Method for producing fucosylated oligosaccharides and use thereof

Abstract

A method for producing fucosylated oligosaccharides. The core of the method lies in converting GDP-D-rhamnose into GDP-L-fucose by using GDP-D-rhamnose-3,5-epimerase, wherein the GDP-D-rhamnose is obtained by means of converting GDP-D-mannose as a substrate via GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose reductase. The expression of GDP-D-mannose-4,6-dehydratase, GDP-4-keto-6-deoxy-D-mannose reductase, GDP-D-rhamnose-3,5-epimerase, and fucosyltransferase in genetically modified host cells enables the production of fucosylated oligosaccharides via the described pathway.

Description

Methods for producing fucosylated oligosaccharides and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. CN202410437877.0, filed on April 11, 2024, which is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biotechnology, and in particular to methods for producing fucosylated oligosaccharides and uses thereof. BACKGROUND

[0004] Human milk oligosaccharides (HMOs) are the third largest component in human milk after lactose and fat, consisting of more than 200 oligosaccharides. Among them, 2’-fucosyllactose (2’-FL) and 3-fucosyllactose (3-FL) are fucosylated oligosaccharides, and are the most abundant in human milk, accounting for about 35% of total HMOs, and have extremely important physiological functions, including affecting the composition of intestinal microbiota, resisting the adhesion of pathogenic bacteria, regulating immune function, and promoting brain development, etc.

[0005] Due to the high application value of 2’-FL, it has also become one of the most widely studied oligosaccharides. The preparation of 2’-FL is mainly through biological synthesis methods, including microbial fermentation synthesis and enzymatic synthesis. Microbial fermentation method has attracted the attention of many scholars at home and abroad due to its mild reaction conditions, low production cost, and small environmental pollution, and has become a research hotspot for the production of 2’-FL.

[0006] Fucosyltransferases can catalyze the transfer of L-fucose from a GDP-L-fucose donor substrate to an acceptor substrate (such as lactose) to synthesize fucosylated oligosaccharides such as 2’-FL and 3-FL. Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce GDP-L-fucose and express exogenous fucosyltransferases to synthesize fucosylated oligosaccharides such as 2’-FL and 3-FL (Bioresour. Technol. 2023, 374: 128818; ACS Synth. Biol. 2023, 12(1): 238-248; CN 107849577; WO2012 / 112777A1; WO2015175801A1; Microb. Cell Fact. 2022, 21(1): 110). The core of the metabolic pathways of these strains is to convert GDP-D-mannose to GDP-4-keto-6-deoxy D-mannose by GDP-D-mannose 4,6-dehydratase (GMD), and then further generate GDP-L-fucose by GDP-L-fucose synthetase (WcaG), and then use fucosyltransferases to catalyze the synthesis of 2’-FL and 3-FL from GDP-L-fucose and lactose. In addition, only Conagen has created a new 2’-FL production route through in vitro multi-enzyme cascade, and the core of the route is to synthesize GDP-L-fucose from GDP-L-galactose by GMD and WcaG (WO 2022 / 040411). Overall, the production method of fucosylated oligosaccharides such as 2’-FL is currently rarely studied, and it is necessary to explore new production pathways to provide new ideas for the efficient production of fucosylated oligosaccharides.

[0007] D-rhamnose is an important component of the surface polysaccharides of many pathogenic bacteria, including Pseudomonas aeruginosa, and GDP-D-rhamnose is the precursor of D-rhamnose (Curr. Opin. Struct. Biol. 2000, 10(6): 687-696). Similar to GDP-L-fucose, the biosynthesis of GDP-D-rhamnose also involves two proteins: GMD and GDP-4-keto-6-deoxy-D-mannose reductase (RMD). First, GMD can convert GDP-D-mannose to GDP-4-keto-6-deoxy-D-mannose, and then RMD reduces the latter to GDP-D-rhamnose (Eur. J Biochem. 2002, 269(2): 593-601). GMD is widely present in nature, and it is involved in the first step of L-fucose, 6-deoxy-D-talose, and D-rhamnose biosynthesis, so there have been many reports on GMD from different sources. In contrast, only a few RMDs have been confirmed to reduce GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose, including AtRMD from Aneurinibacillus thermoaerophilus and PaRMD from Pseudomonas aeruginosa (FEBS J. 2009, 276(10): 2686-2700; J. Biol. Chem. 2001, 276(8): 5577-5583). In addition, although there have been reports of carbonyl reductases that can catalyze the asymmetric reduction of GDP-4-keto-6-deoxy-D-mannose analogs, they do not use GDP-4-keto-6-deoxy-D-mannose as a substrate, and the specific activity needs to be explored (Biochemistry. 2022, 61: 2138-2147; Angew Chem Int Ed Engl. 2008, 47(51): 9814-59).

[0008] GDP-mannose 3,5-epimerase (GME) can catalyze the reversible isomerization of the 3,5-position groups of GDP-D-mannose to generate GDP-L-galactose. AtGME from Arabidopsis thaliana and OsGME from Oryza sativa have been intensively studied, and even the crystal structure of AtGME protein has been resolved (Phytochemistry. 2006, 67(4): 338-346; J. Am. Chem. Soc. 2005, 127(51): 18309-18320; Biotechnol. Adv. 2021, 48: 107705.). In 2019, Gevaert et al. also reported a microbial source of GME, i.e. MfGME from Methylacidiphilum fumariolicum (Int. J Mol. Sci. 2019, 20(14): 3530). However, there is no report on the substrate spectrum of GME catalysis at present. SUMMARY

[0009] In one aspect, the present application provides a genetically modified host cell comprising genes of a GDP-D-mannose synthesis pathway, and comprising a gene encoding a GDP-D-mannose-4,6-hydrolase (GMD), a gene encoding a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose, a gene encoding a GDP-D-rhamnose-3,5-epimerase, and a gene encoding a fucosyltransferase capable of transferring a fucose residue to a recipient substrate, thereby synthesizing the fucosylated oligosaccharide.

[0010] 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 Gram-negative bacterium or a Gram-positive bacterium. In some embodiments, the host cell is a bacterium of the genus Escherichia, Corynebacterium, Bacillus, Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Pseudomonas. In some embodiments, the host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, or Yarrowia lipolytica.

[0011] In some embodiments, the fucosyltransferase is any one, any two, any three, or four of: an a-1,2-fucosyltransferase, an a-1,3-fucosyltransferase, an a-1,4-fucosyltransferase, and an a-1,3 / 4-fucosyltransferase. In some embodiments, the a-1,2-fucosyltransferase is capable of using lactose, lacto-N-tetraose (LNT), or lacto-N-neotetraose (LNnT) as acceptor substrate. In some embodiments, the a-1,2-fucosyltransferase is an a-1,2-fucosyltransferase derived from Helicobacter pylori, E. coli 0128, E. coli 0126, Bacteroides fragilis, Azospirillum lipoferum, H. mustelae, H. billis, Campylobacter jejuni, Bacteroides vulgatus, or Prevotella sp., or a functional variant thereof. In some embodiments, the a-1,3-fucosyltransferase is capable of using lactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or 2’-fucosyllactose (2’-FL) as acceptor substrate. In some embodiments, the a-1,3-fucosyltransferase is an a-1,3-fucosyltransferase derived from Helicobacter pylori, H. hepaticus, H. billis, H. trogontum, H. typhlonius, Bacteroides fragilis, or Akkermansia muciniphila, or a functional variant thereof. In some embodiments, the a-1,3 / 4-fucosyltransferase is capable of using lactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or 2’-fucosyllactose (2’-FL) as acceptor substrate. In some embodiments, the a-1,3 / 4-fucosyltransferase is an a-1,3 / 4-fucosyltransferase derived from Helicobacter pylori, or a functional variant thereof. In some embodiments, the a-1,4-fucosyltransferase is capable of using lactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or 2’-fucosyllactose (2’-FL) as acceptor substrate.

[0012] In some embodiments, the GDP-D-mannose-4,6-hydrolase is GMD derived from Escherichia coli, or a functional variant thereof.

[0013] In some embodiments, the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose is a GDP-4-keto-6-deoxy D-mannose reductase (RMD). In some embodiments, the GDP-4-keto-6-deoxy D-mannose reductase is RMD derived from Pseudomonas aeruginosa, 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 is a reductase comprising an amino acid sequence as set forth in SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 37, or a functional variant thereof.

[0014] In some embodiments, the GDP-D-rhamnose-3,5-epimerase is a GDP-mannose 3,5-epimerase (GME) derived from Oryza sativa, GME derived from Arabidopsis thaliana, or a functional variant thereof.

[0015] In some embodiments, the fucosylated oligosaccharide belongs to human milk oligosaccharides (HMOs). In some embodiments, the host cell is capable of providing a recipient substrate inside the cell. In some embodiments, the host cell is capable of transporting a recipient substrate into the cell, or the host cell is capable of synthesizing the recipient substrate from an exogenous precursor through a synthetic pathway contained inside the cell. In some embodiments, the recipient substrate is lactose or a lactose derivative. In some embodiments, the lactose derivative is lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or 2'-fucosyllactose (2'-FL).

[0016] In some embodiments, the host cell comprises a gene encoding a lactose permease. In some embodiments, the lactose permease is a lactose permease derived from Escherichia coli or Kluyveromyces lactis, or a functional variant thereof.

[0017] In some embodiments, the fucosylated oligosaccharide comprises 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopen-taose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofucopentaose I (LNnFP-I), lacto-N-neofucopentaose III (LNnFP-III), lacto-N-neofucopentaose V (LNnFP-V), lacto-N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNDFH-II).

[0018] Another aspect of the application provides a method of producing a fucosylated oligosaccharide, comprising culturing any of the preceding host cells under conditions suitable for producing the breast milk oligosaccharide, to transfer a fucose residue to a recipient substrate, thereby synthesizing the fucosylated lactose.

[0019] In some embodiments, the fucosylated oligosaccharide is a human milk oligosaccharide (HMO). In some embodiments, the fucosylated oligosaccharide comprises 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopen-taose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofucopentaose I (LNnFP-I), lacto-N-neofucopentaose III (LNnFP-III), lacto-N-neofucopentaose V (LNnFP-V), lacto-N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNDFH-II).

[0020] In some embodiments, the culture medium used to culture the host cell comprises at least one carbon source. In some embodiments, the culture medium used to culture the host cell is supplemented with a recipient substrate or a precursor capable of being synthesized into the recipient substrate by a synthetic pathway comprised within the cell. In some embodiments, the culture medium used to culture the host cell is supplemented with lactose or a precursor capable of being synthesized into lactose by a synthetic pathway comprised within the cell. In some embodiments, the method further comprises recovering the fucosylated oligosaccharide from the culture medium.

[0021] Another aspect of the application provides the use of any of the foregoing host cells in the production of fucosylated oligosaccharides. In some embodiments, the fucosylated oligosaccharides are human milk oligosaccharides (HMOs). In some embodiments, the fucosylated oligosaccharides include 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopen-taose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofucopentaose I (LNnFP-I), lacto-N-neofucopentaose III (LNnFP-III), lacto-N-neofucopentaose V (LNnFP-V), lacto-N-difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNDFH-II). BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1: Exemplary 2'-FL synthesis route.

[0023] Figure 2: Analysis profile of GDP-D-rhamnose and GDP-L-fucose reaction solution.

[0024] Figure 3: Analysis profile of 2'-FL produced by Cg2FL-11 and standard control profile.

[0025] Figure 4: Analysis profile of EcGMD reaction solution.

[0026] Figure 5: Analysis profile of DdahC reaction solution. DETAILED DESCRIPTION

[0027] Unless defined otherwise, 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 application belongs.

[0028] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict between the present specification and the incorporated references, the present specification shall control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0029] The terms "about" and "approximately" when used with a numerical variable, generally mean that the value of the variable and the entire range of values of the variable are within the measurement or experimental error (e.g., 95% confidence interval of the mean) or within a wider range of specified values (e.g., ±5% or ±10%).

[0030] The term "comprising" or variations such as "comprise", "comprises" or "comprised of is to be construed as meaning that the listed steps or options are to be optionally included in the described combinations. "Consisting essentially of means excluding any step or option not listed, but allowing the inclusion of non- specified steps or options that do not materially affect the basic and novel characteristics of the claimed application. The term "comprising" particular steps or options also covers "consisting of and "consisting essentially of the particular steps or options.

[0031] When referring to a numerical range, it is to be understood that the specific values of the upper and lower limits of the range are explicitly disclosed, as well as all intermediate ranges included therein, such as the intermediate ranges between any upper or lower limit and any intermediate value, or between any two intermediate values. Also, any intermediate ranges, sub-ranges, and all individual values described in the numerical range can be excluded from the numerical range.

[0032] The term "and / or" should be understood to mean either one or any combination of the listed items that are connected by the term.

[0033] The term "gene" refers to a nucleotide sequence encoding a gene product. The gene product can be a protein or a ribonucleic acid.

[0034] The term "nucleic acid", "nucleic acid sequence" or "polynucleotide" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, including DNA or RNA, including linear or circular DNA or RNA.

[0035] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. Enzymes in the present application are proteins that are capable of catalyzing a chemical reaction of a substrate.

[0036] The terms "genetically engineered", "genetically modified", "engineered", "engineered", "recombinant" refer to the alteration of the sequence of a polypeptide, a polynucleotide or the sequence of genes comprised by a host cell by artificial manipulation, so as to comprise sequences that do not naturally occur in the polypeptide, polynucleotide or host cell. When a host cell is "engineered" to display a certain characteristic (e.g. to comprise a gene encoding a certain protein, to express a certain protein, or to overexpress a certain gene), it is meant that the host cell did not have this characteristic prior to the engineering, but has this characteristic after the engineering.

[0037] The term "host cell" refers to any cell containing an exogenous nucleic acid sequence.

