RU2818835C2 - Fucosyltransferases and their use for obtaining fucosylated oligosaccharides - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of producing fucosylated oligosaccharide, including the following stages: a) providing at least one genetically modified cell that expresses a polypeptide capable of exhibiting β-1,3-N-acetylglucosaminyltransferase activity to transfer N-acetylglucosamine to lactose, expresses a polypeptide capable of exhibiting β-1,3-galactosyltransferase or β-1,4-galactosyltransferase activity leading to the formation of LNT or LNnT, and the said cell is genetically modified to express a heterologous fucosyltransferase, wherein the said heterologous fucosyltransferase is capable of transferring a fucose residue from a donor substrate to an acceptor molecule, wherein the said acceptor molecule is lactotetraose selected from LNT or LNnT; wherein the heterologous fucosyltransferase is selected from the group consisting of polypeptides represented by any of the following SEQ ID NO: 17–30, and functional polypeptide fragments represented by any of the following SEQ ID NO: 17–30; b) culturing the said at least one genetically modified cell in the presence of at least one carbon source and under conditions suitable for the at least one genetically modified cell to transfer a fucose residue from a donor substrate to an acceptor molecule; and c) optionally, isolating the said fucosylated oligosaccharide.

EFFECT: invention makes it possible to obtain fucosylated oligosaccharides with a high degree of efficiency.

9 cl, 8 dwg, 6 tbl, 6 ex

Description

The present invention relates to new fucosyltransferases and their use for the production of fucosylated oligosaccharides.

Prior Art

To date, approximately 200 structurally varying breast milk oligosaccharides (HMOs) have been identified. The basis of these HMOs is the disaccharide lactose, and they carry additional monosaccharide residues, mainly N-acetyl-glucosamine, fucose, sialic acid and galactose. The concentration and composition of HMO in breast milk varies among individuals and throughout the lactation period, from 20 g/L in colostrum to 5-10 g/L in mature milk.

The milk of women belonging to the so-called “secretory phenotype” has a high content of α-1,2-fucosylated HMOs. These women express the FUT2 gene, which encodes the so-called “fucosyltransferase 2”. The most common HMOs in their milk are 2'-fucosyllactose (2'-FL; Fuc(α1-2)Gal(β1-4)Glc) and lacto-N-fucopentaose-I (LNFP-I; Fuc(α1-2 )Gal(β1-3)GlcNAc(β1 -3)Gal(β1-4)Glc).

Breast milk oligosaccharides are not digested as they pass through the intestines of infants. Due to their constant presence in the baby's intestines, they have a beneficial effect on children. More specifically, HMOs have been shown to be prebiotics because they serve as a carbon source for symbiotic microorganisms of the genus Bifidobacterium, Bacteroides and Lactobacillus. Thus, CMOs support the proliferation of these microorganisms in the intestines of infants.

Breast milk oligosaccharides also directly reduce pathogen colonization of the infant's intestine because they prevent said pathogens from adhering to glycan structures on the surface of the intestinal mucosa. HMOs act as a decoy due to their structural similarity to epithelial tissue surface glycans and inhibit pathogen invasion, thereby reducing the risk of infection.

Alpha-1,2-fucosylated HMOs have been shown to protect against infections caused by Campylobacter jejuni, the causative agent of most common cases of bacterial diarrhea. α-1,2-Fucosylated HMOs are also associated with protection against heat-stable toxin-induced diarrhea in Escherichia coli. In addition, the risk of infection with diarrhea-mediated caliciviruses is reduced by the high content of α-1,2-fucosylated HMOs in breast milk. HMO, especially fucosylated HMO lacto-N-fucopentaose V (LNFP-V; Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]Glc, bind(s) with the carbohydrate-binding site of toxin A from Clostridium difficile, which is the most common cause of nosocomial diarrhea. Thus, HMOs appear to prevent C. difficile toxin A from interacting with cellular receptors. In addition, the adhesion of Pseudomonas aeruginosa to epithelial tissue cells was significantly inhibited by 2′-FL and 3-fucosyllactose (3-FL; Gal(β1-4)[Fuc(α1-3)]Glc). The binding of noroviruses (Norfolk-like viruses, NLV), which are the main cause of acute gastroenteritis, to blood group antigens is prevented by the action of α-1,2-fucosylated HMOs, as well as α-1,3-fucosylated HMOs. This indicates the ability of these HMOs to inhibit the binding of the norovirus capsid to host receptor glycans.

In view of the known beneficial properties of HMOs and in particular fucosylated HMOs, it is desirable to develop an economically viable method for their synthesis. Biotechnological methods for producing HMO using bacteria whose metabolism has been modified are described. Various fucosyltransferases have been described for the production of fucosylated oligosaccharides using genetically modified bacteria.

In the case of obtaining 2'-fucosyllactose (2'-FL), α-1,2-fucosyltransferases WbgL from E. coli O126 and FucT2 from Helicobacter pylori (EP 2 479 263 B1), α-1,2-fucosyltransferases WbIA from Vibrio are described cholerae O22, FutD from H. bilis ATCC (American Type Culture Collection) 437879, FutE from H, cinaede CCUG (University of Gothenburg Culture Collection) 18818, FutN from Bacteroides vulgatus ATCC 8482, FutO from Bacteroides ovatus ATCC 8483, WbgN from E. coli O55:H7, Bft1 and Bft3 from Bacteroides fragilis NCTC (National Type Culture Collection) 9343 (WO 2014/018596 A2) and α-1,2-fucosyltransferase FucT2 from H. pylori for the synthesis of saccharides of Lewis Y and Lewis B antigens (US 6670160 B2).

In the case of obtaining 3-fucosyllactose, α-1,3-fucosyltransferase Amuc from Akkermansia muciniphila and FucT6 and FucT7 from Bacteroides fragilis (EP 2 439 264 A1), α-1,3-fucosyltransferase FutA from H. pylori (US 2014/0120611 A1). In addition, WO 2016/040531 A1 describes α-1,3-fucosyltransferase CafC from B. nordii CL02T12C05 for the synthesis of 3-fucosyllactose and lactodifucotetraose and CafD from N. hepaticus ATCC 51449 for the production of LNnFP-III (lacto-N-neofucopentaose-III ).

However, it is known in the art that glycosyltransferases, including fucosyltransferases, can vary greatly in terms of kinetics, substrate specificity, affinity for donor substrates and acceptor molecules, stability and solubility. In addition, the choice of fucosyltransferase to mediate the desired fucosylation reaction significantly influences the final yield of the desired fucosylated oligosaccharide. For example, from WO 2014/018596 A1 it is clear that the bacterium E. coli producing WbgL synthesized 2'-FL and was also capable of synthesizing lactodifucotetraose (LDFT), while E. coli producing WbsJ from E. coli or WbIA from V. cholerae was able to stimulate the synthesis of 2'-FL, but did not synthesize LDFT.

In addition, the production of more complex fucosylated oligosaccharides, such as fucosylated tetrasaccharides, fucosylated pentasaccharides, fucosylated hexasaccharides or even fucosylated heptasaccharides, is only known on a small scale.

Because of these disadvantages, there is a need for additional fucosyltransferases that exhibit faster kinetics, higher affinity for nucleotide sugar donors, and/or diverse specificities for acceptor molecules. There is a particular need for fucosyltransferases that can be used in the industrial production of complex fucosylated human milk oligosaccharides, i.e. in fucosyltransferases that are capable of fucosylating tri-, tetra-, penta- or even hexasaccharides and/or have activity sufficient to produce commercially viable amounts of the desired fucosylated oligosaccharide.

