CN120818475A

CN120818475A - Method for producing fucosylated oligosaccharides and its application

Info

Publication number: CN120818475A
Application number: CN202410437877.0A
Authority: CN
Inventors: 陈铖; 沈伟; 钱润泽; 黄恩玲; 刁刘洋
Original assignee: Ketaiya Biotechnology Shanghai Co ltd
Current assignee: Ketaiya Biotechnology Shanghai Co ltd
Priority date: 2024-04-11
Filing date: 2024-04-11
Publication date: 2025-10-21
Also published as: WO2025214489A1

Abstract

The invention discloses a method for producing fucosylated oligosaccharides. The method is characterized in that GDP-D-rhamnose is converted into GDP-L-fucose by using GDP-D-rhamnose-3, 5-epimerase, and the GDP-D-rhamnose is obtained by converting GDP-D-mannose-4, 6-dehydratase and GDP-4-keto-6-deoxidized D-mannose reductase by using GDP-D-mannose as a substrate. 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, fucosylated oligosaccharides may be produced by the above-described pathways.

Description

Method for producing fucosylated oligosaccharides and use thereof

Technical Field

The present invention relates to the field of biotechnology, in particular to a method for producing fucosylated oligosaccharides and uses thereof.

Background

Breast milk oligosaccharides (human milk oligosaccharides, HMOs) are the third largest component of human breast milk with levels inferior to lactose and fat, consisting of more than 200 oligosaccharides. Among them, 2' -fucosyllactose (2 ' -fucosyllactose,2' -FL) and 3-fucosyllactose (3-fucosyllactose, 3-FL) are fucosylated oligosaccharides, which are most abundant in breast milk and account for about 35% of total HMO, and have extremely important physiological functions including affecting the composition of intestinal microbiota, resisting adhesion of pathogenic bacteria, regulating immune function, promoting brain development, etc.

2' -FL is also one of the most widely studied oligosaccharides because of its great application value. The preparation of 2' -FL is mainly performed by biosynthesis methods including microbial fermentation synthesis and enzymatic synthesis. The microbial fermentation method has attracted attention of a plurality of scholars at home and abroad due to the advantages of mild reaction conditions, low production cost, small environmental pollution and the like, and has become a research hotspot for 2' -FL production.

Fucosyltransferases catalyze the transfer of L-fucose from a GDP-L-fucose donor substrate to an acceptor substrate (e.g., lactose) to synthesize fucosylated oligosaccharides such as 2' -FL and 3-FL. Coli, saccharomyces cerevisiae, corynebacterium glutamicum, bacillus subtilis, etc. have been engineered to produce GDP-L-fucose and express exogenous fucosyltransferases to synthesize fucosylated oligosaccharides (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). such as 2'-FL and 3-FL, the core of these bacterial strain metabolic pathways is the conversion of GDP-D-mannose to GDP-4-keto-6-deoxy-D-mannose by GDP-D-mannose-4, 6-dehydratase, GMD using GDP-D-mannose-4, 6-dehydratase, GMD, followed by further production of GDP-L-fucose by GDP-L-fucose synthase (GDP-fucose synthetase, wcaG), and the synthesis of 2' -FL and 3-FL by the catalysis of GDP-L-fucose and lactose using fucosyltransferases. In addition, only Conagen company has created a new 2' -FL production route by means of in vitro multi-enzyme cascade, the core of which is the synthesis of GDP-L-fucose by GMD and WcaG using GDP-L-galactose as substrate (WO 2022/040411). In general, the production methods of fucosylated oligosaccharides such as 2' -FL have little research at present, and a new production path needs to be searched for, thus providing a new idea for efficient production thereof.

D-rhamnose is an important component of surface polysaccharides of a variety of pathogenic bacteria including Pseudomonas aeruginosa, and GDP-D-rhamnose is a synthetic precursor of D-rhamnose (Curr. Opin. Structure. Biol.2000,10 (6): 687-696). GDP-D-rhamnose is similar to GDP-L-fucose in that its biosynthesis involves two proteins, GMD and GDP-4-keto-6-deoxy-D-mannose reductase (GDP-4-keyo-6-deoxy-D-mannose reductase, RMD). Firstly GMD is able to 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 involves the first step in biosynthesis of L-fucose, 6-deoxy-D-talose and D-rhamnose, and thus related studies on GMD from different sources have been reported. In contrast, only a few RMDs have been demonstrated to reduce GDP-4-keto-6-deoxy D-mannose to GDP-D-rhamnose, atRMD from aerophilic thiobacillus (Aneurinibacillus thermoaerophilus) and PaRMD from pseudomonas aeruginosa (Pseudomonas aeruginosa), respectively (FEBS j 2009,276 (10): 2686-2700; j.biol. Chem.2001,276 (8): 5577-5583). In addition, carbonyl reductases which catalyze the asymmetric reduction of GDP-4-keto-6-deoxy D-mannose analogues have been reported, but specific activities have not been investigated using GDP-4-keto-6-deoxy D-mannose as a substrate (biochemistry.2022, 61:2138-2147;Angew Chem Int Ed Engl.2008,47 (51): 9814-59).

GDP-mannose-3, 5-epimerase (GDP-mannose, 5-epimerase, GME) is capable of catalyzing the reversible isomerization of the 3, 5-group of GDP-D-mannose to GDP-L-galactose. AtGME from Arabidopsis thaliana (Arabidopsis thaliana) and OsGME from rice (Oryza sativa) have been studied intensively, and even AtGME protein crystal structures have been resolved for (Phytochemistry.2006,67(4):338-346;J.Am.Chem.Soc.2005,127(51):18309-18320;Biotechnol.Adv.2021,48:107705.).2019 years, gevaert et al reported that GME from microorganism, namely MfGME from volcanic fumarole Methylophilus (Methylacidiphilum fumariolicum) (int.J mol. Sci.2019,20 (14): 3530). However, there is currently no report on GME catalytic substrate spectra.

Disclosure of Invention

In one aspect, the invention provides a genetically modified host cell comprising a GDP-D-mannose synthesis pathway gene and comprising a gene encoding a GDP-D-mannose-4, 6-dehydratase (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 receptor substrate, thereby synthesizing the fucosylated oligosaccharide.

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, wherein the host cell is Escherichia coli, corynebacterium glutamicum, bacillus subtilis, saccharomyces cerevisiae, or yarrowia lipolytica.

In some embodiments, the fucosyltransferase is any one, any two, any three, or four selected from the group consisting of an alpha-1, 2-fucosyltransferase, an alpha-1, 3-fucosyltransferase, an alpha-1, 4-fucosyltransferase, and an alpha-1, 3/4-fucosyltransferase. In some embodiments, the α -1, 2-fucosyltransferase is capable of using lactose, milk-N-tetraose (LNT), or milk-N-neotetraose (LNnT) as a acceptor substrate. In some embodiments, the α -1, 2-fucosyltransferase is an α -1, 2-fucosyltransferase derived from helicobacter pylori (Helicobacter pylori), escherichia coli O128, escherichia coli O126, bacteroides fragilis (Bacteroides fragilis), azospirillum lipogenic (Azospirillum lipoferum), helicobacter ferret (h.mustelae), helicobacter bile (h.bilis), campylobacter jejuni (Campylobacter jejuni), bacteroides vulgare (Bacteroides vulgatus) or prasugrel sp, or a functional variant thereof. In some embodiments, the α -1, 3-fucosyltransferase is capable of using lactose, milk-N-tetraose (LNT), milk-N-neotetraose (LNnT), or 2 '-fucosylated lactose (2' -FL) as a acceptor substrate. In some embodiments, the alpha-1, 3-fucosyltransferase is an alpha-1, 3-fucosyltransferase derived from helicobacter pylori (Helicobacter pylori), helicobacter hepaticum (h. Hepatous), helicobacter bile (h. Billis), helicobacter longus (h. Strogontum), helicobacter cecum (h. Tyrosponius), bacteroides fragilis (Bacteroides fragilis), or akkermansia viscosa (AKKERMANSIA MUCINIPHILA), or a functional variant thereof. In some embodiments, the α -1, 3/4-fucosyltransferase is capable of using lactose, milk-N-tetraose (LNT), milk-N-neotetraose (LNnT), or 2 '-fucosylated lactose (2' -FL) as a acceptor substrate. In some embodiments, the alpha-1, 3/4-fucosyltransferase is an alpha-1, 3/4-fucosyltransferase derived from helicobacter pylori (Helicobacter pylori), or a functional variant thereof. In some embodiments, the α -1, 4-fucosyltransferase is capable of using lactose, milk-N-tetraose (LNT), milk-N-neotetraose (LNnT), or 2 '-fucosylated lactose (2' -FL) as a acceptor substrate.

In some embodiments, the GDP-D-mannose-4, 6-dehydratase is GMD derived from escherichia coli, 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 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 (Pseudomonas aeruginosa), or a functional variant thereof. In some embodiments, the reductase enzyme capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D rhamnose is a reductase enzyme comprising the amino acid sequence 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.

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

In some embodiments, the fucosylated oligosaccharide belongs to Human Milk Oligosaccharide (HMO). In some embodiments, the host cell is capable of providing a receptor substrate within the cell. In some embodiments, the host cell is capable of transporting the receptor substrate into the cell, or the host cell is capable of synthesizing the receptor substrate from an externally applied precursor material via a synthetic pathway contained within the cell. In some embodiments, the acceptor substrate is lactose or a lactose derivative. In some embodiments, the lactose derivative is milk-N-tetraose (LNT), milk-N-neotetraose (LNnT), or 2 '-fucosylated lactose (2' -FL).

In some embodiments, the host cell comprises a gene encoding lactose permease. In some embodiments, the lactose permease is a lactose permease derived from escherichia coli or kluyveromyces lactis, or a functional variant thereof.

In some embodiments, the fucosylated oligosaccharides include 2 '-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), milk-N-fucose I (LNFP-I), milk-N-fucose II (LNFP-II), milk-N-fucose V (LNFP-V), milk-N-neofucose I (LNnFP-I), milk-N-neofucose III (LNnFP-III), milk-N-neofucose V (LNnFP-V), milk-N-difucose hexose I (LNDFH-I), milk-N-difucose II (LNDFH-II).

Another aspect of the invention provides a method of producing a fucosylated oligosaccharide comprising culturing any of the foregoing host cells under conditions suitable for production of the breast milk oligosaccharide to transfer fucose residues to a recipient substrate to synthesize the fucosylated lactose.

In some embodiments, the fucosylated oligosaccharide belongs to Human Milk Oligosaccharide (HMO). In some embodiments, the fucosylated oligosaccharides include 2 '-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), milk-N-fucose I (LNFP-I), milk-N-fucose II (LNFP-II), milk-N-fucose V (LNFP-V), milk-N-neofucose I (LNnFP-I), milk-N-neofucose III (LNnFP-III), milk-N-neofucose V (LNnFP-V), milk-N-difucose hexose I (LNDFH-I), milk-N-difucose II (LNDFH-II).

In some embodiments, the medium used to culture the host cell comprises at least one carbon source. In some embodiments, the medium used to culture the host cell has added to it a receptor substrate or a precursor substance capable of synthesizing the receptor substrate via a synthetic pathway contained within the cell. In some embodiments, the medium used to culture the host cells is supplemented with lactose or a precursor substance capable of synthesizing lactose via a synthetic pathway contained within the cell. In some embodiments, the method further comprises recovering the fucosylated oligosaccharide from the culture medium.

In a further aspect the invention provides the use of any of the aforementioned host cells for the production of a fucosylated oligosaccharide. In some embodiments, the fucosylated oligosaccharide belongs to Human Milk Oligosaccharide (HMO). In some embodiments, the fucosylated oligosaccharides include 2 '-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), milk-N-fucose I (LNFP-I), milk-N-fucose II (LNFP-II), milk-N-fucose V (LNFP-V), milk-N-neofucose I (LNnFP-I), milk-N-neofucose III (LNnFP-III), milk-N-neofucose V (LNnFP-V), milk-N-difucose hexose I (LNDFH-I), milk-N-difucose II (LNDFH-II).

Drawings

FIG. 1 illustrates an exemplary 2' -FL synthesis route.

FIG. 2 analysis spectra of GDP-D-rhamnose and GDP-L-fucose reaction solutions.

FIG. 3 is a graph showing the analysis of 2' -FL produced by Cg2FL-11 and a standard control.