[0038] The term "functional variant" when applied to a polypeptide or protein refers to a polypeptide having at least 70%, at least 80%, at least 85%, 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%, or at least 99% sequence identity to the parent polypeptide and having the same or substantially the same function as the parent polypeptide. A functional variant can also refer to a polypeptide having one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) insertions, deletions, and / or substitutions of amino acids as compared to the parent polypeptide and having the same or substantially the same function as the parent polypeptide.

[0039] The term "sequence identity" refers to the percentage of nucleotide or amino acid residues of two or more sequences that are the same when the sequences are aligned for maximum sequence comparison, i.e., taking into account gaps and insertions. Alignment and calculation of percent sequence identity of two sequences can be performed by appropriate computer programs known in the art. These programs include, but are not limited to, BLAST, ALIGN, ClustalW, EMBOSS Needle, etc. One example of a local alignment program is BLAST (Basic Local Alignment Search Tool), which is available on the web page of the National Center for Biotechnology Information, currently found at http: / / www.ncbi.nlm.nih.gov / / . It was first described by Altschul et al. (1990) J. Mol. Biol. 215; 403. Biol. 215; 403-410, 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 can be accessed at http: / / www.ebi.ac.uk / Tools / psa / .

[0040] The term "exogenous" with respect to a host cell means a substance or molecule that originates from or is produced by a source other than the host cell. An "exogenous gene" or "exogenous enzyme" means a nucleic acid that is not a gene or enzyme naturally present in the cell and that is introduced into the cell by artificial means. The sequence of an exogenous gene or exogenous enzyme can be identical to or different from an endogenous sequence naturally present in the cell. A gene or enzyme that is different from an endogenous sequence naturally present in the cell can be referred to as a heterologous gene or heterologous enzyme, which originates from a strain or species different from the host cell. As used herein, the term "exogenous gene" or "exogenous enzyme" includes "heterologous gene" or "heterologous enzyme" unless otherwise indicated.

[0041] The term "endogenous" means a gene or protein (e.g., a wild-type gene or wild-type enzyme) or synthetic pathway that is naturally present in a host cell.

[0042] The term "naturally occurring" means a nucleotide sequence, amino acid sequence, complex, pathway, or cell that exists in nature and has not been intentionally modified by man.

[0043] The term "derived from", when applied to a protein or gene sequence, means that the protein or gene sequence has the same structure or sequence as a protein or gene sequence naturally present in a particular organism, and is not limited to being isolated directly from the organism.

[0044] The term "overexpress" means that the expression of a gene product or polypeptide in a host cell is greater after genetic modification than before genetic modification. The term "overexpress" can also mean any detectable expression of a particular gene product introduced into a host cell, if the host cell did not contain the particular gene product before genetic modification.

[0045] The term "vector" means a tool that allows or facilitates the transfer of a nucleic acid fragment from one environment to another, such as a host cell. A vector allows another nucleic acid fragment to be inserted therein for the purpose of replication of the inserted fragment.

[0046] The term "expression vector" means a vector used to express a gene product, which typically includes one or more expression control sequences for controlling and regulating transcription and / or translation of a gene sequence that can be expressed.

[0047] The term "expression cassette" means a nucleotide sequence that includes a relevant nucleic acid under the control of and operably linked to an appropriate promoter or other regulatory element, so as to be transcribed in a host cell.

[0048] The term "operably linked" means that the regulatory sequences necessary for expression of a coding sequence are placed in the DNA molecule in proper positions relative to the coding sequence, so as to affect the expression of the coding sequence.

[0049] The term "Human Milk Oligosaccharides" (HMO) refers to carbohydrates consisting of 3-10 monosaccharides linked by glycosidic bonds, which are the third most abundant solid component in human milk after fat and lactose. The basic structure of HMOs consists of five basic monosaccharides, namely: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and N-acetylneuraminic acid (NeuAc or Neu5c).

[0050] The term "fucosylated oligosaccharide" is an oligosaccharide having a fucose residue. Such oligosaccharides are neutral. Exemplary fucosylated oligosaccharides include 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-fucopentaose (e.g., lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyl lacto-N-hexose, fucosyl lacto-N-neohexose, difucosyl lacto-N-hexaose I, and / or difucosyl lacto-N-neohexaose II, and the like.

[0051] The term "fucosyltransferase" refers to a polypeptide that is capable of catalyzing the transfer of a fucose residue from a donor substrate to an acceptor substrate. The donor substrate of a fucosyltransferase is typically GDP-L-fucose. The acceptor substrate includes oligosaccharides, glycopeptides, glycoproteins, and glycolipids. Typically, the fucose residue is transferred to an N-acetylglucosamine residue, an N-acetylgalactosamine residue, a galactose residue, a fucose residue, a sialic acid residue, or a glucose residue of, for example, an oligosaccharide, or a sugar moiety of a glycopeptide or glycolipid. The term "fucosyltransferase" is to be understood herein to include the wild-type fucosyltransferase and functional variants thereof that are also capable of catalyzing the transfer of a fucose residue from a donor substrate to an acceptor substrate, i.e., they also have fucosyltransferase activity.

[0052] The term "donor substrate" with respect to the transfer of a fucose residue from a donor substrate to an acceptor molecule refers to a molecule comprising a fucose residue that is catalytically transferred by a fucosyltransferase to a particular acceptor substrate. In the present application, the donor substrate is GDP-L-fucose. The term "acceptor substrate" refers to a molecule that receives a fucose residue from a donor substrate in a reaction catalyzed by a fucosyltransferase.

[0053] The term "precursor" refers to a compound that is a starting material or an intermediate in a biosynthetic pathway of a compound. These intermediates include exogenously added compounds, or compounds that are endogenously produced by a cell.

[0054] The term "functional" or the term "capable of" when used to describe the activity or function of an enzyme means that the enzyme will exhibit the particular activity or function under suitable reaction conditions. The enzyme can not exhibit the activity or function in the absence of suitable reaction conditions, but does exhibit the activity or function when suitable reaction conditions are present. Suitable reaction conditions include the presence of a suitable donor substrate, the presence of a suitable acceptor molecule, the presence of necessary cofactors, a pH within an appropriate range, a suitable temperature, and the like.

[0055] Unless otherwise indicated, herein nucleic acids are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxyl orientation, except that the end of the linearized sequence is positioned for convenience.

[0056] The present application provides a new synthetic pathway of human milk oligosaccharides, in particular a synthetic pathway of fucosylated oligosaccharides in human milk oligosaccharides. Specifically, the present application provides a new intracellular synthetic pathway of GDP-L-fucose, which can be used as a donor substrate to synthesize fucosylated oligosaccharides by transferring the fucose in the donor substrate to a suitable acceptor substrate through a fucosyltransferase in the cell.

[0057] The present inventors found that GDP-D-rhamnose is an isomer of GDP-L-fucose, and the only difference between them is the conformation of the 3,5 groups, which can be theoretically converted to each other through an isomerization reaction. The present inventors explored the substrate spectrum of GDP-mannose 3,5-epimerase (GME) and found that GDP-mannose 3,5-epimerase can catalyze the reversible isomerization of GDP-D-rhamnose and GDP-L-fucose, and has the activity of GDP-D-rhamnose 3,5-epimerase (GRE). Expressing GDP-D-mannose 4,6-dehydratase (GMD), a reductase capable of catalyzing 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 GDP-D-rhamnose 3,5-epimerase (GRE) in a host cell (such as Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and other microorganisms) can synthesize GDP-L-fucose from GDP-D-mannose through a new pathway. An exemplary production pathway of fucosylated oligosaccharide 2'-FL is shown in FIG. 1.

[0058] To achieve production of fucosylated oligosaccharides, the present invention provides a host cell genetically modified to comprise a GDP-D-mannose synthesis pathway gene, and to comprise

[0059] i) a gene encoding a GDP-D-mannose-4,6-lyase (GMD),

[0060] ii) a gene encoding a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose,

[0061] iii) a gene encoding a GDP-D-rhamnose-3,5-epimerase (GRE), and

[0062] iv) a gene encoding a fucosyltransferase capable of transferring a fucose residue to a recipient substrate to synthesize a fucosylated oligosaccharide.

[0063] The host cell of the present invention is capable of expressing the enzymes encoded by the above genes in a functional form, and is capable of synthesizing the fucosylated oligosaccharide within the cell. Any one of the enzymes encoded by the above genes can be constitutively expressed or inducibly expressed in the host cell.

[0064] In some embodiments, any two or three of i), ii), and iii) are not the same gene. In some embodiments, the GDP-D-mannose-4,6-lyase (GMD) and the reductase capable of catalyzing 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-lyase (GMD) and the GDP-D-rhamnose-3,5-epimerase (GRE) are not encoded by the same gene, and / or the reductase capable of catalyzing 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.

[0065] The host cell of the present invention comprises a GDP-D-mannose synthesis pathway gene to synthesize GDP-D-mannose within the host cell. By "GDP-D-mannose synthesis pathway" is meant a series of reactions controlled and catalyzed by enzymes, the result of which is the synthesis of GDP-D-mannose. Typically, "GDP-D-mannose synthesis pathway" means a series of reactions controlled and catalyzed by enzymes that synthesize GDP-D-mannose from a carbon source. By "GDP-D-mannose synthesis pathway gene" is meant the encoding genes for the enzymes comprised in the GDP-D-mannose synthesis pathway, which genes can express the enzymes in the host cell, thereby catalyzing the synthesis of GDP-D-mannose.

[0066] One or more of the genes in the GDP-D-mannose synthesis pathway can be endogenous or exogenous. In some embodiments, the host cell used naturally has a GDP-D-mannose synthesis pathway and is capable of synthesizing GDP-D-mannose within the cell, in which case all of the genes of the GDP-D-mannose synthesis pathway comprised by the host cell can be endogenous. In some embodiments, the host cell used can not naturally have a GDP-D-mannose synthesis pathway, e.g., lacks one or more enzymes in the GDP-D-mannose synthesis pathway, in which case the missing one or more enzymes can be provided by introducing an exogenous enzyme gene into the host cell for expression of the missing one or more enzymes in the host cell. The exogenous enzyme gene can be comprised in a free expression vector introduced into the host cell or integrated into the chromosome of the host cell.

[0067] Fructose-6-phosphate is an intermediate in carbon source metabolism present in almost all organisms, including bacterial cells. Common carbon sources, such as glucose, glycerol, etc., can be metabolized within the cell to generate fructose-6-phosphate. GDP-D-mannose can be further synthesized from fructose-6-phosphate as a precursor. In bacterial cells, an exemplary GDP-D-mannose synthesis pathway can include a synthesis pathway from a carbon source to fructose-6-phosphate and a synthesis pathway from fructose-6-phosphate to GDP-D-mannose. An exemplary synthesis pathway from fructose-6-phosphate to GDP-D-mannose includes reactions catalyzed by three enzymes: (1) synthesis of mannose-6-phosphate from fructose-6-phosphate, catalyzed by mannose-6-phosphate isomerase (ManA); (2) synthesis of mannose-1-phosphate from mannose-6-phosphate, catalyzed by phosphomannomutase (ManB); and (3) synthesis of GDP-D-mannose from mannose-1-phosphate, catalyzed by mannose-1-phosphate guanylyltransferase (ManC). Most bacterial cells contain enzymes capable of performing the above three steps, such as common E. coli, C. glutamicum, and B. subtilis, etc. However, as metabolic pathways can differ among different bacterial species, when the host cell used lacks one or more enzymes in the GDP-D-mannose synthesis pathway, the missing one or more enzymes can be provided by introducing an exogenous enzyme gene into the host cell for expression of the missing one or more enzymes in the host cell. The exogenous enzyme gene can be comprised in a free expression vector introduced into the host cell or integrated into the chromosome of the host cell.

[0068] The host cell of the application further comprises a gene encoding a GDP-D-mannose-4,6-hydrolase (GMD) to express a GDP-D-mannose-4,6-hydrolase (GMD) in the host cell. The GMD is capable of catalyzing the conversion of GDP-D-mannose to GDP-4-keto-6-deoxy D-mannose. The gene encoding a GDP-D-mannose-4,6-hydrolase (GMD) can be endogenous or exogenous. When the host cell used lacks an endogenous GDP-D-mannose-4,6-hydrolase (GMD) gene, an exogenous GMD gene can be introduced into the host cell to express GMD in the host cell. The exogenous GMD gene can be comprised in a episomal expression vector introduced into the host cell or be integrated into the chromosome of the host cell. In some embodiments, the GMD is a GMD derived from E. coli (EcGMD) or a functional variant thereof. In some embodiments, the GMD derived from E. coli (EcGMD) comprises an amino acid sequence as set forth in SEQ ID NO: 41.

[0069] The host cell of the application further comprises a gene encoding a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose to express a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose in the host. The term "reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose" as it is used herein refers to a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose. One of skill in the art can readily determine whether a polypeptide has the activity of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose by enzyme-catalyzed chemical reactions, for example, by adding the polypeptide under appropriate conditions in the presence of the substrate (GDP-4-keto-6-deoxy D-mannose) and the necessary cofactor (e.g. NADPH) and detecting whether the product (i.e. GDP-D-rhamnose) is formed, for example, by mass spectrometry.

[0070] The gene encoding the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose can be endogenous or exogenous. When the host cell used lacks an endogenous reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose, an exogenous gene encoding a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose can be introduced into the host cell to express a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose in the host cell. The exogenous gene encoding a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose can be contained in a episomal expression vector introduced into the host cell or integrated into the chromosome of the host cell.

