[0048] In some embodiments, the α-1,2-fucosyltransferase variant of the present invention may contain one or more conserved substitutions of amino acids relative to the wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase.

[0049] In some embodiments, the variants comprise amino acid substitutions selected from any of the groups shown in Table 1 below:

[0050] Table 1

[0051] It should be understood that in this invention, when references are made to α-1,2-fucosyltransferase variants containing amino acid changes at certain positions, changes at other positions are not excluded. For any unmentioned position, the amino acid at that position may or may not be changed relative to the parent peptide. In some embodiments, the α-1,2-fucosyltransferase variant contains only the listed amino acid changes relative to the parent peptide and does not contain any amino acid changes at unlisted positions.

[0052] The present invention also relates to a polynucleotide encoding the α-1,2-fucosyltransferase or a variant thereof of the present invention. The polynucleotide may be DNA or RNA, and may be a single-stranded or double-stranded polynucleotide.

[0053] The present invention also relates to a vector comprising a polynucleotide encoding the α-1,2-fucosyltransferase or a variant thereof of the present invention.

[0054] In some embodiments, the vector is an expression vector. The expression vector can be used to introduce a foreign gene (e.g., the gene encoding α-1,2-fucosyltransferase or a variant thereof, as described above) into a host cell to express the protein encoded by that foreign gene in the host cell. The vector can be linear or circular, and can be single-stranded or double-stranded. The vector can be a free vector or an integrated vector. The vector can be a self-replicating vector. The vector can be, for example, a plasmid vector, a phage vector, a bacterial artificial chromosome, a transposon-based vector, or a CRISPR / Cas-based vector. The vector can also be a suicide vector, such as a suicide plasmid vector.

[0055] The expression vector may contain a foreign gene expression cassette, which may include a foreign gene and a regulatory sequence operatively linked thereto, the regulatory sequence guiding the expression of the foreign gene in a suitable host cell. The regulatory sequence may include, but is not limited to, promoters, enhancers, terminators, and other expression control elements. The promoter may be a constitutive promoter to enable sustained expression of the foreign gene, or an inducible promoter to induce expression of the foreign gene upon the addition of an inducer. The vector may also contain one or more selectable marker genes that allow convenient selection of transformed, transfected, or transduced cells, such as genes providing resistance to antibiotics or heavy metals and / or negative selection marker genes (e.g., the sacB gene).

[0056] The appropriate vector can be selected based on different purposes (e.g., autonomous replication within the host cell or integration into the host cell genome) and / or different host cells. Vectors and regulatory sequences suitable for introducing exogenous proteins into different host cells are well known to those skilled in the art. For example, for *Escherichia coli*, vectors that can be used include, but are not limited to, pJC1, pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, or plasmids based on them; for *Corynebacterium glutamicum*, vectors that can be used include, but are not limited to, pBL1, pEKEx1, pEKEx2, pXMJ19, pJC1, pHM1519, pVWEx1, pZ8-1, pECTAC-K99, pECTAC-XK99E, pECTAC-XC99E, pECTAC-XT99A, pNG2, pAPE12 plasmid vectors or plasmid vectors based on them, pK18mobsacB, or a suicide plasmid vector based on it; for *Bacillus subtilis*, vectors that can be used include, but are not limited to, pHT43, pUB110, ... pE194, pUCX05-bgaB, pWB980, pHP13, pBE2, pHP13, pHP13-43, pHT01, pHT304, pMK3, pHCMC05, pMA5, pHY300PLK, or pMUTIN4; for yeast, the vectors that can be used include, but are not limited to, pPIC9, pPIC9k, pHIL-S1, pPICza, pYAM75P6, pHIL-D2, pA0815, and pPIC3. K, pPICZ, pHWO10, pGAPZ, pGAPZa, pYES2, pYES2 / NT, pYES2 / CT, pYES3, pYES6, pYCplac22-GFP, pAUR123, p RS303TEF, pRS304, pRS305, pRS306, pY13TEF, pY14TEF, pY15TEF, pY16TEF, pSH47, pLacZi, pHIS2 or pGAD42. Promoters suitable for use in bacteria include, but are not limited to, inducible promoters such as Ptac, Plac, and Ptrc, or constitutive promoters such as Psod, PcspB, Ptuf, and PgapA. They may also include other promoters such as Pcg2195 (Wei L, et al., Appl Microbiol Biotechnol. 2018 May; 102(9):4117-4130, the full text of which is incorporated herein by reference) or variants of these promoters. In some embodiments, the promoters Psod, Pcg2195, or Pgap are used to express exogenous genes in host cells.In some implementations, the promoter used to express the foreign gene in the host cell is selected from SEQ ID NO:71-73.

[0057] The present invention also relates to expressing the aforementioned α-1,2-fucosyltransferase or a variant thereof, or a host cell containing the aforementioned polynucleotide or the aforementioned vector, for the purpose of expressing the α-1,2-fucosyltransferase or a variant thereof, or for further synthesizing fucosyl oligosaccharides within the cell.

[0058] The host cell can be a prokaryotic or eukaryotic cell. Suitable host cells include bacteria, yeast cells, archaea, fungal cells, insect cells, plant cells, and animal cells, including mammalian cells (such as human cells and cell lines). In some embodiments, the host cell is a microbial cell, which may include bacteria, yeast cells, archaea, fungal cells, etc. The microorganism may be a GRAS (Generally Recognized As Safe) microorganism. The bacteria may be, for example, Gram-negative or Gram-positive bacteria.The bacteria may be, for example, Escherichia bacteria, such as Escherichia coli; Corynebacterium bacteria, such as Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium crenatum, Corynebacterium thermoaminogenes, Corynebacterium aminogenes, etc.; Bacillus bacteria, such as Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus cereus, Bacillus stearothermophilus, Bacillus megaterium, etc. The following bacteria are included: *Lactobacillus* (e.g., *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus delbrueckii*, *Lactococcus lactis*); *Bifidobacterium*; *Streptococcus*; *Lactococcus*; *Streptmyces*; *Pseudomonas* (e.g., *Pseudomonas aeruginosa*); *Clostridium*; *Brevibacillus*; *Enterococcus*; *Pediococcus*; *Leuconostoc*, etc.Yeast cells can be, for example, yeast cells from the genera *Saccharomyces*, *Saccharomycopsis*, *Pichia*, *Hansenula*, *Kluyveromyces*, *Yarrowia*, *Rhodotorula*, or *Schizosaccharomyces*, such as *Saccharomyces cerevisiae*, *Yarrowia lipolytica*, *Candida utilis*, or *Pichia pastoris*.

[0059] The α-1,2-fucosyltransferase or a variant thereof of the present invention can be used to produce target fucosyl oligosaccharides.

[0060] In some embodiments, the α-1,2-fucosyltransferase or a variant thereof can be used to catalyze the transfer of fucose from a donor substrate to an acceptor substrate to synthesize 2'-fucosylated lactose.

[0061] In some embodiments, two or more glycosyltransferases may be used to synthesize the target fucosyl oligosaccharide through two or more sequential glycosyltransfer reactions, wherein the product of the preceding glycosyltransfer reaction may serve as the acceptor substrate for the subsequent glycosyltransfer reaction.

[0062] The glycosyltransferase may be one or more selected from fucosyltransferases (e.g., α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,4-fucosyltransferase, α-1,3 / 4-fucosyltransferase and / or α-1,6-fucosyltransferase), galactosyltransferases (e.g., β-1,3-galactosyltransferase, β-1,4-galactosyltransferase), acetylglucosamine transferases (e.g., β-1,3-N-acetylglucosamine transferase), and sialyltransferases (e.g., α-2,3-sialyltransferase and / or α-2,6-sialyltransferase).

[0063] In some embodiments, the two or more glycosyltransferases include the α-1,2-fucosyltransferase of the present invention or a variant thereof, and also include at least one glycosyltransferase having a different activity (or catalyzing a different reaction) than the α-1,2-fucosyltransferase of the present invention or a variant thereof.

[0064] The donor substrate of the glycosyltransferase can be an activated monosaccharide. Examples of activated monosaccharides include, but are not limited to, GDP-L-fucose, GDP-mannose, UDP-N-acetylgalactosamine, UDP-glucose, UDP-galactose, CMP-sialic acid, and / or UDP-N-acetylglucosamine.

[0065] The acceptor substrates of the glycosyltransferase include, but are not limited to, one or more of lactose, lact-N-tetrasaccharide (LNT), lact-N-neotetrasaccharide (LNnT), and 2'-fucosylated lactose.

[0066] The target fucoidan oligosaccharides include, but are not limited to, 2'-fucosylated lactose (2'-FL), difucosylated lactose-N-tetrasaccharide (DFL), lact-N-fucopentose I (LNFP-I), lact-N-fucopentose II (LNFP-II), lact-N-fucopentose III (LNFP-III), lact-N-fucopentose VI (LNFP-VI), lact-N-fucopentose V (LNFP-V), lact-N-neofofucopentose I (LNnFP-I), and lact-N-neofofucopentose... One or more of the following: lactopentose II (LNnFP-II), lacto-N-neofopentose III (LNnFP-III), lacto-N-neofopentose VI (LNnFP-VI), lacto-N-neofopentose V (LNnFP-V), lacto-N-difucohexose I (LNDFH-I), lacto-N-difucohexose II (LNDFH-II), lacto-N-difucohexose III (LNDFH-III), and fucodisialyllactose-N-hexasaccharide (FDSLNH).

[0067] In some implementations, the target fucosyl oligosaccharide can be a fucosyl oligosaccharide obtained by transferring glycosyl groups from the donor substrate to 2'-FL using an additional glycosyltransferase, with 2'-FL as the acceptor substrate.

[0068] The synthesis of the target fucosyl oligosaccharide can be carried out intracellularly, extracellularly, or under cell-free conditions. In some embodiments, the α-1,2-fucosyltransferase or a variant thereof described in this invention can be contacted with a donor substrate and an acceptor substrate to transfer fucosyl groups from the donor substrate to the acceptor substrate, thereby obtaining the target fucosyl oligosaccharide. In some embodiments, two or more glycosyltransferases (one of which is the α-1,2-fucosyltransferase or a variant thereof described in this invention) may be contacted with their donor and acceptor substrates to transfer fucosylate from the donor substrate of the α-1,2-fucosyltransferase or a variant thereof to the acceptor substrate, thereby sequentially carrying out two or more reactions catalyzed by each glycosyltransferase to transfer glycosylate from its donor substrate to its acceptor substrate, ultimately synthesizing the target fucosyl oligosaccharide, wherein at least one of the two or more reactions is a reaction catalyzed by the α-1,2-fucosyltransferase or a variant thereof described in this invention to transfer fucosylate from the enzyme's donor substrate to the acceptor substrate.

[0069] The α-1,2-fucosyltransferase or a variant thereof, or other glycosyltransferase, may be provided in the form of a purified polypeptide or in a host cell expressing the enzyme and capable of secreting it extracellularly. The donor substrate may be provided in a purified form or in a form capable of intracellular production and extracellular transport to the host cell. The acceptor substrate may be provided in a purified form or in a form capable of intracellular production and extracellular transport to the host cell.

[0070] The synthesis of the target fucosyl oligosaccharide can occur intracellularly. To this end, the present invention provides a genetically modified host cell capable of synthesizing the target fucosyl oligosaccharide intracellularly. The genetically modified host cell is capable of expressing the α-1,2-fucosyltransferase of the present invention or a variant thereof. The genetically modified host cell may further express at least one glycosyltransferase with a different activity (or catalyzes a different reaction) than the α-1,2-fucosyltransferase of the present invention or a variant thereof, to synthesize other structurally more complex fucosyl oligosaccharides.

[0071] To synthesize a target fucosyl oligosaccharide within a host cell, the host cell should be able to provide a donor substrate and / or an acceptor substrate intracellularly. The donor substrate may be provided extracellularly (e.g., added to a culture medium) and transported into the cell, or synthesized intracellularly from a precursor (e.g., a carbon source different from the donor substrate) via an intracellular synthetic pathway containing the donor substrate. The acceptor substrate may be provided extracellularly (e.g., added to a culture medium) and transported into the cell, or synthesized intracellularly from a precursor (e.g., a carbon source different from the acceptor substrate) via an intracellular synthetic pathway containing the acceptor substrate.

[0072] The genes for one or more enzymes involved in the aforementioned receptor substrate and / or donor substrate synthesis pathways can be endogenous or exogenous within the host cell. In some embodiments, the host cell used naturally possesses the aforementioned receptor substrate and / or donor substrate synthesis pathways, enabling intracellular synthesis of these pathways. In this case, all genes involved in the aforementioned receptor substrate and / or donor substrate synthesis pathways contained in the host cell can be endogenous. In some embodiments, the host cell used may not naturally possess the aforementioned receptor substrate and / or donor substrate synthesis pathways, for example, lacking one or more enzymes in these pathways. In this case, the missing one or more enzymes can be provided by introducing exogenous enzyme genes into the host cell to express the missing one or more enzymes in the host cell. The exogenous enzyme genes can be contained in a free expression vector introduced into the host cell or integrated into the host cell's chromosome.

[0073] Those skilled in the art are familiar with various glycosyltransferases and their donor and acceptor substrates, and know how to construct synthetic or transport pathways for donor and acceptor substrates in host cells.

[0074] In some embodiments, the donor substrate is GDP-L-fucose. The genetically modified host cell may contain genes for the GDP-L-fucose synthesis pathway to synthesize GDP-L-fucose as the donor substrate, and the α-1,2-fucosyltransferase or a variant thereof expressed by the host cell may transfer fucose from GDP-L-fucose to the acceptor substrate to synthesize fucosyl oligosaccharides.

[0075] GDP-L-fucose can be synthesized through various pathways, including de novo synthesis and salvage pathways. The de novo synthesis pathway exists in some prokaryotes (e.g., *Escherichia coli*, *Corynebacterium glutamicum*) and eukaryotes. It utilizes carbon sources such as glycerol, glucose, and sucrose to produce fructose-6-phosphate via intracellular metabolic pathways. Fructose-6-phosphate is then catalyzed by mannose-6-phosphate isomerase (ManA) to produce mannose-6-phosphate, which is then catalyzed by phospmannose mutase (ManB) to produce mannose-1-phosphate. Mannose-1-phosphate guanylate transferase (ManC) catalyzes the transfer of nucleotides from GTP to mannose-1-phosphate to generate GDP-mannose. GDP-mannose is then catalyzed by GDP-mannose-4,6-dehydratase (GMD) to produce GDP-4-keto-6-deoxy-D-mannose, which is further catalyzed by GDP-L-fucose synthase (GFS) to produce GDP-L-fucose.

[0076] Fructose-6-phosphate is an intermediate product of carbon source metabolism found in almost all organisms, including bacterial cells. In some embodiments, genetically modified host cells may contain genes for this de novo synthesis pathway, specifically genes containing mannose-6-phosphate isomerase (ManA), phosphogannal mutase (ManB), mannose-1-phosphate guanylate transferase (ManC), GDP-mannose-4,6-dehydratase (GMD), and GDP-L-fucose synthase (GFS), to express these enzymes in the host cell, thereby enabling the synthesis of GDP-L-fucose.