FIG. 4 is a chart of the analysis of the EcGMD reaction solution.

FIG. 5 shows the analysis of DdacC reaction solution.

Detailed Description

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 invention belongs.

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

The terms "about" and "approximately" when used with a digital variable generally mean that the value of the variable and the total value of the variable are within a measured or experimental error (e.g., 95% confidence interval of the average) or within a wider range of specified values (e.g., ±5% or±10%).

The term "comprising" or variations thereof, such as "comprising," "having," "including," means including the stated step or element, but not excluding any other step or element. "consisting of" means that steps or elements not listed are not included. "consisting essentially of" means that steps or elements that do not materially affect the basic and novel characteristics of the claimed invention are not excluded. The term "comprising" a particular step or element and variations thereof also include "consisting of" and "consisting essentially of" the particular step or element.

When referring to a numerical range, it is intended that the upper and lower limits of the range be specifically disclosed, as well as all intervening ranges encompassed therein, for example, intermediate ranges between the upper or lower limit and any intermediate value or between any two intermediate values thereof. Also, any intervening ranges, subranges, and any individual value described in that numerical range may be excluded from the numerical range.

The term "and/or" should be understood as any one element or combination of any number of elements connected by the term.

The term "gene" refers to a nucleotide sequence encoding a gene product. The gene product may be a protein or ribonucleic acid.

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

The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. The enzyme of the present invention is a protein capable of catalyzing a chemical reaction of a substrate.

The terms "genetically modified," "engineered," "recombinant" are those that have been altered by manual manipulation to alter the sequence of a polypeptide, polynucleotide, or to alter the sequence of a gene comprised by a host cell so that it comprises a sequence that does not naturally occur in the polypeptide, polynucleotide, or host cell. When a host cell is "engineered" to exhibit a characteristic (e.g., comprise a gene encoding a particular protein, express a particular protein, or overexpress a particular gene), it is meant that the host cell does not have the characteristic prior to the transformation, but has the characteristic after the transformation.

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

The term "functional variant", when applied to a polypeptide or protein, refers to a polypeptide that has 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 a parent polypeptide and has the same or substantially the same function as the parent polypeptide. Functional variants may also refer to polypeptides having one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acid insertions, deletions, and/or substitutions as compared to a parent polypeptide and having the same or substantially the same function as the parent polypeptide.

The term "sequence identity" refers to the percentage of identical nucleotide or amino acid residues at corresponding positions of two or more sequences when the sequences are aligned to maximize sequence matching, i.e., to allow for gaps and insertions. The alignment of sequences and calculation of percent sequence identity can be performed by appropriate computer programs known in the art. These programs include, but are not limited to BLAST, ALIGN, clustalW, EMBOSS Needle, and the like. An example of a local alignment program is BLAST (basic local alignment search tool), which is available from the Web pages of the national center for Biotechnology information, and is currently found in http:// www.ncbi.nlm.nih.gov/, which is described for the first time in Altschul et al (1990) J.mol.biol.215;403.Biol.215;403-410. The Needleman-Wunsch based 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), can be referred to in http:// www.ebi.ac.uk/Tools/psa.

The term "exogenous" is relative to a host cell and refers to a substance or molecule derived from or produced outside the host cell. "exogenous gene" or "exogenous enzyme" refers to a nucleic acid that is not a naturally occurring gene or enzyme in a cell, but is introduced into the cell from outside the cell by artificial means. The sequence of the exogenous gene or exogenous enzyme may be the same or different from the endogenous sequence naturally present in the cell. Genes or enzymes that differ from endogenous sequences naturally present in the cell may be referred to as heterologous genes or heterologous enzymes, which originate from a different strain or species than the host cell. Herein, unless otherwise indicated, when referring to "foreign gene" or "foreign enzyme", the case of "heterologous gene" or "heterologous enzyme" is included.

The term "endogenous" refers to a gene or protein (e.g., wild-type gene or wild-type enzyme) or synthetic pathway that naturally occurs in a host cell.

The term "naturally occurring" refers to a nucleotide sequence, amino acid sequence, complex, pathway, or cell that occurs in nature without human manipulation.

The term "derived from," when applied to a protein or gene sequence, refers to a protein or gene sequence that is derived from a particular organism, and refers to the protein or gene sequence having the same structure or sequence as the protein or gene sequence naturally occurring in the organism, and is not limited to being isolated directly from the organism.

The term "over-expression" refers to the greater expression of a gene product or polypeptide in a host cell after genetic engineering than before genetic engineering. The term "over-expression" may also mean any detectable expression resulting from the introduction of a particular gene product into a host cell if the host cell does not contain the particular gene product prior to genetic engineering.

The term "vector" refers to a means for allowing or facilitating the transfer of a nucleic acid fragment from one environment to another, such as a host cell. The vector allows insertion of another nucleic acid fragment therein to effect replication of the inserted fragment.

The term "expression vector" refers to a vector for expressing a gene product, which typically includes one or more expression control sequences for controlling and regulating transcription and/or translation of the gene sequence capable of expressing the product.

The term "expression cassette" refers to a nucleotide sequence comprising a nucleic acid of interest under the control of and operably linked to an appropriate promoter or other regulatory element to transduce the nucleic acid of interest in a host cell.

The term "operably linked" refers to the placement of regulatory sequences necessary for the expression of a coding sequence in a DNA molecule in a position relative to the coding sequence so as to affect the expression of the coding sequence.

The term "breast milk oligosaccharide" (Human Milk Oligosaccharides, HMO) refers to a carbohydrate formed by connecting 3-10 monosaccharides through glycosidic bonds, and is the third solid component of breast milk with the content ranking behind fat and lactose. The basic structure of breast milk oligosaccharide consists of five basic monosaccharides, namely D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc) and N-acetylneuraminic acid (NeuAc or Neu5 c), respectively.

The term "fucosylated oligosaccharide" is an oligosaccharide having fucose residues. The oligosaccharide is neutral. Exemplary fucosylated oligosaccharides include 2' -fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-fucose (e.g., lacto-N-fucose I, lacto-N-fucose II, lacto-N-fucose III, lacto-N-fucose V), lacto-N-fucose, lacto-N-difucose hexose I, fucosyllactose-N-hexose, fucosyllactose-N-neohexose, difucosyllactose-N-hexose I, difucosyllactose-N-neohexose II, and the like.

The term "fucosyltransferase" refers to a polypeptide capable of catalyzing the transfer of a fucose residue from a donor substrate to an acceptor substrate. The donor substrate for fucosyltransferases is typically GDP-L-fucose. Receptor substrates include oligosaccharides, glycopeptides, glycoproteins, and glycolipids. Typically, the fucose residue is transferred to, for example, an N-acetylglucosamine residue, an N-acetylgalactosamine residue, a galactose residue, a fucose residue, a sialic acid residue or a glucose residue of an oligosaccharide, or a glycomoiety of a glycoprotein or glycolipid. The term "fucosyltransferase" is herein understood as comprising said wild-type fucosyltransferase as well as functional variants thereof, which functional variants are also capable of catalyzing the transfer of fucose residues from a donor substrate to an acceptor substrate, i.e. they are also fucosyltransferase active.

In terms of transferring fucose residues from a donor substrate to an acceptor molecule, the term "donor substrate" refers to a molecule comprising fucose residues, wherein the fucose comprised therein is catalytically transferred to a specific acceptor substrate by a fucosyltransferase. In the present invention, 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.

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

The term "functional" or the term "capable" when used in describing the activity or function of an enzyme means that the enzyme will exhibit a particular activity or function under the appropriate reaction conditions. The enzyme may not exhibit this activity or function when the appropriate reaction conditions are not provided, but the enzyme exhibits this activity or function when the appropriate reaction conditions are provided. Suitable reaction conditions include the presence of a suitable donor substrate, the presence of a suitable acceptor molecule, the presence of necessary cofactors, pH values in the appropriate range, suitable temperatures, etc.

Unless otherwise indicated, in this context, nucleic acids are written in a 5 'to 3' direction from left to right, while amino acid sequences are written in an amino-to-carboxy-terminal direction from left to right.

The present invention provides a novel pathway for the synthesis of human milk oligosaccharides, in particular fucosylated oligosaccharides in human milk oligosaccharides. In particular, the present invention provides a novel intracellular GDP-L-fucose synthesis pathway, wherein the GDP-L-fucose is used as a donor substrate, and fucose can be transferred to a suitable donor substrate by a fucosyltransferase in cells to synthesize fucosylated oligosaccharides.

The inventors found that GDP-D-rhamnose is an isomer of GDP-L-fucose, and that only the 3, 5-position groups are conformationally different between the two, and that the interconversion can be theoretically achieved by isomerization reactions. The present inventors have studied the substrate spectra of GDP-mannose-3, 5-epimerase (GDP-mannose, 3,5-epimerase, GME) and found that GDP-mannose-3, 5-epimerase is capable of catalyzing the reversible isomerization of GDP-D-rhamnose and GDP-L-fucose, which has the activity of GDP-D-rhamnose-3,5-epimerase (GDP-D-rhamnose-3, 5-epimerase, GRE). GDP-D-mannose-4, 6-dehydratase (GDP-D-mannose, 6-dehydratase, GMD), reductase (e.g., GDP-4-keto-6-deoxy-D-mannose reductase (GDP-4-keto-6-deoxy-D-mannose reductase) (GDP-4-keto-6-deoxy-D-mannose reductase, RMD)) and GDP-D-rhamnose-3,5-epimerase (GRE) are expressed in host cells (e.g., microorganisms such as E.coli, saccharomyces cerevisiae, corynebacterium glutamicum, etc.), GDP-L-fucose can be synthesized from GDP-D-mannose by a novel route. An exemplary production route for the fucosylated oligosaccharide 2' -FL is shown in FIG. 1.

To achieve the production of fucosylated oligosaccharides, the invention provides a genetically modified host cell comprising a GDP-D-mannose synthesis pathway gene and comprising

I) A gene encoding GDP-D-mannose-4, 6-dehydratase (GMD),

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

Iii) Genes encoding GDP-D-rhamnose-3, 5-epimerase (GRE), and

Iv) a gene encoding a fucosyltransferase capable of transferring fucose residues to a receptor substrate to synthesize fucosylated oligosaccharides.

The host cell of the invention is capable of expressing the functional enzyme encoded by the above gene and of synthesizing the fucosylated oligosaccharide in the cell.

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-dehydratase (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-dehydratase (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.

The host cells of the present invention comprise a GDP-D-mannose synthesis pathway gene to synthesize GDP-D-mannose in the host cell. The "GDP-D-mannose synthesis pathway" refers to a series of reactions controlled and catalyzed by enzymes, the result of which is the synthesis of GDP-D-mannose. Typically, the "GDP-D-mannose synthesis pathway" refers to a series of reactions that are enzymatically controlled and catalyzed to synthesize GDP-D-mannose from a carbon source. "GDP-D-mannose synthesis pathway gene" refers to genes encoding enzymes contained in the GDP-D-mannose synthesis pathway, which genes can express the enzymes in a host cell, thereby catalyzing the synthesis of GDP-D-mannose.

One or more of the GDP-D-mannose synthesis pathway genes may be endogenous or exogenous, respectively. In some embodiments, the host cell used naturally has a GDP-D-mannose synthesis pathway, capable of intracellular synthesis of GDP-D-mannose, where the host cell comprises all genes of the GDP-D-mannose synthesis pathway that may be endogenous. In some embodiments, the host cell used may not naturally have a GDP-D-mannose synthesis pathway, e.g., lack one or more enzymes of the GDP-D-mannose synthesis pathway, in which case the one or more enzymes that are lacking may be provided by introducing an exogenous enzyme gene into the host cell to express the one or more enzymes that are lacking in the host cell. The exogenous enzyme gene may be contained in an episomal expression vector introduced into the host cell or integrated into the chromosome of the host cell.