[0071] In some embodiments, the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose is a GDP-4-keto-6-deoxy D-mannose reductase (RMD), such as RMD derived from Pseudomonas aeruginosa (PaRMD) or RMD derived from Aneurinibacillus thermoaerophilus (AtRMD) (FEBS J. 2009, 276(10): 2686-2700; J. Biol. Chem. 2001, 276(8): 5577-5583; the entire contents of which are 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 is DnmV derived from Streptomyces peucetius, DdahC derived from Campylobacter jejuni, C4 reductase derived from Campylobacter jejuni HS10A serotype (HS10A), C4 reductase derived from Campylobacter jejuni HS41B serotype (HS41B) (Biochemistry. 2022, 61: 2138-2147; Angew Chem Int Ed Engl. 2008, 47(51): 9814-59; the entire contents of these references are 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 comprises an amino acid sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39, 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 comprises an amino acid sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 37, or a functional variant thereof.

[0072] The host cell of the present application also comprises a gene encoding a GDP-D-rhamnose-3,5-epimerase (GRE) to express a GDP-D-rhamnose-3,5-epimerase (GRE) in the host cell. The term "GDP-D-rhamnose-3,5-epimerase (GRE)" refers to an enzyme capable of catalyzing the conversion of GDP-D-rhamnose to GDP-L-fucose. One skilled in the art can readily determine whether a polypeptide is a GDP-D-rhamnose-3,5-epimerase by enzyme-catalyzed chemical reactions, for example, by adding the polypeptide under appropriate conditions in the presence of the substrate (GDP-D-rhamnose) and detecting whether the product (i.e., GDP-L-fucose) is formed, which can be detected, for example, by mass spectrometry.

[0073] The gene encoding a GDP-D-rhamnose-3,5-epimerase (GRE) can be endogenous or exogenous. When the host cell used lacks an endogenous GDP-D-rhamnose-3,5-epimerase (GRE), an exogenous GRE gene can be introduced into the host cell to express GRE in the host cell. The exogenous GRE gene can be comprised in a episomal expression vector introduced into the host cell or integrated into the chromosome of the host cell.

[0074] GDP-mannose-3,5-epimerase (GME) not only can catalyze the conversion of GDP-D-mannose to GDP-L-fucose, but also has the activity of catalyzing the conversion of GDP-D-rhamnose to GDP-L-fucose, such as GME derived from Arabidopsis thaliana (AtGME), GME derived from Oryza sativa (OsGME), GME derived from Methylacidiphilum fumariolicum (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 entire contents of these documents are incorporated herein by reference), and thus can be used as GDP-D-rhamnose-3,5-epimerase (GRE) in the present application. Thus, in some embodiments, the GDP-D-rhamnose-3,5-epimerase (GRE) comprises a GDP-mannose-3,5-epimerase (GME). In other embodiments, the GDP-D-rhamnose-3,5-epimerase (GRE) can comprise any polypeptide that can catalyze the conversion of GDP-D-rhamnose to GDP-L-fucose. In some embodiments, the GRE is a GRE derived from Arabidopsis thaliana (e.g., GME derived from Arabidopsis thaliana (AtGME)), a GRE derived from Oryza sativa (e.g., GME derived from Oryza sativa (OsGME)), a GRE derived from Methylacidiphilum fumariolicum (e.g., GME derived from Methylacidiphilum fumariolicum (MfGME)), or a functional variant thereof (which should have the ability to catalyze the conversion of GDP-D-rhamnose to GDP-L-fucose). In some embodiments, the GDP-D-rhamnose-3,5-epimerase (GRE) comprises an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or a functional variant thereof.In some embodiments, the GDP-D-rhamnose-3,5-epimerase (GRE) comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or a functional variant thereof.

[0075] The host cell of the present application also comprises a gene encoding a fucosyltransferase to express a fucosyltransferase in the host cell. A fucosyltransferase is capable of catalyzing the transfer of a fucose residue from a donor substrate to an acceptor substrate, forming an a-1,2-, a-1,3-, a-1,4-, or a-1,6-glycosidic bond between the fucose and the sugar moiety of the acceptor substrate, thereby synthesizing a fucosylated oligosaccharide. There are a variety of different fucosylated oligosaccharides present in human milk oligosaccharides (HMOs), including but not limited to fucosyllactose, lacto-N-fucopentaose, lacto-N-neofucopentaose, lacto-N-neodifucohexaose, and the like. A typical donor substrate is GDP-L-fucose. The acceptor substrate typically comprises a galactose moiety, a glucose moiety, and / or an N-acetylglucosamine moiety, and more commonly the acceptor substrate includes lactose or a lactose derivative that has a structure that allows it to form a fucosylated oligosaccharide upon linkage to a fucose moiety catalyzed by a fucosyltransferase. The lactose derivative that can serve as an acceptor substrate can be, for example, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or 2’-fucosyllactose (2’-FL).

[0076] The gene encoding a fucosyltransferase can be endogenous or exogenous. When the host cell used lacks an endogenous fucosyltransferase, an exogenous fucosyltransferase gene can be introduced into the host cell to express a fucosyltransferase in the host cell. The exogenous fucosyltransferase gene can be comprised in a episomal expression vector introduced into the host cell or integrated into the chromosome of the host cell.

[0077] In microbial fermentation production, different fucosylated oligosaccharides can be obtained using different acceptor substrates and different fucosyltransferases with GDP-L-fucose as the donor substrate. For example, 2’-fucosyllactose (2’-FL) can be synthesized using lactose as the acceptor substrate and a-1,2-fucosyltransferase; 3-fucosyllactose (3-FL) can be synthesized using lactose as the acceptor substrate and a-1,3-fucosyltransferase. (Bioresour. Technol. 2023, 374:128818; ACS Synth. Biol. 2023, 12(1):238-248; CN 107849577; WO2012 / 112777A1; WO2015175801A1; Microb. Cell Fact. 2022, 21(1):110; the entire contents of these documents are incorporated herein by reference)

[0078] Di-fucosylated lactose (DFL, which can also be referred to as lacto-N- difucotetraose (LDFT)) can be synthesized using a-1,2-fucosyltransferase to catalyze the synthesis of the intermediate 2’-FL using lactose as the acceptor substrate, and then using a-1,3 / 4-fucosyltransferase to catalyze the synthesis of the intermediate 2’-FL using the intermediate 2’-FL as the acceptor substrate (Zhang A et al., Metab Eng. 2021; 66: 12-20; Liang S et al., J Agric Food Chem. 2024 Feb 28; 72(8): 4367-4375; the entire contents of these documents are incorporated herein by reference).

[0079] Other more complex fucosylated oligosaccharides can also be synthesized using GDP-L-fucose as the donor substrate, using different acceptor substrates and different fucosyltransferases. For example, lacto-N-fucopentaose I (LNFP-I) can be synthesized using a-1,2-fucosyltransferase to catalyze the a-1,2-fucosylation of galactose using lacto-N-tetraose (LNT) as the acceptor substrate. Lacto-N-fucopentaose II (LNFP-II) can be synthesized using a-1,4-fucosyltransferase to catalyze the a-1,4-fucosylation of N-acetylglucosamine using lacto-N-tetraose (LNT) as the acceptor substrate. Lacto-N-fucopentaose V (LNFP-V) can be synthesized using a-1,3-fucosyltransferase to catalyze the a-1,3-fucosylation of glucose using lacto-N-tetraose (LNT) as the acceptor substrate. Lacto-N- neofucopentaose I (LNnFP-I) can be synthesized using a-1,2-fucosyltransferase to catalyze the a-1,2-fucosylation of galactose using lacto-N-neotetraose (LNnT) as the acceptor substrate. Lacto-N-neofucopentaose III (LNnFP-III) can be synthesized using a-1,3-fucosyltransferase to catalyze the a-1,3-fucosylation of N-acetylglucosamine using lacto-N-neotetraose (LNnT) as the acceptor substrate. Lacto-N-neofucopentaose V (LNnFP-V) can be synthesized using a-1,3-fucosyltransferase to catalyze the a-1,3-fucosylation of glucose using lacto-N-neotetraose (LNnT) as the acceptor substrate. (Tomotoshi Sugita et al., Journal of Biotechnology, Volume 361, 2023, Pages 110-118; WO2019 / 008133; the entire contents of these documents are incorporated herein by reference)

[0080] Thus, for different target fucosylated oligosaccharides, host cells comprising different fucosyltransferases can be constructed to achieve the synthesis of the target fucosylated oligosaccharides.

[0081] In some embodiments, the exogenous fucosyltransferase expressed in the host cell can be any one, any two, any three or four selected from the group consisting of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,4-fucosyltransferase and α-1,3 / 4-fucosyltransferase.

[0082] 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. 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. 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. The term "α-1,4-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor substrate to form an α-1,4-glycosidic bond between the fucose residue and the sugar moiety of the acceptor molecule.

[0083] Fucosyltransferases have substrate selectivity, and the acceptor substrate range of different fucosyltransferases can vary. In the host cells and methods of the present application, the synthesis of a particular fucosylated oligosaccharide of interest is achieved by the pairing of both the acceptor substrate and the fucosyltransferase capable of catalyzing the reaction of the acceptor substrate. The selection of the fucosyltransferase is related to the fucosylated oligosaccharide of interest to be synthesized and the selected acceptor substrate. The fucosyltransferase included in the host cell should be capable of catalyzing the transfer of a fucose residue to the acceptor substrate provided in the host cell to synthesize the fucosylated oligosaccharide of interest. For example, when the fucosylated oligosaccharide of interest is 2'-FL, the acceptor substrate is lactose, and the fucosyltransferase included in the host cell for the production of 2'-FL is an a-1,2-fucosyltransferase capable of using lactose as the acceptor substrate; when the fucosylated oligosaccharide of interest is 3-FL, the acceptor substrate is lactose, and the fucosyltransferase included in the host cell for the production of 3-FL is an a-1,3-fucosyltransferase capable of using lactose as the acceptor substrate; when the fucosylated oligosaccharide of interest is DFL, the synthesis is in two stages, the acceptor substrates are lactose and 2'-FL, respectively, and the fucosyltransferases included in the host cell for the production of DFL include an a-1,2-fucosyltransferase capable of using lactose as the acceptor substrate and an a-1,3 / 4-fucosyltransferase capable of using 2'-FL as the acceptor substrate; when the fucosylated oligosaccharide of interest is LNFP-I, LNFP-II, or LNFP-V, the acceptor substrate is LNT, and the fucosyltransferases included in the host cell for the production of these fucosylated oligosaccharides of interest are an a-1,2-fucosyltransferase, an a-1,4-fucosyltransferase, and an a-1,3-fucosyltransferase, respectively, capable of using LNT as the acceptor substrate; when the fucosylated oligosaccharide of interest is LNnFP-I, LNnFP-III, or LNFP-V, the acceptor substrate is LNnT, and the fucosyltransferases included in the host cell for the production of these fucosylated oligosaccharides of interest are an a-1,2-fucosyltransferase, an a-1,3-fucosyltransferase, respectively, capable of using LNnT as the acceptor substrate.

[0084] In some embodiments, the fucosyltransferase expressed in the host cell is capable of using lactose or a lactose derivative (e.g., LNT, LNnT, or 2'-FL) as the acceptor substrate. Those skilled in the art know how to select a fucosyltransferase suitable for the target product and the corresponding acceptor substrate. Methods for identifying the catalytic ability of a fucosyltransferase for a particular acceptor substrate are well known to those skilled in the art and can be achieved by common enzyme catalysis reactions and detection of product formation.

[0085] In some embodiments, the a-1,2-fucosyltransferase used in the present application is capable of using lactose, LNT, LNnT as acceptor substrate. The a-1,2-fucosyltransferase used in the present application can be derived from bacteria, fungi, plants, animals (e.g., mammals, such as humans). Exemplary a-1,2-fucosyltransferases include FutC derived from Helicobacter pylori, WbsJ derived from Escherichia coli 0128, WbgL derived from Escherichia coli 0126, WcfB and WcfW derived from Bacteroides fragilis, SAMT derived from Azospirillum lipoferum, FutL derived from H. mustelae, FutF derived from H. billis, FutG derived from Campylobacter jejuni, FutN derived from Bacteroides vulgatus, FutW derived from Prevotella sp. (Bioresour. Technol. 2023, 374:128818; ACS Synth. Biol. 2023, 12(1):238-248; CN 107849577; WO2012 / 112777A1; WO2015175801A1; WO2019025485A1; Microb. Cell Fact. 2022, 21(1):110; Tomotoshi Sugita et al., Journal of Biotechnology, Volume 361, 2023, Pages 110-118; WO2019 / 008133; the entire contents of these documents are incorporated herein by reference), or functional variants thereof. In some embodiments, the a-1,2-fucosyltransferase used in the present application is a-1,2-fucosyltransferase derived from Helicobacter pylori. In some embodiments, the a-1,2-fucosyltransferase used in the present application comprises an amino acid sequence as set forth in SEQ ID NO: 7.

[0086] In some embodiments, the a-1,3-fucosyltransferase used in the present application is capable of using lactose, LNT, LNnT, or 2’-FL as acceptor substrate. The a-1,3-fucosyltransferase used in the present application can be derived from bacteria, fungi, plants, animals (e.g., mammals, such as humans). Exemplary a-1,3-fucosyltransferases include FutA or FutB derived from Helicobacter pylori, FutJ (Hh0072) and FutK (Hh1776) derived from H. hepaticus, FutE derived from H. billis, FutD derived from H. trogontum, FutH derived from H. typhlonius, FutM, FucT6, or FucT7 derived from Bacteroides fragilis, Amuc_0760 derived from Akkermansia muciniphila (WO2012 / 112777; WO2019025485A1; EP2439264A1; WO2010 / 142305; Tomotoshi Sugita et al., Journal of Biotechnology, Volume 361, 2023, Pages 110-118; WO2019 / 008133; the entire contents of these documents are incorporated herein by reference), or functional variants thereof. In some embodiments, the a-1,3-fucosyltransferase used in the present application is a-1,3-fucosyltransferase derived from Helicobacter pylori. In some embodiments, the a-1,3-fucosyltransferase used in the present application comprises an amino acid sequence as set forth in SEQ ID NO: 43.