In an attempt to resolve this problem, the inventors searched protein databases and nucleotide sequence databases for data relating to as yet unknown fucosyltransferases. Putative fucosyltransferases derived from hits identified through database searches were analyzed for fucosyltransferase activity in the corresponding polypeptides. Based on this approach, as yet unknown fucosyltransferases were identified that use lactotetraose as an acceptor molecule for fucosylation.

Brief description of the invention

New fucosyltransferases derived from bacterial cells have been proposed. To exhibit their fucosyltransferase activity, these fucosyltransferases use lactotetraose as an acceptor molecule. These new fucosyltransferases can be used to synthesize fucosylated oligosaccharides based on LNT (lacto-N-tetraose) and/or LNnT (lacto-N-neotetraose).

According to the first aspect, a method for producing fucosylated oligosaccharides is provided, wherein a genetically modified cell is used to produce said fucosylated oligosaccharide. Said genetically modified cell is genetically modified to express a heterologous fucosyltransferase that is capable of transferring a fucose residue from a donor substrate to an acceptor molecule, said acceptor molecule being lactotetraose.

According to a second aspect, a genetically modified cell is provided for use in a process for producing fucosylated oligosaccharides. Said genetically modified cell is genetically modified to express a heterologous fucosyltransferase that is capable of transferring a fucose residue from a donor substrate to an acceptor molecule, said acceptor molecule being lactotetraose.

According to a third aspect, a recombinant nucleic acid molecule is provided for the expression of a heterologous fucosyltransferase when accumulated in a cell, wherein said fucosyltransferase is capable of catalyzing the transfer of a fucose residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is lactotetraose.

According to a fourth aspect, fucosyltransferases are provided that are capable of transferring a fucose residue from a donor substrate to an acceptor molecule, said acceptor molecule being lactotetraose.

A fifth aspect provides the use of a fucosyltransferase capable of transferring a fucose residue from a donor substrate to an acceptor molecule, said acceptor molecule being lactotetraose, to produce fucosylated oligosaccharides.

According to the sixth aspect, a method is provided for the production of fucosylated oligosaccharides using in vitro biocatalysis, which uses a fucosyltransferase, wherein said fucosyltransferase is capable of transferring a fucose residue from a donor substrate to an acceptor molecule.

According to a seventh aspect, there are provided fucosylated oligosaccharides produced by the method of the first aspect or the method of the sixth aspect.

According to the eighth aspect, the use of fucosylated oligosaccharides according to the seventh aspect for the preparation of a food composition is proposed.

According to the ninth aspect, there is provided a food composition containing at least one fucosylated oligosaccharide according to the seventh aspect.

Brief description of graphic materials

In FIG. 1 shows a schematic representation of the plasmid map of the expression vector pINT-malE-fucT-zeo, which was used for heterologous expression of nucleotide sequences encoding putative fucosyltransferases in E. coli.

In FIG. 2 shows chromatograms of liquid chromatography/mass spectrometry (LC/MS) analyzes for type 1 (core structure: Gal(β1,3)GlcNAc) and type 2 fucosylated products (core structure: Gal(β1,4)GlcNAc) .

In FIG. Figure 2a shows chromatograms for fucosylated LNT and LNnT derivatives.

In FIG. 2b shows chromatograms for a mixture of LNFP-III and LNnFP-V, which were synthesized in in vitro reactions using cell extracts containing heterologously expressed fucosyltransferase from B. fragilis, i.e. FucT109 (top panel), compared with chromatograms for sugar standards.

In FIG. 3 shows a schematic representation of the metabolic pathways for the production of fucosylated oligosaccharides based on lacto-N-tetraose and lacto-N-neotetraose in E. coli.

In FIG. Figure 4 shows the results of thin layer chromatography (TLC) analyzes of culture supernatants of lacto-N-fucopentaose-producing E. coli strains containing pINT-malE-fucT109-zeo.

In FIG. Figure 5 shows a histogram that demonstrates the production of extracellular LNFP-I using E. coli (strain No. 993) after integration of different fucT genes into the chromosome.

In FIG. 6 is a bar graph that demonstrates the production of LNFP-I using E. coli (strain #1772) using glucose as the carbon source in a 1 L fermentation process.

In FIG. 7 is a graph illustrating the degradation of LNT-2 (lacto-N-triose-2) and lactose by hydrolases expressed in E. coli strain No. 1886.

In FIG. Figure 8 shows a thin layer chromatography result illustrating the time-dependent cleavage of LNT by β-1,3-galactosidase Bga42A.

Detailed description

According to a first aspect, a method for producing fucosylated oligosaccharides is provided, comprising the steps of:

a) providing at least one genetically modified cell that is genetically modified to express a heterologous fucosyltransferase, wherein said heterologous fucosyltransferase is capable of transferring a fucose residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is lactotetraose;

b) culturing at least one genetically modified cell in the presence of at least one carbon source and under conditions suitable for the at least one genetically modified cell to transfer a fucose residue from a donor substrate to an acceptor molecule; And

c) optionally releasing the fucosylated oligosaccharide.

In the method of the first aspect, a genetically modified cell is proposed. The term "genetically modified" as used herein refers to modification of the genetic basis of a cell using molecular biology techniques. Modification of the genetic basis of a cell may include the transfer of genes within and/or across species boundaries, insertion, deletion, substitution and/or modification of nucleotides, triplets, genes, open reading frames, promoters, enhancers, terminators and other nucleotide sequences that mediate and /or regulating gene expression. Modification of the genetic basis of a cell aims to create a genetically modified organism that has specific desired properties. Genetically modified cells may contain one or more genes that are not present in the native (non-genetically modified) form of the cell. Methods for introducing exogenous nucleic acid molecules and/or inserting exogenous nucleic acid molecules (recombinant, heterologous) into a structure carrying the hereditary information of a cell for the purpose of inserting, deleting or changing the nucleotide sequence of the structure carrying the genetic information of a cell are known to a person skilled in the art. Genetically modified cells may contain one or more genes that are present in the native form of the cell, and these genes are modified and reintroduced into the cell by artificial means. The term "genetically modified" also includes cells that contain a nucleic acid molecule that is endogenous to that cell and that is modified without removing the nucleic acid molecule from that cell. Such modifications include modifications made using gene replacement, site-specific mutations and related techniques.

A genetically modified cell is a prokaryotic cell or a eukaryotic cell. Suitable cells include yeast cells, bacteria, archaebacteria, fungal cells, insect cells, plant cells and animal cells, including mammalian cells (such as human cells and cell lines).

In a further and/or alternative embodiment, the prokaryotic cell is a bacterial cell, preferably selected from a genus selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, Bifidobacterium, sporolactobacillus spp., Micromonospora spp., Micrococcus spp., Rhodococcus spp. and Pseudomonas. Suitable bacterial species are Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, Bacillus circulans, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Citrobacter f reundii, Clostridium cellulolyticum, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Corynebacterium glutamicum, Enterococcus faecium, Enterococcus thermophiles, Escherichia coli, Erwinia herbicola (Pantoea agglomerans), Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, obacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus , Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, Lactococcus lactis, Pantoea citrea, Pectobacterium carotovorum, Proprionibacterium freudenreichii, Pseudomonas fluorescens, Pseudomonas aeruginosa, Streptococcus thermo philes and Xanthomonas campestris.

In a further and/or alternative embodiment, the eukaryotic cell is a yeast cell, an insect cell, a plant cell, or a mammalian cell. The yeast cell is preferably selected from the group consisting of Saccharomyces sp., in particular Saccharomyces cerevisiae, Saccharomycopsis sp., Pichia sp., in particular Pichia pastoris, Hansenula sp., Kluyveromyces sp., Yarrowia sp., Rhodotorula sp, and Schizosaccharomyces sp.