Fructose-6-phosphate is an intermediate product of the metabolism of carbon sources found in almost all organisms (including bacterial cells), and common carbon sources such as glucose, glycerol, etc. can be metabolized in cells to produce fructose-6-phosphate. GDP-D-mannose may be further synthesized with fructose-6-phosphate as a precursor. In bacterial cells, exemplary GDP-D-mannose synthesis pathways may include the synthesis pathway from a carbon source to fructose-6-phosphate and the synthesis pathway from fructose-6-phosphate to GDP-D-mannose. An exemplary synthetic 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 phosphomannose mutase (ManB), and (3) synthesis of GDP-D-mannose from mannose-1-phosphate, catalyzed by mannose-1-guanylate transferase (ManC). Most bacterial cells contain enzymes capable of performing the above three steps, such as E.coli, corynebacterium glutamicum, and Bacillus subtilis, among others. However, since metabolic pathways in different bacterial species may also differ, when the host cell used lacks one or more enzymes of the GDP-D-mannose synthesis pathway, the lacking one or more enzymes may be provided by introducing exogenous enzyme genes into the host cell to express the lacking one or more enzymes in the host cell. The exogenous enzyme gene may be contained in an episomal expression vector introduced into the host cell or integrated into the chromosome of the host cell.

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

The host cell of the present invention further comprises a reductase gene encoding a polypeptide 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 described herein refers to a reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D rhamnose. One skilled in the art can readily determine by an enzymatically catalyzed chemical reaction whether a polypeptide has the activity of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D rhamnose, e.g., by adding the polypeptide in the presence of a substrate (GDP-4-keto-6-deoxy D-mannose) and a necessary cofactor (e.g., NADPH) under suitable conditions and detecting the formation of a product (i.e., GDP-D rhamnose), which may be detected, e.g., by mass spectrometry.

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

In some embodiments, 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 (RMD), such as RMD (PaRMD) derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa) or RMD (AtRMD) derived from Achromobacter thermophilum (Aneurinibacillus thermoaerophilus) (FEBS J2009, 276 (10): 2686-2700; J.biol. Chem.2001,276 (8): 5577-5583; incorporated herein by reference in its entirety), 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 wave (Streptomyces peucetius), ddahC derived from Campylobacter jejuni (Campylobacter jejuni), C4 reductase (HS 10A) derived from Campylobacter jejuni (Campylobacter jejuni) HS10A serotype, C4 reductase (HS 41B) derived from Campylobacter jejuni (Campylobacter jejuni) HS41B serotype (biochemistry.2022, 61:2138-2147;Angew Chem Int Ed Engl.2008,47 (51): 9814-59; these documents are incorporated herein by reference in their entirety), or a functional variant thereof. In some embodiments, the reductase enzyme capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D rhamnose comprises the amino acid sequence 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 enzyme capable of catalyzing the conversion of GDP-4-keto-6-deoxy D-mannose to GDP-D rhamnose comprises an amino acid sequence 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.

The host cells of the invention also comprise a gene encoding GDP-D-rhamnose-3, 5-epimerase (GRE) to express 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 an enzyme-catalyzed chemical reaction, for example, by adding the polypeptide in the presence of a substrate (GDP-D-rhamnose) under suitable conditions and detecting the formation of a product (i.e., GDP-L-fucose) to determine whether it has the activity of catalyzing the conversion of GDP-D-rhamnose to GDP-L-fucose, the formation of which can be detected, for example, by mass spectrometry.

The gene encoding GDP-D-rhamnose-3, 5-epimerase (GRE) may be endogenous or exogenous. When the host cell used lacks 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 may be contained in an episomal expression vector introduced into the host cell or integrated into the chromosome of the host cell.

GDP-mannose-3, 5-epimerase (GME) is capable of catalyzing not only the conversion of GDP-D mannose to GDP-L fucose but also has an activity of catalyzing the conversion of GDP-D-rhamnose to GDP-L-fucose, such as GME (AtGME) derived from Arabidopsis thaliana (Arabidopsis thaliana), GME (OsGME) derived from rice (Oryza sativa), GME(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; derived from volcanic aerosolium methyl acidophilus (Methylacidiphilum fumariolicum), which are incorporated herein by reference in their entirety, and thus can be used in the present invention as GDP-D-rhamnose-3, 5-epimerase (GRE). Thus, in some embodiments, the GDP-D-rhamnose-3, 5-epimerase (GRE) comprises GDP-mannose-3, 5-epimerase (GME). In other embodiments, the GDP-D-rhamnose-3, 5-epimerase (GRE) may comprise any polypeptide capable of catalyzing the conversion of GDP-D-rhamnose to GDP-L-fucose. In some embodiments, the GRE is an arabidopsis (Arabidopsis thaliana) -derived GRE (e.g., arabidopsis (Arabidopsis thaliana) -derived GME (AtGME)), a rice (Oryza sativa) -derived GRE (e.g., oryza sativa-derived GME (OsGME)), a volcanic aerothricini (Methylacidiphilum fumariolicum) -derived GRE (e.g., volcanic aerothricini (Methylacidiphilum fumariolicum) -derived GME (MfGME)), or a functional variant thereof (the functional variant 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 the amino acid sequence 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 the amino acid sequence set forth in SEQ ID NO. 1 or SEQ ID NO.3, or a functional variant thereof.

The host cells of the invention also comprise a gene encoding a fucosyltransferase to express the fucosyltransferase in the host cell. Fucosyltransferases are capable of catalyzing the transfer of fucose residues from a donor substrate to an acceptor substrate, forming alpha-1, 2-, alpha-1, 3-, alpha-1, 4-or alpha-1, 6-glycosidic linkages between fucose and the sugar moiety of the acceptor substrate, thereby synthesizing fucosylated oligosaccharides. There are a number of different fucosylated oligosaccharides in breast milk oligosaccharides (HMOs), including but not limited to fucosyllactose, lacto-N-fucose, lacto-N-neofucopyranose, lacto-N-neodifucose hexose, 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 common acceptor substrates include lactose or lactose derivatives having a structure that allows them to form fucosylated oligosaccharides upon attachment to the fucose moiety via the catalysis of a fucosyltransferase. Lactose derivatives which can be used as acceptor substrates can be, for example, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) or 2 '-fucosylated lactose (2' -FL).

The gene encoding the fucosyltransferase may be endogenous or exogenous. When the host cell used lacks endogenous fucosyltransferases, exogenous fucosyltransferase genes can be introduced into the host cell to express the fucosyltransferases in the host cell. The exogenous fucosyltransferase gene may be contained in an episomal expression vector introduced into the host cell or integrated into the chromosome of the host cell.

In microbial fermentation production, GDP-L-fucose is used as a donor substrate, and different acceptor substrates and different fucosyltransferases are used to obtain different fucosylated oligosaccharides. For example, 2 '-fucosyllactose (2' -FL) may be synthesized catalytically using alpha-1, 2-fucosyltransferase and 3-fucosyllactose (3-FL) may be synthesized catalytically using alpha-1, 3-fucosyltransferase and .(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; may be used as acceptor substrates, the disclosures of which are incorporated herein by reference in their entirety

The di-fucosylated lactose (DFL, which may also be referred to as lacto-N-di-fucose (LDFT)) may be synthesized as an acceptor substrate from lactose using alpha-1, 2-fucosyltransferase catalysis to synthesize intermediate 2'-FL, and then using alpha-1, 3/4-fucosyltransferase to synthesize (Zhang A et al.,Metab Eng.2021;66:12-20;Liang S et al.,J Agric Food Chem.2024Feb 28;72(8):4367-4375; using this intermediate 2' -FL as an acceptor substrate, the entire contents of these documents are incorporated herein by reference.

Other more complex fucosylated oligosaccharides may also be synthesized using GDP-L-fucose as a donor substrate, using different acceptor substrates and different fucosyltransferases. For example, lacto-N-fucose I (LNFP-I) can be synthesized using lacto-N-tetraose (LNT) as acceptor substrate, using alpha-1, 2-fucosyltransferase to catalyze the alpha-1, 2-fucosylation of galactose. lacto-N-fucose II (LNFP-II) can be synthesized using lacto-N-tetraose (LNT) as acceptor substrate, using alpha-1, 4-fucosyltransferase to catalyze the alpha-1, 4-fucosylation of N-acetylglucosamine. lacto-N-fucose V (LNFP-V) can be synthesized using lacto-N-tetraose (LNT) as acceptor substrate, using alpha-1, 3-fucosyltransferase to catalyze the alpha-1, 3-fucosylation of glucose. lacto-N-neofucoidan I (LNnFP-I) can be synthesized from lacto-N-neotetraose (LNnT) as acceptor substrate by alpha-1, 2-fucosyltransferase catalyzed alpha-1, 2-fucosylation of galactose. lacto-N-neofucose III (LNnFP-III) can be synthesized using alpha-1, 3-fucosyltransferase catalyzed alpha-1, 3-fucosylation of N-acetylglucosamine with lacto-N-neotetraose (LNnT) as acceptor substrate. lacto-N-neofucose V (LNnFP-V) can be synthesized using alpha-1, 3-fucosyltransferase-catalyzed alpha-1, 3-fucosylation of glucose with lacto-N-neotetraose (LNnT) as 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)

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

In some embodiments, the exogenous fucosyltransferase expressed in the host cell may be any one, any two, any three, or four selected from the group consisting of an alpha-1, 2-fucosyltransferase, an alpha-1, 3-fucosyltransferase, an alpha-1, 4-fucosyltransferase, and an alpha-1, 3/4-fucosyltransferase.

The term "alpha-1, 2-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor substrate to form an alpha-1, 2-glycosidic bond between a fucose residue and a sugar moiety of the acceptor substrate. The term "alpha-1, 3-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor substrate to form an alpha-1, 3-glycosidic bond between a fucose residue and a sugar moiety of the acceptor substrate. The term "alpha-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 alpha-1, 3/4-glycosidic bond between a fucose residue and a sugar moiety of the acceptor substrate. The term "alpha-1, 4-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor substrate to form an alpha-1, 4-glycosidic bond between a fucose residue and a sugar moiety of an acceptor molecule.

Fucosyltransferase has the advantages of substrate selectivity and high yield, the receptor substrate ranges for different fucosyltransferases may vary. In the host cells and methods of the invention, synthesis of a particular target fucosylated oligosaccharide is achieved by a combination of both a receptor substrate and a fucosyltransferase capable of catalyzing a reaction of the receptor substrate. The selection of the fucosyltransferase is related to the target fucosylated oligosaccharide to be synthesized and the selected acceptor substrate. The fucosyltransferase contained within the host cell should be capable of catalyzing the transfer of fucose residues to the acceptor substrate provided within the host cell to synthesize the target fucosylated oligosaccharide. For example, when the target fucosylated oligosaccharide is 2' -FL, the acceptor substrate is lactose, and the fucosyltransferase contained in the host cell for producing 2' -FL is α -1, 2-fucosyltransferase capable of using lactose as the acceptor substrate; when the target fucosylated oligosaccharide is DFL, the synthesis is divided into two stages, the acceptor substrate is lactose and 2' -FL, the host cell for producing DFL comprises alpha-1, 2-fucosyltransferase capable of taking lactose as acceptor substrate and alpha-1, 3/4-fucosyltransferase capable of taking 2' -FL as acceptor substrate, when the target fucosylated oligosaccharide is LNFP-I, LNFP-II or LNFP-V, the acceptor substrate is LNT, the host cell for producing the target fucosylated oligosaccharide comprises alpha-1, 2-fucosyltransferase capable of taking lactose as acceptor substrate, alpha-1, 4-fucosyltransferase capable of taking 2' -FL as acceptor substrate, and alpha-3/4-fucosyltransferase capable of taking LNT as acceptor substrate, when the target fucosylated oligosaccharide is LNFP-I, LNFP-II or LNFP-V, the host cell for producing the target fucosylated oligosaccharide comprises fucosyltransferase capable of taking LNT as acceptor substrate, alpha-1, 2-fucosyltransferase capable of taking LNT as acceptor substrate, alpha-1, 4-fucosyltransferase and alpha-3/4-fucosyltransferase capable of taking LNT as acceptor substrate, respectively, and the LNT-52-5-fucosyltransferase capable of taking LNT as acceptor substrate, respectively, the LNT-52-5-3-fucosyltransferase capable of taking LNFP-II or LNFP-III, respectively, the target fucosylated oligosaccharide is LNFP-3-L, and the host cell for producing the target fucosyltransferase, alpha-1, 3-fucosyltransferase.

In some embodiments, the fucosyltransferase expressed in the host cell is capable of having lactose or a lactose derivative (e.g., LNT, LNnT, or 2' -FL) as a receptor substrate. Those skilled in the art know how to select a fucosyltransferase suitable for the desired product and the corresponding acceptor substrate. Methods for identifying the catalytic ability of a fucosyltransferase to a particular acceptor substrate are well known to those skilled in the art and can be accomplished by conventional enzymatic reactions and detection of product formation.