[0077] Most cells naturally contain the GDP-D-mannose synthesis pathway, which includes genes for mannose-6-phosphate isomerase (ManA), mannose phosphate mutase (ManB), and mannose-1-phosphate guanylate transferase (ManC). Examples include common bacteria such as *Escherichia coli*, *Corynebacterium glutamicum*, and *Bacillus subtilis*. However, metabolic pathways can differ between species. When a host cell lacks one or more enzymes in the GDP-D-mannose synthesis pathway, these missing enzymes can be provided by introducing exogenous enzyme genes into the host cell to express them. These exogenous enzyme genes can be contained in a free expression vector introduced into the host cell or integrated into the host cell's chromosome.

[0078] Some species, such as *Escherichia coli*, naturally contain the biosynthetic pathway from GDP-D-mannose to GDP-L-fucose, i.e., they contain genes for GDP-mannose-4,6-dehydratase (GMD) and GDP-L-fucose synthase (GFS), while other species, such as *Bacillus licheniformis* and *Corynebacterium glutamicum*, do not contain these genes. When the host cell used lacks one or more enzymes in the GDP-D-mannose to GDP-L-fucose biosynthetic pathway (i.e., GDP-mannose-4,6-dehydratase (GMD) and / or GDP-L-fucose synthase (GFS)), the missing enzymes can be provided by introducing exogenous enzyme genes into the host cell to express the missing enzymes in the host cell. The exogenous enzyme gene can be contained in a free expression vector introduced into the host cell or integrated into the host cell's chromosome.

[0079] In some embodiments, the GDP-mannose-4,6-dehydratase (GMD) is GDP-mannose-4,6-dehydratase (EcGMD) derived from *E. coli* or a functional variant thereof. In some embodiments, the GDP-mannose-4,6-dehydratase (EcGMD) derived from *E. coli* comprises the amino acid sequence shown in SEQ ID NO:61. In some embodiments, the GDP-L-fucose synthase (GFS) is GDP-L-fucose synthase (WcaG) derived from *E. coli* or a functional variant thereof. In some embodiments, the GDP-L-fucose synthase (WcaG) derived from *E. coli* comprises the amino acid sequence shown in SEQ ID NO:63.

[0080] The salvage pathway utilizes L-fucose, which is free in the cytoplasm and is derived from extracellular or lysosomal degradation products, as a substrate. L-fucose is first phosphorylated by fucokinase to form fucose-1-phosphate, and then catalyzed by GDP-L-fucosylationase to generate GDP-L-fucose.

[0081] Genetically modified host cells can contain genes for this salvage pathway, namely, genes containing fucokinase and GDP-L-fucopyrophosphorylase, to express these enzymes in the host cells, thereby enabling the synthesis of GDP-L-fucose using L-fucose as a carbon source.

[0082] In some implementations, the functions of fucokinase and GDP-L-fucopyrophosphorylase can be achieved by a single enzyme, such as a fucokinase / GDP-L-fucopyrophosphorylase bifunctional enzyme, like the fucokinase / GDP-L-fucopyrophosphorylase bifunctional enzyme Fkp or a functional variant thereof from Bacteroides fragilis.

[0083] In some implementations, the genetically modified host cell may contain genes for the salvage pathway, namely, genes for fucokinase and GDP-L-fucopyrophosphorylase (e.g., a bifunctional enzyme of fucokinase / GDP-L-fucopyrophosphorylase), to express the enzyme in the host cell, thereby enabling the synthesis of GDP-L-fucose from added fucose.

[0084] When the host cells used lack fucokinase and GDP-L-fucosylationase, these enzymes can be provided by introducing exogenous enzyme genes into the host cells to express the enzymes. The exogenous enzyme genes can be contained in a free expression vector introduced into the host cells or integrated into the host cell's chromosome.

[0085] Recently, a new GDP-L-fucose synthesis pathway was also discovered, disclosed in application number CN.

[0086] Patent No. 202410437877.0 (the entire contents of which are incorporated herein by reference) is cited. In this novel GDP-L-fucose synthesis pathway, the synthesis of GDP-mannose is the same as the aforementioned de novo synthesis pathway, i.e., fructose-6-phosphate is generated from carbon sources such as glycerol, glucose, and sucrose via intracellular metabolic pathways. Fructose-6-phosphate is then catalyzed by mannose-6-phosphate isomerase (ManA) to generate mannose-6-phosphate, which is then catalyzed by phospmannose mutase (ManB) to generate mannose-1-phosphate. Mannose-1-phosphate guanylate transferase (ManC) catalyzes the transfer of nucleotides from GTP to mannose-1-phosphate to generate GDP-mannose. However, the synthetic pathway from GDP-mannose to GDP-L-fucose differs from the aforementioned de novo synthetic pathway, including: (i) GDP-mannose is catalyzed by GDP-mannose-4,6-dehydratase (GMD) to generate GDP-4-keto-6-deoxy-D-mannose; (ii) GDP-4-keto-6-deoxy-D-mannose is catalyzed by a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose to generate GDP-D-rhamnose; and (iii) GDP-D-rhamnose is catalyzed by GDP-D-rhamnose-3,5-epimerase (GRE) to synthesize GDP-L-fucose.

[0087] The enzymes involved in any two or three of (i), (ii), and (iii) may not be the same enzyme. For example, the GDP-D-mannose-4,6-dehydratase (GMD) and the reductase that catalyzes the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose are not encoded by the same gene; the GDP-D-mannose-4,6-dehydratase (GMD) and the GDP-D-rhamnose-3,5-epimerase (GRE) are not encoded by the same gene; and / or the reductase that catalyzes the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose and the GDP-D-rhamnose-3,5-epimerase (GRE) are not encoded by the same gene.

[0088] The host cells express: (i) GDP-D-mannose-4,6-dehydratase (GMD), (ii) reductases that catalyze the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose (such as GDP-4-keto-6-deoxy-D-mannose reductase (RMD)), and (iii) GDP-D-rhamnose-3,5-epimerase (GRE) that can synthesize GDP-L-fucose from GDP-D-mannose.

[0089] In some implementations, the genetically modified host cell may contain the novel GDP-L-fucose synthesis pathway genes, specifically, genes containing mannose-6-phosphate isomerase (ManA), mannose phosphate mutase (ManB), mannose-1-phosphate guanylate transferase (ManC), GDP-mannose-4,6-dehydratase (GMD), a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose, and GDP-D-rhamnose-3,5-epimerase (GRE), to express these enzymes in the host cell, thereby enabling the synthesis of GDP-L-fucose.

[0090] When the host cell used lacks one or more of the following enzymes: GDP-mannose-4,6-dehydratase (GMD), a reductase that catalyzes the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose, and GDP-D-rhamnose-3,5-epimerase (GRE), the missing enzyme can be provided by introducing a foreign enzyme gene into the host cell to express the missing enzyme in the host cell. The foreign enzyme gene can be contained in a free expression vector introduced into the host cell or integrated into the host cell's chromosome.

[0091] In some embodiments, the GDP-mannose-4,6-dehydratase (GMD) is a GMD derived from Escherichia coli (EcGMD) or a functional variant thereof.

[0092] In some embodiments, the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose can be GDP-4-keto-6-deoxy-D-mannose reductase (RMD), such as the RMD (PaRMD) derived from Pseudomonas aeruginosa or the RMD (AtRMD) derived from Aneurinibacillus thermoaerophilus (FEBS J.2009,276(10):2686-2700; J.Biol.Chem.2001,276(8):5577-5583; the full text of which is incorporated herein by reference), or a functional variant thereof. In some embodiments, the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose may be DdahC derived from Campylobacter jejuni (Biochemistry. 2022, 61:2138-2147; Angew Chem Int Ed Engl. 2008, 47(51):9814-59; the full text of these references is incorporated herein by reference) or a functional variant thereof. In some embodiments, the DdahC derived from Campylobacter jejuni comprises the amino acid sequence shown in SEQ ID NO:67.

[0093] In some embodiments, the GDP-D-rhamnose-3,5-epimerase (GRE) may be GDP-mannose-3,5-epimerase (GME). GDP-mannose-3,5-epimerase (GME) can catalyze the conversion of GDP-D-mannose to GDP-L-fucose, and also has the activity of catalyzing the conversion of GDP-D-rhamnose to GDP-L-fucose. For example, GME derived from Arabidopsis thaliana (AtGME), GME derived from rice (Oryza sativa) (OsGME), and GME derived from volcanic fumarole methyl acidophilus (MfGME) (Phytochemistry. 2006, 67(4):338-346; J. Am. Chem. Soc. 2005, 127(51):18309-18320; Biotechnol. Adv. 2021, 48:107705; Int. J Mol. Sci. 2019, 20(14):3530; the full text of these references is incorporated herein by reference. Therefore, it can be used as GDP-D-rhamnose-3,5-epimerase (GRE) in this invention. In some embodiments, the GDP-D-rhamnose-3,5-epimerase (GRE) may be the above-mentioned GDP-mannose-3,5-epimerase (GME) or a functional variant thereof. In some embodiments, the GDP-D-rhamnose-3,5-epimerase / GDP-mannose-3,5-epimerase derived from rice (Oryza sativa) contains the amino acid sequence shown in SEQ ID NO:69.

[0094] In some embodiments, the host cell of the present invention is able to provide lactose intracellularly as the acceptor substrate of the α-1,2-fucosyltransferase.

[0095] In some embodiments, the host cell of the present invention contains a gene encoding a lactose transporter to express the lactose transporter. The lactose transporter is capable of transporting lactose from the culture medium into the cell to provide lactose intracellularly. The gene encoding the lactose transporter can be endogenous or exogenous. The lactose transporter can be a lactose permease. When the host cell used lacks an endogenous lactose transporter, a gene encoding an exogenous lactose transporter can be introduced into the host cell to enable the host cell to contain and express the exogenous lactose transporter. The exogenous lactose transporter gene can be contained in a free expression vector introduced into the host cell or integrated into the chromosome of the host cell. In some embodiments, the lactose permease contained in the host cell can be, for example, lactose permease LacY derived from *Escherichia coli* or lactose permease Lac12 derived from *Kluyveromyces lactis*, or functional variants thereof. In some embodiments, the lactose permease (LacY) derived from Escherichia coli comprises the amino acid sequence shown in SEQ ID NO:65.

[0096] In some embodiments, the host cell can also synthesize lactose intracellularly without the addition of exogenous lactose. In some embodiments, the host cell naturally possesses the ability to synthesize lactose from carbon sources other than lactose, potentially enabling endogenous lactose synthesis. In some embodiments, the host cell can be genetically modified to express enzymes for synthesizing lactose from other carbon sources (e.g., glucose), such as expressing β-1,4-galactosyltransferase to achieve intracellular lactose synthesis. β-1,4-galactosyltransferase catalyzes the reaction of galactose and glucose to produce lactose. Examples of β-1,4-galactosyltransferase include Pm1141 of Pasteurella multocida and Lex1 of Aggregatibacter aphrophilus NJ8700 (WO2015 / 150328; the entire text of which is incorporated herein by reference). When host cells are cultured using a carbon source that can be converted into glucose through cellular metabolism, the host cells are able to produce free glucose from the carbon source within the cells, which is then catalyzed by β-1,4-galactosyltransferase to synthesize lactose. The carbon source can be, for example, glucose, sucrose, glycerol, fructose, xylose, cellulose, molasses, corn syrup, galactose, methanol, pyruvate, succinic acid, or any other carbon source that can be converted into glucose through cellular metabolism.

[0097] In some embodiments, the host cell is a genetically modified Corynebacterium glutamicum containing endogenous GDP-D-mannose synthesis pathway genes and genetically modified to include genes encoding exogenous GDP-mannose-4,6-dehydratase (GMD), exogenous GDP-L-fucose synthase (GFS), and genes encoding the α-1,2-fucosyltransferase or a variant thereof of the present invention. In a more preferred embodiment, the genetically modified Corynebacterium glutamicum further contains a gene encoding exogenous lactose permease. In some embodiments, the host cell is used for the synthesis of 2'-FL.

[0098] In some embodiments, the host cell is a genetically modified Corynebacterium glutamicum containing endogenous GDP-D-mannose synthesis pathway genes and genetically modified to include genes encoding exogenous GDP-D-mannose-4,6-dehydratase (GMD), exogenous reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose, exogenous GDP-D-rhamnose-3,5-epomerase (GRE), and genes encoding the α-1,2-fucosyltransferase or a variant thereof of the present invention. In a more preferred embodiment, the genetically modified Corynebacterium glutamicum further contains a gene encoding exogenous lactose permease. In some embodiments, the host cell is used for the synthesis of 2'-FL.

[0099] In some embodiments, the host cell is a genetically modified *E. coli* containing an endogenous gene for the GDP-D-mannose synthesis pathway, an endogenous gene encoding GDP-mannose-4,6-dehydratase (GMD), and an endogenous gene encoding an exogenous GDP-L-fucose synthase (GFS), which is genetically modified to include a gene encoding the α-1,2-fucosyltransferase of the present invention or a variant thereof for the synthesis of 2'-FL. In some embodiments, the gene encoding β-galactosidase LacZ in the genetically modified *E. coli* is knocked out and / or the gene encoding UDP-glucose lipotransferase WcaJ is knocked out.

[0100] In some embodiments, the host cell is a genetically modified *E. coli* containing endogenous GDP-D-mannose synthesis pathway genes, which are genetically modified to include genes encoding exogenous GDP-D-mannose-4,6-dehydratase (GMD), exogenous reductases catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose, exogenous GDP-D-rhamnose-3,5-epomerase (GRE), and genes encoding the α-1,2-fucosyltransferase of the present invention or variants thereof, for the synthesis of 2'-FL. In some embodiments, the genes encoding β-galactosidase LacZ, GDP-L-fucose synthase WcaG, and / or UDP-glucose lipotransferase WcaJ are knocked out in the genetically modified *E. coli*.

[0101] In some embodiments, the genetically modified host cell may further contain the gene for α-1,3-fucosyltransferase or α-1,3 / 4-fucosyltransferase to express α-1,3-fucosyltransferase or α-1,3 / 4-fucosyltransferase in the host cell. This α-1,3-fucosyltransferase or α-1,3 / 4-fucosyltransferase catalyzes the transfer of fucose from GDP-L-fucoose to 2'-FL synthesized in the host cell, generating difucosyllactose-N-tetrasaccharide (DFL). The α-1,3-fucosyltransferase or α-1,3 / 4-fucosyltransferase can be provided by introducing a foreign enzyme gene into the host cell. This foreign enzyme gene may be contained in a free expression vector introduced into the host cell or integrated into the host cell's chromosome.