A genetically modified cell is genetically engineered to express a heterologous fucosyltransferase. The term "heterologous" as used herein refers to a nucleotide sequence, nucleic acid molecule or polypeptide that is foreign to a cell or organism, i.e. to a nucleotide sequence, nucleic acid molecule or polypeptide that does not occur naturally in a specified cell or organism. "Heterologous sequence" or "heterologous nucleic acid" or "heterologous polypeptide" as used herein means anything just listed that comes from a source foreign to that particular host cell (eg, another species) or, if comes from the same source, it is modified from its original form. Thus, the heterologous nucleic acid operably linked to a promoter is derived from a source different from that of the promoter or, if originated from the same source, is modified from its original form. A heterologous sequence can be stably introduced into the genome of a host microorganism cell by, for example, transfection, transformation, conjugation or transduction, thereby causing the host cell to be genetically modified. Methods may be used which will depend on the host cell and the sequence to be introduced. Various methods are known to those skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, ^2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Accordingly, a “heterologous polypeptide” is a polypeptide that does not naturally occur in a given cell, and a “heterologous fucosyltransferase” is a fucosyltransferase that does not naturally occur in a given cell.

The term "fucosyltransferase" as used herein refers to polypeptides that are capable of catalyzing the transfer of a fucose moiety from a donor substrate to an acceptor molecule. The donor substrate for transfer of the fucose residue to the acceptor molecule is usually guanosine diphosphate-L-fucose (GDP-L-fucose). Suitable acceptor molecules for fucose residues include oligosaccharides, glycopeptides, glycoproteins and glycolipids. Typically, the fucose residue is transferred, for example, to 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 saccharide moiety of a glycoprotein or glycolipid. It is also understood that the term "fucosyltransferase" as used herein includes functional variants of said novel fucosyltransferase, functional fragments of said fucosyltransferases and functional fragments of said functional variants. The term "functional" indicates that these variants and fragments are also capable of catalyzing the transfer of a fucose residue from a donor substrate to an acceptor molecule, i.e. they may have fucosyltransferase activity.

The term "functional fragment" as used herein refers to a polypeptide that is truncated relative to the native fucosyltransferase and wherein the fragment is capable of having the same fucosyltransferase activity as the native polypeptide from which the fragment is derived.

The term "functional variant" as used herein refers to a polypeptide that is capable of having the same fucosyltransferase activity as the naturally occurring polypeptide from which the derivative is derived, but which has an altered amino acid sequence compared to the naturally occurring polypeptide.

Heterologous fucosyltransferase is capable of transferring a fucose residue from a donor substrate to an acceptor molecule. The term "capable of anything" in relation to a heterologous fucosyltransferase refers to the fucosyltransferase activity of the heterologous fucosyltransferase and the fact that suitable reaction conditions are required for the heterologous fucosyltransferase to exhibit enzymatic activity. In the absence of suitable reaction conditions, the heterologous fucosyltransferase does not exhibit its enzymatic activity, but retains its enzymatic activity and exhibits its enzymatic activity when suitable reaction conditions are restored. Suitable reaction conditions include the presence of a suitable donor substrate, the presence of suitable acceptor molecules, the presence of important cofactors such as, for example, monovalent or divalent ions, a pH value in the appropriate range, a suitable temperature, and the like. It is not necessary to satisfy all optimal values of the factors influencing the enzymatic reaction catalyzed by the heterologous fucosyltransferase, but the reaction conditions must be such that the heterologous fucosyltransferase exhibits its enzymatic activity. Accordingly, the term “capable of anything” excludes any conditions under which the enzymatic activity of the heterologous fucosyltransferase is irreversibly impaired, and also excludes the heterologous fucosyltransferase being affected by any such conditions. On the contrary, the term "capable of something" means that the fucosyltransferase is enzymatically active, i.e. exhibits its fucosyltransferase activity if suitable reaction conditions are provided (when all the requirements necessary for the fucosyltransferase to exhibit its enzymatic activity are provided).

Fucosyltransferases can promote the formation of α-1,2-, α-1,3-, α-1,4-, or α-1,6-glycosidic bonds between the fucose residue and the saccharide moiety in the acceptor molecule. Accordingly, the term "alpha-1,2-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor molecule to form an alpha-1,2 linkage between the fucose residue and the saccharide residue of the acceptor molecule. The term "alpha-1,3-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor molecule to form an alpha-1,3 bond between the fucose residue and the saccharide residue of the acceptor molecule. The term "alpha-1,4-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor molecule to form an alpha-1,4 bond between the fucose residue and the saccharide residue of the acceptor molecule; and the term "alpha-1,6-fucosyltransferase" refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate to an acceptor molecule to form an alpha-1,6 bond between the fucose residue and the saccharide residue of the acceptor molecule.

The term “donor substrate,” as applied to the transfer of a fucose residue from a donor substrate to an acceptor molecule, refers to a molecule containing a fucose residue, which molecule is used by a heterologous fucosyltransferase as a source of fucose to be transferred to a particular acceptor molecule. Typically the donor substrate is GDP-fucose.

The term "acceptor molecule" as used herein refers to a molecule that receives a fucose residue from a donor substrate as a result of the enzymatic activity of a heterologous fucosyltransferase. As used herein, the term "acceptor molecule" more specifically refers to a molecule consisting of or containing a saccharide moiety. Unless otherwise specified, the term "scavenger molecule" as used herein refers to lactotetraose.

Heterologous fucosyltransferase is capable of transferring a fucose residue to lactotetraose as an acceptor molecule. The term "lactotetraose" as used herein refers to a tetrasaccharide, i.e. an oligosaccharide consisting of 4 monosaccharide residues, with the tetrasaccharide containing a lactose motif (Gal(β1,4)Glc) at its reducing end.

In one embodiment, lactotetraose is selected from the group consisting of lacto-N-tetraose (LNT;Gal(β1,3)GlcNAc(β1,3)Gal(β1,4)Glc) and lacto-N-neotetraose (LNnT;Gal( β1,3)GlcNAc(β1,4)Gal(β1,4)Glc). The enzymatic activity of the heterologous fucosyltransferase contributes to the production of fucosylated oligosaccharide, more specifically fucosylated lactotetraose, i.e. lactofucopentaose. Said lactofucopentaose is a pentasaccharide preferably selected from the group consisting of lacto-N-fucopentaose I (LNFP-I), lacto-N-neofucopentaose I (LNnFP-I), lacto-N-fucopentaose II (LNFP II), lacto-N -neofucopentaose III (LNnFP-III), lacto-N-fucopentaose V (LNFP-V) and lacto-N-neofucopentaose V (LNnFP-V).

Polypeptides that have been identified in the genome of various bacterial species that are capable of exhibiting fucosyltransferase activity to transfer a fucose residue from a donor substrate to lactotetraose are shown in Table 1.

Table 1. Fucosyltransferases capable of transferring a fucose residue from a donor substrate to lactotetraose. Amino acid (aa) sequences of the fucosyltransferases used in the Examples section, and nucleotide (nt) sequences encoding these amino acid sequences and used for the expression of fucosyltransferases in accordance with the section

Examples are provided in the last two columns of this table by indicating their corresponding SEQ ID NO.

Thus, in an additional and/or alternative embodiment, the heterologous fucosyltransferase is selected from the group consisting of polypeptides represented by any of SEQ ID NO: +-30, functional variant polypeptides represented by any of SEQ ID NO: 16-30, functional fragments of polypeptides, represented by any of SEQ ID NO: 16-30, and functional variants of functional fragments of polypeptides represented by any of SEQ ID NO: 16-30. Thus, the heterologous fucosyltransferase is selected from the group of polypeptides represented by any of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO : 30, functional polypeptide fragments represented by any of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, functional variant polypeptides represented by any of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, and functional options functional polypeptide fragments represented by any of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.