[0087] In some embodiments, the a-1,3 / 4-fucosyltransferase used in the present application is capable of using lactose, LNT, LNnT, or 2’-FL as acceptor substrate. The a-1,3 / 4-fucosyltransferase used in the present application can be derived from bacteria, fungi, plants, animals (e.g., mammals such as humans). Exemplary a-1,3 / 4-fucosyltransferases include a-1,3 / 4-fucosyltransferase derived from Helicobacter pylori (Zhang A et al., Metab Eng. 2021; 66: 12-20; Liang S et al., J Agric Food Chem. 2024 Feb 28; 72(8): 4367-4375; the entire contents of these documents are incorporated herein by reference), or functional variants thereof.

[0088] In some embodiments, the a-1,4-fucosyltransferase used in the present application is capable of using lactose, LNT, LNnT, or 2’-FL as acceptor substrate. The a-1,4-fucosyltransferase used in the present application can be derived from bacteria, fungi, plants, animals (e.g., mammals such as humans). Exemplary a-1,4-fucosyltransferases include a-1,4-fucosyltransferase as described in Tomotoshi Sugita et al., Journal of Biotechnology, Volume 361, 2023, Pages 110-118 and WO2019 / 008133 (the entire contents of these documents are incorporated herein by reference), or functional variants thereof.

[0089] In some embodiments, to improve the fucosyltransfer ability of the host cell, more than 1 copy (e.g., 2, 3, or more copies) of the exogenous fucosyltransferase gene can be introduced into the host cell. The more than 1 copy of the exogenous fucosyltransferase gene can have the same sequence or different sequences. The more than 1 copy of the exogenous fucosyltransferase gene can be contained in the same expression cassette or in different expression cassettes in the host cell. The more than 1 copy of the exogenous fucosyltransferase gene can be located in a free vector (e.g., a plasmid, such as using a multi-copy plasmid or containing multiple copies in one plasmid) in the host cell or respectively integrated into different sites of the host cell genome. In some embodiments, the host cell can contain more than 1 copy of the exogenous a-1,2-fucosyltransferase gene, for example, more than 1 copy of the a-1,2-fucosyltransferase derived from Helicobacter pylori.

[0090] Synthesis of fucosylated oligosaccharides requires the involvement of acceptor substrates, and in some embodiments, the host cell is capable of providing an acceptor substrate for the fucosyltransferase catalyzed reaction intracellularly, for example, by transporting the acceptor substrate into the cell, or by synthesizing the acceptor substrate intracellularly. The kind of acceptor substrate is related to the target fucosylated oligosaccharide to be synthesized and the specific fucosyltransferase contained in the host cell, and the synthesis of a specific target fucosylated oligosaccharide is achieved by the combination of both the acceptor substrate and the fucosyltransferase having catalytic ability to the acceptor substrate. The acceptor substrate provided intracellularly should be capable of accepting the fucose residue catalyzed by the fucosyltransferase expressed in the host cell to synthesize the target fucosylated oligosaccharide.

[0091] In some embodiments, the host cell of the present application comprises a transporter protein capable of transporting an acceptor substrate into the cell, or comprises an intracellular synthesis pathway capable of synthesizing an acceptor substrate using an exogenous precursor (e.g., an exogenous carbon source). In some embodiments, the acceptor substrate is lactose or a lactose derivative (e.g., LNT, LNnT, or 2’-FL). In some embodiments, the exogenous precursor is lactose or lacto-N-triose II (LNT-2) (WO 2019 / 008133; incorporated by reference in its entirety).

[0092] In some embodiments, the acceptor substrate is lactose or a lactose derivative synthesized using lactose as a precursor, the structure of which allows it to generate a fucosylated oligosaccharide after being linked to a fucose moiety via fucosyltransferase catalysis. In some embodiments, the lactose derivative is capable of being synthesized intracellularly by an enzyme-catalyzed reaction using lactose as a precursor. In some embodiments, the lactose derivative can be, for example, LNT, LNnT, or 2’-FL.

[0093] In some embodiments, the host cell naturally has a transporter protein gene capable of transporting an acceptor substrate into the cell, or a synthesis pathway gene capable of synthesizing an acceptor substrate using an exogenous precursor (e.g., an exogenous carbon source). For example, E. coli has a lactose permease gene, which can naturally transport lactose into the cell, at which point the acceptor substrate can be transported into the cell using the endogenous enzyme of the host cell or synthesized in the cell using an exogenous carbon source.

[0094] In some embodiments, the host cell can be genetically engineered to introduce an exogenous transporter gene that can be used to transport the acceptor substrate into the cell or an exogenous synthesis pathway gene that can synthesize the acceptor substrate from an exogenously supplied precursor (e.g., an exogenously supplied carbon source), and the desired acceptor substrate is transported to or synthesized in the cell by expressing these exogenous genes, for example, when the host cell is not naturally capable of transporting the acceptor substrate into the cell or is not naturally capable of endogenously synthesizing the acceptor substrate, or when the host cell naturally has an enzyme gene that is capable of transporting the acceptor substrate into the cell but it is still desired to utilize other carbon sources as precursors for the synthesis of the acceptor substrate in the cell.

[0095] In some embodiments, the host cell of the present application is capable of providing lactose in the cell to provide lactose as the acceptor substrate or to synthesize the acceptor substrate.

[0096] In some embodiments, the host cell of the present application comprises a gene encoding a lactose transporter to express a lactose transporter. The lactose transporter is capable of transporting lactose from the culture medium into the cell to provide lactose in the cell. 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 make the host cell comprise the gene encoding the exogenous lactose transporter and express the exogenous lactose transporter. The exogenous lactose transporter gene can be comprised in a episomal expression vector introduced into the host cell or integrated into the chromosome of the host cell. In some embodiments, the lactose permease that the host cell comprises can be, for example, a lactose permease from E. coli (LacY) or a lactose permease from Kluyveromyces lactis (Lac12). In some embodiments, the E. coli lactose permease comprises an amino acid sequence as set forth in SEQ ID NO: 9.

[0097] In some embodiments, the host cell can also synthesize lactose intracellularly without the addition of exogenous lactose. In some embodiments, the host cell naturally has the ability to synthesize lactose from other carbon sources other than lactose, possibly performing endogenous synthesis of lactose. In some embodiments, the host cell can be genetically engineered to express enzymes for the synthesis of lactose from other carbon sources, such as glucose, for example, to express a b-1,4-galactosyltransferase to enable intracellular synthesis of lactose. A b-1,4-galactosyltransferase is capable of catalyzing the production of lactose from galactose and glucose. Examples of b-1,4-galactosyltransferase can be Pm1141 of Pasteurella multocida and Lex1 of Aggregatibacter aphrophilus NJ8700 (WO2015 / 150328; incorporated by reference herein in its entirety). When the host cell is cultured with a carbon source that is capable of being metabolized by the cell to glucose, the host cell is capable of producing free glucose intracellularly from the carbon source and catalyzing the synthesis of lactose by the b-1,4-galactosyltransferase. The carbon source can be, for example, glucose, sucrose, glycerol, fructose, xylose, cellulose, molasses, corn syrup, galactose, methanol, pyruvate, succinate, or any other carbon source that can be metabolized by the cell to glucose.

[0098] In some embodiments, the acceptor substrate for the fucosyltransferase can be synthesized from lactose as a precursor through enzyme-catalyzed reactions. For example, the acceptor substrate LNnT for the synthesis of LNnFP-I, LNnFP-III, LNnFP-V can be synthesized from lactose, UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-galactose (UDP-Gal) through the catalysis of b-1,3-N-acetylglucosaminyltransferase and b-1,4-galactosyltransferase. The acceptor substrate LNT for the synthesis of LNFP-I, LNFP-II, LNFP-V can be synthesized from lactose, UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-galactose (UDP-Gal) through the catalysis of b-1,3-N-acetylglucosaminyltransferase and b-1,3-galactosyltransferase (Tomotoshi Sugita et al., Journal of Biotechnology, Volume 361, 2023, Pages 110-118; Hu M et al., J Agric Food Chem. 2022; 70(28):8704-8712; incorporated by reference herein in their entirety). UDP-GlcNAc, UDP-Gal, lactose can be endogenously produced by the host cell or exogenously introduced.

[0099] In some embodiments, the host cell does not have an endogenous β-1,3-N- acetylglucosaminyltransferase gene, an endogenous exogenous β-1,4-galactosyltransferase gene, an endogenous β-1,3-N-acetylglucosaminyltransferase gene, and / or an endogenous β-1,3-galactosyltransferase gene, and such a host cell, in addition to being able to provide lactose within the cell (e.g., by expressing a lactose transport protein gene or by expressing an intracellular synthesis pathway gene that can synthesize lactose from an exogenous precursor), can be genetically engineered to introduce an exogenous β-1,3-N-acetylglucosaminyltransferase gene and an exogenous β-1,4-galactosyltransferase gene, or to introduce an exogenous β-1,3-N-acetylglucosaminyltransferase gene and an exogenous β-1,3-galactosyltransferase gene. These exogenous enzyme genes can be contained in episomal expression vectors introduced into the host cell or can be integrated into the chromosome of the host cell. Thus, in some embodiments, to produce more complex fucosylated oligosaccharides, the host cells of the present application can further include enzyme genes for producing suitable acceptor substrates, e.g., a β-1,3-N-acetylglucosaminyltransferase gene and a β-1,4-galactosyltransferase gene, or a β-1,3-N-acetylglucosaminyltransferase gene and a β-1,3-galactosyltransferase gene. Any one or more of these enzyme genes can be endogenous or exogenous.

[0100] The supply of UDP-galactose can be obtained from the host cell's own metabolic pathway, which can include, for example, phosphoglucomutase, UTP-glucose-1-phosphate- uridyltransferase, and UDP-glucose-4-epimerase catalyzed synthesis. The supply of UDP- galactose can also be obtained by providing galactose in the host cell culture medium, which is taken up by the cell, phosphorylated to galactose-1-phosphate, and converted to UDP- galactose. The supply of UDP-N-acetylglucosamine can also be obtained from the host cell's own metabolic pathway.

[0101] Different cells possess different intrinsic capabilities for the above-mentioned enzymes, and one skilled in the art will understand that when a host cell naturally possesses one or more of the above-mentioned enzymes, the endogenous enzyme of the host cell can be used without the need for introduction from an exogenous source, or the endogenous enzyme gene can be engineered to be overexpressed or inactivated to more favorably facilitate the synthesis of fucosylated oligosaccharides. For enzymes that the host cell does not naturally possess, a heterologous enzyme gene needs to be introduced. One skilled in the art is able to judge for a particular host cell which enzymes need to be introduced and which enzymes can use the endogenous enzyme of the host cell.

[0102] For example, for the most common host cells such as E. coli, C. glutamicum, B. subtilis, S. cerevisiae, etc., E. coli naturally has GDP-D-mannose-4,6-dehydratase (GMD), so when E. coli is used as a host cell, its endogenous GMD gene can be used, or its GMD gene can also be overexpressed to increase the synthesis of fucosylated oligosaccharide precursors. However, microorganisms such as B. subtilis, C. glutamicum, S. cerevisiae, etc. naturally do not have GDP-D-mannose-4,6-dehydratase, so a heterologous GMD gene needs to be introduced additionally.

[0103] For the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose into GDP-D-rhamnose, GDP-D-rhamnose-3,5-epimerase (GRE), fucosyltransferase, E. coli, C. glutamicum, B. subtilis, S. cerevisiae all naturally do not have these enzymes, and a heterologous enzyme gene needs to be introduced additionally.

[0104] E. coli and B. subtilis naturally have lactose transport proteins, so when lactose needs to be transported into the host cell, there is no need to introduce a corresponding enzyme from the outside. However, C. glutamicum and S. cerevisiae naturally do not have lactose transport proteins, so when lactose needs to be transported into the host cell, a heterologous enzyme gene needs to be introduced additionally.

[0105] As will be appreciated by those skilled in the art, the host cells of the present application can also be further engineered to optimize the production of the fucosylated oligosaccharides. For example, the host cells of the present application can also be further engineered to increase the production capacity of intracellular GDP-L-fucose. In some embodiments, the host cells can be engineered to overexpress one or more enzymes in the GDP-L-fucose synthesis pathway. In some embodiments, the host cells can be engineered to overexpress one or more genes in the GDP-D-mannose synthesis pathway. In some embodiments, the one or more genes in the GDP-D-mannose synthesis pathway include a phosphomannose mutase (ManB) gene and / or a mannose-1 -phosphate guanylyltransferase (ManC) gene. The overexpression can be achieved by introducing additional exogenous genes encoding the enzymes into the host cells, which can have the same or different sequences from the endogenous genes in the host cells having the same function, which can increase the copy number of the genes encoding the enzymes in the host cells, thereby increasing the expression level of the enzymes. The overexpression can also be achieved by engineering the regulatory sequences (e.g., promoters) operably linked to the genes encoding the enzymes in the host cells, for example, replacing the original promoters (e.g., the promoters of the genes naturally present in the host cells) with stronger promoters, or introducing mutations in the promoter regions or inserting new regulatory elements to enhance the activities thereof. As will be appreciated by those skilled in the art, the term "stronger promoter" refers to a promoter having stronger promoter activity, e.g., having stronger ability to bind to the transcription initiation complex and / or having stronger RNA polymerase binding ability, relative to the original promoters in the host cells (e.g., the promoters of the genes naturally present in the host cells). As is known to those skilled in the art, the use of stronger promoters can increase the expression level of the genes operably linked to the promoters (e.g., increase the expression level of the proteins encoded by the genes). In some embodiments, the stronger promoter refers to a promoter having a -10 region comprising TATAAT. In some embodiments, the host cells are engineered to have a -10 region sequence of the phosphomannose mutase (ManB) gene and / or the mannose-1 -phosphate guanylyltransferase (ManC) gene comprising TATAAT.