In some embodiments, the α -1, 2-fucosyltransferases used in the present invention are capable of having lactose, LNT, LNnT as acceptor substrates. The alpha-1, 2-fucosyltransferases used in the present invention may be of bacterial, fungal, plant, animal (e.g. mammalian, such as human) origin. Exemplary α -1, 2-fucosyltransferases include FutC derived from helicobacter pylori (Helicobacter pylori), wbsJ derived from escherichia coli O128, wbgL derived from escherichia coli O126, wcfB and WcfW derived from bacteroides fragilis (Bacteroides fragilis), SAMT derived from azospirillum adipogenesis (Azospirillum lipoferum), futL derived from helicobacter ferret (h.mustelae), futF derived from helicobacter bile (h.bilis), futG derived from campylobacter jejuni (Campylobacter jejuni), futN derived from bacteroides vulgare (Bacteroides vulgatus), FutW(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; derived from Prevotella sp.) or functional variants thereof. In some embodiments, the alpha-1, 2-fucosyltransferase used in the present invention is an alpha-1, 2-fucosyltransferase derived from helicobacter pylori (Helicobacter pylori). In some embodiments, the alpha-1, 2-fucosyltransferase used in the present invention comprises an amino acid sequence as set forth in SEQ ID NO. 7.

In some embodiments, the α -1, 3-fucosyltransferases used in the present invention are capable of using lactose, LNT, LNnT, or 2' -FL as acceptor substrates. The alpha-1, 3-fucosyltransferases used in the present invention may be of bacterial, fungal, plant, animal (e.g. mammalian, such as human) origin. Exemplary α -1, 3-fucosyltransferases include FutA or FutB derived from helicobacter pylori (Helicobacter pylori), futJ (Hh 0072) and FutK (Hh 1776) derived from helicobacter (h. Hepaticus), futE derived from helicobacter (h. Bilis), futD derived from helicobacter (h. Strogontum), futH derived from helicobacter cecum (h. Tyrosponius), futM, fucT6 or FucT7 derived from bacteroides fragilis (Bacteroides fragilis), Amuc_0760(WO2012/112777;WO2019025485A1;EP2439264A1;WO2010/142305;Tomotoshi Sugita et al.,Journal of Biotechnology,Volume 361,2023,Pages110-118;WO2019/008133; derived from ackermannia viscosa (AKKERMANSIA MUCINIPHILA), or functional variants thereof. In some embodiments, the alpha-1, 3-fucosyltransferase used in the present invention is an alpha-1, 3-fucosyltransferase derived from helicobacter pylori (Helicobacter pylori). In some embodiments, the alpha-1, 3-fucosyltransferase used in the present invention comprises the amino acid sequence shown as SEQ ID NO. 43.

In some embodiments, the α -1, 3/4-fucosyltransferases used in the present invention are capable of using lactose, LNT, LNnT, or 2' -FL as acceptor substrates. The alpha-1, 3/4-fucosyltransferases used in the present invention may be of bacterial, fungal, plant, animal (e.g. mammalian, such as human) origin. Exemplary alpha-1, 3/4-fucosyltransferases include alpha-1, 3/4-fucosyltransferases (Zhang A et al.,Metab Eng.2021;66:12-20;Liang S et al.,J Agric Food Chem.2024Feb 28;72(8):4367-4375; derived from helicobacter pylori (Helicobacter pylori), which are incorporated herein by reference in their entirety), or functional variants thereof.

In some embodiments, the α -1, 4-fucosyltransferases used in the present invention are capable of using lactose, LNT, LNnT, or 2' -FL as acceptor substrates. The alpha-1, 4-fucosyltransferases used in the present invention may be of bacterial, fungal, plant, animal (e.g. mammalian, such as human) origin. Exemplary α -1, 4-fucosyltransferases include α -1, 4-fucosyltransferases as described in Tomotoshi Sugita et al, journal of Biotechnology, volume 361,2023,Pages 110-118 and WO2019/008133 (the entire disclosures of which are incorporated herein by reference), or functional variants thereof.

In some embodiments, to increase the fucosylation capacity of a host cell, more than 1 copy (e.g., 2, 3, or more copies) of an exogenous fucosyltransferase gene may be introduced into the host cell. The more than 1 copy of the exogenous fucosyltransferase gene may have the same sequence or different sequences. The more than 1 copy of the exogenous fucosyltransferase gene may be contained in the same expression cassette in the host cell or in different expression cassettes. The more than 1 copy of the exogenous fucosyltransferase gene may be located in an episomal vector (e.g., a plasmid, such as using a multicopy plasmid or including multiple copies in one plasmid) in the host cell or integrated into the host cell genome at different sites, respectively. In some embodiments, the host cell may comprise more than 1 copy of the exogenous alpha-1, 2-fucosyltransferase gene, such as more than 1 copy of the alpha-1, 2-fucosyltransferase from helicobacter pylori (Helicobacter pylori).

The synthesis of fucosylated oligosaccharides requires the participation of a receptor substrate, and in some embodiments, the host cell is capable of providing a receptor substrate for a fucosyltransferase-catalyzed reaction within the cell, for example, by transporting the receptor substrate into the cell, or synthesizing the receptor substrate within the cell. The kind of the 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 the specific target fucosylated oligosaccharide is achieved by the combination of the acceptor substrate and the fucosyltransferase having catalytic ability to the acceptor substrate. The acceptor substrate provided within the host cell should be capable of accepting fucosyl residues that are catalytically transferred by the fucosyltransferase expressed within the host cell to synthesize the fucosylated oligosaccharide of interest.

In some embodiments, host cells of the invention comprise a transporter capable of transporting a receptor substrate into a cell, or comprise an intracellular synthesis pathway capable of synthesizing a receptor substrate using an additional precursor species (e.g., an additional 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 additional precursor material is lactose or milk-N-trisaccharide II (LNT-2) (WO 2019/008133; incorporated herein by reference in its entirety).

In some embodiments, the acceptor substrate is lactose or a lactose derivative synthesized from lactose as a precursor, the lactose derivative having a structure that allows it to form a fucosyl oligosaccharide upon attachment to a fucosyl moiety via fucosyl transferase catalysis. In some embodiments, the lactose derivatives are capable of being synthesized in a host cell by an enzyme-catalyzed reaction using lactose as a precursor. In some embodiments, the lactose derivative may be, for example, LNT, LNnT, or 2' -FL.

In some embodiments, the host cell naturally has a transporter gene capable of transporting the receptor substrate into the cell or a synthesis pathway gene capable of synthesizing the receptor substrate using an additional precursor species (e.g., an additional carbon source). For example, E.coli has a lactose permease gene that can naturally transport lactose into the cell, where it can be transported into the cell by the host cell's endogenous enzymes or synthesized in the cell by an additional carbon source.

In some embodiments, the host cell may be genetically engineered to introduce exogenous transporter genes useful for transporting a receptor substrate into the cell or exogenous synthesis pathway genes capable of synthesizing the receptor substrate from an externally added precursor substance (e.g., an externally added carbon source), to transport or synthesize a desired receptor substrate into the cell by expressing these exogenous genes, e.g., when the host cell is unable to natively transport the receptor substrate into the cell or is unable to natively endogenously synthesize the receptor substrate, or when the host cell naturally has an enzyme gene capable of transporting the receptor substrate into the cell, it is desirable to synthesize the receptor substrate in the cell using other carbon sources as precursors.

In some embodiments, the host cells of the invention are capable of providing lactose within the cell, to provide lactose as a receptor substrate, or to provide lactose for synthesis of a receptor substrate.

In some embodiments, a host cell of the invention comprises a gene encoding a lactose transporter protein to express the lactose transporter protein. Lactose transporters are capable of transporting lactose from the medium into the cell to provide lactose within the cell. The gene encoding lactose transporter may be endogenous or exogenous. The lactose transporter may be lactose permease. When the host cell used lacks an endogenous lactose transporter, a gene encoding an exogenous lactose transporter may be introduced into the host cell such that the host cell comprises the gene encoding the exogenous lactose transporter and expresses the exogenous lactose transporter. The exogenous lactose transporter gene may be contained in an episomal expression vector introduced into the host cell or integrated into the chromosome of the host cell. In some embodiments, the lactose permease comprised by the host cell may be, for example, lactose permease (LacY) from escherichia coli or lactose permease Lac12 from kluyveromyces lactis (Kluyveromyces lactis). In some embodiments, the E.coli lactose permease comprises the amino acid sequence shown as SEQ ID NO. 9.

In some embodiments, the host cell may 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 than lactose, possibly with endogenous synthesis of lactose. In some embodiments, the host cell may be genetically engineered to express enzymes for the synthesis of lactose from other carbon sources (e.g., glucose), such as expressing β -1, 4-galactosyltransferase to effect the synthesis of lactose within the cell. Beta-1, 4-galactosyltransferase is capable of catalyzing galactose and glucose to lactose. Examples of beta-1, 4-galactosyltransferases may be Pm1141 of Pasteurella multocida (Pasteurella multocida) and Lex1 of Agrobacter acidophilus (Aggregatibacter aphrophilus) NJ8700 (WO 2015/150328; incorporated herein by reference in its entirety). When host cells are cultured with a carbon source capable of being converted to glucose by cellular metabolism, the host cells are able to produce free glucose from the carbon source within the cell and to catalyze the synthesis of lactose by beta-1, 4-galactosyltransferase. The carbon source may be, for example, glucose, sucrose, glycerol, fructose, xylose, cellulose, molasses, corn syrup, galactose, methanol, pyruvic acid, succinic acid or any other carbon source convertible into glucose by cellular metabolism.

In some embodiments, lactose may be a precursor to the synthesis of the acceptor substrate for the fucosyltransferase by an enzyme-catalyzed reaction. For example, the acceptor substrate LNnT for the synthesis LNnFP-I, LNnFP-III, LNnFP-V, can be synthesized starting from lactose, UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-galactose (UDP-Gal) by catalysis of beta-1, 3-N-acetylglucosaminyl transferase and beta-1, 4-galactosyltransferase. The receptor substrates LNT for the synthesis of LNFP-I, LNFP-II, LNFP-V can be synthesized (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; by catalysis of beta-1, 3-N-acetylglucosaminyl transferase and beta-1, 3-galactosyltransferase starting from lactose, UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-galactose (UDP-Gal), the entire contents of which are incorporated herein by reference. UDP-GlcNAc, UDP-Gal, lactose may be produced endogenously by the host cell or introduced exogenously.

In some embodiments, the host cell does not have an endogenous β -1, 3-N-acetylglucosaminyl transferase gene, an endogenous exogenous β -1, 4-galactosyltransferase gene, an endogenous β -1, 3-N-acetylglucosaminyl transferase gene, and/or an endogenous β -1, 3-galactosyltransferase gene, and such host cell, in addition to being capable of providing lactose within the cell (e.g., by expressing a lactose transporter gene or by expressing an intracellular synthetic pathway gene that can synthesize lactose from an exogenous precursor substance), may be genetically engineered to introduce an exogenous β -1, 3-N-acetylglucosaminyl transferase gene and an exogenous β -1, 4-galactosyltransferase gene, or to introduce an exogenous β -1, 3-N-acetylglucosaminyl transferase gene and an exogenous β -1, 3-galactosyltransferase gene. These exogenous enzyme genes may be contained in episomal expression vectors introduced into the host cell or integrated into the chromosome of the host cell. Thus, in some embodiments, to produce more complex fucosylated oligosaccharides, the host cell of the invention may further comprise enzyme genes for producing suitable acceptor substrates, such as a β -1, 3-N-acetylglucosaminyl transferase gene and a β -1, 4-galactosyltransferase gene, or a β -1, 3-N-acetylglucosaminyl transferase gene and a β -1, 3-galactosyltransferase gene. Any one or more of these enzyme genes may be endogenous or exogenous.

The supply of UDP-galactose may be obtained from metabolic pathways of the host cell itself, which may include, for example, phosphoglucomutase, UTP-glucose-1-phosphate-uridyltransferase and UDP-glucose-4-epimerase catalyzed synthesis. The supply of UDP-galactose may also be achieved by providing galactose in the host cell culture medium, which is phosphorylated to galactose-1-phosphate and then converted to UDP-galactose by the cells upon uptake of galactose. The supply of UDP-N-acetylglucosamine can also be obtained from the host cell's own metabolic pathways.