In an additional and/or alternative embodiment, the heterologous fucosyltransferase is encoded by a nucleic acid molecule containing

1) a nucleotide sequence, which is represented by any of SEQ ID NO: 1-15;

2) a nucleotide sequence having at least 80% sequence identity with one of the nucleotide sequences, which is represented by any of SEQ ID NO: 1-15, preferably over the entire length of the sequence;

3) a nucleotide sequence encoding a polypeptide having an amino acid sequence, which is represented by any of SEQ ID NO: 16-30;

4) a nucleotide sequence encoding a polypeptide having an amino acid sequence that has at least 80% identity with any of the amino acid sequences represented by any of SEQ ID NO: 16-30;

5) a nucleotide sequence encoding a functional fragment of any of the polypeptides corresponding to 3) and 4); or

6) in this case, the nucleic acid molecule hybridizes with the complementary chain of the nucleic acid molecule corresponding to 1), 2), 3), 4) or 5), under stringent conditions.

The expression "SEQ ID NO: 1-15" refers to the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

"Stringe hybridization" refers, for example, to: hybridization in 4 x SSC (sodium chloride/sodium citrate solution) at 65°C and subsequent repeated washes in 0.1 x SSC at 65°C for a total of approximately 1 hour. Less stringent hybridization conditions are, for example: hybridization in 4x SSC at 37°C and subsequent repeated washings in 1x SSC at room temperature. "Stringent hybridization conditions" may also mean: hybridization at 68°C in 0.25 M sodium phosphate, pH 7.2, with 7% (w/v) SDS (sodium dodecyl sulfate), 1 mM EDTA (ethylenediaminetetheracetic acid) and 1% (w/v) BSA (bovine serum albumin) for 16 hours followed by washing twice with 2xSSC and 0.1% (w/v) SDS at 68°C.

The nucleotide sequence encoding the heterologous fucosyltransferase may be a linear nucleic acid molecule or a circular nucleic acid molecule. Additionally and/or alternatively, the nucleotide sequence encoding the heterologous fucosyltransferase may be present as an extrachromosomal nucleic acid molecule or may be integrated into the chromosomal nucleic acid molecule(s) or at least one of the nucleic acid molecule(s). a chromosome of nucleic acid molecules, wherein said chromosomal nucleic acid molecule may be a linear or circular (bacterial chromosome) nucleic acid molecule.

The at least one genetically modified cell is cultured in the presence of at least one carbon source.

As used herein, the term “culturing” means growing a cell in a fermentation medium and under conditions that permit and are suitable for obtaining the desired fucosylated oligosaccharide(s). Several suitable fermentation media and cell culture conditions will be readily available to one skilled in the art after reading the description of this invention based on the technical and expert skill of the skilled person.

In a further and/or alternative embodiment, the at least one carbon source is selected from the group consisting of glycerol, sucrose, glucose, galactose, fructose, molasses, lactose, xylose, cellulose, pyruvate, succinate, synthetic carbon monoxide and any other carbon source, and energy, which can be metabolized by the genetically modified cell to produce the desired fucosylated oligosaccharide. In this context, it should be understood that any other, preferably inexpensive, substrates can be used as a carbon source for fermentation, and one skilled in the art will readily be able to use a carbon source suitable within the scope of the present invention to grow the microorganism for large-scale production of the desired monosaccharide. In a preferred embodiment of the production of fucosylated oligosaccharides, lactose is supplied to the fermentation medium, in particular if the genetically modified cell is not able to synthesize lactose itself. In a further and/or alternative embodiment of the production of fucosylated oligosaccharides, fucose is supplied to the fermentation medium, in particular if the genetically modified cell is not able to synthesize fucose itself. Supplying fucose to the fermentation medium can enhance the intracellular synthesis of GDP-fucose through the use of the fucose utilization pathway or fucose utilization system.

The at least one genetically modified cell is cultured under conditions that are suitable for the at least one genetically modified cell to undergo transfer of a fucose residue from a donor substrate to an acceptor molecule by the action of a heterologous fucosyltransferase. To produce a fucosylated oligosaccharide, at least one genetically modified cell is cultured in a fermentation medium that contains sufficient amounts of nutrients to ensure that at least one cell is metabolically active, thereby expressing a heterologous fucosyltransferase and thereby ensuring that the cell has sufficient amounts of donor material. substrate and acceptor molecules for the manifestation of the enzymatic activity of heterologous fucosyltransferase. To provide conditions suitable for at least one genetically modified cell to transfer a fucose residue from a donor substrate to an acceptor molecule using the activity of its heterologous fucosyltransferase, the fermentation medium must have, among other things, a suitable temperature, a suitable pH, a suitable the amount of oxygen dissolved in the fermentation medium, as well as the appropriate osmolarity. Suitable values may vary and depend on the type of cell being cultured. Suitable values can be readily determined by one skilled in the art.

In a further and/or alternative embodiment, culturing at least one genetically modified cell under conditions suitable for the at least one genetically modified cell to transfer a fucose moiety from a heterologous fucosyltransferase donor substrate to an acceptor molecule includes the step of supplying exogenous lactose to fermentation environment during the cultivation of at least one genetically modified cell. This allows at least one genetically modified cell to assimilate said exogenously supplied lactose for endogenous synthesis of lactotetraose. Said endogenously synthesized lactotetraose can then serve as an acceptor substrate for heterologous fucosyltransferase.

In a further and/or alternative embodiment, culturing the at least one genetically modified cell under conditions suitable for the at least one genetically modified cell to undergo transfer of a fucose moiety from a heterologous fucosyltransferase donor substrate to an acceptor molecule comprises the endogenous synthesis of lactose at least at least one genetically modified cell. In one embodiment, endogenous lactose synthesis may occur due to the natural ability of the genetically modified cell to synthesize lactose. Additionally and/or alternatively, endogenous lactose synthesis is mediated by an overexpressing heterologous β-1,4-galactosyltransferase in a genetically modified cell. Thus, the genetically modified cell is also genetically modified with the ability to overexpress, compared to the genetically unmodified progenitor cell, the gene for said heterologous β-1,4-galactosyltransferase. The gene for this heterologous β-1,4-galactosyltransferase encodes a β-1,4-galactosyltransferase that catalyzes the formation of lactose from galactose and glucose. Examples of suitable β-1,4-galactosyltransferases are selected from the group consisting of Pm 1141 from Pasteurella multocida (Accession No. AEC04686) and Lex1 from Aggregatibacter aphrophilus NJ8700 (Accession No. AK965832).

In a further and/or alternative embodiment, culturing the at least one genetically modified cell under conditions suitable for the at least one genetically modified cell to transfer a fucose moiety from a heterologous fucosyltransferase donor substrate to an acceptor molecule includes the step of supplying lacto-N -triose-2 (LNT-2) into the fermentation medium during the cultivation of at least one genetically modified cell, wherein at least one said genetically modified cell contains (1) β-1,3-galactosyltransferase or β-1,4 -galactosyltransferase and (2) α-1,2- and/or α-1,3-fucosyltransferase as a glycosyltransferase. LNT-2 is a trisaccharide that can be produced with reasonable efficiency. Supply of LNT-2 to the fermentation medium allows at least one genetically modified cell to utilize said exogenously supplied LNT-2 as a precursor for the endogenous synthesis of LNT or LNnT, which in turn can serve as acceptor molecules for heterologous fucosyltransferase.

According to a further and/or alternative embodiment of the method for producing fucosylated oligosaccharide, the method comprising the step of culturing at least one genetically modified cell is a continuous fermentation method or a batch fermentation method, preferably a fed-batch fermentation method.