[0102] To enhance the production of the target fucosyl oligosaccharide, the host cell of the present invention can be further modified to enhance the supply of donor substrates (such as GDP-L-fucose), acceptor substrates (such as lactose), and / or fucosyltransferases, for example, by enhancing the synthesis of intracellular donor substrates and / or acceptor substrates, enhancing the function of fucosyltransferases, and / or enhancing the function of transport proteins that transport acceptor substrates into the cell, so as to optimize the production of fucosyl oligosaccharides.

[0103] Enhancing the supply of donor and / or acceptor substrates may include enhancing the activity of one or more enzymes involved in the synthesis pathway of the donor and / or acceptor substrates, or eliminating or attenuating competitive metabolic pathways of the donor and / or acceptor substrates and their intermediate metabolites. A competitive metabolic pathway refers to other metabolic pathways involved by these intermediate metabolites or the donor and / or acceptor substrates that are not included in the synthesis pathway of the donor and / or acceptor substrates or the target fucosylated oligosaccharide. Eliminating or attenuating competitive metabolic pathways of the donor and / or acceptor substrates or the intermediate metabolites includes eliminating or attenuating the activity of one or more enzymes involved in the competitive metabolic pathway.

[0104] Genetic modifications that enhance enzyme activity may include increasing the enzyme's activity and / or overexpressing the enzyme. Increasing enzyme activity may, for example, include mutating an endogenous enzyme gene to enhance its activity, or replacing the endogenous enzyme gene with an exogenous enzyme gene having higher activity. The exogenous enzyme may be an enzyme derived from a species different from the host cell, or a mutant of the wild-type enzyme. Overexpressing the enzyme may, for example, include increasing the copy number of the enzyme gene, and / or replacing the enzyme's natural promoter with a promoter having a higher expression level. In some embodiments, increasing the copy number of the enzyme gene includes additionally introducing an exogenous enzyme gene into the host cell without altering the expression of the endogenous enzyme gene. The exogenous enzyme gene may be one, two, three, or more copies, and may have the same or different sequences as the endogenous enzyme. For example, the exogenous enzyme may be derived from the same species as the host cell, or the exogenous enzyme may have higher activity than the endogenous enzyme, for example, an enzyme derived from a species different from the host cell, or a mutant of the wild-type enzyme.

[0105] Eliminating or attenuating the effect of the enzyme includes genetically modifying the host cell to partially or completely inactivate the endogenous gene encoding the enzyme. This includes, but is not limited to: knocking out or knocking down the endogenous gene, for example, deleting all or part of the sequence of the endogenous gene, mutating the endogenous gene, or inserting a foreign sequence into the endogenous gene, so as to cause the enzyme activity encoded by the endogenous gene to be lost or attenuated. Genetic modification to partially or completely inactivate the endogenous gene encoding the enzyme may also include: knocking out or knocking down the natural regulatory sequence (e.g., the natural promoter) of the endogenous gene, for example, deleting all or part of the sequence of the regulatory sequence, mutating the regulatory sequence, or inserting a foreign sequence into the regulatory sequence, so as to partially or completely inactivate the regulatory sequence, thereby eliminating or reducing the expression of the endogenous gene.

[0106] In some embodiments, the host cells of the present invention can be further modified to increase intracellular GDP-L-fucose production capacity. In some embodiments, the host cells can be modified to overexpress enzymes in the GDP-L-fucose synthesis pathway. In some embodiments, the host cells can be modified to overexpress one or more genes in the GDP-D-mannose synthesis pathway genes. In some embodiments, one or more genes in the GDP-D-mannose synthesis pathway genes include the phosphogmannose mutase (ManB) gene and / or the mannose-1-phospguanylate transferase (ManC) gene. This overexpression can be achieved by introducing additional exogenous genes encoding the enzyme into the host cells. The introduced exogenous genes may have the same or different sequences as endogenous genes with the same function in the host cells, which can increase the copy number of the gene encoding the enzyme in the host cells, thereby increasing the expression level of the enzyme. The overexpression can also be achieved by modifying the regulatory sequence (e.g., the promoter) operatively linked to the gene encoding the enzyme in the host cell, for example, by replacing the original promoter (e.g., the promoter of the gene naturally present in the host cell) with a stronger promoter, or by introducing mutations or inserting new regulatory elements into the promoter region to enhance its activity. Those skilled in the art will understand that the term "stronger promoter" refers to a promoter with stronger promoter activity relative to the original promoter in the host cell (e.g., the promoter of the gene naturally present in the host cell), such as a stronger ability to bind to the transcription initiation complex and / or a stronger RNA polymerase binding ability. As is known to those skilled in the art, using a stronger promoter can increase the expression level of the gene operatively linked to the promoter (e.g., increase the expression level of the protein encoded by the gene). In some embodiments, the -10 or -35 region of the promoter can be modified to obtain a stronger promoter. In some embodiments, the stronger promoter refers to a promoter whose -10 region contains TATAAT and / or whose -35 region contains TTGGCA. In some embodiments, the host cell is modified such that the -10 region sequence of the promoter of the phosphogmannose mutase (ManB) gene and / or the -35 region contains TATAAT and / or TTGGCA.

[0107] In some implementations, the host cell can be engineered to eliminate or attenuate the activity of enzymes that catalyze the conversion of GDP-L-fucose into substances other than fucosyl oligosaccharides (such as colacid). For example, the host cell can be engineered to be a nonfunctional UDP-glucose lipotransferase WcaJ or to have attenuated UDP-glucose lipotransferase WcaJ.

[0108] In some implementations, the host cell can be further engineered to eliminate or attenuate the competitive metabolic pathway of lactose. For example, the host cell can be engineered to have a non-functional galactosidase LacZ or a galactosidase LacZ with reduced activity.

[0109] In some embodiments, the host cell can be further modified to eliminate or attenuate a pre-existing GDP-L-fucose synthesis pathway, enabling the host cell to synthesize GDP-L-fucose via an alternative pathway. For example, the activity of an enzyme (e.g., GDP-L-fucose synthase WcaG) that catalyzes the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-L-fucose can be eliminated or attenuated, allowing the host cell to synthesize GDP-L-fucose solely through the aforementioned novel pathway. For instance, the host cell can be modified to have a non-functional GDP-L-fucose synthase WcaG or a less active version of it.

[0110] In some implementations, when the host cell does not naturally contain one or more enzymes in the target fucosyl oligosaccharide synthesis pathway, and it is necessary to introduce exogenous genes for these enzymes into the host cell, for any one enzyme, more than one copy (e.g., two, three or more copies) of the exogenous enzyme gene can be introduced into the host cell to enhance the function of the enzyme.

[0111] In some embodiments, to enhance the fucosyltransferase capacity of host cells, more than one copy (e.g., two, three, or more copies) of the gene for the α-1,2-fucosyltransferase or a variant thereof of the present invention may be introduced into the host cell. The more than one copy of the α-1,2-fucosyltransferase or a variant thereof may have the same sequence or different sequences. The more than one copy of the α-1,2-fucosyltransferase or a variant thereof may be contained in the same expression cassette or in different expression cassettes within the host cell. The more than one copy of the α-1,2-fucosyltransferase or a variant thereof may be located in a free vector (e.g., a plasmid, such as a multi-copy plasmid or containing multiple copies in a single plasmid) within the host cell or may be integrated separately into different sites in the host cell genome.

[0112] Methods for genetically modifying host cells are well known to those skilled in the art. Genetic modification techniques can be used to introduce free exogenous nucleic acid sequences (e.g., in the form of plasmids) into host cells, insert exogenous nucleic acid sequences or delete endogenous nucleic acid sequences into the host cell genome, or replace a segment of endogenous nucleic acid sequence in the host cell genome with a segment of exogenous nucleic acid sequence, in order to alter the genes of the host cell and thus change its phenotype.

[0113] Expressing a foreign protein (such as one or more enzymes mentioned above) in a host cell can include introducing a foreign gene into the host cell to express the protein encoded by that foreign gene. This can be achieved, for example, by introducing a nucleic acid sequence containing the foreign gene (e.g., a vector) into the host cell. The nucleic acid sequence containing the foreign gene can be linear or circular, and can be single-stranded or double-stranded. The vector can be a self-replicating vector. The vector can be a free vector or an integrative vector. The vector can be, for example, a plasmid vector, a phage vector, a bacterial artificial chromosome, a transposon-based vector, or a CRISPR / Cas-based vector. The vector can also be a suicide vector, such as a suicide plasmid vector.

[0114] The vector may contain a foreign gene expression cassette, which may include a foreign gene and a regulatory sequence operatively linked thereto, the regulatory sequence of which directs the expression of the foreign gene in a suitable host cell. The regulatory sequence may include, but is not limited to, promoters, enhancers, terminators, and other expression control elements. The promoter may be a constitutive promoter to enable sustained expression of the foreign gene, or an inducible promoter to induce expression of the foreign gene upon the addition of an inducer. The vector may also contain one or more selectable marker genes that allow convenient selection of transformed, transfected, or transduced cells, such as genes providing resistance to antibiotics or heavy metals and / or negative selection marker genes (e.g., the sacB gene).

[0115] The foreign gene, after being introduced into the host cell, can exist in and be expressed from a free vector (e.g., a free plasmid), for example, by introducing an expression vector (e.g., a plasmid vector) containing a foreign gene expression cassette into the host cell. The foreign gene can also be integrated into the host cell's genome for expression, for example, by introducing an integrative plasmid vector containing the foreign gene (the plasmid may, for example, contain homologous arms for integration into the host cell genome via homologous recombination), a phage vector, a CRISPR / Cas system, or a transposon system (e.g., the Piggybac or Sleeping Beauty system). The foreign gene can be randomly integrated into the host cell, or it can be integrated into a suitable site in the host cell (e.g., via homologous recombination or a CRISPR / Cas system). The suitable site can be, for example, a safe harbor site known in the art, or a deleted endogenous gene site in the host cell. In Corynebacterium glutamicum, exogenous genes can be integrated into the ISCg1 (e.g., ISCg1a, ISCg1b, ISCg1c and / or ISCg1e) and / or ISCg2 (e.g., ISCg2b, ISCg2c, ISCg2d, ISCg2e and / or ISCg2f) loci, cg0554 locus, poxB locus, and / or tnp21a (ISCg21a) locus in the Corynebacterium glutamicum chromosome. Optionally, these genomes can be knocked out and the exogenous gene introduced at the knockout site. The exogenous gene can be contained in an expression cassette, which is randomly or targetedly integrated into the host cell genome. Alternatively, the exogenous gene can be targeted and integrated into a suitable site in the host cell to express the exogenous gene using endogenous regulatory sequences within the host cell. In some embodiments, the exogenous gene can be codon-optimized for the host cell.

[0116] Methods for expressing multiple foreign genes in host cells are well known to those skilled in the art. For example, a separate transcription unit can be constructed for each foreign gene, each with its own promoter and capable of being transcribed into its own mRNA. Appropriate promoters can be selected for different genes as needed. As another example, two or more (e.g., two, three, or four) foreign genes can be included in the same transcription unit, sharing the same promoter. Each of the multiple foreign genes in the same transcription unit may have a ribosome binding site (RBS) to facilitate individual translation of each gene, with multiple genes transcribed and translated in a polycistronic manner. Multiple transcription units can be contained in different vectors or in the same vector. The transcription units can be introduced into host cells using the methods described above.

[0117] Appropriate methods for introducing exogenous nucleic acid sequences (e.g., vectors) into host cells are known to those skilled in the art, including but not limited to calcium phosphate transfection, protoplast fusion, electroporation, liposomes, lipid nanoparticles, microinjection, naked DNA or RNA (e.g., mRNA) transfection, plasmid vector transformation, phage vector transduction, etc.

[0118] The aforementioned gene-editing techniques can also be used to knock out or knock down endogenous genes, so that the host cell does not have the functional protein encoded by the endogenous gene or to reduce the activity of the protein encoded by the endogenous gene, for example, by deleting all or part of the endogenous gene sequence, mutating the endogenous gene, or inserting an exogenous sequence into the endogenous gene. These gene-editing techniques can also be used to change the regulatory sequence of the endogenous gene (e.g., promoter) to increase or decrease the expression of the endogenous gene. Those skilled in the art can select appropriate methods to gene-edit the host cell according to the host cell and the exogenous or endogenous nucleic acid sequence used (A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (1989) and Ausubel et al, eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1997)). In some implementations, partial or complete deletion of the endogenous gene can be achieved by introducing an integrative plasmid vector (which may contain homologous arms to integrate into a designated site in the host cell genome via homologous recombination) or a targeted genome editing technology (such as the CRISPR / Cas system) into the host cell, or by replacing or deleting part or all of the natural promoter to achieve partial or complete deletion of the natural promoter.

[0119] The present invention also provides a method for producing fucoidan oligosaccharides, comprising culturing the genetically modified host cells of the present invention under appropriate conditions to synthesize the fucoidan oligosaccharides. In some embodiments, the method further includes recovering the fucoidan oligosaccharides.

[0120] The recombinant Corynebacterium glutamicum can be cultured using any suitable culture medium, such as those well known to those skilled in the art. In some embodiments, the culture medium contains at least one carbon source, which may be selected from, but is not limited to, glucose, sucrose, glycerol, fructose, lactose, xylose, cellulose, molasses, corn syrup, galactose, methanol, acetic acid, lactic acid, pyruvate, and / or succinic acid. In some embodiments, the carbon source may be supplemented at appropriate times during the culture of the host cells.

[0121] The culture medium may contain substances capable of generating the donor substrate via a synthetic pathway within the host cell, acting as a precursor (e.g., a carbon source different from the donor substrate). In some embodiments, the host cell may utilize the aforementioned carbon source to generate GDP-L-fucose via an intracellular synthetic pathway. In some embodiments, the culture medium may contain fucose to enable host cells comprising a salvage pathway for GDP-L-fucose synthesis to utilize fucose to synthesize the donor substrate GDP-L-fucose.

[0122] The culture medium may contain a receptor substrate or a substance capable of producing the receptor substrate via a synthetic pathway within the host cell, acting as a precursor (e.g., a carbon source different from the receptor substrate). In some embodiments, the receptor substrate is lactose. In some embodiments, the precursor is a non-lactose carbon source (e.g., glucose or a carbon source capable of being converted to glucose by cellular metabolism, such as glucose, sucrose, glycerol, fructose, xylose, cellulose, molasses, corn syrup, galactose, methanol, acetic acid, lactic acid, pyruvate, succinic acid, or any other carbon source that can be converted to glucose by cellular metabolism). Correspondingly, the host cell used contains a transporter protein gene capable of transporting the receptor substrate into the cell, or contains a synthetic pathway gene capable of synthesizing the receptor substrate using the precursor. In some embodiments, the receptor substrate (e.g., lactose) or precursor may be supplemented into the culture medium at appropriate times during the culture of the host cell.

[0123] When a host cell contains a gene encoding one or more enzymes that is expressed in an inducible manner, an inducer is added during cell culture to induce the expression of the enzyme.