The inherent ability of the various cells to possess the enzymes described above varies, and it will be appreciated by those skilled in the art that when the host cell naturally possesses one or more of the enzymes described above, the endogenous enzymes of the host cell may be used without introduction from an external source, or the endogenous enzyme genes may be engineered to be overexpressed or inactivated to further facilitate the synthesis of fucosylated oligosaccharides. For enzymes that are not naturally present in the host cell, additional introduction of heterologous enzyme genes is required. Those skilled in the art will be able to determine which enzymes require additional introduction for a particular host cell and which enzymes may use the host cell's endogenous enzymes.

For example, for most common host cells such as E.coli, corynebacterium glutamicum, bacillus subtilis, saccharomyces cerevisiae, etc., E.coli naturally has GDP-D-mannose-4, 6-dehydratase (GMD), so when E.coli is used as the host cell, its endogenous GMD gene may be used, or its GMD gene may also be overexpressed to increase the synthesis of fucosylated oligosaccharide precursors. However, microorganisms such as Bacillus subtilis, corynebacterium glutamicum and Saccharomyces cerevisiae do not naturally possess GDP-D-mannose-4, 6-dehydratase, and thus, it is necessary to introduce a heterologous GMD gene.

For the reductase capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-D rhamnose, GDP-D-rhamnose-3, 5-epimerase (GRE), fucosyltransferase, E.coli, corynebacterium glutamicum, bacillus subtilis, saccharomyces cerevisiae, which are not naturally present, it is necessary to introduce a heterologous enzyme gene.

Coli and bacillus subtilis naturally have lactose transporters and therefore, when lactose is required to be transported into the host cell, the corresponding enzyme need not be introduced from an external source. While corynebacterium glutamicum and saccharomyces cerevisiae do not naturally possess lactose transporters, additional introduction of heterologous enzyme genes is required when lactose transport into the host cell is desired.

Those skilled in the art will appreciate that the host cells of the invention may be further engineered to optimize the production of fucosylated oligosaccharides. For example, the host cells of the invention may be further engineered to increase the intracellular GDP-L-fucose production capacity. In some embodiments, the host cell may be engineered to overexpress an enzyme in the GDP-L-fucose synthesis pathway. In some embodiments, the host cell may be engineered to overexpress one or more of the GDP-D-mannose synthesis pathway genes. In some embodiments, the one or more genes of the GDP-D-mannose synthesis pathway gene comprises a phosphomannose mutase (ManB) gene and/or a mannose-1-guanylate transferase (ManC) gene. The overexpression may be achieved by introducing an additional foreign gene encoding the enzyme into the host cell, and the introduced foreign gene may have the same or different sequence from an endogenous gene having the same function in the host cell, which may increase the copy number of the gene encoding the enzyme in the host cell, thereby increasing the expression amount of the enzyme. The overexpression may also be achieved by engineering regulatory sequences (e.g., promoters) in the host cell that are operably linked to the gene encoding the enzyme, such as replacing an original promoter (e.g., the promoter of the gene naturally occurring in the host cell) with a stronger promoter, or introducing mutations or inserting new regulatory elements in the promoter region to enhance its activity. As will be appreciated by those skilled in the art, the term "stronger promoter" refers to a promoter that has stronger promoter activity, e.g., has a stronger ability to bind to a transcription initiation complex and/or has a stronger ability to bind to an RNA polymerase than a promoter native to the host cell (e.g., a promoter of the gene naturally occurring in the host cell). As known to those skilled in the art, the use of a stronger promoter can increase the level of expression of a gene operably linked to the promoter (e.g., increase the amount of expression of a protein encoded by the gene). In some embodiments, the stronger promoter refers to a promoter whose-10 region comprises TATAAT. In some embodiments, the host cell is engineered such that the-10 region sequence of the promoter of the phosphomannose mutase (ManB) gene and/or the mannose-1-guanylate transferase (ManC) gene comprises TATAAT.

In some embodiments, the host cell may also be engineered to prevent depletion of the intracellular GDP-L-fucose pool. For example, the host cells of the invention may be further engineered to eliminate or attenuate other metabolic pathways for GDP-L-fucose, such as to eliminate or attenuate enzymes capable of catalyzing the conversion of GDP-L-fucose to substances other than fucosylated oligosaccharides, such as colanic acid. For example, the host cell may be engineered to have no functional UDP-glucose lipid carrier transferase WcaJ or to have reduced activity UDP-glucose lipid carrier transferase WcaJ.

The host cells of the invention may be further engineered to eliminate or attenuate other metabolic pathways of lactose, which are other metabolic pathways besides the synthesis of fucosylated oligosaccharides catalyzed by fucosyltransferase with lactose as a receptor substrate. For example, the host cell may be engineered to have no functional galactosidase LacZ or to have a reduced activity of galactosidase LacZ.

The host cells of the invention may be further engineered to eliminate or attenuate other synthetic pathways of GDP-L-fucose originally present in the host cells, such as eliminating or attenuating enzymes capable of catalyzing the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-L-fucose (e.g., GDP-L-fucose synthase WcaG), such that the host cells synthesize GDP-L-fucose via the synthetic pathways of the invention. For example, the host cell may be engineered to have no functional GDP-L-fucose synthase WcaG or to have a GDP-L-fucose synthase WcaG with reduced activity.

In the present invention, the host cell may 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 (e.g., human cells and cell lines). In some embodiments, the host cell is a microbial cell, which may include bacteria, yeast cells, archaebacteria, fungal cells, and the like. The microorganism may be a GRAS (generally regarded as safe) microorganism. The bacteria may be, for example, gram-negative or gram-positive bacteria. The bacteria may be, for example, escherichia bacteria such as Escherichia coli (ESCHERICHIA COLI), corynebacterium bacteria such as Corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium beijing (Corynebacterium pekinense), corynebacterium crenatum (Corynebacterium crenatum), corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes), corynebacterium thermoaminogenes, Corynebacterium ammoniagenes (Corynebacterium aminogenes) and the like; bacillus bacteria such as Bacillus subtilis (Bacillus subtilis), bacillus licheniformis (Bacillus licheniformis), bacillus coagulans (Bacillus coagulans), bacillus cereus (Bacillus stearothermophilus), bacillus stearothermophilus (Bacillus stearothermophilus), Bacillus megaterium (Bacillus megaterium) and the like, lactobacillus bacteria such as Lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus casei (Lactobacillus casei), Lactobacillus delbrueckii (Lactobacillus delbrueckii), lactobacillus plantarum (Lactococcus lactis), bifidobacterium (Bifidobacterium) bacteria, streptococcus (Streptococcus) bacteria, lactobacillus (Lactobacillus) bacteria, streptomyces (STREPTMYCES) bacteria, pseudomonas (Pseudomonas) bacteria, such as Pseudomonas aeruginosa (Pseudomonas aeruginosa), clostridium (Clostridium) bacteria, brevibacterium (Brevibacillus) bacteria, enterococcus (Enterococcus) bacteria, pediococcus) bacteria, leuconostoc (Leuconostoc) bacteria, and the like. The yeast cell may be, for example, a yeast cell of Saccharomyces (Saccharomyces), fuscoporia (Saccharomycopsis), pichia (Pichia), hansenula (Hansenula), kluyveromyces (Kluyveromyces), yarrowia (Yarrowia), rhodotorula (Rhodotorula) or Schizosaccharomyces (Schizosaccharomyces), such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Yarrowia lipolytica (Yarrowia lipolytica), candida utilis (Candida) or Pichia pastoris.

Methods for genetically engineering host cells are well known to those skilled in the art. Genetic engineering techniques may be used to introduce a episomal exogenous nucleic acid sequence (e.g., in the form of a plasmid) into a host cell or to insert an exogenous nucleic acid sequence into the host cell genome or to delete an endogenous nucleic acid sequence, or to replace an endogenous nucleic acid sequence in the host cell genome with an exogenous nucleic acid sequence, to effect a change in the host cell's gene and thus its phenotype. For example, an exogenous gene may be introduced into a host cell to express a protein (one or more enzymes as mentioned above) encoded by the exogenous gene in the host cell, which may be accomplished, for example, by introducing into the host cell a vector comprising the exogenous gene, which may be in linear or circular form, and may be single-stranded or double-stranded. The vector may be a self-replicating vector. The vector may be an episomal vector or an integrative vector. The vector may be, for example, a plasmid vector, a phage vector, a bacterial artificial chromosome, a transposon-based vector, or a CRISPR/Cas system-based vector, etc. The exogenous gene may be present in and expressed from an episomal vector (e.g., episomal plasmid) upon introduction into the host cell, for example, by introducing into the host cell a plasmid vector comprising an exogenous gene expression cassette. The exogenous gene expression cassette can include an exogenous gene and a regulatory sequence operably linked thereto. Regulatory sequences may include, but are not limited to, promoters, enhancers, terminators, and other expression control elements. Promoters may be constitutive or inducible. The exogenous gene may also be integrated into the genome of the host cell to effect expression, for example, by introducing into the host cell an integrative plasmid vector comprising the exogenous gene (which plasmid may, for example, comprise a homology arm for integration into the host cell genome by homologous recombination), a phage vector, a CRISPR/Cas system, or a transposon system (e.g., piggybac or a Sleeping Beauty system). The expression cassette comprising the exogenous gene may be integrated into the host cell genome, or the exogenous gene may be integrated into a suitable site in the host cell to express the exogenous gene using regulatory sequences endogenous to the host cell. Suitable methods for introducing exogenous nucleic acid sequences (e.g., vectors) into host cells are known to those of skill in the art and include, but are not limited to, calcium phosphate transfection, protoplast fusion, electroporation, liposomes, lipid nanoparticles, microinjection, naked DNA or RNA (e.g., mRNA) transfection, plasmid vector transformation, phage vector transduction, and the like. These genetic engineering techniques may also be used to knock-out or knock-down an endogenous gene such that the host cell does not have the functionality of the protein encoded by the endogenous gene or such that the activity of the protein encoded by the endogenous gene is reduced. 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 engineering techniques may also be used to alter the regulatory sequences (e.g., promoters) of an endogenous gene to increase expression of the endogenous gene. Those skilled in the art are able to select appropriate methods for genetically engineering host cells based on the host cells and the exogenous or endogenous nucleic acid sequences used (A Laboratory Manual(2nd Ed.),Vols.1-3,Cold Spring Harbor Laboratory(1989)and Ausubel et al,eds.,Current Protocols in Molecular Biology,John Wiley&Sons,Inc.,New York(1997)).

In some embodiments, the host cell is a genetically modified corynebacterium glutamicum comprising an endogenous GDP-D-mannose synthesis pathway gene, and genetically modified to comprise a gene encoding an exogenous GDP-D-mannose-4, 6-dehydratase (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 a more preferred embodiment, the genetically modified corynebacterium glutamicum further comprises a gene encoding an exogenous lactose permease. In a more preferred embodiment, 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 a more preferred embodiment, the fucosyltransferase is an alpha-1, 2-fucosyltransferase or an alpha-1, 3-fucosyltransferase derived from helicobacter pylori.

In some embodiments, the host cell is a genetically modified escherichia coli comprising an endogenous GDP-D-mannose synthesis pathway gene and 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 fucosyltransferase. In a more preferred embodiment, the gene encoding β -galactosidase LacZ is knocked out, the gene encoding GDP-L-fucose synthase WcaG is knocked out, and/or the gene encoding UDP-glucose lipid carrier transferase WcaJ is knocked out in the genetically modified E.coli. In a more preferred embodiment, 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 a more preferred embodiment, the fucosyltransferase is an alpha-1, 2-fucosyltransferase or an alpha-1, 3-fucosyltransferase derived from helicobacter pylori.

The host cells and methods described herein can be used to produce fucosylated oligosaccharides, particularly those belonging to HMOs, such as those comprising a lactose moiety and a fucose moiety, which can be synthesized starting from lactose and GDP-L-fucose 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-fucose, lacto-N-neofucose, lacto-N-neodifucose, and the like, such as 2 '-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucose I (LNFP-I), lacto-N-fucose II (LNFP-II), lacto-N-fucose V (LNFP-V), lacto-N-neofucose I (LNnFP-I), lacto-N-neofucose III (LNnFP-III), lacto-N-neofuce V (LNnFP-V), lacto-N-difucose I (LNH-I), lacto-N-difucose II (DFH-II), and the like.