Thus, according to an embodiment wherein the cultivation is a continuous fermentation method, that is, a method in which at least one carbon source is continuously added to the fermentation medium during the cultivation step of the genetically modified cell, and in which the medium is continuously removed during the fermentation process fermentation. As a result of the continuous addition of a carbon source during the cultivation step, continuous and efficient production of the oligosaccharide is achieved.

In an embodiment where the cultivation is a batch fermentation method, a closed culture system is used with a specific nutrient composition at the start of fermentation and specific temperatures, pressures, aeration conditions and other conditions to optimize growth. The fermentation process neither adds nutrients nor removes waste products during cell culture.

Fed-batch fermentation refers to a technique in which one or more than one nutrient (substrate) is introduced into the bioreactor during the culture process, and in which the product(s) remain in the bioreactor until the end of the cycle, or in which at least one batch The fermentation medium, including cells and product(s), is removed from the bioreactor during the fermentation process. Portions of the fermentation medium can be removed from the bioreactor several times and/or at different time intervals during the fermentation process.

In a further and/or alternative embodiment, a method for producing a fucosylated oligosaccharide comprises recovering the desired fucosylated oligosaccharide from a production cell culture. As used herein, the term “recovery” means isolating, collecting, purifying, selecting, or otherwise separating an oligosaccharide produced by a host microorganism of the invention from a culture of the host microorganism. The term "purification" as used herein refers to the removal of at least a substantial amount of impurities and unwanted compounds. These impurities and undesirable compounds (unwanted by-products) are cells, ions and salts, other saccharides other than the desired lactofucopentaose, for example, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, especially lactotetraose, and other pentasaccharides other than the desired lactofucopentaose.

In an additional and/or alternative embodiment, recovery and/or purification of the fucosylated oligosaccharide comprises a step selected from the group consisting of (1) crystallization of the fucosylated oligosaccharide from a solution of said fucosylated oligosaccharide and (2) spray drying of the fucosylated oligosaccharide. These steps provide the fucosylated oligosaccharide in crystalline or amorphous form.

In an additional and/or alternative embodiment, at least one glycosidase is used to extract or purify the desired fucosylated oligosaccharides, wherein the at least one glycosidase is used to break down interfering and/or undesirable impurities or by-products, unused starting substrates and intermediates formed in process of obtaining the desired oligosaccharide. By using at least one glycosidase, it is achieved that, for example, other (oligo)saccharides different from the desired fucosylated oligosaccharide can be metabolized, wherein other (oligo)saccharides are produced in at least one genetically modified cell or are produced in at least one at least one genetically modified cell during the synthesis of the desired fucosylated oligosaccharide, and other oligosaccharides interfere with the purification step of the desired oligosaccharide.

The at least one glycosidase can either be added externally to the fermentation medium at the end of the fermentation process or endogenously synthesized by the at least one genetically modified cell.

The addition of at least one glycosidase to the fermentation medium is advantageous if the genetically modified cell does not synthesize one or more glycosidases, for example, because the endogenous genes of the genetically modified cell encoding the one or more glycosidases are deleted, or because the expression of endogenous genes, encoding the specified one or more glycosidases is disrupted.

In one embodiment, where at least one glycosidase is added to the fermentation medium, the at least one glycosidase is produced by at least one other cell other than the genetically modified cell to produce a fucosylated oligosaccharide, and the at least one other said cell is further added into the fermentation medium to express the gene(s) encoding at least one glycosidase.

In this embodiment, the at least one glycosidase expressed by the at least one other cell is either a native glycosidase of said one other cell or a heterologous glycosidase, wherein said other cell is stably transfected with the ability to express the heterologous glycosidase, and wherein the heterologous glycosidase is expressed in this other cell is inducible. Preferably, the heterologous glycosidase is encoded by a nucleotide sequence that is stably integrated into the genome of at least one other cell.

This embodiment is particularly suitable in a continuous fermentation process for producing a fucosylated oligosaccharide, where, for example, two separate vessels or containers are provided for the fermentation, one vessel/container being used for carrying out the oligosaccharide synthesis reaction and the second vessel/container being primarily used for culturing cells that express heterologous glycosidase.

In a further and/or alternative embodiment, the at least one glycosidase expressed by at least one other cell is an intracellular glycosidase. Thus, at least one glycosidase expressed by at least one cell is located within the at least one cell. Thus, said at least one other cell captures unwanted impurities, by-products, unused starting substrates and/or intermediates formed during the production of the desired oligosaccharide, whereby the captured compounds are degraded by the action of at least one intracellular glycosidase.

In an alternative embodiment, the at least one glycosidase expressed by the at least one other cell is secreted by the at least one other cell into the fermentation medium. Undesired impurities, by-products, unused starting substrates and/or intermediates formed during the process of obtaining the desired oligosaccharide are then broken down in the fermentation medium. This embodiment is advantageous in that at least one other cell is unable to take up unwanted contaminants, by-products, unused starting substrates and/or intermediates generated during the production of the desired oligosaccharide.

In a preferred embodiment, the glycosidase catalyzes the breakdown of lactose. A suitable glycosidase for the breakdown of lactose is β-1,4-galactosidase LacZ from E. coli. A suitable glycosidase for hydrolysis of the LNT-2 intermediate is β-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum JCM1254. A suitable glycosidase for hydrolysis of the LNT intermediate is β-1,3-galactosidase Bga42A from Bifidobacterium longum subsp. infantis.

In a further and/or alternative embodiment, a genetically modified cell that also produces at least one glycosidase, or at least one other cell that produces at least one glycosidase, expresses at least one glycosidase as a result of external influence, for example, through induced by changing the expression temperature or through substrate-induced expression. This means that the expression of at least one glycosidase is suppressed during the synthesis of the desired fucosylated oligosaccharide and can be induced, for example, by changing temperature or by adding an inducer such as IPTG (isopropyl-β-D-thiogalactoside) at the end of the fermentation process . Expression of the glycosidase will be induced once a sufficient and/or substantially maximum amount of oligosaccharide has been produced during cultivation of the genetically modified cell. The expressed glycosidases will then degrade unwanted saccharide intermediates, substrates, etc., leaving the medium substantially free of intermediate saccharides or substrates that would otherwise interfere with or complicate the purification of the desired oligosaccharide. Several suitable agents for inducible expression are known in the art (see, eg, Sambrook et al., 1989, supra), and one skilled in the art will be able to employ an agent suitably suited for the desired oligosaccharide.

The term “regulated” in the context of the present invention in relation to a gene generally means a gene whose transcription can be regulated in a desired manner, for example, suppressed or activated, i.e. the amount of protein synthesized encoded by a regulated gene differs, for example as a result of attenuation/suppression or activation, from the case of an unregulated gene.

In a further embodiment, monosaccharides resulting from the breakdown of unwanted saccharide intermediates, substrates, etc., can be metabolized by the genetically modified cell.

According to the second aspect, a genetically modified cell is provided for the production of a fucosylated oligosaccharide or for use in a method for its production. Said genetically modified cell is genetically modified to express a heterologous fucosyltransferase that is capable of transferring a fucose residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is lactotetraose.

The term "genetically modified" as used herein in relation to a host cell indicates that the host cell replicates a heterologous or recombinant nucleic acid molecule and/or expresses a peptide or protein encoded by the heterologous nucleotide sequence (i.e., nucleotide sequence , "foreign to the specified cell"). Genetically modified cells may contain genes that are not found in the native (non-recombinant) form of the cell. Genetically modified cells may also contain genes found in the cell's native form, where such genes are modified and reintroduced into the cell by artificial means. The term also includes cells that contain a nucleic acid molecule endogenous to the cell that is modified without removing the nucleic acid from the cell; such modifications include modifications made using gene replacement, site-specific mutations and related techniques. Accordingly, a "recombinant polypeptide" is a polypeptide that is produced by a genetically modified cell.