[0106] In some embodiments, the host cells can also be engineered to prevent depletion of the intracellular GDP-L-fucose pool. For example, the host cells of the present application can be further engineered to eliminate or attenuate other metabolic pathways of GDP-L-fucose, for example, to eliminate or attenuate enzymes capable of catalyzing the conversion of GDP-L-fucose to substances other than fucosylated oligosaccharides (e.g., colanic acid). For example, the host cells can be engineered to have no functional UDP-glucose lipid carrier transferase WcaJ or to have attenuated activity of UDP-glucose lipid carrier transferase WcaJ.

[0107] The host cell of the present application can be further engineered to eliminate or attenuate other metabolic pathways for lactose, i.e. other than the synthesis of fucosylated oligosaccharides catalyzed by a fucosyltransferase using lactose as acceptor substrate. For example, the host cell can be engineered to have no functional or have attenuated activity of the galactosidase LacZ.

[0108] The host cell of the present application can be further engineered to eliminate or attenuate other synthetic pathways for GDP-L-fucose that are naturally present in the host cell, e.g. to eliminate or attenuate an enzyme capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-L-fucose (e.g. the GDP-L-fucose synthase WcaG), so that the host cell synthesizes GDP-L-fucose via the synthetic pathway of the present application. For example, the host cell can be engineered to have no functional or have attenuated activity of the GDP-L-fucose synthase WcaG.

[0109] In the present application, the host cell can be a prokaryotic cell or a 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 can include bacteria, yeast cells, archaea, fungal cells, and the like. The microorganism can be a GRAS (Generally Regarded as Safe) microorganism. The bacteria, for example, can be a 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, and the like; Bacillus bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus cereus, Bacillus stearothermophilus, Bacillus megaterium, and the like; Lactobacillus bacteria such as Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactococcus lactis; Bifidobacterium bacteria; Streptococcus bacteria; Lactococcus bacteria; Streptmyces bacteria; Pseudomonas bacteria such as Pseudomonas aeruginosa; Clostridium bacteria; Brevibacillus bacteria; Enterococcus bacteria; Pediococcus bacteria; Leuconostoc bacteria; and the like.The yeast cell can for example be a yeast cell of the genus Saccharomyces, Saccharomycopsis, Pichia, Hansenula, Kluyveromyces, Yarrowia, Rhodotorula or Schizosaccharomyces, for example Saccharomyces cerevisiae, Yarrowia lipolytica, Candida utilis or Pichia pastoris.

[0110] Methods for genetically modifying host cells are well known to those skilled in the art. Genetic modification techniques can be used to introduce an exogenous nucleic acid sequence into a host cell (e.g., in the form of a plasmid), or to insert an exogenous nucleic acid sequence into the host cell genome or delete an endogenous nucleic acid sequence, or replace an endogenous nucleic acid sequence in the host cell genome with an exogenous nucleic acid sequence, to alter the genotype of the host cell and thereby alter its phenotype. For example, an exogenous gene can be introduced into a host cell to express a protein encoded by the exogenous gene (such as one or more enzymes as mentioned above), which can be achieved, for example, by introducing into the host cell a vector comprising the exogenous gene, which can be in linear or circular form, and which can be single- or double-stranded. The vector can be a self-replicating vector. The vector can be episomal or integrative. The vector can be, for example, a plasmid vector, a bacteriophage vector, a bacterial artificial chromosome, a transposon-based vector, or a CRISPR / Cas system-based vector, etc. The exogenous gene can exist in and be expressed from an episomal vector (e.g., an episomal plasmid) after being introduced into the host cell, which can be achieved, for example, by introducing into the host cell a plasmid vector comprising an exogenous gene expression cassette. The exogenous gene expression cassette can include the exogenous gene and regulatory sequences operably linked thereto. Regulatory sequences can include, but are not limited to, promoters, enhancers, terminators, and other expression control elements. The promoter can be constitutive or inducible. The exogenous gene can also be integrated into the genome of the host cell for expression, which can be achieved, for example, by introducing into the host cell an integrative plasmid vector comprising the exogenous gene (which can comprise homology arms for integration into the host cell genome by homologous recombination), a bacteriophage vector, a CRISPR / Cas system, or a transposon system (e.g., Piggybac or Sleeping Beauty system). The expression cassette comprising the exogenous gene can be integrated into the host cell genome, or the exogenous gene can be integrated into a suitable site in the host cell to utilize the host cell’s endogenous regulatory sequences to express the exogenous gene. Suitable methods for introducing an exogenous nucleic acid sequence (e.g., a vector) into a host cell 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, bacteriophage vector transduction, etc. These genetic modification techniques can also be used to knock out or knock down an endogenous gene, so that the host cell does not have a functional protein encoded by the endogenous gene or the activity of the protein encoded by the endogenous gene is attenuated. This can be achieved, for example, by deleting all or part of the sequence of the endogenous gene, mutating the endogenous gene, or inserting an exogenous sequence into the endogenous gene. These genetic modification techniques can also be used to alter the regulatory sequences (e.g., promoters) of an endogenous gene to increase the expression of the endogenous gene.Those skilled in the art can select an appropriate method to genetically modify 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)).

[0111] Suitable vectors and regulatory sequences for use with various host cells can be chosen by one of ordinary skill in the art depending on the host cell used. For example, for E. coli, vectors comprising but not limited to pJC1, pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, or plasmids based thereon can be used; for C. glutamicum, vectors comprising but not limited to pBL1, pEKEx1, pEKEx2, pXMJ19, pJC1, pHM1519, pVWEx1, pZ8-1, pECTAC-K99, pECTAC-XK99E, pECTAC-XC99E, pECTAC-XT99A, pNG2, pAPE12 plasmid vector or plasmid vectors based thereon, pK18mobsacB or suicide plasmid vectors based thereon can be used; for B. subtilis, vectors comprising but not limited to pHT43, pUB110, pE194, pUCX05-bgaB, pWB980, pHP13, pBE2, pHP13, pHP13-43, pHT01, pHT304, pMK3, pHCMC05, pMA5, pHY300PLK or pMUTIN4 can be used; for yeast, vectors comprising but not limited to pPIC9, pPIC9k, pHIL-S1, pPICza, pYAM75P6, pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa, pYES2, pYES2 / NT, pYES2 / CT, pYES3, pYES6, pYCplac22-GFP, pAUR123, pRS303TEF, pRS304, pRS305, pRS306, pY13TEF, pY14TEF, pY15TEF, pY16TEF, pSH47, pLacZi, pHIS2 or pGAD42 can be used.Promoters suitable for use in bacteria include, but are not limited to, inducible promoters such as Ptac, Plac, Ptrc, and the like, or constitutive promoters such as Psod, PcspB, Ptuf, and PgapA, and can also include other promoters such as Pcg2195 and the like (Wei L, et al., Appl Microbiol Biotechnol. 2018 May; 102(9): 4117-4130, hereby incorporated by reference in its entirety); PdapA and variants thereof such as Pdap-A16 (Vasicova P, et al., J Bacteriol. 1999 Oct; 181(19): 6188-91, hereby incorporated by reference in its entirety); and Pdap-A16-1 and variants thereof Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, and the like (Duan, Yanting et al., ACS Synthetic Biology. 2021, 10, 38-48, hereby incorporated by reference in its entirety). In some embodiments, the promoters Psod, Pcg2195, and / or Pgap are used to express an exogenous gene in a host cell. In some embodiments, a promoter having a nucleic acid sequence selected from SEQ ID NOs: 51-53 is used to express an exogenous gene in a host cell.

[0112] In some embodiments, the host cell is a genetically modified C. glutamicum that comprises an endogenous GDP-D-mannose synthesis pathway gene and is genetically modified to comprise a gene encoding an exogenous GDP-D-mannose-4,6-lyase (GMD), a gene encoding an exogenous reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose, a gene encoding an exogenous GDP-D-rhamnose-3,5-epimerase (GRE), and a gene encoding an exogenous fucosyltransferase. In more preferred embodiments, the genetically modified C. glutamicum further comprises a gene encoding an exogenous lactose permease. In more preferred embodiments, the fucosyltransferase is an alpha-1,2-fucosyltransferase or an alpha-1,3-fucosyltransferase, and the host cell is used to synthesize 2’-FL or 3-FL. In more preferred embodiments, the fucosyltransferase is an alpha-1,2-fucosyltransferase or an alpha-1,3-fucosyltransferase derived from H. pylori.

[0113] In some embodiments, the host cell is a genetically modified E. coli that comprises an endogenous GDP-D-mannose synthesis pathway gene and is genetically modified to comprise a gene encoding an exogenous reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose, a gene encoding an exogenous GDP-D-rhamnose-3,5-epimerase (GRE), and a gene encoding an exogenous rhamnosyltransferase. In more preferred embodiments, the genetically modified E. coli has a gene encoding beta-galactosidase LacZ knocked out, a gene encoding GDP-L-fucose synthetase WcaG knocked out, and / or a gene encoding UDP-glucose lipid carrier transferase WcaJ knocked out. In more preferred embodiments, the rhamnosyltransferase is an alpha-1,2- rhamnosyltransferase or an alpha-1,3-rhamnosyltransferase, and the host cell is used to synthesize 2’-FL or 3-FL. In more preferred embodiments, the rhamnosyltransferase is an alpha-1,2- rhamnosyltransferase or an alpha-1,3-rhamnosyltransferase derived from H. pylori.

[0114] The host cells and methods described herein can be used to produce fucosylated oligosaccharides, particularly those that are HMOs, such as those that comprise a lactose moiety and a fucose moiety, which can be synthesized using lactose and GDP-L-fucose as starting materials and / or substrates. Fucosylated oligosaccharides that can be produced using the host cells and methods described herein include, but are not limited to, fucosyllactose, lacto-N-fucopentaose, lacto-N-neofucopentaose, lacto-N-neodifucohexaose, and the like, such as 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofucopentaose I (LNnFP-I), lacto-N-neofucopentaose III (LNnFP-III), lacto-N-neofucopentaose V (LNnFP-V), lacto-N-difucohexaose I (LNDFH-I), and / or lacto-N-difucohexaose II (LNDFH-II), and the like.

[0115] The present application also provides methods of producing fucosylated oligosaccharides, comprising culturing the genetically engineered host cells of the present application under suitable conditions to synthesize the fucosylated oligosaccharides.

[0116] In some embodiments, at least one carbon source is included in the culture medium, which can be selected from, but not limited to, glucose, sucrose, glycerol, fructose, lactose, xylose, cellulose, molasses, corn syrup, galactose, methanol, pyruvate, succinate, and the like. The carbon source can be supplemented at appropriate times during the cultivation of the host cell.

[0117] A receptor substrate or a substance capable of producing a receptor substrate as a precursor substance through a synthetic pathway included in the host cell can be included in the culture medium. In some embodiments, the receptor substrate is lactose or a lactose derivative (e.g., LNT, LNnT, or 2'-FL). In some embodiments, the precursor substance is lactose, lacto-N-triose II, or a carbon source other than lactose (e.g., glucose or a carbon source capable of being metabolized into glucose by the cell, which can be glucose, sucrose, glycerol, fructose, xylose, cellulose, molasses, corn syrup, galactose, methanol, pyruvate, succinate, or any other carbon source that can be metabolized into glucose by the cell). Correspondingly, the host cell used includes a transporter gene capable of transporting the receptor substrate into the cell, or a synthetic pathway gene capable of synthesizing the receptor substrate using the precursor substance.

[0118] In some embodiments, lactose can be included in the culture medium to provide a receptor substrate for fucosyltransferase. Correspondingly, the host cell used includes a gene encoding a lactose transporter. In this case, lactose can directly accept a fucose as a receptor substrate to synthesize a fucosylated oligosaccharide, for example, in the production of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), or difucosyllactose (DFL). In some embodiments, lactose serves as a precursor substance to synthesize a receptor substrate inside the cell. Correspondingly, the host cell used includes a gene encoding a lactose transporter and a synthetic pathway gene capable of synthesizing a receptor substrate using lactose as a precursor substance, for example, in the production of lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-neofucopentaose I (LNnFP-I), lacto-N-neofucopentaose III (LNnFP-III), lacto-N-neofucopentaose V (LNnFP-V), lacto-N-difucohexaose I (LNDFH-I), and / or lacto-N-difucohexaose II (LNDFH-II), etc. Lactose can be supplemented at appropriate times during the cultivation of the host cell.

[0119] When a gene encoding one or more enzymes included in the host cell is expressed in an inducible manner, an inducer is added during the cell cultivation to induce the expression of the enzyme(s).

[0120] The host cell culture can be a batch fermentation, i.e., using a closed culture system with a specific medium at the start of the fermentation and using specific temperature, pressure, aeration, and other environmental conditions to optimize growth, without the addition of nutrients or withdrawal of fermentation broth during the incubation of the cells. The host cell culture can also be a fed-batch fermentation, i.e., where nutrients are added intermittently or continuously during the fermentation, but without withdrawal of fermentation broth. The host cell culture can also be a continuous fermentation, i.e., where nutrients are added continuously and fermentation broth is withdrawn continuously, such that the volume of culture in the fermentation system is maintained constant. The host cell fermentation can also be a combination of two or three of the above-mentioned fermentation modes.

[0121] The genetically engineered host cells of the present application are cultured under conditions suitable for the production of the fucosylated oligosaccharides. Suitable conditions include suitable temperature, pH, dissolved oxygen, osmotic pressure, and other conditions, etc. Suitable conditions can vary depending on the type of host cell, which can be readily determined by one skilled in the art.

[0122] In some embodiments, the production method further comprises recovering the synthesized product, i.e., the fucosylated oligosaccharides, from the culture medium. The term "recovering" refers to isolating or further purifying the fucosylated oligosaccharides produced by the host cells of the present application from other components in the host cell culture. The term "purifying" refers to removing impurities and unwanted byproducts, such as cells, ions, salts, other saccharides other than the desired fucosylated oligosaccharides.