The invention also provides a method of producing a fucosylated oligosaccharide, the method comprising culturing a genetically engineered host cell of the invention under suitable conditions to synthesize the fucosylated oligosaccharide.

In some embodiments, the medium comprises at least one carbon source that may be selected from, but is not limited to, glucose, sucrose, glycerol, fructose, lactose, xylose, cellulose, molasses, corn syrup, galactose, methanol, pyruvic acid, succinic acid, and the like. The carbon source may be supplemented at an appropriate time during the cultivation of the host cell.

The medium may contain a receptor substrate or a substance capable of producing the receptor substrate as a precursor substance via a synthetic pathway contained within the host cell. In some embodiments, the acceptor substrate is lactose or a lactose derivative (e.g., LNT, LNnT, or 2' -FL). In some embodiments, the precursor material is lactose, milk-N-trisaccharide II, or a non-lactose carbon source (e.g., glucose or a carbon source capable of being converted to glucose by cellular metabolism, such as may be glucose, sucrose, glycerol, fructose, xylose, cellulose, molasses, corn syrup, galactose, methanol, pyruvic acid, succinic acid, or any other carbon source capable of being converted to glucose by cellular metabolism). Correspondingly, the host cell used comprises a transporter gene capable of transporting the receptor substrate into the cell, or comprises a synthesis pathway gene capable of synthesizing the receptor substrate using the precursor substance.

In some embodiments, lactose may be included in the medium to provide a recipient substrate for the fucosyltransferase. Accordingly, the host cell used comprises a gene encoding a lactose transporter. In this case, lactose may directly accept fucose as a acceptor substrate to synthesize fucosylated oligosaccharides, for example in the production of 2 '-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL) or Difucosyllactose (DFL). In some embodiments, lactose is used as a precursor to synthesize the receptor substrate in the cell. Accordingly, the host cell used comprises a gene encoding lactose transporter and a synthesis pathway gene capable of synthesizing a receptor substrate with lactose as a precursor substance, for example, in the production of lacto-N-fucose I (LNFP-I), lacto-N-fucose II (LNFP-II), lacto-N-fucose V (LNFP-V), lacto-N-neofucose I (LNnFP-I), lacto-N-neofucose III (LNnFP-III), lacto-N-neofucose V (LNnFP-V), lacto-N-difucose I (LNDFH-I), lacto-N-difucose II (LNDFH-II) and the like. Lactose may be supplemented at an appropriate time during the cultivation of the host cells.

When the gene encoding one or more enzymes contained in the host cell is expressed in an inducible manner, an inducer is added during cell culture to induce the expression of the enzymes.

The cultivation of the host cells may be a batch fermentation, i.e. the use of a closed culture system with a specific medium at the beginning of the fermentation and the use of specific temperatures, pressures, aeration and other environmental conditions to optimize the growth, without adding nutrients during the cultivation of the cells and without letting out the fermentation broth. The host cell culture may also be fed-batch fermentation, i.e., the intermittent or continuous first addition of nutrients during fermentation, but without the evolution of fermentation broth. The host cell may also be cultured by continuous fermentation, i.e., by continuously adding nutrients during the fermentation process and continuously discharging the fermentation broth, thereby maintaining a constant amount of broth in the fermentation system. The fermentation of the host cell may also be a combination of two or three of the above fermentation modes.

The genetically engineered host cells of the invention are cultured under conditions suitable for the production of fucosylated oligosaccharides. Suitable conditions include suitable temperature, pH, dissolved oxygen, osmotic pressure, and other conditions. Depending on the different types of host cells, the suitable conditions may vary, which can be readily determined by a person skilled in the art.

In some embodiments, the production method further comprises recovering the synthesized product, i.e., the fucosylated oligosaccharide, from the culture medium. The term "recovering" refers to isolating or further purifying the fucosylated oligosaccharides produced by the host cell of the invention from other components in the host cell culture. The term "purification" refers to the removal of impurities and unwanted byproducts, such as cells, ions, salts, other sugars than the desired fucosylated oligosaccharides.

The product may be recovered from the culture medium and/or from the host cell itself. The product may be recovered, for example, from the culture medium or from the supernatant of the cell lysate. Cell lysis may be performed by chemical or physical methods well known in the art.

Purification can be carried out by techniques well known to those skilled in the art. For example, the product may be purified from the culture medium by methods well known to those skilled in the art, such as by column chromatography using an activated carbon step and eluting with 35-50% ethanol, by an ethanol gradient, or by size exclusion. Purity may be assessed by any known method, such as thin layer chromatography or other electrophoresis or chromatographic techniques generally known in the art.

The invention is further illustrated by the following examples, which should not be construed as limiting the invention.

The reagents used in the examples below were commercially available products unless otherwise specified. The experimental methods of molecular biology not specifically described in the examples were carried out with reference to the specific methods listed in J.Sambrook, molecular Cloning: A Laboratory Manual, third Edition, or according to the kit and product instructions.

EXAMPLE 1 cultivation of Corynebacterium glutamicum and method for detecting products

Two different media were used to culture Corynebacterium glutamicum, seed medium LBHI and fermentation medium FM20, respectively.

The seed culture medium LBHI comprises 2.5g/L yeast powder, 5g/L peptone, 5g/L NaCl and 18.5g/L brain heart infusion broth.

The FM20 component of the fermentation medium comprises 50g/L glucose, 20g/L lactose, 4g/L peptone, 2g/L yeast powder, (NH ₄)₂SO₄ g/L, urea 5g/L,KH₂PO₄ 1g/L,K₂HPO₄ 1g/L,MgSO₄ 0.25g/L,MOPS 42g/L,CaC1₂10mg/L, biotin 0.2mg/L protocatechuic acid) 0.03mg/L,FeSO₄·7H₂O 10mg/L,MnSO₄·H₂O 10mg/L,ZnSO₄·7H₂O 1mg/L,CuSO₄ 0.2mg/L,NiCl₂·6H₂O 0.02mg/L,pH7.0).

The seed medium LBHI was sterilized by autoclaving (121 ℃ C., 20 min). The fermentation medium FM20 was filter sterilized using a 0.22 μm filter. The medium was made selective by adding kanamycin when necessary. The solid plate can be prepared by adding 2% of agar into the culture medium.

During fermentation of the Corynebacterium glutamicum strain, colonies on the plates were picked up into seed medium for overnight culture. The bacterial liquid after overnight culture was inoculated into a 96-well plate containing a fermentation medium at an inoculum size of 5%, and cultured at 30℃for 48 hours. The plate was centrifuged and the supernatant was aspirated to determine the titer of each substance in the supernatant after culture.

The analysis and detection of substances such as GDP-D-mannose (GDP-D-mannose), GDP-D-rhamnose (GDP-D-rhamnose) and GDP-L-fucose (GDP-L-fucose) were carried out by UPLC-MS of Agilent. The sample is separated by a chromatographic column, ionized in an ion source and detected by a mass spectrometer to determine the concentration of each species. The column used was Agilent hilic-z 2.7 μm,3.0 x 150mM, mobile phase 10mM ammonium acetate (pH 9.6) -acetonitrile (V/V=25%/75%) at a flow rate of 0.35mL/min, sample volume of 1 μl, column temperature of 35 ℃, mass spectrometer detector ion source ESI, scanning mode SIM, polarity negative mode.

Analytical detection of 2'-FL, 3-FL was performed by Agilent's UPLC-MS. The sample is separated by a chromatographic column, ionized in an ion source and detected by a mass spectrometer to determine the concentration of each species. The column used was ACQUITY UPLC BEH Amide 1.7.7 μm, 2.1X105 mM or other equivalent column, the mobile phase was 10mM ammonium acetate (pH9.6) -acetonitrile (V/V=30%/70%), the flow rate was 0.3mL/min, the sample injection volume was 1. Mu.L, the column temperature was 35 ℃, the mass spectrometer ion source was ESI, the scanning mode was SIM, and the polarity was positive mode.

EXAMPLE 2 cultivation of recombinant E.coli and method for protein expression and purification

The culture of the escherichia coli is carried out by using an LB culture medium, wherein the culture medium comprises 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl.

And (3) carrying out gene synthesis on the protein sequence to be expressed, wherein a synthetic vector is pRSFDuet-1 plasmid, and the synthetic sequence is positioned behind the histidine tag of the first multiple cloning site. The recombinant plasmid is introduced into E.coli BLR (DE 3) to construct recombinant E.coli. Recombinant E.coli was inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured with shaking at 37℃and 200 rpm. When the absorbance density (OD ₆₀₀) of the culture solution reaches 0.6-0.8, the culture temperature is adjusted to 16 ℃, and isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 0.2mM is added to induce protein expression. And (3) after the culture is continued for 24 hours, centrifugally collecting thalli by using a high-speed refrigerated centrifuge, and obtaining engineering strain wet cells for efficiently expressing target proteins.

Protein purification by nickel affinity chromatography, the buffers used for purification included:

20mM PBS,500mM NaCl,10mM imidazole, 2mM beta-mercaptoethanol, pH 7.4;

20mM PBS,500mM NaCl,500mM imidazole, 2mM beta-mercaptoethanol, pH 7.4;

And the solution C is 20mM PBS,150mM NaCl,1mM dithiothreitol with the pH value of 7.4.

The specific purification method comprises 1) carrying out ultrasonic disruption after resuspension of the wet cells by the solution A, obtaining supernatant of disruption solution by centrifugal separation, 2) balancing a nickel column by using the solution A with the volume of 10 times of the column, then filtering the disruption supernatant by a filter membrane, loading the sample, 3) completely flowing out the supernatant, adding a mixed solution (5% solution B) with the volume of 5 times of the column to wash out the impurity protein, 4) eluting target protein adsorbed by nickel by using the mixed solution (40% solution B) with the volume of 5 times of the column, and 5) carrying out centrifugal concentration on the collected target protein by using a10 kDa ultrafiltration tube. And 6) adding 10mL of C solution when the concentration volume is less than 0.5mL, and carrying out centrifugal concentration again, wherein 6) the buffer solution is replaced by the buffer solution after 3 times of reciprocation, and most of imidazole is removed to obtain the target protein pure enzyme. Quick freezing the pure enzyme in liquid nitrogen, and storing in a refrigerator at-80 ℃. Protein concentration was measured using an ultra-micro spectrophotometer NanoDrop one, detecting the ultraviolet absorbance of the protein at 280 nm.

EXAMPLE 3 GDP-mannose-3, 5-epimerase (GME) catalyzes the reversible isomerization of GDP-D-rhamnose and GDP-L-fucose

Since GDP-mannose-3, 5-epimerase (GME) was discovered in the last 70 th century, researchers have performed protein physicochemical property studies and catalytic mechanism analyses on GME of different origins. These GMEs include AtGME (SEQ ID NO:1, whose gene sequence is SEQ ID NO: 2) derived from Arabidopsis thaliana (Arabidopsis thaliana), mfGME (SEQ ID NO:3, whose gene sequence is SEQ ID NO: 4) derived from Fuscoporia obliqua (Methylacidiphilum fumariolicum), and OsGME (SEQ ID NO:5, whose gene sequence is SEQ ID NO: 6) derived from Oryza sativa. However, no relevant search has been reported for GME catalytic substrate spectra, and whether GME can catalyze isomerization reactions of GDP-D-rhamnose and GDP-L-fucose is unknown.

The AtGME gene sequence, mfGME gene sequence and OsGME gene sequence were subjected to gene synthesis. The synthetic vector is pRSFDuet-1 plasmid, the synthetic sequence is located behind the histidine tag of the first multiple cloning site, and the recombinant plasmid is named pRSFDuet-AtGME, pRSFDuet-MfGME, pRSFDuet-OsGME. Pure enzymes AtGME, mfGME and OsGME were obtained as described in example 2. And then, GDP-D-rhamnose and GDP-L-fucose are respectively taken as substrates, and the activities of the three GMEs are detected through in vitro enzymatic reactions.