Accordingly, by the expression “genetically modified cell” is meant a cell that has been transformed or transfected.

Thus, the nucleotide sequences used in the present invention may, for example, be contained in a vector with which cells of a host microorganism are to be stably transformed/transfected, or which are otherwise to be introduced into cells of a host microorganism.

Methods for creating "recombinant DNA", including isolation, synthesis, purification and amplification of genetic material used to transform or transfect selected host cells, are known to one skilled in the art. Thus, it is generally known how to transfect cells using “hybrid” viral or circular plasmid DNA that includes selected exogenous (ie, foreign or “heterologous”) nucleotide sequences. These techniques, known in the art, involve creating a transfection vector by enzymatic cleavage of circular viral or plasmid DNA to produce linear DNA chains. Selected foreign DNA strands, typically containing sequences encoding the desired protein product, are prepared in linear form using exactly the same/similar enzymes. Linear viral or plasmid DNA is incubated with foreign DNA in the presence of ligating enzymes capable of carrying out the reconstruction process and forming “hybrid” vectors containing a selected segment of exogenous DNA “spliced” into a viral or circular DNA plasma ide.

A genetically modified cell is a prokaryotic cell or a eukaryotic cell. Suitable cells include yeast cells, bacteria, archaebacteria, fungi, insect cells, plant cells and animal cells, including mammalian cells (such as human cells and cell lines).

In a further and/or alternative embodiment, the prokaryotic cell is a bacterial cell, preferably selected from a genus selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, Bifidobacterium, Sporolactobacillus spp., Micromonospora spp., Micrococcus spp., Rhodococcus spp. and Pseudomonas. Suitable bacterial species include Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, Bacillus circulans, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Citrobacter fre undii, Clostridium cellulolyficum, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Corynebacterium glutamicum, Enterococcus faecium, Enterococcus thermophiles, Escherichia coli, Erwinia herbicola (Pantoea agglomerans), Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, obacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus , Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, Lactococcus lactis, Pantoea citrea, Pectobacterium carotovorum, Proprionibacterium freudenreichii, Pseudomonas fluorescens, Pseudomonas aeruginosa, Streptococcus thermo philes and Xanthomonas campestris.

In a further and/or alternative embodiment, the eukaryotic cell is a yeast cell, an insect cell, a plant cell, or a mammalian cell. The yeast cell is preferably selected from the group consisting of Saccharomyces sp., in particular Saccharomyces cerevisiae, Saccharomycopsis sp., Pichia sp., in particular Pichia pastoris, Hansenula sp., Kluyveromyces sp., Yarrowia sp., Rhodotorula sp. and Schizosaccharomyces sp.

A genetically modified cell is genetically modified to express a heterologous fucosyltransferase capable of transferring a fucose residue from a donor substrate to an acceptor molecule, lactotetraose. In an additional and/or alternative embodiment, the heterologous fucosyltransferase is selected from the group consisting of polypeptides represented by any of SEQ ID NO: 16-30, functional variants of polypeptides represented by any of SEQ ID NO: 16-30, functional fragments of polypeptides represented by any of SEQ ID NO: 16-30, and functional variants of functional polypeptide fragments represented by any of SEQ ID NO: 16-30.

In an additional and/or alternative embodiment, the heterologous fucosyltransferase is encoded by a nucleic acid molecule containing

1) a nucleotide sequence, which is represented by any of SEQ ID NO: 1-15;

2) a nucleotide sequence having at least 80% sequence identity with one of the nucleotide sequences, which is represented by any of SEQ ID NO: 1-15, preferably over the entire length of the sequence;

3) a nucleotide sequence encoding a polypeptide having an amino acid sequence, which is represented by any of SEQ ID NO: 16-30;

4) a nucleotide sequence encoding a polypeptide having an amino acid sequence that has at least 80% identity with any of the amino acid sequences represented by any of SEQ ID NO: 16-30;

5) a nucleotide sequence encoding a functional fragment of any of the polypeptides corresponding to 3) and 4); or

6) in this case, the nucleic acid molecule hybridizes with the complementary chain of the nucleic acid molecule corresponding to 1), 2), 3), 4) or 5), under stringent conditions.

The nucleotide sequence encoding the heterologous fucosyltransferase may be present as a linear or circular extrachromosomal nucleic acid molecule in a genetically modified cell, or may be integrated into a chromosomal nucleic acid molecule of the cell, wherein said chromosomal nucleic acid molecule may be linear or circular ( bacterial chromosome) nucleic acid molecule.

The genetically modified cell is capable of synthesizing GDP-fucose, which is required for a reaction catalyzed by a heterologous polypeptide capable of exhibiting fucosyltransferase activity to transfer a fucose residue from a donor substrate to lactotetraose to produce the desired fucosylated oligosaccharide, since GDP-fucose serves as a donor substrate for the fucose residue , transferred to lactotetraose by the action of heterologous fucosyltransferase. Thus, in one embodiment, the genetically modified cell is also genetically modified to have the ability to produce increased intracellular GDP-fucose compared to the cell before it was genetically modified.

In an additional and/or alternative embodiment, the provision of an intracellular supply of GDP-fucose for the production of fucosylated oligosaccharides is achieved by the genetically modified cell also being genetically modified such that said cell expresses or overexpresses a gene encoding bifunctional fucokinase/L-fucose-1- phosphate guanyltransferase (Fkp), preferably a gene encoding a bifunctional fucokinase/L-fucose-1-phosphate-guanyltransferase (Fkp) from Bacteroides fragilis (Accession No. AY849806), which is capable of catalyzing the conversion of L-fucose to GDP-fucose. To obtain the desired fucosylated oligosaccharide, it is preferable that the cell fermentation process introduces L-fucose to the genetically modified cell.

In an additional and/or alternative embodiment, the GDP-fucose for the synthesis of the desired fucosylated oligosaccharide can be taken from the cells' own metabolism of GDP-fucose using a "de novo pathway". To increase the intracellular supply of GDP-fucose through the "de novo pathway", the genetically modified cell must also be genetically modified to express or overexpress, relative to the pre-genetically modified cell, at least one of the phosphomannomutase-encoding genes mannose-1-phosphate guanosyltransferase, GDP-mannose-4,6-dehydratase and GDP-L-fucose synthase. In a preferred embodiment, the cell is genetically modified to overexpress all four of these genes.

In a further and/or alternative embodiment, the genetically modified cell is also genetically modified to exhibit enhanced import of exogenous L-fucose across its cell membrane. Preferably, the genetically modified cell is also genetically modified to express or overexpress, relative to the progenitor cell before it was genetically modified, one nucleotide sequence selected from the group consisting of nucleotide sequences encoding the major facilitated transporter FucP from E . coli MG1655 (accession number AIZ90162), nucleotide sequences encoding functional variants of the main facilitated transporter FucP from £. co//, nucleotide sequences encoding functional fragments of the main facilitated transport transporter FucP from E. coli, and nucleotide sequences encoding functional variants of functional fragments of the main facilitated transport transporter FucP from E. coli. Expression or overexpression of the major facilitated transporter FucP, functional variants and/or functional fragments thereof in a genetically modified cell enhances the uptake of exogenous L-fucose by the cell across its cell membrane.