[0123] 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 supernatant of the culture medium or cell lysate. Cell lysis can be performed by chemical or physical methods known in the art.

[0124] Purification can be performed by techniques known to those skilled in the art. For example, the product can be purified from the culture medium by methods known to those skilled in the art, such as by column chromatography using an activated charcoal step and elution with 35-50% ethanol, by ethanol gradient, or by size exclusion. Purity can be assessed by any known method, such as thin layer chromatography or other electrophoretic or chromatographic techniques generally known in the art.

[0125] The present application is further described by the following examples, which should not be construed as limiting the present application.

[0126] The reagents used in the following examples are commercially available unless otherwise specified. The molecular biology experimental methods not specifically described in the examples are performed according to the specific methods listed in J. Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition, or according to the instructions of the kits and products.

[0127] Example 1 Culture of C. glutamicum and method for detecting products

[0128] Two different media were used to culture C. glutamicum, seed medium LBHI and fermentation medium FM20.

[0129] The components of seed medium LBHI are as follows: yeast extract 2.5 g / L, peptone 5 g / L, NaCl 5 g / L, brain heart infusion broth 18.5 g / L.

[0130] The components of fermentation medium FM20 are as follows: glucose 50 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.

[0131] Seed medium LBHI was sterilized by autoclaving (121°C, 20 min). Fermentation medium FM20 was sterilized by filtration through a 0.22 μm filter. If necessary, the medium was made selective by the addition of kanamycin. Solid plates were prepared by the addition of 2% agar to the medium.

[0132] For fermentation of C. glutamicum, colonies from plates were picked into seed medium and incubated overnight. The overnight culture was inoculated into 96-well plates containing fermentation medium at a 5% inoculation level and incubated at 30°C for 48 h. The plates were centrifuged and the supernatant was removed and assayed for the concentration of each substance in the supernatant.

[0133] The analysis and detection of GDP-D-mannose, GDP-D-rhamnose, GDP-L-fucose and other substances are carried out by Agilent UPLC-MS. The sample is separated by a chromatographic column, ionized in an ion source and detected by a mass spectrometer mass analyzer to determine the concentration of each substance. The chromatographic column used is Agilent hilic-z 2.7 μm, 3.0*150 mm, the mobile phase is 10 mM ammonium acetate (pH 9.6)-acetonitrile (V / V=25% / 75%), the flow rate is 0.35 mL / min, the injection volume is 1 μL, the column temperature is 35°C, the mass spectrometer detector ion source is ESI, the scanning mode is SIM, and the polarity is negative mode.

[0134] The analysis and detection of 2'-FL and 3-FL are carried out by Agilent UPLC-MS. The sample is separated by a chromatographic column, ionized in an ion source and detected by a mass spectrometer mass analyzer to determine the concentration of each substance. The chromatographic column used is ACQUITY UPLC BEH Amide 1.7 μm, 2.1*150 mm or other equivalent chromatographic column, the mobile phase is 10 mM ammonium acetate (pH 9.6)-acetonitrile (V / V=30% / 70%), the flow rate is 0.3 mL / min, the injection volume is 1 μL, the column temperature is 35°C, the mass spectrometer detector ion source is ESI, the scanning mode is SIM, and the polarity is positive mode.

[0135] Culture of recombinant E. coli and protein expression and purification method

[0136] LB medium is used for the culture of E. coli, and the specific components are as follows: 10 g / L of proteose peptone, 5 g / L of yeast powder, and 10 g / L of NaCl.

[0137] The protein sequence to be expressed is synthesized by gene synthesis, and the synthesis vector is pRSFDuet-1 plasmid. The recombinant plasmid is introduced into E. coli BLR(DE3) to construct a recombinant E. coli. The recombinant E. coli is inoculated into LB liquid medium containing 50 μg / mL of kanamycin and cultured at 37°C, 200 rpm. When the optical density (OD 600 ) of the culture solution reaches 0.6-0.8, the culture temperature is adjusted to 16°C, and the final concentration of isopropyl-β-D-thiogalactoside (IPTG) is 0.2 mM to induce protein expression. After 24 h of continuous culture, the bacterial cells are collected by high-speed refrigerated centrifugation to obtain wet cells of the engineering strain with high expression of the target protein.

[0138] Protein purification is carried out by nickel affinity chromatography, and the buffers used for purification include:

[0139] A solution: 20 mM PBS, 500 mM NaCl, 10 mM imidazole, 2 mM β-mercaptoethanol, pH 7.4;

[0140] B solution: 20 mM PBS, 500 mM NaCl, 500 mM imidazole, 2 mM β-mercaptoethanol, pH 7.4;

[0141] C solution: 20 mM PBS, 150 mM NaCl, 1 mM dithiothreitol, pH 7.4.

[0142] The specific purification method is as follows: 1) the wet cells obtained above are resuspended with A solution and subjected to ultrasonic disruption, and the supernatant of the disrupted solution is obtained by centrifugal separation; 2) the nickel column is equilibrated with 10 column volumes of A solution, and then the supernatant of the disrupted solution is filtered with a filter membrane and then loaded; 3) after the supernatant is completely eluted, 5 column volumes of a mixture of A and B solutions (5% B solution) are added to wash away impurities; 4) 5 column volumes of a mixture of A and B solutions (40% B solution) are used to elute the target protein adsorbed by nickel; 5) the collected target protein is concentrated by centrifugation with a 10 kDa ultrafiltration tube. When the volume of the concentrated solution is less than 0.5 mL, 10 mL of C solution is added, and the solution is concentrated again by centrifugation; 6) the buffer replacement is completed by repeating the above steps for 3 times, most of the imidazole is removed, and the target protein is obtained. The pure enzyme is quickly frozen in liquid nitrogen and stored in a refrigerator at -80°C. The protein concentration is determined by using an ultramicro spectrophotometer NanoDrop one to detect the ultraviolet absorption of the protein at 280 nm.

[0143] Example 3 GDP-mannose-3,5-epimerase (GME) catalyzing the reversible isomerization reaction of GDP-D-rhamnose and GDP-L-fucose

[0144] Since GDP-mannose-3,5-epimerase (GME) was discovered in the 1970s, researchers have studied the physicochemical properties and catalytic mechanisms of GMEs from different sources. These GMEs include AtGME (SEQ ID NO: 1, whose gene sequence is SEQ ID NO: 2) from Arabidopsis thaliana, MfGME (SEQ ID NO: 3, whose gene sequence is SEQ ID NO: 4) from Methylacidiphilum fumariolicum, and OsGME (SEQ ID NO: 5, whose gene sequence is SEQ ID NO: 6) from Oryza sativa. However, there have been no reports on the exploration of the catalytic substrate spectrum of GME, and it is unknown whether GME can catalyze the isomerization of GDP-D-rhamnose and GDP-L-fucose.

[0145] The AtGME gene sequence, MfGME gene sequence, and OsGME gene sequence were synthesized. The synthesis vector was pRSFDuet-1 plasmid, and the synthesis sequence was located after the first multiple cloning site histidine tag. The recombinant plasmids were named pRSFDuet-AtGME, pRSFDuet-MfGME, and pRSFDuet-OsGME. According to the method shown in Example 2, AtGME, MfGME, and OsGME protein pure enzymes were obtained. Then, the activities of the three GMEs were detected by in vitro enzymatic reaction using GDP-D-rhamnose and GDP-L-fucose as substrates, respectively.

[0146] The reversible isomerization reaction route of GDP-D-rhamnose and GDP-L-fucose catalyzed by GME is as follows:

[0147] The specific reaction conditions are as follows: 50 mM phosphate buffer (pH 7.5), 0.5 g / L GDP-D-rhamnose or GDP-L-fucose, and 1 g / L GME pure enzyme. In the control experiment, only the protein was not added, and the other conditions were kept the same. The reaction was carried out at 30°C and 400 rpm for 5 h, and then 5 times the volume of organic solvent (acetonitrile:methanol = 1:1) was added to quench the reaction. After centrifugation to remove the protein, the product was analyzed according to the method described in Example 1.

[0148] Among AtGME, MfGME and OsGME, OsGME from Oryza sativa showed the best reaction performance. As shown in Figure 2, with GDP-D-rhamnose as the substrate, OsGME can generate GDP-L-fucose. With GDP-L-fucose as the substrate, OsGME can also generate GDP-D-rhamnose. OsGME realizes the reversible isomerization conversion of GDP-D-rhamnose and GDP-L-fucose. The substrate actually catalyzed by GME in the present application is GDP-D-rhamnose, and therefore the enzyme represented by GDP-mannose-3, 5-epimerase (GME) in the present application, which can catalyze the conversion of GDP-D-rhamnose to GDP-L-fucose, is named as GDP-D-rhamnose-3, 5-epimerase (GRE). The above-mentioned AtGME, MfGME and OsGME can also be respectively referred to as AtGRE, MfGRE and OsGRE. If GDP-D-mannose-4, 6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose reductase (RMD) and GDP-D-rhamnose-3, 5-epimerase (GRE) are introduced into the strain in vivo, it is extremely possible to realize the synthesis of 2'-FL through a brand-new metabolic pathway.

[0149] Example 4 Construction of Corynebacterium glutamicum Recombinant Strain HpfutC-lacY-ManB-ManC

[0150] The α-1, 2-fucosyltransferase HpfutC gene sequence (SEQ ID NO: 8, the amino acid sequence of the enzyme is SEQ ID NO: 7) of Helicobacter pylori and the lactose permease LacY gene sequence (SEQ ID NO: 10, the amino acid sequence of the enzyme is SEQ ID NO: 9) of Escherichia coli were subjected to gene synthesis. The gene synthesis vector used pUC57 plasmid, and the recombinant plasmids were named as pUC57-HpfutC and pUC57-LacY, respectively.

[0151] The homologous arms HpfutC-lacY-Up, HpfutC-lacY-Down, the promoter Psod (SEQ ID NO: 51) and Pcg2195 (SEQ ID NO: 52) were obtained by PCR amplification using the primer pairs HpfutC-lacY-U-F / HpfutC-lacY-U-R, HpfutC-lacY-D-F / HpfutC-lacY-D-R, Psod-F-1 / Psod-R-1, Pcg2195-F-1 / Pcg2195-R-1, respectively, with Corynebacterium glutamicum ATCC 13032 genome as the template (PCR system used 2xPhanta Max Master Mix, (Dye Plus), Vazyme); the lactose permease LacY gene fragment was obtained by PCR amplification using the primer pair LacY-F / LacY-R with pUC57-LacY as the template (PCR system used 2xPhanta Max Master Mix, (Dye Plus), Vazyme); the a-1,2-fucosyltransferase HpfutC gene fragment was obtained by PCR amplification using the primer pair HpfutC-F / HpfutC-R with pUC57-HpfutC as the template; the plasmid pK18mobsacB was digested with restriction endonucleases EcoR I and Xba I to obtain the linearized fragment line-pK18mobsacB. The above seven fragments: HpfutC-lacY-Up, Pcg2195, HpfutC, Psod, LacY, HpfutC-lacY-Down, line-pK18mobsacB were assembled seamlessly using the recombination cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, Catalog No: C113-02), and the reaction system and reaction conditions were in accordance with the instructions of the kit. After the seamless assembly was completed, the Trans1 T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-HpfutC-lacY. The verified plasmid was electroporated into Corynebacterium glutamicum ATCC 13032, and the genes encoding HpfutC and LacY were introduced into the poxB site of the Corynebacterium glutamicum ATCC 13032 genome by using kanamycin resistance and sacB gene counter-screening. To enable the strain to have sufficient fucosyltransferase activity, the a-1,2-fucosyltransferase HpfutC gene sequence was inserted into the cg0554 site and tnp2b site of the Corynebacterium glutamicum genome by a similar method.On this basis, the same sacB gene counter-screening method is used to replace the phosphomannomutase ManB promoter-10 region sequence "TAGGAT" with "TATAAT", and the mannose-1-phosphate guanylyltransferase ManC promoter-10 region sequence "TAAAGT" with "TATAAT", in order to improve the supply of 2'-FL precursor GDP-L-fucose. Thus, the basic strain HpfutC-LacY-ManB-ManC is obtained.

[0152] Table 1 Primers used for constructing the recombinant strain HpfutC-LacY-ManB-ManC of Corynebacterium glutamicum

[0153] Example 5 Construction of 2'-FL production strain

[0154] RMD (PaRMD) derived from Pseudomonas aeruginosa (SEQ ID NO: 11, the gene sequence of which is SEQ ID NO: 12) and RMD (AtRMD) derived from Aneurinibacillus thermoaerophilus (SEQ ID NO: 13, the gene sequence of which is SEQ ID NO: 14) have been confirmed to catalyze the asymmetric reduction of GDP-4-keto-6-deoxy D-mannose. In addition, several documents have reported that carbonyl reductases can catalyze the asymmetric reduction of GDP-4-keto-6-deoxy D-mannose analogues (Biochemistry. 2022, 61: 2138-2147; Angew Chem Int Ed Engl. 2008, 47(51): 9814-59). This example investigates whether these reductases have activity for GDP-4-keto-6-deoxy D-mannose, and the specific reductase information is shown in Table 2. These reductases are gene synthesized, and the gene synthesis vector is pUC57, and the recombinant plasmids are named pUC57-1, pUC57-2, pUC57-3, pUC57-4, pUC57-5, pUC57-6, pUC57-7, pUC57-8, pUC57-9, pUC57-10, pUC57-11, pUC57-12, pUC57-13, pUC57-14, pUC57-15.

[0155] Table 2 Information of GDP-4-keto-6-deoxy D-mannose reductase to be screened

[0156] wherein the gene sequences of GerK1, DnmV, UrdZ3, LanZ3, Mydl, UrdR, TyID, ChmD, DdahC, HS10A, HS15, HS41B, and HS53 are 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, and SEQ ID NO: 40, respectively.