[0124] Host cell culture can be carried out in several ways: batch fermentation, where a closed culture system with a specific culture medium is used at the start of fermentation, and specific temperature, pressure, aeration, and other environmental conditions are used to optimize growth. No nutrients are added during cell culture, and no fermentation broth is released. Fed-batch fermentation, where nutrients are added intermittently or continuously during fermentation, but no fermentation broth is released. Continuous fermentation, where nutrients are continuously added and fermentation broth is continuously released, thus maintaining a constant volume of culture medium in the fermentation system. Host cell fermentation can also be a combination of two or three of the above methods.

[0125] The genetically modified host cells of this invention are cultured under conditions suitable for the production of fucoidan oligosaccharides. Suitable conditions include appropriate temperature, pH, dissolved oxygen levels, osmotic pressure, and other factors. Depending on the type of host cell, suitable conditions may vary, which can be readily determined by those skilled in the art.

[0126] Typically, the target fucosyl oligosaccharide can be naturally transported extracellularly by the host cell. In some embodiments, the production method further includes recovering the synthesized product, i.e., the fucosyl oligosaccharide, from the culture medium and / or from the host cell. The term "recovery" refers to isolating or further purifying the fucosyl oligosaccharide produced by the host cell of the present invention, separating it from other components in the host cell culture. The term "purification" refers to removing impurities and unwanted byproducts, such as cells, ions, salts, and sugars other than the desired fucosyl oligosaccharide.

[0127] The product can be recovered from the culture medium and / or from the host cells themselves. For example, the product can be recovered from the culture medium or the supernatant of the cell lysate. Cell lysis can be performed using chemical or physical methods known in the art.

[0128] Purification can be performed using techniques known to those skilled in the art. For example, the product can be purified from the culture medium using methods known to those skilled in the art, such as column chromatography with activated carbon, elution with a concentration gradient of ethanol, or size exclusion chromatography or ion exchange chromatography. Purity can be assessed by any known method, such as thin-layer chromatography or other electrophoresis or chromatography techniques commonly known in the art.

[0129] Terminology definition:

[0130] The term "gene" refers to the nucleotide sequence that encodes a gene product. The gene product can be a protein or ribonucleic acid.

[0131] The terms “nucleic acid,” “nucleic acid sequence,” or “polynucleotide” refer to a single- or double-stranded polymer of deoxynucleotide or ribonucleotide bases, including DNA or RNA, including linear or circular DNA or RNA.

[0132] The terms "peptide" and "protein" are used interchangeably, referring to polymers of amino acid residues. The enzyme in this invention is a protein capable of catalyzing a chemical reaction of its substrate.

[0133] The term "glycosyltransferase" refers to an enzyme that catalyzes the transfer of a monosaccharide moiety from an activated nucleotide monosaccharide ("glycosyl donor substrate") to a glycosyl acceptor substrate. It is responsible for the biosynthesis of disaccharides, oligosaccharides, and polysaccharides.

[0134] The term "glycosyltransfer reaction" refers to the reaction in which a monosaccharide moiety is transferred from an activated nucleotide monosaccharide ("glycosyl donor substrate") and linked to a glycosyl acceptor substrate by a glycosidic bond, catalyzed by glycosyltransferases.

[0135] The term "fucosyltransferase" refers to a polypeptide capable of catalyzing the transfer of fucose residues from a donor substrate to an acceptor substrate. The donor substrate for fucosyltransferases is typically GDP-L-fucose. Acceptor substrates include oligosaccharides, glycopeptides, glycoproteins, and glycolipids. Typically, fucose residues are transferred to, for example, N-acetylglucosamine residues, N-acetylgalactosamine residues, galactose residues, fucose residues, sialic acid residues, or glucose residues of oligosaccharides, or the sugar moiety of glycoproteins or glycolipids. The term "fucosyltransferase" here should be understood to include the wild-type fucosyltransferase and its functional variants, which are also capable of catalyzing the transfer of fucose residues from a donor substrate to an acceptor substrate, i.e., they also possess fucosyltransferase activity.

[0136] The term "α-1,2-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor substrate to form an α-1,2-glycosidic bond between the fucose residue and the sugar moiety of the acceptor substrate.

[0137] The term "α-1,3-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor substrate to form an α-1,3-glycosidic bond between the fucose residue and the sugar moiety of the acceptor substrate.

[0138] The term "α-1,3 / 4-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor substrate to form an α-1,3 / 4-glycosidic bond between the fucose residue and the sugar moiety of the acceptor substrate.

[0139] The term "donor substrate," in the context of transferring fucose residues from a donor substrate to an acceptor molecule, refers to a molecule containing fucose residues, typically an active molecule containing fucose residues, wherein the fucose is transferred to a specific acceptor substrate by a fucosyltransferase. In this invention, the donor substrate may be GDP-L-fucose.

[0140] The term "acceptor substrate" refers to a molecule that receives glycosyl groups from a donor substrate in a reaction catalyzed by glycosyltransferases.

[0141] The term "precursor" refers to a compound that serves as a starting material or intermediate in the biosynthetic pathway of a compound. These intermediates include exogenously added compounds or compounds produced endogenously within the cell.

[0142] The term "functionality," or the term "capability" when used to describe the activity or function of an enzyme, means that the enzyme will exhibit a specific activity or function under suitable reaction conditions. The enzyme may not exhibit this activity or function when suitable reaction conditions are not present, but it will exhibit it when suitable reaction conditions are met. Suitable reaction conditions include: the presence of a suitable donor substrate, the presence of a suitable acceptor molecule, the presence of necessary cofactors, a suitable pH range, and a suitable temperature, etc.

[0143] The term "variant" refers to a polypeptide that has one or more amino acid insertions, deletions, and / or substitutions relative to the parent polypeptide. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; insertion means adding one or more amino acids (e.g., 1-5) adjacent to an amino acid occupying a position. A variant retains at least one activity of the parent polypeptide, but may have variations at the activity level; for example, a variant may remain unchanged or improve upon at least one activity or property relative to the parent polypeptide.

[0144] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci USA 94:412-417 (1997); the full text of which is incorporated herein by reference).

[0145] The term "parent" refers to a polypeptide that has been modified to produce a variant, and the parent can be a naturally occurring (wild-type) polypeptide or a mutant thereof.

[0146] When applied to peptides or proteins, the term "functional variant" refers to a peptide that shares at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with its parent peptide and has the same or substantially the same function as the parent peptide. A functional variant may also refer to a peptide that has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acid insertions, deletions, and / or substitutions compared to its parent peptide and has the same or substantially the same function as the parent peptide.

[0147] The term "sequence identity" refers to the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences, when sequences are aligned to maximize sequence matching, taking into account gaps and insertions. Sequence alignment and the calculation of sequence identity percentages can be performed using appropriate computer programs known in the art. These programs include, but are not limited to, BLAST, ALIGN, ClustalW, EMBOSS Needle, etc. For example, global sequence alignment can be performed based on the Needleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D. (1970), "A general method applicable to the search for similarities in the amino acid sequence of two proteins", Journal of Molecular Biology 48(3): 443-53), which is available at http: / / www.ebi.ac.uk / Tools / psa / and can be performed using default parameters. Local alignment can be performed using BLAST (Basic Local Alignment Search Tool), which was first described in Altschul et al. (1990) J.Mol.Biol. 215; 403. Biol. 215; 403-410. The BLAST alignment tool is available from the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / / ), and default parameters can be used for alignment, for example.

[0148] The term "corresponding to..." refers to the position in the reference amino acid sequence that corresponds to a specific position in the reference amino acid sequence when the queried amino acid sequence is optimally aligned with the reference amino acid sequence. In this invention, the positions of the amino acids in the described α-1,2-fucosyltransferase variants are determined based on the amino acid sequence shown in SEQ ID NO:59, a reference amino acid sequence.

[0149] In this document, amino acids are represented by conventional single-letter or three-letter names. As is known to those skilled in the art, the single-letter names for amino acids are as follows: A for alanine; C for cysteine; D for aspartic acid; E for glutamic acid; F for phenylalanine; G for glycine; H for histidine; I for isoleucine; K for lysine; L for leucine; M for methionine; N for asparagine; P for proline; Q for glutamine; R for arginine; S for serine; T for threonine; V for valine; W for tryptophan; and Y for tyrosine.

[0150] In this paper, protein variants are represented as follows: original amino acid, position, substituted amino acid, written in the order of origin. For example, amino acid substitution can be represented as "Q258S", where 258 indicates that the substitution occurs at the 258th amino acid, the Q preceding 258 is the amino acid that occupied that position before the substitution, and the S following 258 is the amino acid that occupied that position after the substitution. Multiple mutations are connected by a plus sign (+) or a forward slash ( / ), for example, "Q258S / N64D" indicates that glutamine (Q) is substituted for serine (S) at position 258 and asparagine (N) is substituted for aspartic acid (D) at position 64.

[0151] The term "wild-type" refers to a naturally occurring organism or cell, or a nucleic acid sequence present in a naturally occurring organism or cell, or a protein expressed by a naturally occurring organism or cell.

[0152] The term "naturally occurring" refers to nucleic acid sequences, amino acid sequences, complexes, pathways, or cells that exist in nature without human intervention.

[0153] The term "derived from" when applied to a protein or gene sequence refers to a protein or gene sequence that is derived from a specific organism, meaning that the protein or gene sequence has the same structure or sequence as proteins or gene sequences naturally present in that organism, and is not limited to being directly isolated from that organism.

[0154] The term "vector" refers to a tool that allows or facilitates the transfer of nucleic acid fragments from one environment to another (such as a host cell). Vectors allow the insertion of another nucleic acid fragment to achieve replication of the inserted fragment.

[0155] The term "expression vector" refers to a vector used to express a gene product, which typically includes one or more expression control sequences for controlling and regulating the transcription and / or translation of a gene sequence that can express the product.

[0156] The term "expression cassette" refers to a nucleotide sequence containing relevant nucleic acids that are controlled by and operatively linked to appropriate promoters or other regulatory elements to enable transcription of the relevant nucleic acids in a host cell.

[0157] The term "transcription unit" refers to a nucleic acid sequence containing one or more genes to be transcribed. Genes within a transcription unit are operatively linked to each other in a manner such that all genes within the unit are under the transcriptional control of the same promoter and / or enhancer, thereby enabling the transcription of more than one protein or product.

[0158] The term "operable linking" refers to placing a regulatory sequence necessary for the expression of a coding sequence in the appropriate position within the DNA molecule relative to the coding sequence, thereby affecting the expression of the coding sequence.

[0159] The term "host cell" refers to any cell containing a foreign nucleic acid sequence. In this invention, the host cell is Corynebacterium glutamicum containing a foreign nucleic acid sequence.

[0160] The term "recombinant" when used to refer to cells, nucleic acids, proteins, or vectors indicates that the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells that have been modified in this way. Therefore, recombinant cells express genes that are not present in the natural (non-recombinant) form of the cell; or express natural genes that are otherwise abnormally, insufficiently, or not expressed at all.

[0161] The terms "genetic modification," "genetic alteration," "engineering," and "modification" refer to the artificial alteration of the sequence of polypeptides, polynucleotides, or the genetic sequence contained in a host cell to include sequences not naturally present in the polypeptide, polynucleotide, or host cell. When a host cell is "modified" to exhibit a certain characteristic (e.g., containing a gene encoding a specific protein, expressing a specific protein, or overexpressing a specific gene), it means that the host cell did not possess that characteristic before modification but acquires it after modification.

[0162] The term "synthetic pathway" refers to a series of reactions controlled and catalyzed by enzymes that result in the synthesis of a chemical substance.

[0163] The term "synthetic pathway gene" refers to the gene encoding an enzyme that catalyzes a series of reactions in a synthetic pathway. These genes can be expressed in the host cell, thereby catalyzing the synthesis of a specific chemical substance.

[0164] The term "exogenous" is relative to the host cell and refers to substances or molecules that originate from or are produced outside the host cell. "Exogenous gene" or "exogenous enzyme" refers to nucleic acids that are not naturally present in the cell but are introduced into the cell artificially. The sequence of an exogenous gene or exogenous enzyme may be the same as or different from the naturally occurring endogenous sequence in the cell. Genes or enzymes that differ from the naturally occurring endogenous sequence in the cell are called heterologous genes or heterologous enzymes, and they may originate from strains or species that are the same as or different from the host cell. In this text, unless otherwise stated, the use of "exogenous gene" or "exogenous enzyme" includes the case of "heterologous gene" or "heterologous enzyme."

[0165] The term "endogenous" refers to genes or proteins (e.g., wild-type genes or wild-type enzymes) or synthetic pathways that are naturally present in host cells.

[0166] The term "human milk oligosaccharides (HMO)," also known as human milk oligosaccharides, refers to carbohydrates composed of 3 to 10 monosaccharides linked by glycosidic bonds. It is the third most abundant solid component in breast milk, after fat and lactose. The basic structure of human milk oligosaccharides consists of five basic monosaccharides: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and N-acetylneuraminic acid (NeuAc or Neu5c).

[0167] The term "fucosyl oligosaccharide" refers to an oligosaccharide (such as a carbohydrate composed of 3 to 10 monosaccharides linked by glycosidic bonds) having fucose residues. This oligosaccharide is neutral. Exemplary fucosyl oligosaccharides include 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, lact-N-fucopentose (e.g., lact-N-fucopentose I, lact-N-fucopentose II, lact-N-fucopentose III, lact-N-fucopentose V), lact-N-fucohexose, lact-N-difucohexose I, fucosyllactose-N-hexose, fucosyllactose-N-neohexose, difucosyllactose-N-hexose I, difucosyllactose-N-neohexose II, etc. The fucosyl oligosaccharide in this invention can be human milk oligosaccharide, i.e., the fucosyl oligosaccharide contained in breast milk.

[0168] The term "intracellular" in this document refers to the reaction and product formation of the fucosyl oligosaccharide synthesis occurring within the same host cell. That is, the glycosyltransferase, donor substrate, and acceptor substrate used to synthesize the fucosyl oligosaccharide are provided within the same host cell, which has a complete synthetic pathway for the synthesis of the fucosyl oligosaccharide.

[0169] The term "extracellular" herein refers to the reaction for synthesizing fucosyl oligosaccharides, and the formation of the product, occurring in an environment outside the cell, wherein the glycosyltransferases, donor substrates, and acceptor substrates used to synthesize the fucosyl oligosaccharides can be provided by different cells, in the form of purified sugars and / or purified proteins, and / or in the form of cell lysate mixtures or cell extracts. When provided by cells, the glycosyltransferases, donor substrates, and / or acceptor substrates can be secreted or transported extracellularly.

[0170] The term "cell-free condition" in this document refers to a reaction for the synthesis of fucosyl oligosaccharides, the formation of the product, occurring in a cell-free environment, wherein the glycosyltransferases, donor substrates, and acceptor substrates used to synthesize the fucosyl oligosaccharides are provided in the form of purified sugars and / or purified proteins and / or in the form of cell-disrupted mixtures or cell extracts.

[0171] The term "knockout" refers to the manipulation of genes to prevent cells or organisms from producing the functional products encoded by those genes.

[0172] The term "knockdown" refers to the process of manipulating genes to reduce the functional activity of functional products encoded by said genes produced by cells or organisms.