In a further and/or alternative embodiment, the genetically modified cell is also genetically modified to prevent depletion of GDP-fucose within the cell. In one embodiment, the cell is genetically modified such that expression of the gene encoding the enzyme WcaJ, which catalyzes the first step of colanic acid synthesis, is disrupted or inactivated, preferably such that the WcaJ gene is at least partially removed from the genetic information of the cell, or such that the nucleotide the sequence of the WcaJ gene is changed, as a result of which transcription of the gene encoding WcaJ is impossible. In a complementary and/or alternative approach, the nucleotide sequence of the gene encoding WcaJ has been altered such that an enzymatically inactive polypeptide is encoded by an altered WcaJ gene, for example, such that a stop codon is introduced into the open reading frame, resulting in the expression of a truncated variant of WcaJ representing is a non-functional fragment, or such that the nucleotide sequence of the WcaJ gene is altered such that the polypeptide encoded by said altered WcaJ gene differs from wild-type WcaJ at one or more amino acid residues, rendering the resulting polypeptide enzymatically inactive.

In a further and/or alternative embodiment, the genetically modified cell is also genetically modified such that the fucl and/or fucK genes encoding L-fucose isomerase and L-fuculose kinase, respectively, are deleted, the nucleotide sequence of the fucl and/or fucK genes is changed to irreversibly inactivate the enzymatic activity of the corresponding polypeptide(s), or such that the expression of the fucl and/or fucK genes is impaired. Abrogation of intracellular synthesis of Fucl and/or FucK eliminates fucose catabolism in the corresponding cell, thereby increasing the amount of fucose that will be available for the formation of GDP-fucose.

In a further embodiment, the genetically modified cell is also genetically modified such that the cell (1) does not express one or more polypeptides that cleave within the cell one or more precursors of the desired fucosylated oligosaccharide intended to be produced, or (2) expresses one or more polypeptides having an altered amino acid sequence and/or length, compared to its naturally occurring homolog, to adversely affect the activity of such an enzyme that cleaves within the cell one or more precursors of the desired fucosylated oligosaccharide intended to be produced.

The term “precursor” as used herein in relation to the desired fucosylated oligosaccharides refers to compounds that are intermediates in the biosynthetic pathway of the desired fucosylated oligosaccharide to be produced. These intermediates include endogenous compounds, e.g. compounds that are produced and may be naturally present in the host cell even when their synthesis in the host bacterial cell is enhanced by genetic modification of the host.

Moreover, in a further and/or alternative embodiment, the genetically modified cell is genetically modified so that it does not contain enzymatically active β-galactosidase.

Additionally, in a further and/or alternative embodiment, the genetically modified cell is genetically modified to lack a functional LacZ gene or to contain a functional LacZ gene that is tightly regulated for expression and that is not expressed during the fermentation process to produce a fucosylated oligosaccharide.

Additionally and/or alternatively, the genetically modified cell is also genetically modified such that the cell does not contain or express polypeptides having the enzymatic activity of hydrolyzing a precursor other than lactose to the desired fucosylated oligosaccharide, e.g., LNT-2 , LNT or LNnT, or larger derivatives of LNT and LNnT. To this end, a genetically modified cell is also genetically modified such that the genome of the cell does not contain a nucleotide sequence encoding a polypeptide capable of catalyzing the hydrolysis of another specified precursor of the desired fucosylated oligosaccharide, or so that the expression of genes encoding such proteins is regulated such that they are not expressed during fermentation to produce fucosylated oligosaccharide.

In a further and/or alternative embodiment, the genetically modified cell contains at least one nucleotide sequence encoding a polypeptide capable of exhibiting β-1,3-N-acetylglucosaminyltransferase activity. The genetically modified cell of this embodiment is capable of expressing a polypeptide capable of exhibiting β-1,3-N-acetylglucosaminyltransferase activity.

Preferably, the genetically modified cell expresses said polypeptide capable of exhibiting β-1,3-N-acetylglucosaminyltransferase activity. More preferably, said genetically modified cell contains a polypeptide capable of exhibiting β-1,3-N-acetylglucosaminyltransferase activity. Preferably, said at least one nucleotide sequence encoding a polypeptide capable of exhibiting β-1,3-N-acetylglucosaminyltransferase activity is a heterologous nucleic acid sequence, i.e. is a nucleotide sequence that does not naturally exist in the non-genetically modified precursor of a genetically modified cell. Due to the expression of a polypeptide capable of exhibiting β-1,3-N-acetylglucosaminyltransferase activity, N-acetylglucosamine can be added to the acceptor substrate lactose in the host cell when said polypeptide has its β-1,3-N-acetylglucosaminyltransferase activity, thereby cells is formed LNT-2.

In a further and/or alternative embodiment, the polypeptide capable of exhibiting β-1,3-N-acetylglucosaminyltransferase activity for the transfer of N-acetylglucosamine to lactose is β-1,3-N-acetylglucosaminyltransferase, which may be selected from the group consisting of LgtA from Neisseria meningitidis MC58 (accession no. NP_274923) and β-1,3-N-acetylglucosaminyltransferase from Pasteurella multocida subsp. Multocida, strain HN06 (accession no. PMCN06_0022).

In a further and/or alternative embodiment, the genetically modified cell contains at least one nucleotide sequence encoding a polypeptide capable of exhibiting β-1,3-galactosyltransferase activity or β-1,4-galactosyltransferase activity. The genetically modified cell of this embodiment may express a polypeptide capable of exhibiting β-1,3-galactosyltransferase activity or β-1,4-galactosyltransferase activity. Preferably, the genetically modified cell expresses said polypeptide capable of exhibiting β-1,3-galactosyltransferase activity or β-1,4-galactosyltransferase activity. More preferably, said genetically modified cell contains a polypeptide capable of exhibiting β-1,3-galactosyltransferase activity or β-1,4-galactosyltransferase activity. Preferably, said at least one nucleotide sequence encoding a polypeptide capable of exhibiting β-1,3-galactosyltransferase activity or β-1,4-galactosyltransferase activity is a heterologous nucleic acid sequence, i.e. is a nucleotide sequence that does not naturally exist in the non-genetically modified precursor of a genetically modified cell. By expressing a polypeptide capable of exhibiting β-1,3-galactosyltransferase activity or β-1,4-galactosyltransferase activity, galactosylation of LNT-2 can occur in a genetically modified cell to form LNT or LNnT, respectively, within the cell.

In a further and/or alternative embodiment, the polypeptide capable of exhibiting β-1,3-galactosyltransferase activity to galactosylate LNT-2 to produce LNT is a β-1,3-galactosyltransferase selected from the group consisting of β-1,3- galactosyltransferase WbdO, derived from Salmonella entehca (accession no. AY730594), and β-1,3-galactosyltransferase, encoded by a gene selected from the group consisting of wbgO from E. coli O55:H7 (accession no. BAG 11838), furA from Lutiella nitroferrum (FuraDRAFT_0419), and functional fragments of the indicated β-1,3-galactosyltransferases.

In an additional and/or alternative embodiment, the polypeptide capable of exhibiting β-1,4-galactosyltransferase activity to galactosylate LNT-2 to produce LNnT is a β-1,4-galactosyltransferase selected from the group consisting of LgtB from Neisseria meningitidis (No. accession AAF42257), Lex1 from Aggregatibarter aphrophilus NJ8700 (accession number YP_003008647), GaIT from Kingella denitriUcans ATCC 33394 (accession number HMPREF9098_2407), GatD from Pasteurella multocida M1404 (accession number GQ444331), GalT from Bacterioidis fragilis NCTC 9343 (Accession No. BF9343_0585), IsgD from Haemophilus influenza (accession no. AAA24981), GalT from Helicobacter pylori (accession no. AB035971) and functional fragments of the indicated p-1,4-galactosyltransferases.

Intracellular synthesis of LNT or LNnT or their larger derivatives in a genetically modified cell requires UDP-galactose (UDP stands for uridine diphosphate) and UDP-N-acetylglucosamine.