The GME catalyzed reversible isomerization reaction route for GDP-D-rhamnose and GDP-L-fucose is as follows:

Specific reaction conditions were 50mM phosphate buffer (pH 7.5), 0.5g/L GDP-D-rhamnose or GDP-L-fucose, 1g/L GME purified enzyme. No protein was added in the control experiments and other conditions remained the same. The reaction was continued at 30 ℃ at 400rpm for 5 hours, and then 5 volumes of an organic solvent (acetonitrile: methanol=1:1) was added to the reaction solution to quench the reaction. After removal of the proteins by centrifugation, the products were analyzed as described in example 1.

OsGME from rice (Oryza sativa) showed the best reactivity among AtGME, mfGME and OsGME. As shown in FIG. 2, using GDP-D-rhamnose as a substrate OsGME may produce GDP-L-fucose. When GDP-L-fucose is used as a substrate, osGME can also produce GDP-D-rhamnose. OsGME effects the reversible isomerisation conversion of GDP-D-rhamnose with GDP-L-fucose. In the invention, the substrate actually catalyzed by the GME is GDP-D-rhamnose, so that an enzyme which is represented by GDP-mannose-3, 5-epimerase (GME) and can catalyze GDP-D-rhamnose to GDP-L-fucose is named GDP-D-rhamnose-3, 5-epimerase (GRE). The above-described AtGME, mfGME, and OsGME may also be referred to as AtGRE, mfGRE, and OsGRE, respectively. 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 a strain, it is highly possible to achieve 2' -FL synthesis through an entirely novel metabolic pathway.

EXAMPLE 4 construction of recombinant strain HpfutC-lacY-ManB-ManC of Corynebacterium glutamicum

The alpha-1, 2-fucosyltransferase HpfutC gene sequence (SEQ ID NO:8, the amino acid sequence of which is SEQ ID NO: 7) of helicobacter pylori (Helicobacter pylori) and the lactose permease LacY gene sequence (SEQ ID NO:10, the amino acid sequence of which is SEQ ID NO: 9) of Escherichia coli (ESCHERICHIA COLI) were subjected to gene synthesis. As the gene synthesis vector, pUC57 plasmid was used, and the recombinant plasmids were designated pUC57-HpfutC and pUC57-LacY, respectively.

The genome of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 is used as a template, and PCR amplification is performed by using a primer pair 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 to obtain homology arms HpfutC-lacY-Up, hpfutC-lacY-Down, The promoter Psod (SEQ ID NO: 51) and Pcg2195 (SEQ ID NO: 52) (PCR system using 2X Phanta Max Master Mix, (Dye Plus), vazyme), the lactose permease LacY gene fragment obtained by PCR amplification of LacY-F/LacY-R using pUC57-LacY as template (PCR system using 2X Phanta Max Master Mix, (Dye Plus), vazyme), the alpha-1, 2-fucosyltransferase HpfutC gene fragment obtained by PCR amplification of pUC57-HpfutC using primer pair HpfutC-F/HpfutC-R, and the linearized fragment line-pK18mobsacB obtained by digestion of plasmid pK18mobsacB with restriction endonucleases EcoRI and Xba I. The 7 fragments HpfutC-lacY-Up, pcg2195, hpfutC, psod, lacY, hpfutC-lacY-Down and line-pK18mobsacB are subjected to seamless assembly by using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, vazyme, cat# C113-02), a reaction system and reaction conditions are all carried out by referring to the kit instruction, and after the seamless assembly is completed, a Trans 1T 1 competent cell is transformed to obtain a recombinant plasmid pK18mobsacB-HpfutC-lacY. The verified plasmid was electrotransformed into Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032, and the genes encoding HpfutC and LacY were introduced at the poxB locus of the ATCC 13032 genome of Corynebacterium glutamicum (Corynebacterium glutamicum) using kanamycin resistance followed by reverse screening of sacB genes. In order to provide the strain with sufficient fucosyltransferase capacity, the alpha-1, 2-fucosyltransferase HpfutC gene sequence was inserted in a similar manner into the c.glutamicum genome cg0554 site and the tnp2b site, respectively. on this basis, the same sacB gene reverse screening mode is adopted to replace the mannose phosphate mutase ManB promoter-10 region sequence "TAGGAT" with "TATAAT", and replace the mannose-1-guanylate phosphate transferase ManC promoter-10 region sequence "TAAAGT" with "TATAAT", so as to improve the supply of 2' -FL precursor GDP-L-fucose. Thus, a base strain HpfutC-LacY-ManB-ManC was obtained.

TABLE 1 construction of primers for recombinant strains HpfutC-LacY-ManB-ManC of Corynebacterium glutamicum

EXAMPLE 52 construction of the FL production Strain

RMD (PaRMD) from Pseudomonas aeruginosa (Pseudomonas aeruginosa) (SEQ ID NO:11, SEQ ID NO: 12) and RMD (AtRMD) from Bacillus stearothermophilus (Aneurinibacillus thermoaerophilus) (SEQ ID NO:13, SEQ ID NO: 14) have been shown to catalyze the asymmetric reduction of GDP-4-keto-6-deoxy-D-mannose. Furthermore, several publications have reported that carbonyl reductase enzymes catalyze asymmetric reduction of GDP-4-keto-6-deoxy D-mannose analogs (biochemistry.2022, 61:2138-2147;Angew Chem Int Ed Engl.2008,47 (51): 9814-59). This example examined whether these reductases are active on GDP-4-keto-6-deoxy-D-mannose, and specific reductase information is shown in Table 2. These reductases were subjected to gene synthesis with pUC57 as the vector and the recombinant plasmid was designated as 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.

TABLE 2 information on GDP-4-keto-6-deoxyD-mannose reductase to be screened

Wherein GerK, dnmV, urdZ3, lanZ3, mydl, urdR, tyID, chmD, ddahC, HS, 10A, HS, HS41B, HS53 have the gene sequences of SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40.

The promoters Pgap (SEQ ID NO: 53), pcg2195 and Psod (PCR system using 2X Phanta Max Master Mix, (Dye Plus), vazyme) were obtained by PCR amplification using the Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 genome as a template and the primer pairs Pgap-F-2/Pgap-R-2, pcg2195-F-2/Pcg2195-R-2, psod-F-2/Psod-R-2 of Table 3, respectively. The gene fragment encoding GDP-D-mannose-4, 6-dehydratase EcGMD (SEQ ID NO:41, whose gene sequence is SEQ ID NO: 42) was obtained by PCR amplification using the Escherichia coli (E.coli) MG1655 genome as a template and the primer pairs EcGMD-F and EcGMD-R shown in Table 3. PCR amplification was performed using pRSFDuet-OsGME as a template and the primer pairs OsGME-F and OsGME-R shown in Table 3 to obtain a gene fragment encoding GDP-D-rhamnose-3, 5-epimerase OsGME. PCR was performed using 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 a template and the corresponding primer set 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 shown in Table 3 to obtain gene fragments encoding PaRMD, atRMD, gerK, dnmV, urdZ3, lanZ, mydl, urdR, tyID, chmD, ddahC, HS10A, HS15, HS41B, HS53 (PCR system was 2X Phanta Max Master Mix, (Dye Plus), vazyme), and plasmid pJC1 was digested with restriction enzymes BamH I and Sal I to obtain linearized fragment line-pJC1. The fragments Pgap, pcg2195, psod, ecGMD, osGME, line-pJC1 and different reductase gene fragments are respectively subjected to seamless assembly by using recombinant cloning kits (ClonExpress Multis One Step Cloning Kit, vazyme, goods number: C113-02), a reaction system and reaction conditions are carried out by referring to kit specifications, and Trans 1T 1 competent cells are transformed after the seamless assembly is completed, so that recombinant plasmids are obtained:

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.

TABLE 32 construction of primers for construction of FL producer strains

The recombinant plasmids are sequentially and electrically transformed into a basic strain HpfutC-LacY-ManB-ManC to respectively obtain recombinant strains of corynebacterium glutamicum 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.

The correspondence between the strain and the transformed recombinant plasmid is shown in the following table:

Example 6 production of 2' -FL Using recombinant strains of Corynebacterium glutamicum containing different reductases

Recombinant strains Cg2FL-1 to Cg2FL-15 of Corynebacterium glutamicum were cultured and analyzed as in example 1. Among the 15 recombinant strains selected, the production of 2' -FL was detected in a plurality, with the highest yield of Cg2FL-11 reaching 78mg/L (Table 4). The corresponding reductase is reductase DdahC from Campylobacter jejuni. The resulting 2' -FL had a detectable molecular weight of 511[ M+Na ] ⁺ as determined by LC-MS analysis, and the retention time and molecular weight were consistent with the standards (FIG. 3). It was thus confirmed that the metabolic pathway contemplated in example 3 enabled synthesis of 2' -FL.

TABLE 4 production of 2' -FL by recombinant strains of Corynebacterium glutamicum containing different reductases

Strain	2' -FL yield (mg/L)
		Cg2FL-1	0.5
Cg2FL-4	12
		Cg2FL-11	78
Cg2FL-12	70
		Cg2FL-14	76

EXAMPLE 7 construction of recombinant Corynebacterium glutamicum control Strain without GDP-D-rhamnose-3, 5-epimerase

PCR was performed sequentially and separately using pUC57-1 through Puc57-15 of example 5 as a template and using the primer pairs of Table 5 to obtain gene fragments of different reductases (PCR system using 2X Phanta Max Master Mix, (Dye Plus), vazyme). The gene fragments of different reductases are respectively assembled with Pgap, pcg2195, ecGMD and line-pJC1 gene fragments obtained in example 5 by using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, vazyme, cat# C113-02), the reaction system and the reaction conditions are carried out by referring to the kit instruction, and the Trans 1T 1 competent cells are transformed after the seamless assembly is completed 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.

TABLE 5 primers used in control strain construction

The recombinant plasmids pJC1-Pgap_ EcGMD-Pcg2195_1 to pJC1-Pgap_ EcGMD-Pcg2195_15 were respectively electrically transformed into the base strains HpfutC-LacY-ManB-ManC obtained as described above, and control recombinant strains were obtained in sequence 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.

Recombinant strains Cg2FL-16 to Cg2FL-30 of Corynebacterium glutamicum were cultured and analyzed as in example 1. According to the result of the product analysis, cg2FL-29 produced 3.1 mg/L2' -FL, and reductase HS41B exhibited weak epimerization activity. In addition, none of the other control strains showed 2' -FL productivity, indicating that most of the active reductases are monofunctional proteins, including DdahC, with only reducing activity and not isomerising activity on GDP-4-keto-6-deoxy-D-mannose. At this time, GME is necessary for the production of 2' -FL.

Example 8 in vitro Multi-enzyme Cascade Generation of GDP-D-rhamnose

Further, the GDP-D-mannose is converted to GDP-rhamnose by an in vitro enzymatic cascade coupled EcGMD and DdahC. Using Escherichia coli (E.coli) MG1655 genome and pUC57-11 as templates, PCR amplification was performed using the primers shown in Table 6 to obtain EcGMD-1 gene fragment and DdahC gene fragment, respectively (PCR system was 2X Phanta Max Master Mix, (Dye Plus), vazyme), and plasmid pRSFDuet-1 was digested with restriction enzymes BamHI and SalI to obtain linearized fragment line-pRSFDuet. And (3) using a recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, vazyme, product number: C113-02), respectively performing seamless assembly on the EcGMD-1 gene fragment, the DdahC gene fragment and the line-pRSFDuet gene fragment, performing a reaction system and reaction conditions by referring to a kit instruction, and transforming the Trans 1T 1 competent cells after the seamless assembly is completed to obtain the recombinant plasmid pRSFDuet-EcGMD, pRSFDuet-DdahC. EcGMD pure enzyme and DdahC pure enzyme were obtained according to the procedure shown in example 2.

TABLE 6 EcGMD and primers for construction of DdahC protein expression plasmid

SEQ ID NO	Primer name	Primer sequences
			136	EcGMD-F-1	tcaccacagccaggatccaatgtcaaaagtcgctctcatc
137	EcGMD-R-1	atgcggccgcaagcttgtcgacttatgactccagcgcgat
			138	DdahC-F-3	caccacagccaggatccaatgatgcagaaggactctaa
139	DdahC-R-3	atgcggccgcaagcttgtcgacttactggcggatattctg

GDP-D-mannose was first converted to GDP-4-keto-6-deoxy-D-mannose by EcGMD under the specific reaction conditions of 50mM phosphate buffer (pH 7.5), 0.5g/L GDP-D-mannose, 0.5mM oxidized coenzyme II,5mM magnesium chloride, 1g/L EcGMD pure enzyme. The reaction was continued at 30 ℃ at 400rpm for 3 hours, after which time a certain amount of the reaction solution was sucked, and the reaction was quenched by adding 5 volumes of an organic solvent (acetonitrile: methanol=1:1). After removal of the proteins by centrifugation, the products were analyzed as described in example 1.