[0173] The term "eliminating or weakening the effect of an enzyme" means that the reaction catalyzed by the enzyme is eliminated or weakened, which can be specifically manifested as the absence of reaction products catalyzed by the enzyme or a reduction in the amount of products generated by the reaction catalyzed by the enzyme.

[0174] The term "enhancing enzyme action" refers to the enhancement of the reaction catalyzed by the enzyme, which can be specifically manifested as an increase in the amount of product generated by the reaction catalyzed by the enzyme.

[0175] The term "enzyme activity" refers to an enzyme's ability to catalyze the conversion of a substrate into a product, which can be evaluated by the amount of product produced. The term "overexpression" refers to the expression level of a gene product or polypeptide in a host cell after genetic modification, which is higher than before the modification. "Overexpression" can also refer to any detectable expression caused by the introduction of a specific gene product into the host cell, even if the host cell did not contain the specific gene product before the modification.

[0176] The term "mannose-6-phosphate isomerase" refers to the enzyme that catalyzes the conversion of fructose-6-phosphate to mannose-6-phosphate. The mannose-6-phosphate isomerase found in *Escherichia coli* and *Corynebacterium glutamicum* can be referred to as ManA.

[0177] The term "phosphogannatase" refers to the enzyme that catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate. Phosphogannatase in *Escherichia coli* and *Corynebacterium glutamicum* can be referred to as ManB.

[0178] The term "mannose-1-phosphate guanosine transferase" refers to an enzyme that catalyzes the transfer of nucleotides from GTP to mannose-1-phosphate to produce GDP-mannose. In *Escherichia coli* and *Corynebacterium glutamate*, mannose-1-phosphate guanosine transferase may be referred to as ManC.

[0179] The term "GDP-mannose-4,6-dehydratase" refers to the enzyme that catalyzes the production of GDP-mannose from GDP-mannose to GDP-4-keto-6-deoxy-D-mannose. The GDP-mannose-4,6-dehydratase in *E. coli* may be referred to as EcGMD.

[0180] The term "GDP-L-fucose synthase" refers to the enzyme that catalyzes the production of GDP-L-fucose from GDP-4-keto-6-deoxy-D-mannose. In Escherichia coli, GDP-L-fucose synthase may be referred to as WcaG.

[0181] The term "fucokinase / GDP-L-fucosylationase bifunctional enzyme" refers to an enzyme that catalyzes the conversion of L-fucose to fuco-1-phosphate, which in turn further generates GDP-L-fucose. The fucokinase / GDP-L-fucosylationase bifunctional enzyme of *Bacteroides fragilis* can be referred to as Fkp.

[0182] The terms "reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose" and "GDP-4-keto-6-deoxy-D-mannose reductase" both refer to enzymes capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose. The term "reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose" also includes DdahC derived from Campylobacter jejuni.

[0183] The term "GDP-D-rhamnose-3,5-epimerase" refers to the enzyme that catalyzes the conversion of GDP-D-rhamnose to GDP-L-fucose. This can include GDP-mannose-3,5-epimerase (GME). Specifically, "GDP-mannose-3,5-epimerase" refers to the enzyme that catalyzes the conversion of GDP-D-mannose to GDP-L-fucose. GDP-mannose-3,5-epimerase derived from rice can be referred to as OsGME.

[0184] The term "β-galactosidase" refers to an enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. In Escherichia coli, β-galactosidase can be referred to as LacZ.

[0185] The term "UDP-glucose lipotransferase" refers to the enzyme that converts GDP-L-fucose into colacid. The UDP-glucose lipotransferase in *E. coli* can be referred to as WcaJ.

[0186] The term "lactose transporter" refers to any protein expressed in microorganisms that can transport lactose across the cell membrane. The lactose permease in *E. coli* can also be called LacY.

[0187] It should be understood that for enzymes with specific activities, the sequences of homologous proteins in different organisms (including different species or different strains of the same species) are readily available to those skilled in the art, for example, by searching public databases (e.g., GenBank).

[0188] Enzymes and their encoding genes with the same biological activity are known in the art to have different names, sometimes related to the microorganism from which the enzyme originates. The enzymes described herein are intended to cover enzymes with the defined functions, and unless otherwise stated, should cover enzymes with the defined functions from any organism (including microorganisms, animals, etc.).

[0189] In this invention, it should be understood that when an abbreviation is shown in parentheses after the name of an enzyme, the abbreviation should not be regarded as a limitation on a specific sequence or specific source of the enzyme, but is merely an example for reference and understanding.

[0190] Unless otherwise stated, nucleic acids are written from left to right in the 5' to 3' direction in this document, and amino acid sequences are written from left to right in the direction from the amino terminus to the carboxyl terminus.

[0191] The present invention is further described through the following embodiments, which should not be construed as limiting the present invention.

[0192] Unless otherwise specified, all reagents used in the following examples are commercially available products. Molecular biology experimental methods not specifically described in the examples were performed according to the specific methods listed in J. Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition, or according to the kit and product instructions.

[0193] Example 1: Culture and Detection of Corynebacterium glutamicum

[0194] The strain was cultured using two different culture media: seed medium LBHI and fermentation medium FM20.

[0195] The seed culture medium LBHI consisted of the following components: yeast extract 10 g / L, peptone 5 g / L, NaCl 10 g / L, and brain heart infusion (BHI) 18.5 g / L.

[0196] The seed culture medium LBHI+K15 (also known as LBHI+kan) consists of the following components: LBHI medium and kanamycin 15 mg / L.

[0197] The components of the LBHI+sucrose medium are as follows: LBHI medium, sucrose 10g / L.

[0198] The fermentation medium FM20 consists of the following components: glucose 30 g / L, lactose 20 g / L, peptone 4 g / L, yeast extract 2 g / L, (NH4)2SO4 10 g / L, urea 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4 0.25 g / L, MOPS 42 g / L, CaCl2 10 mg / L, biotin 0.2 mg / L, protocatechuic acid 0.03 mg / L, FeSO4·7H2O 10 mg / L, MnSO4·H2O 10 mg / L, ZnSO4·7H2O 1 mg / L, CuSO4 0.2 mg / L, NiCl2·6H2O 0.02 mg / L, pH 7.0.

[0199] The seed culture medium was sterilized by autoclaving (121°C, 20 min), and the fermentation medium was sterilized by filtration (0.22 μm filter membrane). If necessary, antibiotics (e.g., kanamycin) were added to make the medium selective. An additional 2% agar was added to the medium when preparing solid plates.

[0200] During fermentation, colonies from the agar plate were picked and cultured overnight in seed culture medium. After overnight culture, the strain was inoculated into 96-well plates containing fermentation medium at a 5% inoculum rate. The wells were incubated at 30°C, 900 rpm, and 80% humidity for 48 hours. The whole culture was then incubated at 80°C for 15 minutes, centrifuged, and the supernatant was collected to determine the product titer of the whole culture. Alternatively, the wells were centrifuged, and the supernatant was collected to determine the product titer in the supernatant after incubation.

[0201] Product titer detection: The titer was determined by HPLC-RID analysis using an Agilent system. The concentration of the substance was determined by analyzing the change in refractive index of the mobile phase as the sample passed through. An Agilent Hilic-Z 4μm, 4.6*250mm column and mobile phase were used to separate all sugars at isocratic flow rates. The mobile phase consisted of 700mL acetonitrile, 300mL ultrapure water, 0.23g ammonium acetate, and 1.5mL 30% ammonia solution. The flow rate was 0.8mL / min, the injection volume was 3μl, the column temperature was 35℃, and the differential detector temperature was 35℃.

[0202] Genetic modification of Corynebacterium glutamicum: The sacB modification method was used. Specifically, a pK18mobsacB-based plasmid was used, and homologous recombination was performed using a sucrase encoded by the Bacillus subtilis sacB gene as an anti-selection marker. This enzyme catalyzes the hydrolysis of sucrose and the synthesis of high-molecular-weight fructose polymers called levans. When the sacB gene is expressed in Corynebacterium glutamicum, the strain cannot grow in sucrose-containing media. The genetic modification procedures were performed according to the reference (Lothar Eggeling and Michael Bott, Handbook of Corynebacterium glutamicum, 1st Edition, 2005).

[0203] Kanamycin resistance and sacB gene reverse screening method for recombinant bacteria (also referred to as the sacB reverse screening method in this paper): Exogenous plasmids were electroporated into the bacterial strain and plated on LBHI+Kan plates. Clones selected from the LBHI+Kan plates were cultured overnight in antibiotic-free LBHI. The overnight culture was then plated onto LBHI+sucrose plates and cultured until clones formed. Clones grown on LBHI+sucrose plates were spotted onto antibiotic-free LBHI plates and LBHI+Kan plates. Clones that did not grow on LBHI+Kan plates but grew on antibiotic-free LBHI plates were confirmed by PCR using primers.

[0204] Example 2: Construction of a recombinant strain of Corynebacterium glutamicum manBC-gmdwcaG

[0205] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, homologous arms manB-Up and manB-Down were obtained by PCR amplification using primer pairs manB-UF / manB-UR and manB-DF / manB-DR (PCR system used: 2×Phanta Max Master Mix (Dye Plus), Vazyme). Plasmid pK18mobsacB was digested with restriction endonucleases EcoRI and XbaI to obtain the linearized fragment line-pK18mobsacB. The above three fragments, manB-Up, manB-Down, and line-pK18mobsacB, were seamlessly assembled using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, catalog number: C113-02). The reaction system and reaction conditions were performed according to the kit instructions. After seamless assembly, the cells were transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-manB. The validated plasmid was electroporated into Corynebacterium glutamicum ATCC13032. Kanamycin resistance and sacB gene reverse screening were used to replace the sequence “TAGGAT” in the -10 region of the manB promoter with “TATAAT” and the sequence “TTCGGA” in the -35 region of the manB promoter with “TTGGCA” to obtain the strain manB with modified -10 and -35 regions of the manB promoter.

[0206] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, homologous arms manC-Up and manC-Down were obtained by PCR amplification using primer pairs manC-UF / manC-UR and manC-DF / manC-DR (PCR system used: 2×Phanta Max Master Mix (Dye Plus), Vazyme). Plasmid pK18mobsacB was digested with restriction endonucleases EcoRI and XbaI to obtain the linearized fragment line-pK18mobsacB. The above three fragments, manC-Up, manC-Down, and line-pK18mobsacB, were seamlessly assembled using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, catalog number: C113-02). The reaction system and reaction conditions were performed according to the kit instructions. After seamless assembly, the cells were transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-manC. The validated plasmid was electroporated into the previously obtained recombinant Corynebacterium glutamicum strain manB. Kanamycin resistance and sacB gene reverse screening were used to replace the sequence “TAAAGT” in the manC promoter-10 region with “TATAAT”, resulting in the strain manBC with the modified manC promoter-10 region.

[0207] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using primer pairs gmdwcaG-UF / gmdwcaG-UR, gmdwcaG-DF / gmdwcaG-DR, and Ptuf-F / Ptuf-R to obtain the upstream homologous arms gmdwcaG-Up, gmdwcaG-Down, and the promoter Ptuf (PCR system used 2×Phanta Max Master Mix, (Dye Plus), Vazyme); using the genome of Escherichia coli MG1655 as a template, PCR amplification was performed using primer pair gmd-F / wcaG-R to obtain the gene fragment gmdwcaG (PCR system used 2×Phanta Max Master Mix, (Dye Plus), Vazyme). Plus), Vazyme); plasmid pK18mobsacB was digested with restriction endonucleases EcoRI and XbaI to obtain the linearized fragment line-pK18mobsacB; the above five fragments: gmdwcaG-Up, gmdwcaG-Down, Ptuf, gmdwcaG, and line-pK18mobsacB were seamlessly assembled using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, catalog number: C113-02). The reaction system and reaction conditions were performed according to the kit instructions. After seamless assembly, Trans1 T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-gmdwcaG. The validated plasmid was electroporated into the previously obtained Corynebacterium glutamicum recombinant strain manBC. Kanamycin resistance and sacB gene back-screening were used to further obtain the basic strain manBC-gmdwcaG with exogenous gmd and wcaG. The polypeptide sequence of Gmd is SEQ ID NO:61, and the gene sequence is SEQ ID NO:62. The polypeptide sequence of WcaG is SEQ ID NO:63, and the gene sequence is SEQ ID NO:64.

[0208] Table 2

[0209] Example 3 Construction of α-1,2-fucosyltransferase-lactose permease expression plasmid

[0210] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, the promoter Psod (SEQ ID NO:71) was amplified by PCR using primer pair Psod-F / Psod-R. The PCR system used was 2×Phanta Max Master Mix (Dye Plus) and Vazyme. Using the genome of Escherichia coli MG1655 as a template, the lactose permease gene fragment LacY was amplified by PCR using primer pair LacY-F / LacY-R (PCR system used was 2×Phanta Max Master Mix (Dye Plus) and Vazyme). Plasmid pJC1 was digested with restriction endonucleases BamHI and SalI to obtain the linearized fragment line-pJC1. The above three fragments: Psod, LacY, and line-pJC1 were used in a recombinant cloning kit (ClonExpress Multis One Step Cloning). The recombinant plasmid pJC1-Psod_LacY was seamlessly assembled using the Vazyme Kit (catalog number: C113-02). The reaction system and conditions were performed according to the kit's instructions. After seamless assembly, the plasmid was transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pJC1-Psod_LacY. The polypeptide sequence of LacY is SEQ ID NO:65, and the gene sequence is SEQ ID NO:66.

[0211] By mining α-1,2-fucosyltransferase from databases, the following polypeptide sequences were obtained: Helicobacter pylori 26695-1MET (SEQ ID NO:1), Campylobacter sp. MIT 12-5580 (SEQ ID NO:3), Bacteroides fragilis (SEQ ID NO:5), Escherichia coli O126 (SEQ ID NO:7), Helicobacter japonicus (SEQ ID NO:9), Helicobacter trogontum (SEQ ID NO:11), Poseidonibacter sp. SJOD-M-5 (SEQ ID NO:13), and Pedobacter sp.The polypeptide sequences of BS3 (SEQ ID NO:15), Christiangramia sabulilitoris (SEQ ID NO:17), Campylobacter hyointestinalis (SEQ ID NO:19), Cupriavidus plantarum (SEQ ID NO:21), Helicobacter himalayensis (SEQ ID NO:23), Herbaspirillum rubrisubalbicans (SEQ ID NO:25), Bacteroides caccae (SEQ ID NO:27), Campylobacter lari (SEQ ID NO:29), and Pseudomonas fluorescens (SEQ ID NO:15) are listed. The following polypeptide sequences are listed: NO:31), SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:46.The polypeptide sequences are as follows: (SEQ ID NO:47), (SEQ ID NO:49), (SEQ ID NO:51), (SEQ ID NO:53), (SEQ ID NO:55), (SEQ ID NO:57), (SEQ ID NO:58), and (SEQ ID NO:59). Without altering the amino acid sequence of the polypeptide, the gene sequence encoding the polypeptide was replaced with codons preferred by *Bacillus glutamate*. After codon optimization, the resulting gene sequences are SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, and SEQ ID NO:60.