[0157] The promoter Pgap (SEQ ID NO: 53), Pcg2195 and Psod were amplified by PCR using primer pairs Pgap-F-2 / Pgap-R-2, Pcg2195-F-2 / Pcg2195-R-2, Psod-F-2 / Psod-R-2, respectively, with Corynebacterium glutamicum ATCC 13032 genome as the template (PCR system used 2xPhanta Max Master Mix, (Dye Plus), Vazyme). The gene fragment encoding GDP-D-mannose-4,6-dehydratase EcGMD (SEQ ID NO: 41, the gene sequence is SEQ ID NO: 42) was amplified by PCR using primer pairs EcGMD-F and EcGMD-R with E. coli MG1655 genome as the template. The gene fragment encoding GDP-D-rhamnose-3,5-epimerase OsGME was amplified by PCR using primer pairs OsGME-F and OsGME-R with pRSFDuet-OsGME as the template. The gene fragments encoding PaRMD, AtRMD, GerK1, DnmV, UrdZ3, LanZ3, Mydl, UrdR, TyID, ChmD, DdahC, HS10A, HS15, HS41B, HS53 were amplified by PCR using corresponding primer pairs 1-F-1 / 1-R-1, 2-F-1 / 2-R-1, 3-F-1 / 3-R-1, 4-F-1 / 4-R-1, 5-F-1 / 5-R-1, 6-F-1 / 6-R-1, 7-F-1 / 7-R-1, 8-F-1 / 8-R-1, 9-F-1 / 9-R-1, 10-F-1 / 10-R-1, 11-F-1 / 11-R-1, 12-F-1 / 12-R-1, 13-F-1 / 13-R-1, 14-F-1 / 14-R-1, 15-F-1 / 15-R-1, respectively, with pUC57-1, pUC57-2, pUC57-3, pUC57-4, pUC57-5, pUC57-6, pUC57-7, pUC57-8, pUC57-9, pUC57-10, pUC57-11, pUC57-12, pUC57-13, pUC57-14, pUC57-15 as the templates (PCR system used 2xPhanta Max Master Mix, (Dye Plus), Vazyme). The plasmid pJC1 was linearized by restriction enzymes BamH I and Sal I to obtain linear fragment line-pJC1.The fragments Pgap, Pcg2195, Psod, EcGMD, OsGME, line-pJC1 and different reductase gene fragments were respectively assembled by using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, item number: C113-02). The reaction system and reaction conditions were all performed according to the kit instructions. After the seamless assembly was completed, the Trans1 T1 competent cells were transformed to obtain the recombinant plasmid.

[0158] pJC1-Pgap_EcGMD-Pcg2195_1-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_2-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_3-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_4-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_5-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_6-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_7-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_8-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_9-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_10-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_11-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_12-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_13-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_14-Psod_OsGME, pJC1-Pgap_EcGMD-Pcg2195_15-Psod_OsGME.

[0159] Table 3 Primers used for constructing 2’-FL production strains

[0160] The above recombinant plasmids were sequentially electrotransformed into the base strain HpfutC-LacY-ManB-ManC, to obtain recombinant Corynebacterium glutamicum strains Cg2FL-1, Cg2FL-2, Cg2FL-3, Cg2FL-4, Cg2FL-5, Cg2FL-6, Cg2FL-7, Cg2FL-8, Cg2FL-9, Cg2FL-10, Cg2FL-11, Cg2FL-12, Cg2FL-13, Cg2FL-14, Cg2FL-15, respectively.

[0161] The correspondence between the above strains and the transformed recombinant plasmids is shown in the following table:

[0162] Example 6 Production of 2’-FL by recombinant Corynebacterium glutamicum strains containing different reductases

[0163] The recombinant Corynebacterium glutamicum strains Cg2FL-1 to Cg2FL-15 were cultured and analyzed according to the method of Example 1. Among the 15 recombinant strains screened, 2’-FL production was detected in multiple strains, with Cg2FL-11 having the highest yield, reaching 78 mg / L (Table 4). The corresponding reductase was reductase DdahC from Campylobacter jejuni. The 2’-FL produced was analyzed by LC-MS and the molecular weight was found to be 511 [M+Na] + , and the retention time and molecular weight were consistent with those of the standard (Figure 3). This confirmed that the metabolic pathway conceived in Example 3 could achieve 2’-FL synthesis.

[0164] Table 4 2’-FL production yield of recombinant Corynebacterium glutamicum strains containing different reductases

[0165] Example 7 Construction of recombinant Corynebacterium glutamicum control strains not containing GDP-D-rhamnose-3,5-epimerase

[0166] The gene fragments of different reductases were obtained by PCR amplification using the primers in Table 5 and pUC57-1 to pUC57-15 in Example 5 as templates (2x Phanta Max Master Mix, (Dye Plus), Vazyme was used in the PCR system). The gene fragments of different reductases were respectively assembled with the Pgap, Pcg2195, EcGMD, line-pJC1 gene fragments obtained in Example 5 using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, item number: C113-02), and the reaction system and reaction conditions were carried out according to the kit instructions. After the seamless assembly was completed, the Trans1 T1 competent cells were transformed to obtain recombinant plasmids pJC1-Pgap_EcGMD-Pcg2195_1, pJC1-Pgap_EcGMD-Pcg2195_2, pJC1-Pgap_EcGMD-Pcg2195_3, pJC1-Pgap_EcGMD-Pcg2195_4, pJC1-Pgap_EcGMD-Pcg2195_5, pJC1-Pgap_EcGMD-Pcg2195_6, pJC1-Pgap_EcGMD-Pcg2195_7, pJC1-Pgap_EcGMD-Pcg2195_8, pJC1-Pgap_EcGMD-Pcg2195_9, pJC1-Pgap_EcGMD-Pcg2195_10, pJC1-Pgap_EcGMD-Pcg2195_11, pJC1-Pgap_EcGMD-Pcg2195_12, pJC1-Pgap_EcGMD-Pcg2195_13, pJC1-Pgap_EcGMD-Pcg2195_14, pJC1-Pgap_EcGMD-Pcg2195_15.

[0167] Table 5 Primers used for constructing control strains

[0168] The above recombinant plasmids pJC1-Pgap_EcGMD-Pcg2195_1 to pJC1-Pgap_EcGMD-Pcg2195_15 were respectively electro-transformed into the previously obtained base strain HpfutC-LacY-ManB-ManC, to obtain control recombinant strains Cg2FL-16, Cg2FL-17, Cg2FL-18, Cg2FL-19, Cg2FL-20, Cg2FL-21, Cg2FL-22, Cg2FL-23, Cg2FL-24, Cg2FL-25, Cg2FL-26, Cg2FL-27, Cg2FL-28, Cg2FL-29, Cg2FL-30, respectively.

[0169] The correspondence between the above strains and the transformed recombinant plasmids is shown in the following table:

[0170] The recombinant strains Cg2FL-16 to Cg2FL-30 of Corynebacterium glutamicum were cultured and analyzed according to the method of Example 1. According to the product analysis results, Cg2FL-29 produced 3.1 mg / L of 2’-FL, and the reductase HS41B showed weak epimerization activity. In addition, the other control strains did not show 2’-FL production capacity, indicating that most of the active reductases are monofunctional proteins, including DdahC, which only has reduction activity but no isomerization activity for GDP-4-keto-6-deoxy-D-mannose. At this time, GME is necessary for the production of 2’-FL.

[0171] Example 8 In vitro multi-enzyme cascade reaction to produce GDP-D-rhamnose

[0172] Further, coupling EcGMD and DdahC, GDP-D-mannose is converted to GDP-rhamnose through in vitro enzymatic cascade reaction. The EcGMD-1 gene fragment and the DdahC gene fragment were amplified by PCR using the primers in Table 6 with E. coli MG1655 genome, pUC57-11 as templates (PCR system used 2xPhanta Max Master Mix, (Dye Plus), Vazyme); the plasmid pRSFDuet-1 was digested with restriction endonuclease BamH I and Sal I to obtain a linearized fragment line-pRSFDuet. The EcGMD-1 gene fragment, the DdahC gene fragment and the line-pRSFDuet gene fragment were assembled by using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, Cat No: C113-02) respectively, and the reaction system and reaction conditions were in accordance with the instructions of the kit. After the completion of the seamless assembly, the Trans1 T1 competent cells were transformed to obtain the recombinant plasmids pRSFDuet-EcGMD and pRSFDuet-DdahC. The EcGMD pure enzyme and the DdahC pure enzyme were obtained according to the method shown in Example 2.

[0173] Table 6 Primers used for the construction of EcGMD and DdahC protein expression plasmids

[0174] First, GDP-D-mannose is converted to GDP-4-keto-6-deoxy-D-mannose by EcGMD, and the specific reaction conditions are as follows: 50 mM phosphate buffer (pH 7.5), 0.5 g / L GDP-D-mannose, 0.5 mM oxidized coenzyme II, 5 mM magnesium chloride, 1 g / L EcGMD pure enzyme. The reaction was carried out at 30°C, 400 rpm for 3 hours, and then a certain amount of reaction solution was taken and 5 times volume of organic solvent (acetonitrile:methanol = 1:1) was added to quench the reaction. After centrifugation to remove the protein, the product was analyzed according to the method described in Example 1.

[0175] As shown in Figure 4, after 3 hours of reaction, GDP-D-mannose has completely disappeared, and a new peak is generated. Through LC-MS detection analysis, it can be obtained that the molecular weight of the new peak is 586 [M-H] - , which is speculated to be GDP-4-keto-6-deoxy-D-mannose.

[0176] The reaction solution was subjected to ultrafiltration to remove the protein EcGMD, and then 3 mM reduced coenzyme II and 4 g / L of DdahC pure enzyme were added to the filtrate, and the reaction was carried out at 30°C, 400 rpm for 3 hours. After the reaction was completed, 5 times the volume of organic solvent (acetonitrile:methanol = 1:1) was added to quench the reaction. After centrifugation to remove the protein, the product was analyzed according to the method described in Example 1.

[0177] As shown in Figure 5, the GDP-4-keto-6-deoxy-D-mannose generated by EcGMD was completely converted to GDP-D-rhamnose by DdahC. The in vitro experimental results show that GDP-D-mannose can be converted to GDP-D-rhamnose by EcGMD and DdahC. Combined with the results obtained in the above examples, it can be concluded that 2’-FL is generated through a completely new synthesis pathway, i.e., through the pathway shown in Figure 1.

[0178] Example 9 Construction of 3-fucosyllactose-producing strain and production of 3-fucosyllactose

[0179] Referring to the genetic modification method described in Example 4, the α-1,3-fucosyltransferase HpfutA gene (SEQ ID NO: 44, the amino acid sequence of the enzyme is SEQ ID NO: 43) derived from Helicobacter pylori and the lactose permease LacY gene (SEQ ID NO: 10, the amino acid sequence of the enzyme is SEQ ID NO: 9) derived from Escherichia coli were integrated into the genome of Corynebacterium glutamicum ATCC 13032 to obtain the base strain HpfutA-LacY. Referring to the genetic modification method described in Example 5, the GDP-D-mannose-4,6-dehydratase EcGMD gene (SEQ ID NO: 41) derived from Escherichia coli, the GDP-mannose-3,5-epimerase OsGME gene (SEQ ID NO: 5) derived from Oryza sativa, and the GDP-4-keto-6-deoxy D-mannose reductase DdahC gene (SEQ ID NO: 31) derived from Campylobacter jejuni were integrated into the genome of the base strain HpfutA-LacY to construct the 3-fucosyllactose-producing strain Cg3FL-1. According to the method of Example 1, the recombinant strain Cg3FL-1 of Corynebacterium glutamicum was cultured and analyzed, and 45 mg / L of 3-fucosyllactose was detected.

[0180] Example 10 Production of 2’-FL using Escherichia coli as host strain

[0181] The medium composition was as follows: glycerol 40 g / L, lactose 20 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-HCl 10.0 mg / L, Triton 0.1% (v / v), 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.

[0182] The gene knockout and gene insertion related plasmids were constructed using the related technology published by Sheng Yang et al. (Acta. Biochim. Biophys. Sin. 2021, 53(5): 620-627). The required knockout and insertion genes are as follows. The gene encoding β-galactosidase LacZ (SEQ ID NO: 46, the amino acid sequence of its enzyme is SEQ ID NO: 45) in the genome of E. coli MG1655 was knocked out using the related CRISPR-Cas9 technology published by Sheng Yang et al. (Appl. Environ. Microbiol. 2016, 82(12): 3693), and the a-1,2-fucosyltransferase HpfutC gene (SEQ ID NO: 8) and the lactose permease LacY gene (SEQ ID NO: 10) were integrated at the knockout site; the gene encoding GDP-L-fucose synthetase WcaG (SEQ ID NO: 47, the gene sequence is SEQ ID NO: 48) in the genome of E. coli MG1655 was knocked out using CRISPR-Cas9 technology, and the gene encoding GDP-mannose-3,5-epimerase OsGME (SEQ ID NO: 5) was integrated at the knockout site; the gene encoding UDP-glucose lipid carrier transferase WcaJ (SEQ ID NO: 49, the gene sequence is SEQ ID NO: 50) in the genome of E. 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: 31) was integrated at the knockout site, thereby obtaining a 2’-FL production strain Ec2FL-1. When the obtained production strain Ec2FL-1 was fermented, the colonies on the plate were picked into seed culture medium for overnight culture. The bacterial liquid after overnight culture was inoculated into a 96-well plate containing the above-mentioned culture medium at an inoculation amount of 5%, and cultured at 37°C for 48 hours. An appropriate amount of fermentation broth was aspirated, and analyzed according to the method described in Example 1. It was detected that the yield of 2’-FL was 48 mg / L.