As shown in FIG. 4, GDP-D-mannose had completely disappeared after 3 hours of reaction, and a distinct new peak was generated. The new peak molecular weight was 586[ M-H ] ^-, estimated to be GDP-4-keto-6-deoxy-D-mannose, as determined by LC-MS detection analysis.

The reaction solution was subjected to ultrafiltration to remove protein EcGMD, and then 3mM reduced coenzyme II and 4g/L of DdahC pure enzyme were added to the filtrate, followed by reaction at 30℃and 400rpm for 3 hours. After the reaction was completed, the reaction was quenched by adding 5 volumes of an organic solvent (acetonitrile: methanol=1:1). After removal of the proteins by centrifugation, the products were analyzed as described in example 1.

As shown in FIG. 5, the GDP-4-keto-6-deoxy-D-mannose produced by EcGMD is fully converted to GDP-D-rhamnose by DdahC. The results of this in vitro experiment show that GDP-D-mannose can be converted to GDP-D-rhamnose by EcGMD and DdahC. In combination with the results obtained in the examples described above, it was found that 2' -FL was produced by an entirely new synthetic route, i.e., by the route shown in FIG. 1.

EXAMPLE 93 construction of fucosyllactose-producing Strain and production of 3-fucosyllactose

Referring to the genetic modification described in example 4, the gene of alpha-1, 3-fucosyltransferase HpfutA derived from helicobacter pylori (SEQ ID NO:44, the amino acid sequence of which is SEQ ID NO: 43) and the gene of lactose permease LacY derived from Escherichia coli (SEQ ID NO:10, the amino acid sequence of which is SEQ ID NO: 9) were integrated into the genome of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 to obtain a base strain HpfutA-LacY. With reference to the genetic engineering manner described in example 5, the GDP-D-mannose-4, 6-dehydratase EcGMD gene derived from E.coli (SEQ ID NO: 41), the GDP-mannose-3, 5-epimerase OsGME gene derived from rice (Oryza sativa) (SEQ ID NO: 5) and the GDP-4-keto-6-deoxyD-mannose reductase DdahC gene derived from Campylobacter jejuni (Campylobacter jejuni) (SEQ ID NO: 31) were integrated into the genome of the base strain HpfutA-LacY to construct the 3-fucosyllactose producing strain Cg3FL-1. The recombinant strain Cg3FL-1 of Corynebacterium glutamicum was cultured and analyzed as in example 1, and 45mg/L of 3-fucosyllactose was detected.

EXAMPLE 10 production of 2' -FL Using E.coli as host Strain

The culture medium comprises glycerol 40g/L, lactose 20g/L,Na₂HPO₄ 12.8g/L,K₂HPO₄ 3.0g/L,NH₄Cl 2.0g/L,NaCl 0.5g/L,MgSO₄·7H₂O 0.25g/L,CaCl₂·2H₂O 14.7mg/L, thiamine hydrochloride 10.0mg/L, triton 0.1% (v/v), trace metal solution 1.0mL/L(FeCl₃·6H₂O 25.0g/L,CaCl₂·2H₂O 2.3g/L,ZnCl₂ 2.6g/L,Na₂MoO₄·2H₂O 2.6g/L,CuSO₄·5H₂O2.0 g/L,MnSO₄·H₂O 2.5g/L, boric acid 0.7g/L, and pH 6.8.

Gene knockout and gene knockout-related plasmids were constructed using the techniques disclosed in SHENG YANG et al (acta. Biochim. Biophys. Sin.2021,53 (5): 620-627), and the desired knockdown and knockdown genes were as follows. The gene encoding beta-galactosidase LacZ (SEQ ID NO:46, the amino acid sequence of which is SEQ ID NO: 45) in the genome of Escherichia coli (E.coli) MG1655 was knocked out by using the related CRISPR-Cas9 technology (appl. Environ. Microbiol.2016,82 (12): 3693), the gene encoding alpha-1, 2-fucosyltransferase HpfutC (SEQ ID NO: 8) and lactose permease LacY gene (SEQ ID NO: 10) were integrated at the knocked-out site, the gene encoding GDP-L-fucose synthase WcaG (SEQ ID NO:47, the gene sequence of which is SEQ ID NO: 48) was knocked out by using the CRISPR-Cas9 technology, the gene encoding GDP-mannose-3, 5-epimerase OsGME was integrated at the knocked-out site, and the gene encoding GDP-5-glucose oxidase 35F-9 gene was produced by using the gene encoding GDP-L-fucose synthase 35F-45, the gene encoding GDP-5-glucose synthase gene 35F-45 was integrated at the knocked-site, and the gene encoding GDP-35F-45-glucose synthase gene was integrated at the knock-out site (SEQ ID NO: 35) was produced by using the CRISPR-Cas9 technology (SEQ ID NO: 10). When the obtained production strain Ec2FL-1 is fermented, colonies on a plate are picked into a seed culture medium for overnight culture. The overnight cultured bacterial liquid was inoculated into a 96-well plate containing the above medium at an inoculum size of 5%, and cultured at 37℃for 48 hours. An appropriate amount of the fermentation broth was aspirated and analyzed as described in example 1 to detect a yield of 48mg/L of 2' -FL.

Sequence:

SEQ ID NO. 1, atGME, amino acid sequence from Arabidopsis thaliana (Arabidopsis thaliana)

SEQ ID NO. 2, atGME, nucleic acid sequence from Arabidopsis thaliana (Arabidopsis thaliana)

SEQ ID NO. 3, mfGME, amino acid sequence derived from volcanic fumarole methyl acidophilus (Methylacidiphilum fumariolicum)

SEQ ID NO. 4, mfGME, nucleic acid sequence from volcanic fumarole methyl acidophilus (Methylacidiphilum fumariolicum)

SEQ ID NO. 5, osGME from rice (Oryza sativa), amino acid sequence

SEQ ID NO. 6, osGME from rice (Oryza sativa), nucleic acid sequence

SEQ ID NO. 7, alpha-1, 2-fucosyltransferase HpfutC from helicobacter pylori (Helicobacter pylori), amino acid sequence

SEQ ID NO. 8, alpha-1, 2-fucosyltransferase HpfutC from helicobacter pylori (Helicobacter pylori), nucleic acid sequence

SEQ ID NO. 9, lactose permease LacY from E.coli (ESCHERICHIA COLI), amino acid sequence

SEQ ID NO. 10, lactose permease LacY from E.coli (ESCHERICHIA COLI), nucleic acid sequence

SEQ ID NO. 11, RMD (PaRMD) derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa), amino acid sequence

SEQ ID NO. 12, RMD (PaRMD) derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa), nucleic acid sequence

SEQ ID NO. 13, RMD (AtRMD) derived from Pyrobaculum aerophilium (Aneurinibacillus thermoaerophilus), amino acid sequence

SEQ ID NO. 14, RMD (AtRMD) derived from Pyrobaculum aerophilium (Aneurinibacillus thermoaerophilus), nucleic acid sequence

SEQ ID NO. 15, gerK1 from Streptomyces sp KCTC 0041BP, amino acid sequence

SEQ ID NO. 16, gerK1 derived from Streptomyces sp KCTC 0041BP, nucleic acid sequence

SEQ ID NO. 17, dnmV, amino acid sequence from Streptomyces coelicolor (Streptomyces peucetius)

SEQ ID NO. 18, dnmV, nucleic acid sequence from Streptomyces coelicolor (Streptomyces peucetius)

SEQ ID NO. 19, urdZ, amino acid sequence derived from Streptomyces fradiae (Streptomyces fradiae)

SEQ ID NO. 20, urdZ, nucleic acid sequence from Streptomyces fradiae (Streptomyces fradiae)

SEQ ID NO. 21, lanZ, amino acid sequence derived from Streptomyces coelicolor (Streptomyces cyanogenus)

SEQ ID NO. 22, lanZ, nucleic acid sequence from Streptomyces coelicolor (Streptomyces cyanogenus)

SEQ ID NO. 23, mydl, amino acid sequence from Micromonospora gracilis (Micromonospora griseorubida)

SEQ ID NO. 24, mydl, nucleic acid sequence from Micromonospora gracilis (Micromonospora griseorubida)

SEQ ID NO. 25, urdR, amino acid sequence derived from Streptomyces fradiae (Streptomyces fradiae)

SEQ ID NO. 26, urdR, nucleic acid sequence from Streptomyces fradiae (Streptomyces fradiae)

SEQ ID NO. 27, tyID, amino acid sequence derived from Streptomyces fradiae (Streptomyces fradiae)

SEQ ID NO. 28, tyID, nucleic acid sequence from Streptomyces fradiae (Streptomyces fradiae)

SEQ ID NO. 29, chmD, amino acid sequence derived from Streptomyces bikini (Streptomyces bikiniensis)

SEQ ID NO. 30, chmD, nucleic acid sequence from Streptomyces bikini (Streptomyces bikiniensis)

SEQ ID NO. 31, ddahC, amino acid sequence from Campylobacter jejuni (Campylobacter jejuni)

SEQ ID NO. 32, ddahC, nucleic acid sequence from Campylobacter jejuni (Campylobacter jejuni)

SEQ ID NO. 33, HS10A, amino acid sequence from Campylobacter jejuni (Campylobacter jejuni)

SEQ ID NO. 34, HS10A, nucleic acid sequence from Campylobacter jejuni (Campylobacter jejuni)

SEQ ID NO. 35, HS15 from Campylobacter jejuni (Campylobacter jejuni), amino acid sequence

SEQ ID NO. 36, HS15 from Campylobacter jejuni (Campylobacter jejuni), nucleic acid sequence

SEQ ID NO. 37, HS41B, amino acid sequence from Campylobacter jejuni (Campylobacter jejuni)

SEQ ID NO. 38, HS41B, nucleic acid sequence from Campylobacter jejuni (Campylobacter jejuni)

SEQ ID NO. 39, HS53 from Campylobacter jejuni (Campylobacter jejuni), amino acid sequence

SEQ ID NO. 40, HS53 from Campylobacter jejuni (Campylobacter jejuni), nucleic acid sequence

SEQ ID NO. 41, GDP-D-mannose-4, 6-dehydratase EcGMD from E.coli (E.coli), amino acid sequence

SEQ ID NO. 42, GDP-D-mannose-4, 6-dehydratase EcGMD from E.coli (E.coli), nucleic acid sequence

SEQ ID NO. 43, alpha-1, 3-fucosyltransferase HpfutA from helicobacter pylori (Helicobacter pylori), amino acid sequence

SEQ ID NO. 44, alpha-1, 3-fucosyltransferase HpfutA from helicobacter pylori (Helicobacter pylori), nucleic acid sequence

SEQ ID NO. 45, beta-galactosidase LacZ from Escherichia coli (E.coli) MG1655, amino acid sequence

SEQ ID NO. 46, beta-galactosidase LacZ from E.coli (E.coli) MG1655, nucleic acid sequence

SEQ ID NO. 47, GDP-L-fucose synthase WcaG from E.coli (E.coli) MG1655, amino acid sequence

SEQ ID NO. 48, GDP-L-fucose synthase WcaG from E.coli (E.coli) MG1655, nucleic acid sequence

SEQ ID NO. 49, UDP-glucose lipid carrier transferase WcaJ from Escherichia coli (E.coli) MG1655, amino acid sequence

SEQ ID NO. 50, UDP-glucose lipid carrier transferase WcaJ from Escherichia coli (E.coli) MG1655, nucleic acid sequence

SEQ ID NO. 51, promoter Psod derived from Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032

SEQ ID NO. 52, the promoter Pcg2195 from Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032

SEQ ID NO. 53, promoter Pgap derived from Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032

The embodiments of the present invention are not limited to the examples described above, and those skilled in the art can make various changes and modifications in form and detail without departing from the spirit and scope of the present invention, which are considered to fall within the scope of the present invention.

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.

11. The host cell of 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 of 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 include 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).