[0212] The promoter Pcg2195 sequence was added before all α-1,2-fucosyltransferase gene sequences to synthesize the genes. The gene synthesis vector used was the pJC1-Psod_LacY plasmid, and the gene synthesis sequence was located after the restriction site BamHI. The recombinant plasmids were named pJC1-FucT1-LacY, pJC1-FucT2-LacY, pJC1-FucT3-LacY, pJC1-FucT4- LacY, pJC1-FucT5-LacY, pJC1-FucT6-LacY, pJC1-FucT7-LacY, pJC1-FucT8-LacY , pJC1-FucT9-LacY, pJC1-FucT10-LacY, pJC1-FucT11-LacY, pJC1-FucT12-LacY, pJC1-FucT13-LacY, pJC1-FucT14-LacY, pJC1-FucT15-LacY, pJC1-FucT16-LacY, p JC1-FucT17-LacY, pJC1-FucT18-LacY, pJC1-FucT19-LacY, pJC1-FucT20-LacY, pJC1-FucT21-LacY, pJC1-FucT22-LacY, pJC1-FucT23-LacY, pJC1-FucT24-LacY, pJC1-FucT25-LacY, pJC1-FucT26-LacY, pJC1-FucT27-LacY, pJC1-FucT28-LacY, pJC1-FucT29-LacY, pJC1-FucT30-LacY (the recombinant plasmids correspond one-to-one with the aforementioned α-1,2-fucosyltransferase and its gene sequences in the order they are written).

[0213] Table 3

[0214] Example 4: Construction of a recombinant strain of Corynebacterium glutamicum to synthesize 2'-fucosylated lactose

[0215] The recombinant plasmids pJC1-FucT1-LacY, pJC1-FucT2-LacY, pJC1-FucT3-LacY, pJC1-FucT4-LacY, pJC1-FucT5-LacY, pJC1-FucT6-LacY, pJC1-FucT7-LacY, pJC1-FucT8-LacY, pJC1-FucT9-LacY, pJC1-FucT10-LacY, pJC1-FucT11-LacY, pJC1-FucT12-LacY, pJC1-FucT13-LacY, and pJC1-FucT14-LacY were synthesized from the above genes. pJC1-FucT15-LacY, pJC1-FucT16-LacY, pJC1-FucT17-LacY, pJC1-FucT18-LacY, pJC1-FucT19-LacY, pJC1-FucT20-LacY, pJC1-FucT21-LacY, pJ C1-FucT22-LacY, pJC1-FucT23-LacY, pJC1-FucT24-LacY, pJC1-FucT25-LacY, pJC1-FucT26-LacY, pJC1-FucT27-LacY, pJC1-FucT28-LacY, pJC1- FucT29-LacY and pJC1-FucT30-LacY were electroporated into the aforementioned basic strain manBC-gmdwcaG, respectively, to obtain recombinant Corynebacterium glutamicum strains Cg2FL-FucT1, Cg2FL-FucT2, Cg2FL-FucT3, Cg2FL-FucT4, Cg2FL-FucT5, Cg2FL-FucT6, Cg2FL-FucT7, Cg2FL-FucT8, Cg2FL-FucT9, Cg2FL-FucT10, Cg2FL-FucT11, Cg2FL-FucT12, and Cg2FL-FucT13 that synthesize 2'-fucosylated lactose. Cg2FL-FucT14, Cg2FL-FucT15, Cg2FL-FucT16, Cg2FL-FucT17, Cg2FL-FucT18, Cg2FL-FucT19, Cg2FL-FucT20, Cg2FL-FucT21, Cg2FL-FucT22, Cg2FL-FucT23, Cg2FL-FucT24, Cg2FL-FucT25, Cg2FL-FucT26, Cg2FL-FucT27, Cg2FL-FucT28, Cg2FL-FucT29, Cg2FL-FucT30 (the recombinant strains correspond one-to-one with the aforementioned recombinant plasmids in the order they are written).

[0216] The recombinant strains Cg2FL-FucT1, Cg2FL-FucT2, Cg2FL-FucT3, Cg2FL-FucT4, Cg2FL-FucT5, Cg2FL-FucT6, Cg2FL-FucT7, Cg2FL-FucT8, Cg2FL-FucT9, Cg2FL-FucT10, Cg2FL-FucT11, Cg2FL-FucT12, Cg2FL-FucT13, Cg2FL-FucT14, Cg2FL-FucT15, Cg2FL-FucT16, and Cg2FL-FucT1 were stored at -80℃. Glyceryl bacteria 2FL-FucT17, Cg2FL-FucT18, Cg2FL-FucT19, Cg2FL-FucT20, Cg2FL-FucT21, Cg2FL-FucT22, Cg2FL-FucT23, Cg2FL-FucT24, Cg2FL-FucT25, Cg2FL-FucT26, Cg2FL-FucT27, Cg2FL-FucT28, Cg2FL-FucT29, and Cg2FL-FucT30 were inoculated into seed culture medium and cultured and analyzed according to the method in Example 1. The results are shown in Table 4 below.

[0217] Table 4

[0218] Example 5: Construction of a recombinant strain of Corynebacterium glutamicum α-1,2-fucosyltransferase mutant

[0219] Currently, α-1,2-fucosyltransferases generally suffer from poor protein soluble expression and low activity. Poor protein soluble expression can be partly attributed to their poor folding stability, and sequence-based consensus analysis has been widely used to improve protein stability. Consensus analysis compares the target protein sequence with a large number of homologous proteins from different sources. If a certain amino acid is relatively conserved in other homologous proteins but differs from this conserved amino acid in the target protein, then that site is mutated to a more conserved amino acid.

[0220] In this embodiment, the sequence conservation of 20 α-1,2-fucosyltransferases with clear catalytic functions was first analyzed. Combined with the fermentation results mentioned above, non-conserved amino acid residues in the α-1,2-fucosyltransferase CufutC of Cg2FL-FucT30 (i.e., wild-type (WT) α-1,2-fucosyltransferase derived from Ureaplasma urealyticum) were screened from an evolutionary perspective and mutated into conserved residues. Thus, 20 mutation sites were identified, and the specific mutation details are shown in Table 5 below.

[0221] Table 5

[0222] To further enhance the protein activity of CuFutC, amino acids from position 56 to 66, located near the protein's catalytic cavity, were also mutated. Mutants with different mutation combinations were used to construct α-1,2-fucosyltransferase-lactose permease expression plasmids according to the methods described in Examples 3 and 4 above. These plasmids were then electroporated into the basic strain manBC-gmdwcaG obtained in the aforementioned examples, cultured, and analyzed. The following mutant combinations showed improved performance. "Percentage increase in yield relative to WT sequence" refers to the percentage increase in 2'-FL yield relative to Cg2FL-FucT30 in Table 4.

[0223] Table 6

[0224] Example 6: Application of the transferase mutant in strains with a novel 2'-fucosylated lactose synthesis pathway.

[0225] Patent application CN 202410437877.0 (which is incorporated herein by reference in its entirety) discloses a novel pathway for the synthesis of 2'-fucosylated lactose, which allows the synthesis of GDP-L-fucose in cells via a novel pathway different from the conventional de novo synthesis of GDP-L-fucose, followed by the synthesis of 2'-fucosylated lactose from GDP-L-fucose and lactose. This novel pathway for the synthesis of GDP-L-fucose involves: the synthesis of GDP-4-keto-6-deoxy-D-mannose from GDP-D-mannose via GDP-D-mannose-4,6-dehydratase (GMD); the subsequent synthesis of GDP-D-rhamnose from GDP-4-keto-6-deoxy-D-mannose via a reductase capable of converting GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose; and the final synthesis of GDP-L-fucose from GDP-D-rhamnose via GDP-D-rhamnose-3,5-epimerase (GRE). A recombinant Corynebacterium glutamicum strain, Cg2FL-11, was constructed for the production of 2'-fucosyllactose. The construction method of Cg2FL-11 is as follows:

[0226] The gene sequences of α-1,2-fucosyltransferase HpfutC from Helicobacter pylori (SEQ ID NO:2, amino acid sequence of the enzyme is SEQ ID NO:1) and lactose permease LacY from Escherichia coli (SEQ ID NO:66, amino acid sequence of the enzyme is SEQ ID NO:65) were synthesized. The gene synthesis vector used was the pUC57 plasmid, and the recombinant plasmids were named pUC57-HpfutC and pUC57-LacY, respectively.

[0227] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using primer pairs HpfutC-lacY-UF / HpfutC-lacY-UR, HpfutC-lacY-DF / HpfutC-lacY-DR, Psod-F-1 / Psod-R-1, and Pcg2195-F-1 / Pcg2195-R-1 to obtain homologous arms HpfutC-lacY-Up, HpfutC-lacY-Down, promoter Psod (SEQ ID NO:71), and Pcg2195 (SEQ ID NO:72). (The PCR system used was 2×Phanta Max Master Mix, (Dye) Using pUC57-LacY as a template, the lactose permease LacY gene fragment was amplified by PCR using primer pair LacY-F / LacY-R (PCR system used: 2×Phanta Max Master Mix, (Dye Plus), Vazyme); using pUC57-HpfutC as a template, the α-1,2-fucosyltransferase HpfutC gene fragment was amplified by PCR using primer pair HpfutC-F / HpfutC-R; plasmid pK18mobsacB was digested with restriction endonucleases EcoR I and Xba I to obtain the linearized fragment line-pK18mobsacB. The seven fragments mentioned above—HpfutC-lacY-Up, Pcg2195, HpfutC, Psod, LacY, HpfutC-lacY-Down, and line-pK18mobsacB—were seamlessly assembled using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, catalog number: C113-02). The reaction system and conditions were performed according to the kit instructions. After seamless assembly, the plasmid was transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-HpfutC-lacY. The validated plasmid was electroporated into Corynebacterium glutamicum ATCC 13032. Using kanamycin resistance and sacB gene reverse screening, genes encoding HpfutC and LacY were introduced into the poxB site of the Corynebacterium glutamicum ATCC 13032 genome. To ensure that the strain has sufficient fucosyltransferase capacity, the α-1,2-fucosyltransferase HpfutC gene sequence was inserted into the cg0554 and tnp2b sites of the Corynebacterium glutamicum genome in a similar manner.Based on this, the same sacB gene reverse screening method was used to replace the sequence “TAGGAT” in the promoter-10 region of the phosphogannatase ManB with “TATAAT”, and the sequence “TAAAGT” in the promoter-10 region of the mannose-1-phosphate guanylate transferase ManC with “TATAAT”, in order to increase the supply of the 2'-FL precursor GDP-L-fucose. This resulted in the basic strain HpfutC-LacY-ManB-ManC.

[0228] Table 7

[0229] The reductase DdahC (GenBank: AAY17132.1) derived from Campylobacter jejuni was synthesized. Its amino acid sequence is SEQ ID NO:67, its gene sequence is SEQ ID NO:68, its gene synthesis vector is pUC57, and its recombinant plasmid is named pUC57-11.

[0230] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using primers listed in Table 8 for Pgap-F-2 / Pgap-R-2, Pcg2195-F-2 / Pcg2195-R-2, and Psod-F-2 / Psod-R-2 to obtain promoters Pgap (SEQ ID NO:73), Pcg2195 (SEQ ID NO:72), and Psod (SEQ ID NO:71) (PCR system used was 2×Phanta Max Master Mix, (Dye Plus), Vazyme). Using the genome of *Escherichia coli* MG1655 as a template, PCR amplification was performed using primers shown in Table 8 for EcGMD-F and EcGMD-R to obtain the gene fragment encoding GDP-D-mannose-4,6-dehydrase EcGMD (SEQ ID NO:61, gene sequence of which is SEQ ID NO:62). Using pRSFDuet-OsGME as a template, PCR amplification was performed using primers shown in Table 8 for OsGME-F and OsGME-R to obtain the gene fragment encoding GDP-D-rhamnose-3,5-epimerase OsGME (SEQ ID NO:69, gene sequence of which is SEQ ID NO:70). Using pUC57-11 as a template, PCR amplification of the gene fragment encoding DdahC was performed using the corresponding primer pairs 11-F-1 / 11-R-1 shown in Table 8 (PCR system used 2×Phanta Max Master Mix, (Dye Plus), Vazyme); plasmid pJC1 was digested with restriction endonucleases BamHI and SalI to obtain the linearized fragment line-pJC1. The gene fragments Pgap, Pcg2195, Psod, EcGMD, OsGME, line-pJC1, and DdahC were seamlessly assembled using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, catalog number: C113-02). The reaction system and conditions were performed according to the kit instructions. After seamless assembly, the cells were transformed into Trans1 T1 competent cells to obtain the recombinant plasmid: pJC1-Pgap_EcGMD-Pcg2195_11-Psod_OsGME, which contains the following elements: Pgap, Pcg2195, Psod, EcGMD, OsGME, line-pJC1, and DdahC.

[0231] Table 8

[0232] The recombinant plasmid was electroporated into the basic strain HpfutC-LacY-ManB-ManC to obtain the recombinant strain Cg2FL-11 of Corynebacterium glutamicum.

[0233] The recombinant strain Cg2FL-11 of Corynebacterium glutamicum was cultured and analyzed, and its 2'-FL yield reached 78 mg / L. The molecular weight of the generated 2'-FL was determined to be 511 [M+Na] by LC-MS analysis. + The retention time and molecular weight were consistent with the standard, thus confirming that the aforementioned metabolic pathway can achieve the synthesis of 2'-FL.

[0234] Furthermore, a recombinant Corynebacterium glutamicum control strain without GDP-D-rhamnosyl-3,5-epimerase was constructed. Using the aforementioned pUC57-11 as a template, PCR amplification was performed on 11-F-2 / 11-R-2 using the primers in the table above to obtain the DdahC gene (PCR system using 2×Phanta Max Master Mix, (Dye Plus), Vazyme). Using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, catalog number: C113-02), the gene of DdahC was seamlessly assembled with the previously obtained Pgap, Pcg2195, EcGMD, and line-pJC1 gene fragments. The reaction system and reaction conditions were performed in accordance with the kit instructions. After seamless assembly, the cells were transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pJC1-Pgap_EcGMD-Pcg2195_11, which was then electroporated into the previously obtained basic strain HpfutC-LacY-ManB-ManC to obtain the control recombinant strain Cg2FL-26.

[0235] Culture and analysis of the recombinant strain Cg2FL-26 of Corynebacterium glutamicum did not reveal its ability to produce 2'-FL, indicating that Ddahc is a monofunctional protein with only reducing activity and no isomerizing activity towards GDP-4-keto-6-deoxy-D-mannose. In this case, GME is essential for the production of 2'-FL.