[0183] Sequence:

[0184] SEQ ID NO: 1, AtGME derived from Arabidopsis thaliana, amino acid sequence

[0185] SEQ ID NO: 2, AtGME derived from Arabidopsis thaliana, nucleic acid sequence

[0186] SEQ ID NO: 3, MfGME from Methylacidiphilum fumariolicum, amino acid sequence

[0187] SEQ ID NO: 4, MfGME from Methylacidiphilum fumariolicum, nucleic acid sequence

[0188] SEQ ID NO: 5, OsGME from Oryza sativa, amino acid sequence

[0189] SEQ ID NO: 6, OsGME from Oryza sativa, nucleic acid sequence

[0190] SEQ ID NO: 7, a-1,2-fucosyltransferase HpfutC from Helicobacter pylori, amino acid sequence

[0191] SEQ ID NO: 8, a-1,2-fucosyltransferase HpfutC from Helicobacter pylori, nucleic acid sequence

[0192] SEQ ID NO: 9, lactose permease LacY from Escherichia coli, amino acid sequence

[0193] SEQ ID NO: 10, lactose permease LacY from Escherichia coli, nucleic acid sequence

[0194] SEQ ID NO: 11, RMD (PaRMD) from Pseudomonas aeruginosa, amino acid sequence

[0195] SEQ ID NO: 12, RMD (PaRMD) from Pseudomonas aeruginosa, nucleic acid sequence

[0196] SEQ ID NO: 13, RMD from Aneurinibacillus thermoaerophilus (AtRMD), amino acid sequence

[0197] SEQ ID NO: 14, RMD from Aneurinibacillus thermoaerophilus (AtRMD), nucleic acid sequence

[0198] SEQ ID NO: 15, GerKl from Streptomyces sp. KCTC 0041BP, amino acid sequence

[0199] SEQ ID NO: 16, GerKl from Streptomyces sp. KCTC 0041BP, nucleic acid sequence

[0200] SEQ ID NO: 17, DnmV from Streptomyces peucetius, amino acid sequence

[0201] SEQ ID NO: 18, DnmV from Streptomyces peucetius, nucleic acid sequence

[0202] SEQ ID NO: 19, UrdZ3 from Streptomyces fradiae, amino acid sequence

[0203] SEQ ID NO: 20, UrdZ3 from Streptomyces fradiae, nucleic acid sequence

[0204] SEQ ID NO: 21, LanZ3 from Streptomyces cyanogenus, amino acid sequence

[0205] SEQ ID NO: 22, LanZ3 from Streptomyces cyanogenus, nucleic acid sequence

[0206] SEQ ID NO: 23, Mydl from Micromonospora griseorubida, amino acid sequence

[0207] SEQ ID NO: 24, Mydl from Micromonospora griseorubida, nucleic acid sequence

[0208] SEQ ID NO: 25, UrdR from Streptomyces fradiae, amino acid sequence

[0209] SEQ ID NO: 26, UrdR from Streptomyces fradiae, nucleic acid sequence

[0210] SEQ ID NO: 27, TyID from Streptomyces fradiae, amino acid sequence

[0211] SEQ ID NO: 28, TyID from Streptomyces fradiae, nucleic acid sequence

[0212] SEQ ID NO: 29, ChmD from Streptomyces bikiniensis, amino acid sequence

[0213] SEQ ID NO: 30, ChmD from Streptomyces bikiniensis, nucleic acid sequence

[0214] SEQ ID NO: 31, DdahC from Campylobacter jejuni, amino acid sequence

[0215] SEQ ID NO: 32, DdahC from Campylobacter jejuni, nucleic acid sequence

[0216] SEQ ID NO: 33, HS10A from Campylobacter jejuni, amino acid sequence

[0217] SEQ ID NO: 34, HS10A from Campylobacter jejuni, nucleic acid sequence

[0218] SEQ ID NO: 35, HS15 from Campylobacter jejuni, amino acid sequence

[0219] SEQ ID NO: 36, HS15 from Campylobacter jejuni, nucleic acid sequence

[0220] SEQ ID NO: 37, HS41B from Campylobacter jejuni, amino acid sequence

[0221] SEQ ID NO: 38, HS41B from Campylobacter jejuni, nucleic acid sequence

[0222] SEQ ID NO: 39, HS53 from Campylobacter jejuni, amino acid sequence

[0223] SEQ ID NO: 40, HS53 from Campylobacter jejuni, nucleic acid sequence

[0224] SEQ ID NO: 41, GDP-D-mannose-4,6-hydrolase EcGMD from E. coli, amino acid sequence

[0225] SEQ ID NO: 42, GDP-D-mannose-4, 6-dehydratase EcGMD from E. coli, nucleic acid sequence

[0226] SEQ ID NO: 43, a-1,3-fucosyltransferase HpfutA from Helicobacter pylori, amino acid sequence

[0227] SEQ ID NO: 44, a-1,3-fucosyltransferase HpfutA from Helicobacter pylori, nucleic acid sequence

[0228] SEQ ID NO: 45, β-galactosidase LacZ from E. coli MG1655, amino acid sequence

[0229] SEQ ID NO: 46, β-galactosidase LacZ from E. coli MG1655, nucleic acid sequence

[0230] SEQ ID NO: 47, GDP-L-fucose synthetase WcaG from E. coli MG1655, amino acid sequence

[0231] SEQ ID NO: 48, GDP-L-fucose synthetase WcaG from E. coli MG1655, nucleic acid sequence

[0232] SEQ ID NO: 49, UDP-glucose lipid carrier transferase WcaJ from E. coli MG1655, amino acid sequence

[0233] SEQ ID NO: 50, UDP-glucose lipid carrier transferase WcaJ from E. coli MG1655, nucleic acid sequence

[0234] SEQ ID NO: 51, Promoter Psod from Corynebacterium glutamicum ATCC 13032

[0235] SEQ ID NO: 52, promoter Pcg2195 derived from Corynebacterium glutamicum ATCC 13032

[0236] SEQ ID NO: 53, promoter Pgap derived from Corynebacterium glutamicum ATCC 13032

[0237] The embodiments of the present application are not limited to the above-described examples, and various changes and improvements can be made in form and details by those skilled in the art without departing from the spirit and scope of the present application, and these are considered to fall within the scope of the present application.

Claims

1. A genetically modified host cell comprising genes for a GDP-D-mannose synthesis pathway, comprising a gene encoding a 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, a gene encoding a GDP-D-rhamnose-3,5-epimerase, and a gene encoding a fucosyltransferase capable of transferring a fucose residue to an acceptor substrate to synthesize the fucosylated oligosaccharide.

2. The host cell of claim 1, wherein the host cell is a microbial cell.

3. The host cell of claim 1, wherein the host cell is a bacterial or yeast cell.

4. The host cell of claim 1, wherein the host cell is a Gram-negative bacterium or a Gram-positive bacterium.

5. The host cell according to claim 1, wherein the host cell is a bacterium of the genus Escherichia, Corynebacterium, Bacillus, Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, or Pseudomonas.

6. The host cell of claim 1, wherein the host cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae or Yarrowia lipolytica.

7. The host cell of any one of claims 1 to 6, wherein the host cell comprises one or more copies of a gene encoding a fucosyltransferase.

8. The host cell according to any one of claims 1 to 7, wherein the fucosyltransferase is any one, any two, any three or four selected from the group consisting of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,4-fucosyltransferase and α-1,3 / 4-fucosyltransferase.

9. The host cell according to any one of claims 1 to 8, wherein the α-1,2-fucosyltransferase is capable of using lactose, lacto-N-tetraose (LNT) or lacto-N-neotetraose (LNnT) as an acceptor substrate.

10. The host cell of claim 9, wherein the α-1,2-fucosyltransferase is an α-1,2-fucosyltransferase derived from Helicobacter pylori, Escherichia coli O128, Escherichia coli O126, Bacteroides fragilis, Azospirillum lipoferum, H. mustelae, H. billis, Campylobacter jejuni, Bacteroides vulgatus or Prevotella sp., or a functional variant thereof. The host cell according to any one of claims 1 to 10, wherein the α-1,3-fucosyltransferase is capable of using lactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) or 2'-fucosylated lactose (2'-FL) as an acceptor substrate.

12. The host cell of claim 11, wherein the α-1,3-fucosyltransferase is an α-1,3-fucosyltransferase derived from Helicobacter pylori, H. hepaticus, H. billis, H. trogontum, H. typhlonius, Bacteroides fragilis or Akkermansia muciniphila, or a functional variant thereof.

13. The host cell according to any one of claims 1 to 12, wherein the α-1,3 / 4-fucosyltransferase is capable of using lactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) or 2'-fucosylated lactose (2'-FL) as an acceptor substrate. The host cell according to claim 13 , wherein the α-1,3 / 4-fucosyltransferase is an α-1,3 / 4-fucosyltransferase derived from Helicobacter pylori , or a functional variant thereof.

15. The host cell of any one of claims 1 to 12, wherein the α-1,4-fucosyltransferase is capable of using lactose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or 2'-fucosylated lactose (2'-FL) as an acceptor substrate. The host cell according to any one of claims 1 to 15 , wherein the GDP-D-mannose-4,6-dehydratase is a GDP-D-mannose-4,6-dehydratase derived from Escherichia coli , or a functional variant thereof.

17. The host cell according to any one of claims 1 to 16, wherein the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose is GDP-4-keto-6-deoxy-D-mannose reductase.

18. The host cell according to claim 17, wherein the GDP-4-keto-6-deoxy-D-mannose reductase is a GDP-4-keto-6-deoxy-D-mannose reductase derived from Pseudomonas aeruginosa, or a functional variant thereof.

19. The host cell according to any one of claims 1 to 16, wherein the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose is a reductase comprising the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 33 or SEQ ID NO: 37, or a functional variant thereof.

20. The host cell according to any one of claims 1 to 19, wherein the GDP-D-rhamnose-3,5-epimerase is an isomerase comprising the amino acid sequence shown in SEQ ID NO: 5, an isomerase comprising the amino acid sequence shown in SEQ ID NO: 1, or a functional variant thereof.

21. The host cell of any one of claims 1 to 20, wherein the fucosylated oligosaccharides are human milk oligosaccharides (HMOs).

22. The host cell of any one of claims 1-21, wherein the host cell is capable of providing a receptor substrate intracellularly.

23. The host cell of claim 22, wherein the host cell is capable of transporting a receptor substrate into the cell, or the host cell is capable of synthesizing the receptor substrate from an exogenous precursor via a synthetic pathway contained within the cell.

24. The host cell of any one of claims 1-23, wherein the acceptor substrate is lactose or a lactose derivative.

25. The host cell of claim 24, wherein the lactose derivative is lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or 2'-fucosylated lactose (2'-FL).

26. The host cell of any one of claims 1-25, wherein the host cell comprises a gene encoding a lactose permease.

27. The host cell of claim 26, wherein the lactose permease is a lactose permease derived from Escherichia coli or Kluyveromyces lactis, or a functional variant thereof.

28. The host cell of any one of claims 1 to 27, wherein the fucosylated oligosaccharides comprise 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentose I (LNFP-I), lacto-N-fucopentose II (LNFP-II), lacto-N-fucopentose V (LNFP-V), lacto-N-neofucopentose I (LNnFP-I), lacto-N-neofucopentose III (LNnFP-III), lacto-N-neofucopentose V (LNnFP-V), lacto-N-difucohexose I (LNDFH-I), and lacto-N-difucohexose II (LNDFH-II).

29. The host cell of any one of claims 1-28, wherein one or more genes in the GDP-D-mannose synthesis pathway genes are overexpressed.

30. The host cell of claim 29, wherein one or more of the GDP-D-mannose synthesis pathway genes comprises a phosphomannose mutase (ManB) gene and / or a mannose-1-phosphate guanylate transferase (ManC) gene.

31. A method for producing fucosylated oligosaccharides, comprising culturing the host cell according to any one of claims 1 to 28 under conditions suitable for producing the human milk oligosaccharides, so as to transfer fucose residues to an acceptor substrate, thereby synthesizing the fucosylated lactose.

32. The method of claim 31, wherein the fucosylated oligosaccharide is a human milk oligosaccharide (HMO).

33. The method of claim 32, wherein the fucosylated oligosaccharides comprise 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentose I (LNFP-I), lacto-N-fucopentose II (LNFP-II), lacto-N-fucopentose V (LNFP-V), lacto-N-neofucopentose I (LNnFP-I), lacto-N-neofucopentose III (LNnFP-III), lacto-N-neofucopentose V (LNnFP-V), lacto-N-difucohexose I (LNDFH-I), and lacto-N-difucohexose II (LNDFH-II).

34. The method of any one of claims 31 to 33, wherein the medium used to culture the host cell comprises at least one carbon source.

35. The method according to any one of claims 31 to 34, wherein the culture medium used for culturing the host cells is supplemented with a receptor substrate or a precursor substance capable of synthesizing the receptor substrate through a synthetic pathway contained in the cells.

36. The method according to any one of claims 31 to 35, wherein the culture medium used to culture the host cells is supplemented with lactose or a precursor substance capable of synthesizing lactose through a synthetic pathway contained in the cells.

37. The method of any one of claims 31 to 36, further comprising recovering the fucosylated oligosaccharides from the culture medium.

38. Use of the host cell according to any one of claims 1 to 28 in producing fucosylated oligosaccharides.

39. The use according to claim 38, wherein the fucosylated oligosaccharide is human milk oligosaccharide (HMO).

40. The use according to claim 39, wherein the fucosylated oligosaccharides comprise 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentose I (LNFP-I), lacto-N-fucopentose II (LNFP-II), lacto-N-fucopentose V (LNFP-V), lacto-N-neofucopentose I (LNnFP-I), lacto-N-neofucopentose III (LNnFP-III), lacto-N-neofucopentose V (LNnFP-V), lacto-N-difucohexose I (LNDFH-I), and lacto-N-difucohexose II (LNDFH-II).

Images