[0236] In the recombinant strain Cg2FL-11 constructed above, lacY and HpfutC were first knocked out using the sacB reverse screening method. Then, the pJC1-FucT1-LacY and pJC1-FucT30-LacY strains constructed in the previous examples, as well as the expression plasmid carrying the mutated FucT30, were electroporated to obtain the corresponding modified strains. Culture and supernatant product determination were performed as described in Example 1. The relative changes in the product titer of 2'-fucosylated lactose are shown in Table 9 below. The relative changes in product titer were determined as follows: the 2'-FL product titer of NCg2FL-Fuct1 was taken as 100%, and the 2'-FL product titers of the other strains were divided by the product titer of NCg2FL-Fuct1 and then multiplied by 100%.

[0237] Table 9

[0238] Example 7: Methods for Cultivating and Modifying Escherichia coli

[0239] Culture media: The strains were cultured using two different culture media, namely ordinary medium LB and fermentation medium FM21.

[0240] LB medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L. Add 15 g / L agar powder to the solid medium. If antibiotics are used for screening, the corresponding antibiotic concentrations are kanamycin 50 mg / L and spectinomycin 50 mg / L.

[0241] Fermentation medium FM21: glycerol 20.0 g / L, lactose 10 g / L, Na2HPO4 12.8 g / L, K2HPO4 3.0 g / L, NH4Cl 2.0 g / L, NaCl 0.5 g / L, MgSO4·7H2O 0.25 g / L, CaCl2·2H2O 14.7 mg / L, thiamine hydrochloride 10.0 mg / L, trace metal solution 1.0 mL / L (FeCl3·6H2O 25.0 g / L, CaCl2·2H2O 2.3 g / L, ZnCl2 2.6 g / L, Na2MoO4·2H2O 2.6 g / L, CuSO4·5H2O 2.0 g / L, MnSO4·H2O 2.5 g / L, boric acid 0.7 g / L), pH 6.8.

[0242] Genetic modification method: The strain was modified by constructing relevant plasmids and performing corresponding genetic operations using the CRISPR-Cas9 technology described in the relevant literature published by Sheng Yang et al. (Acta. Biochim. Biophys. Sin. 2021, 53(5):620-627) and (Appl. Environ. Microbiol. 2016, 82(12):3693).

[0243] When conducting fermentation tests, colonies from the plates were picked and cultured overnight in seed culture medium. After overnight culture, the strain was inoculated into 96-well plates containing fermentation medium at a 5% inoculum rate. The plates were cultured at 37°C, 900 rpm, and 80% humidity for 48 hours. The entire culture was then incubated at 80°C for 15 minutes, centrifuged, and the supernatant was collected to determine the product titer. The product titer determination was performed according to Example 1.

[0244] Example 8: Application of the transferase mutant in the production of 2'-fucosylated lactose in Escherichia coli

[0245] Using the method described in Example 7, the gene cluster encoding lactose utilization-related gene LacZYA and the gene encoding UDP-glucose lipotransferase WcaJ in the *E. coli* MG1655 genome were first knocked out. Then, the modified strains were obtained by electroporation into the pJC1-FucT1-LacY, pJC1-FucT30-LacY, and expression plasmids carrying the mutated FucT30 constructed in the previous examples. Culture and supernatant product analysis were performed as described in Example 7. The relative changes in the product titer of 2'-fucosylated lactose are shown in Table 10 below.

[0246] Table 10

[0247] Example 9: Application of the transferase mutant in the production of 2'-fucosylated lactose in Escherichia coli using a novel pathway.

[0248] Patent application number CN 202410437877.0 (the entire text of which is incorporated herein by reference) discloses a novel route for the synthesis of 2'-fucosylated lactose.

[0249] A recombinant Escherichia coli strain, Ec2FL-1, was constructed for the production of 2'-fucosylated lactose.

[0250] The construction method of strain Ec2FL-1 is as follows:

[0251] Gene knock-in and gene knockout related plasmids were constructed using the relevant technology published by Sheng Yang et al. (Acta. Biochim. Biophys. Sin. 2021, 53(5): 620-627). The required knockout and knock-in genes are as follows. Using the CRISPR-Cas9 technology published by Sheng Yang et al. (Appl. Environ. Microbiol. 2016, 82(12):3693), the gene encoding β-galactosidase LacZ in the genome of Escherichia coli (E. coli) MG1655 was knocked out, and the α-1,2-fucosyltransferase HpfutC gene (SEQ ID NO:2) and the lactose permease LacY gene (SEQ ID NO:66) were integrated at the knockout site. The gene encoding GDP-L-fucose synthase WcaG (SEQ ID NO:63, its gene sequence is SEQ ID NO:64) in the genome of E. coli (E. coli) MG1655 was knocked out using CRISPR-Cas9 technology, and the gene encoding GDP-mannose-3,5-epimerase OsGME (SEQ ID NO:64) was integrated at the knockout site. NO:70); The gene encoding UDP-glucose lipotransferase WcaJ in the genome of Escherichia coli MG1655 was knocked out using CRISPR-Cas9 technology, and the gene encoding GDP-4-keto-6-deoxy-D-mannose reductase DdahC (SEQ ID NO:67) was integrated into the knockout site to obtain the 2'-FL producing strain Ec2FL-1.

[0252] In strain Ec2FL-1, the fucosyltransferase futC, originally integrated into the chromosome by Helicobacter pylori, was knocked out using the method described in Example 7. Then, the modified strains were obtained by electroporation into the pJC1-FucT1-LacY, pJC1-FucT30-LacY, and expression plasmids carrying the mutated FucT30 constructed in the aforementioned examples. Culture and supernatant product analysis were performed as described in Example 7. The relative changes in the product titer of 2'-fucosyllactose are shown in Table 11 below.

[0253] Table 11

[0254] Partial sequences used in the examples:

[0255] The polypeptide sequence of α-1,2-fucosyltransferase derived from Helicobacter pylori 26695-1 MET (SEQ ID NO:1)

[0256] The gene sequence of α-1,2-fucosyltransferase derived from Helicobacter pylori 26695-1 MET (SEQ ID NO:2)

[0257] The polypeptide sequence of α-1,2-fucosyltransferase derived from Campylobacter sp. MIT 12-5580 (SEQ ID NO:3)

[0258] The gene sequence of α-1,2-fucosyltransferase derived from Campylobacter sp. MIT 12-5580 (SEQ ID NO:4)

[0259] The polypeptide sequence of α-1,2-fucosyltransferase derived from Bacteroides fragilis (SEQ ID NO:5)

[0260] The gene sequence of α-1,2-fucosyltransferase derived from Bacteroides fragilis (SEQ ID NO:6)

[0261] The polypeptide sequence of α-1,2-fucosyltransferase derived from Escherichia coli O126 (SEQ ID NO:7)

[0262] The gene sequence of α-1,2-fucosyltransferase derived from Escherichia coli O126 (SEQ ID NO:8)

[0263] The polypeptide sequence of α-1,2-fucosyltransferase derived from Helicobacter japonicus (SEQ ID NO:9)

[0264] The gene sequence of α-1,2-fucosyltransferase derived from Helicobacter japonicus (SEQ ID NO:10)

[0265] The polypeptide sequence of α-1,2-fucosyltransferase derived from Helicobacter trogontum (SEQ ID NO:11)

[0266] The gene sequence of α-1,2-fucosyltransferase derived from Helicobacter trogontum (SEQ ID NO:12)

[0267] The polypeptide sequence of α-1,2-fucosyltransferase derived from Poseidonibacter sp. SJOD-M-5 (SEQ ID NO:13)

[0268] The gene sequence of α-1,2-fucosyltransferase derived from Poseidonibacter sp. SJOD-M-5 (SEQ ID NO:14)

[0269] The polypeptide sequence of α-1,2-fucosyltransferase derived from Pedobacter sp. BS3 (SEQ ID NO:15)

[0270] The gene sequence of α-1,2-fucosyltransferase derived from Pedobacter sp. BS3 (SEQ ID NO:16)

[0271] The polypeptide sequence of α-1,2-fucosyltransferase derived from Christiangramia sabulilitoris (SEQ ID NO:17)

[0272] The gene sequence of α-1,2-fucosyltransferase derived from Christiangramia sabulilitoris (SEQ ID NO:18)

[0273] The polypeptide sequence of α-1,2-fucosyltransferase derived from Campylobacter hyointestinalis (SEQ ID NO:19)

[0274] The gene sequence of α-1,2-fucosyltransferase derived from Campylobacter hyointestinalis (SEQ ID NO:20)

[0275] The polypeptide sequence of α-1,2-fucosyltransferase derived from the copper-loving bacterium Cupriavidus plantarum (SEQ ID NO:21).

[0276] The gene sequence of α-1,2-fucosyltransferase from the plant copper-loving bacterium Cupriavidus plantarum (SEQ ID NO:22).

[0277] The polypeptide sequence of α-1,2-fucosyltransferase derived from Helicobacter himalayensis (SEQ ID NO:23)

[0278] The gene sequence of α-1,2-fucosyltransferase derived from Helicobacter himalayensis (SEQ ID NO:24)

[0279] The polypeptide sequence of α-1,2-fucosyltransferase derived from Herbaspirillum rubrisubalbicans (SEQ ID NO:25)

[0280] The gene sequence of α-1,2-fucosyltransferase derived from Herbaspirillum rubrisubalbicans (SEQ ID NO:26)

[0281] The polypeptide sequence of α-1,2-fucosyltransferase derived from Bacteroides caccae (SEQ ID NO:27)

[0282] The gene sequence of α-1,2-fucosyltransferase derived from Bacteroides caccae (SEQ ID NO:28)

[0283] The polypeptide sequence of α-1,2-fucosyltransferase derived from Campylobacter lari (SEQ ID NO:29)

[0284] The gene sequence of α-1,2-fucosyltransferase derived from Campylobacter lari (SEQ ID NO:30)

[0285] The polypeptide sequence of α-1,2-fucosyltransferase derived from Pseudomonas fluorescens (SEQ ID NO:31)

[0286] The gene sequence of α-1,2-fucosyltransferase derived from Pseudomonas fluorescens (SEQ ID NO:32)

[0287] The polypeptide sequence of α-1,2-fucosyltransferase derived from Helicobacter muridarum (SEQ ID NO:33)

[0288] The gene sequence of α-1,2-fucosyltransferase derived from Helicobacter muridarum (SEQ ID NO:34)

[0289] The polypeptide sequence of α-1,2-fucosyltransferase derived from Helicobacter mustelae (SEQ ID NO:35)

[0290] The gene sequence of α-1,2-fucosyltransferase derived from Helicobacter mustelae (SEQ ID NO:36)

[0291] The polypeptide sequence of α-1,2-fucosyltransferase derived from Candidatus Melainabacteria bacterium (SEQ ID NO:37)

[0292] The gene sequence of α-1,2-fucosyltransferase derived from Candidatus Melainabacteria bacterium (SEQ ID NO:38)

[0293] The polypeptide sequence of α-1,2-fucosyltransferase from Neocallimastix californiae (SEQ ID NO:39)

[0294] The gene sequence of α-1,2-fucosyltransferase from Neocallimastix californiae (SEQ ID NO:40)

[0295] The polypeptide sequence of α-1,2-fucosyltransferase derived from *Candidatus Falkowbacteria bacterium* RIFOXYA2_FULL_47_19 (SEQ ID NO:41).

[0296] The gene sequence of α-1,2-fucosyltransferase derived from *Candidatus Falkowbacteria bacterium* RIFOXYA2_FULL_47_19 (SEQ ID NO:42).

[0297] The polypeptide sequence of α-1,2-fucosyltransferase derived from Patescibacteria group bacterium (SEQ ID NO:43)

[0298] The gene sequence of α-1,2-fucosyltransferase derived from Patescibacteria group bacterium (SEQ ID NO:44)

[0299] The polypeptide sequence of α-1,2-fucosyltransferase derived from α-proteobacteria bacterium (SEQ ID NO:45)

[0300] The gene sequence of α-1,2-fucosyltransferase derived from α-Proteobacteria bacterium (SEQ ID NO:46)

[0301] The polypeptide sequence of α-1,2-fucosyltransferase derived from Acetobacter sp. (SEQ ID NO:47)

[0302] The gene sequence of α-1,2-fucosyltransferase derived from Acetobacter sp. (SEQ ID NO:48)

[0303] The polypeptide sequence of α-1,2-fucosyltransferase derived from Candidatus Gastranaerophilales bacterium (SEQ ID NO:49)

[0304] The gene sequence of α-1,2-fucosyltransferase derived from Candidatus Gastranaerophilales bacterium (SEQ ID NO:50)

[0305] The polypeptide sequence of α-1,2-fucosyltransferase derived from Parcubacteria group bacterium (SEQ ID NO:51)

[0306] The gene sequence of α-1,2-fucosyltransferase derived from Parcubacteria group bacterium (SEQ ID NO:52).

[0307] The polypeptide sequence of α-1,2-fucosyltransferase derived from Desulfuromonadales bacterium (SEQ ID NO:53)

[0308] The gene sequence of α-1,2-fucosyltransferase derived from *Desulfuromonadales bacterium* (SEQ ID NO:54).

[0309] The polypeptide sequence of α-1,2-fucosyltransferase derived from Lachnospiraceae bacterium (SEQ ID NO:55)

[0310] The gene sequence of α-1,2-fucosyltransferase derived from Lachnospiraceae bacterium (SEQ ID NO:56)

[0311] The polypeptide sequence of artificially constructed α-1,2-fucosyltransferase (SEQ ID NO:57)

[0312] The gene sequence of an artificially constructed α-1,2-fucosyltransferase (SEQ ID NO:58)

[0313] The polypeptide sequence of α-1,2-fucosyltransferase derived from Corynebacterium urealyticum (SEQ ID NO:59)

[0314] The gene sequence of α-1,2-fucosyltransferase derived from Corynebacterium urealyticum (SEQ ID NO:60)

[0315] The polypeptide sequence of GDP-mannose-4,6-dehydratase GMD derived from Escherichia coli MG1655 (SEQ ID NO:61)

[0316] The gene sequence of GDP-mannose-4,6-dehydratase GMD derived from Escherichia coli MG1655 (SEQ ID NO:62).

[0317] The polypeptide sequence of GDP-L-fucosylate synthase WcaG derived from Escherichia coli MG1655 (SEQ ID NO:63).

[0318] The gene sequence of GDP-L-fucosylate synthase WcaG from Escherichia coli MG1655 (SEQ ID NO:64)

[0319] The polypeptide sequence of the lactose permease LacY derived from Escherichia coli MG1655 (SEQ ID NO:65).

[0320] The gene sequence of the lactose permease LacY derived from Escherichia coli MG1655 (SEQ ID NO:66)

[0321] The polypeptide sequence of the reductase DdahC derived from Campylobacter jejuni (SEQ ID NO:67)

[0322] The gene sequence of the reductase DdahC derived from Campylobacter jejuni (SEQ ID NO:68)

[0323] The polypeptide sequence of GDP-mannose-3,5-epimerase / GDP-D-rhamnose-3,5-epimerase derived from rice (Oryza sativa) (SEQ ID NO:69)

[0324] The gene sequence of GDP-mannose-3,5-epimerase / GDP-D-rhamnose-3,5-epimerase derived from rice (Oryza sativa) (SEQ ID NO:70)

[0325] The promoter Psod (SEQ ID NO:71) is derived from Corynebacterium glutamicum ATCC 13032.

[0326] The promoter Pcg219 5 (SEQ ID NO:72) is derived from Corynebacterium glutamicum ATCC 13032.

[0327] The promoter Pgap (SEQ ID NO:73) is derived from Corynebacterium glutamicum ATCC 13032.

[0328] The embodiments of the present invention are not limited to those described above. Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes and improvements to the present invention in form and detail, and these are all considered to fall within the protection scope of the present invention.

Claims

1. An α-1,2-fucosyltransferase variant that, relative to wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase, contains an amino acid substitution of Q258S, and also contains a substitution of one or more amino acids from position 56 to 66.

2. The α-1,2-fucosyltransferase variant of claim 1, wherein the substitution of one or more amino acids at positions 56 to 66 includes substitution of amino acids at one, two, or three positions at positions 60, 62, and 64.

3. The α-1,2-fucosyltransferase variant of claim 2, wherein the amino acid substitution at position 60 is F60A or F60S, the amino acid substitution at position 62 is W62H or W62R, and the amino acid substitution at position 64 is N64D or N64E.

4. The α-1,2-fucosyltransferase variant according to any one of claims 1-3, wherein the variant further comprises one or more amino acid substitutions selected from the group consisting of: V25I, Q203Y, H290W, M210I, and V304I.

5. The α-1,2-fucosyltransferase variant according to any one of claims 1-4, wherein its amino acid sequence has at least 80% sequence identity with the amino acid sequence of wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase.

6. The α-1,2-fucosyltransferase variant according to any one of claims 1-5, wherein, relative to wild-type Corynebacterium urealyticum α-1,2-fucosyltransferase, it comprises any one of the following amino acid substitutions: Q258S / N64D Q258S / N64E Q258S / N64D / V25I Q258S / N64D / F60S Q258S / N64D / F60A Q258S / N64D / W62H Q258S / N64D / Q203Y Q258S / N64D / V25I / W62H Q258S / N64D / V25I / H290W Q258S / N64D / V25I / M210I Q258S / N64D / V25I / V304I Q258S / N64D / F60A / W62R Q258S / N64D / F60S / Q203Y Q258S / N64D / F60A / Q203Y Q258S / N64D / F60A / M210I Q258S / N64D / W62H / M210I Q258S / N64D / W62R / M210I Q258S / N64D / H290W / Q203Y Q258S / N64D / H290W / V304I Q258S / N64D / V25I / F60S / W62R Q258S / N64D / V25I / F60A / W62R Q258S / N64D / V25I / F60A / H290W Q258S / N64D / V25I / F60A / Q203Y Q258S / N64D / V25I / F60S / V304I Q258S / N64D / V25I / F60A / V304I Q258S / N64D / V25I / W62H / H290W Q258S / N64D / V25I / W62R / Q203Y Q258S / N64D / V25I / W62H / V304I Q258S / N64D / V25I / H290W / M210I Q258S / N64D / V25I / Q203Y / V304I Q258S / N64D / F60S / W62H / Q203Y Q258S / N64D / F60S / W62H / M210I Q258S / N64D / F60A / W62R / M210I Q258S / N64D / F60S / W62H / V304I Q258S / N64D / F60A / W62H / V304I Q258S / N64D / F60A / H290W / Q203Y Q258S / N64D / F60A / H290W / M210I Q258S / N64D / F60S / Q203Y / V304I Q258S / N64D / F60S / M210I / V304I Q258S / N64D / W62H / H290W / Q203Y Q258S / N64D / W62R / H290W / Q203Y Q258S / N64D / W62H / H290W / M210I Q258S / N64D / W62H / H290W / V304I Q258S / N64D / W62H / Q203Y / M210I Q258S / N64D / W62R / Q203Y / V304I Q258S / N64D / W62H / M210I / V304I Q258S / N64D / H290W / Q203Y / V304I Q258S / N64D / V25I / F60S / W62H / H290W Q258S / N64D / V25I / F60S / W62H / Q203Y Q258S / N64D / V25I / F60S / W62H / M210I Q258S / N64D / V25I / F60A / W62R / M210I Q258S / N64D / V25I / F60A / W62R / V304I Q258S / N64D / V25I / F60S / H290W / Q203Y Q258S / N64D / V25I / F60A / H290W / Q203Y Q258S / N64D / V25I / F60S / H290W / M210I Q258S / N64D / V25I / F60A / Q203Y / M210I Q258S / N64D / V25I / F60A / Q203Y / V304I Q258S / N64D / V25I / F60A / M210I / V304I Q258S / N64D / V25I / W62H / H290W / V304I Q258S / N64D / V25I / W62R / H290W / V304I Q258S / N64D / V25I / W62H / Q203Y / M210I Q258S / N64D / V25I / W62R / Q203Y / M210I Q258S / N64D / V25I / W62R / Q203Y / V304I Q258S / N64D / V25I / W62H / M210I / V304I Q258S / N64D / V25I / H290W / Q203Y / M210I Q258S / N64D / F60A / W62R / H290W / Q203Y Q258S / N64D / F60S / W62R / H290W / M210I Q258S / N64D / F60S / W62H / H290W / V304I Q258S / N64D / F60S / W62R / H290W / V304I Q258S / N64D / F60A / W62R / H290W / V304I Q258S / N64D / F60S / W62H / Q203Y / V304I Q258S / N64D / F60S / W62R / M210I / V304I Q258S / N64D / F60A / W62H / M210I / V304I Q258S / N64D / F60A / W62R / M210I / V304I Q258S / N64D / F60A / H290W / Q203Y / M210I Q258S / N64D / F60S / H290W / Q203Y / V304I Q258S / N64D / F60A / H290W / Q203Y / V304I Q258S / N64D / F60S / H290W / M210I / V304I Q258S / N64D / F60S / Q203Y / M210I / V304I Q258S / N64D / F60A / Q203Y / M210I / V304I Q258S / N64D / W62R / H290W / Q203Y / V304I Q258S / N64D / W62R / H290W / M210I / V304I Q258S / N64D / W62R / Q203Y / M210I / V304I Q258S / N64D / H290W / Q203Y / M210I / V304I Q258S / N64E / F60S Q258S / N64E / F60A Q258S / N64E / W62R Q258S / N64E / H290W Q258S / N64E / V25I / Q203Y Q258S / N64E / V25I / M210I Q258S / N64E / V25I / V304I Q258S / N64E / F60S / W62R Q258S / N64E / F60A / W62H Q258S / N64E / F60A / W62R Q258S / N64E / F60S / H290W Q258S / N64E / F60A / Q203Y Q258S / N64E / F60S / M210I Q258S / N64E / F60A / V304I Q258S / N64E / W62H / Q203Y Q258S / N64E / W62R / M210I Q258S / N64E / W62R / V304I Q258S / N64E / H290W / Q203Y Q258S / N64E / H290W / V304I Q258S / N64E / Q203Y / M210I Q258S / N64E / Q203Y / V304I Q258S / N64E / V25I / F60S / W62H Q258S / N64E / V25I / F60S / H290W Q258S / N64E / V25I / F60A / M210I Q258S / N64E / V25I / W62H / H290W Q258S / N64E / V25I / W62R / Q203Y Q258S / N64E / V25I / W62R / V304I Q258S / N64E / V25I / Q203Y / M210I Q258S / N64E / F60A / W62R / H290W Q258S / N64E / F60A / W62R / Q203Y Q258S / N64E / F60A / W62H / M210I Q258S / N64E / F60A / W62H / V304I Q258S / N64E / F60S / H290W / M210I Q258S / N64E / F60S / H290W / V304I Q258S / N64E / F60A / Q203Y / M210I Q258S / N64E / F60A / Q203Y / V304I Q258S / N64E / F60S / M210I / V304I Q258S / N64E / F60A / M210I / V304I Q258S / N64E / W62R / H290W / Q203Y Q258S / N64E / W62H / H290W / V304I Q258S / N64E / W62H / Q203Y / V304I Q258S / N64E / H290W / Q203Y / M210I Q258S / N64E / V25I / F60A / W62R / H290W Q258S / N64E / V25I / F60S / W62H / Q203Y Q258S / N64E / V25I / F60A / W62R / Q203Y Q258S / N64E / V25I / F60S / W62R / M210I Q258S / N64E / V25I / F60A / W62R / M210I Q258S / N64E / V25I / F60S / H290W / Q203Y Q258S / N64E / V25I / F60S / H290W / V304I Q258S / N64E / V25I / F60A / Q203Y / M210I Q258S / N64E / V25I / F60A / Q203Y / V304I Q258S / N64E / V25I / F60A / M210I / V304I Q258S / N64E / V25I / W62H / H290W / V304I Q258S / N64E / V25I / W62R / H290W / V304I Q258S / N64E / V25I / W62H / Q203Y / M210I Q258S / N64E / V25I / W62R / Q203Y / M210I Q258S / N64E / V25I / W62H / Q203Y / V304I Q258S / N64E / V25I / W62H / M210I / V304I Q258S / N64E / V25I / W62R / M210I / V304I Q258S / N64E / V25I / H290W / Q203Y / V304I Q258S / N64E / F60S / W62R / H290W / Q203Y Q258S / N64E / F60S / W62H / H290W / M210I Q258S / N64E / F60A / W62R / H290W / M210I Q258S / N64E / F60A / W62H / H290W / V304I Q258S / N64E / F60S / W62R / Q203Y / M210I Q258S / N64E / F60S / W62R / Q203Y / V304I Q258S / N64E / F60A / W62H / Q203Y / V304I Q258S / N64E / F60A / W62R / Q203Y / V304I Q258S / N64E / F60A / W62R / M210I / V304I Q258S / N64E / F60A / H290W / Q203Y / V304I Q258S / N64E / W62R / H290W / Q203Y / M210I Q258S / N64E / H290W / Q203Y / M210I / V304I.

7. A polynucleotide encoding an α-1,2-fucosyltransferase variant as described in any one of claims 1-6.

8. A carrier comprising the polynucleotide as described in claim 7.

9. A host cell expressing the α-1,2-fucosyltransferase variant as described in any one of claims 1-6, or comprising the polynucleotide as described in claim 7 or the vector as described in claim 8.

10. A genetically modified host cell, said host cell expressing α-1,2-fucosyltransferase, and comprising: (1) Genes for the synthesis pathway of the donor substrate of the α-1,2-fucosyltransferase; (2) The gene of the transport protein that transports the receptor substrate of the α-1,2-fucosyltransferase to the host cell, or the gene of the synthetic pathway of the receptor substrate of the α-1,2-fucosyltransferase. The α-1,2-fucosyltransferase is an α-1,2-fucosyltransferase or a variant thereof derived from Escherichia coli O126, Campylobacter hyointestinalis, Helicobacter muridarum, Patescibacteria group bacterium, Acetobacter sp., or Corynebacterium urealyticum, wherein the variant is an α-1,2-fucosyltransferase variant as described in any one of claims 1-6.

11. The host cell of claim 10, wherein the donor substrate is GDP-L-fucose and the acceptor substrate is lactose.

12. The host cell as claimed in claim 10 or 11, wherein the host cell is a microbial cell.

13. The host cell according to any one of claims 10-12, wherein the host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, or Yersinia lipolytica.

14. The host cell according to any one of claims 10-13, wherein the GDP-L-fucose synthesis pathway gene comprises a GDP-D-mannose synthesis pathway gene, a gene encoding GDP-mannose-4,6-dehydratase (GMD), and a gene encoding GDP-L-fucose synthase (GFS); preferably, the GDP-mannose-4,6-dehydratase (GMD) is GDP-mannose-4,6-dehydratase (EcGMD) derived from Escherichia coli or a functional variant thereof, and / or the GDP-L-fucose synthase (GFS) is GDP-L-fucose synthase (WcaG) derived from Escherichia coli or a functional variant thereof.

15. The host cell according to any one of claims 10-14, wherein the GDP-fucose synthesis pathway gene comprises a GDP-D-mannose synthesis pathway gene, a gene encoding GDP-D-mannose-4,6-dehydratase, a gene encoding a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose, and a gene encoding GDP-D-rhamnose-3,5-epimerase; preferably, the GDP-mannose-4,6-dehydratase (GMD) is derived from *Escherichia coli* GMD (EcGMD) or a functional variant thereof, the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose is derived from *Campylobacter jejuni* DdahC or a functional variant thereof, and / or the GDP-D-rhamnose-3,5-epimerase is derived from rice (*Oryza sativa*). GDP-mannose-3,5-epimerase (OsGME) or its functional variants thereof.

16. The host cell according to any one of claims 10-15, wherein the host cell contains a gene encoding a lactose transporter; preferably, the lactose transporter is a lactose permease; more preferably, the lactose permease is a lactose permease derived from Escherichia coli or Kluyveromyces lactis, or a functional variant thereof.

17. The host cell according to any one of claims 10-16, further comprising the gene for α-1,3-fucosyltransferase or α-1,3 / 4-fucosyltransferase.

18. A method for producing fucosyl oligosaccharides, comprising contacting one or more glycosyltransferases with their donor substrate and acceptor substrate to synthesize the fucosyl oligosaccharide, wherein the one or more glycosyltransferases comprise at least α-1,2-fucosyltransferase, said α-1,2-fucosyltransferase being an α-1,2-fucosyltransferase from Corynebacterium urealyticum or a variant thereof, said variant being an α-1,2-fucosyltransferase variant as described in any one of claims 1-6.

19. The method of claim 18, wherein the fucoidosyl oligosaccharide comprises 2'-fucosyllactose (2'-FL) and / or difucosyllactose (DFL).

20. The method of claim 18 or 19, wherein the donor substrate comprises GDP-L-fucose and the acceptor substrate comprises lactose.

21. The method of any one of claims 18-20, wherein the synthesis is carried out in cells, outside cells, or under cell-free conditions.

22. A method for producing fucoidosyl oligosaccharides, comprising culturing a host cell as described in any one of claims 10-17 under conditions suitable for producing the fucoidosyl oligosaccharides, to synthesize the fucoidosyl oligosaccharides.

23. The method of claim 22, wherein the fucoidosyl oligosaccharide comprises 2'-fucosyllactose (2'-FL) and / or difucosyllactose (DFL).

24. The method of claim 22 or 23, wherein the culture medium for culturing the host cells contains at least one carbon source.

25. The method of any one of claims 22-24, wherein the culture medium for culturing the host cells contains lactose or a precursor substance capable of synthesizing lactose via an intracellular synthetic pathway.

26. The method of any one of claims 22-25, the method further comprising recovering the fucosyl oligosaccharide from the culture medium and / or host cells.

27. The use of the host cell as described in any one of claims 10-17 in the production of fucoidosyl oligosaccharides.

28. The application of claim 27, wherein the fucoidosyl oligosaccharide comprises 2'-fucosyllactose (2'-FL) and / or difucosyllactose (DFL).

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