The presence of intracellular UDP-galactose in a genetically modified cell can be achieved by supplying galactose to the genetically modified cell, wherein the cells are cultured in a fermentation medium that contains galactose. Galactose is taken up by the cell, phosphorylated to galactose-1-phosphate and then converted to UDP-galactose. Genes encoding polypeptides carrying enzymatic activities that are necessary for these reactions are well known.

In a further and/or alternative embodiment, the intracellular supply of UDP-galactose can also be provided by enabling the cell's own metabolism, and the cell's own metabolic machinery can be improved by genetically modifying the cell such that, for example, the cell overexpresses UDP-galactose-4' -epimerase or overexpress UDP-galactose-4'-epimerase in combination with glucose-1-phosphate-1-uridinyltransferase.

The presence of intracellular UDP-N-acetylglucosamine in a genetically modified cell can also be ensured by turning on the cell's own metabolism to produce UDP-N-acetylglucosamine. To increase the intracellular supply of UDP-N-acetylglucosamine in a genetically modified cell, the cell may be genetically modified such that one or more of the genes encoding L-glutamine:D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, phosphoglucomutase, and N- acetylglucosamine 1-phosphate uridyltransferase/glucosamine 1-phosphate acetyltransferase are overexpressed.

In a further and/or alternative embodiment, the cell is genetically modified such that catabolism of N-acetylglucose mine in the genetically modified cell is eliminated. As a result of the elimination of cellular catabolism of N-acetylglucosamine, the intracellular level of UDP-N-acetylglucosamine, available for the intracellular synthesis of N-acetylglucosamine, increases.

In a further and/or alternative embodiment, a genetically modified cell for use in the synthesis of complex fucosylated HMOs is capable of incorporating lactose through its cell membrane to store lactose as a starting material to produce the desired fucosylated oligosaccharide. Because of this, the cell can express the endogenous gene encoding lactose permease. In a further and/or alternative embodiment, the cell is genetically modified to contain and express a heterologous lactose permease gene, particularly if the cell does not naturally contain or express a gene encoding lactose permease.

In an additional and/or alternative embodiment, the availability of lactose to produce the desired fucosylated oligosaccharide is provided through the intracellular synthesis of lactose by such a cell. Preferably, this is achieved by the cell expressing an endogenous or recombinant gene encoding a β1-4-galactosyltransferase, which β1-4-galactosyltransferase is capable of transferring the galactose moiety of UDP-galactose to a glucose molecule. Such β1-4-galactosyltransferase can be selected from Pm1141 from Pasteurella multocida (Accession No: AEC04686) or Lex1 from Aggregatibacter aphrophilus NJ8700 (Accession No: YP_003008647).

Thus, in a further and/or alternative embodiment, the genetically modified cell comprises

(1) β-1,3-N-acetylglucosaminyltransferase,

(2) β-1,3-galactosyltransferase or β-1,4-galactosyltransferase; And

(3) α-1,2- and/or α-1,3-fucosyltransferase.

In another embodiment, where a genetically modified cell is cultured to produce a fucosylated oligosaccharide and LNT-2 is added to the fermentation medium as a precursor scavenger molecule, the genetically modified cell contains

(1) β-1,3-galactosyltransferase or β-1,4-galactosyltransferase; And

(2) α-1,2- and/or α-1,3-fucosyltransferase as a glycosyltransferase.

According to a third aspect, recombinant nucleic acid molecules are provided for the expression of heterologous fucosyltransferase in a genetically modified cell. The term "nucleic acid molecule" refers to a single- or double-stranded deoxyribonucleotide unit macromolecule or ribonucleotide unit macromolecule and contains a twisted deoxyribonucleotide unit macromolecule or ribonucleotide unit macromolecule containing one or more known analogues of either naturally occurring or synthetically produced nucleotides .

The recombinant nucleic acid molecule contains a nucleotide sequence encoding a fucosyltransferase selected from the group consisting of polypeptides represented by any of SEQ ID NO: 16-30, functional variants of polypeptides represented by any of SEQ ID NO: 16-30, functional fragments of polypeptides represented by any of SEQ ID NO: 16-30, and functional variants of functional polypeptide fragments represented by any of SEQ ID NO: 16-30.

In an additional and/or alternative embodiment, the nucleotide sequence encoding the fucosyltransferase is selected from the group consisting of:

1) a nucleotide sequence represented by any of SEQ ID NO: 1-15;

2) a nucleotide sequence having sequence identity of at least 80% with one of the nucleotide sequences represented by any of SEQ ID NOs: 1-15, preferably over the entire length of the sequence;

3) a nucleotide sequence encoding a polypeptide having an amino acid sequence, which is represented by any of SEQ ID NO: 16-30;

4) a nucleotide sequence encoding a polypeptide having an amino acid sequence that has at least 80% identity with any of the amino acid sequences represented by any of SEQ ID NO: 16-30;

5) a nucleotide sequence encoding a functional fragment of any of the polypeptides corresponding to 3) and 4); And

6) a nucleic acid molecule that hybridizes with the complementary chain of a nucleic acid molecule corresponding to 1), 2), 3), 4) or 5), under stringent conditions.

Also included within the scope of the present invention under these terms are polymorphic variants, alleles, mutants and interspecies homologues of nucleic acids/polynucleotides and polypeptides having an amino acid sequence having greater than about 60%, 65%, 70%, 75% amino acid sequence identity. 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher amino acid sequence identity, preferably within a region of of at least about 25, 50, 100, 200, 500 or more amino acids, with a polypeptide similar to one of the amino acid sequences among SEQ ID NO: 16-30.

“Variant(s)” as used herein refers to a polynucleotide or polypeptide that is different from the reference polynucleotide or polypeptide, respectively, but retains the essential (enzymatic) properties of the reference polynucleotide or polypeptide. A typical polynucleotide variant differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Changes in the nucleotide sequence can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical polypeptide variant differs in amino acid sequence from another, reference polypeptide. As a rule, the set of differences is limited, so the sequences of the reference polypeptide and the variant are generally very similar and identical in many regions. The amino acid sequences of the variant and the reference polypeptide may differ by one or more substitutions, one or more additions, or deletions in any combination. The replaced or inserted amino acid residue may or may not be a residue encoded by the genetic code. A variant of a polynucleotide or polypeptide may exist in nature, such as an allelic variant, or it may be a variant not known to exist in nature. Variants of polynucleotides and polypeptides unknown in nature can be created by mutagenesis methods, direct synthesis methods and other methods using recombinant technology known to specialists in the art.

In a recombinant nucleic acid molecule, a nucleotide sequence encoding a fucosyltransferase, a functional variant thereof, a functional fragment of a fucosyltransferase, or a functional variant of a functional fragment is operably linked to at least one nucleotide sequence that mediates and/or regulates the expression of the fucosyltransferase, a variant or fragment thereof, provided that a given recombinant nucleic acid molecule is present in the cell.

The term "operably linked" as used herein refers to a functional relationship between a nucleic acid expression control sequence (such as a promoter, operator, enhancer, regulator, transcription factor binding site series, transcription terminator, ribosome binding site) and a second nucleotide sequence, wherein the expression control sequence influences the transcription and/or translation of a nucleic acid corresponding to the second nucleotide sequence. Accordingly, the term “promoter” refers to DNA sequences that typically “precede” a gene in a polymeric DNA molecule and provide the initiation site for transcription into messenger RNA (mRNA). "Regulatory" DNA sequences, typically also located "upstream" of a gene (ie, preceding the gene) in said polymeric DNA molecule, bind proteins that determine the frequency (or extent) of transcription initiation. Collectively referred to as a "promoter/regulator" or "control" DNA sequence, these sequences, which precede a selected gene (or series of genes) on a functional polymeric DNA molecule, act together to determine whether transcription (and possibly expression) of the gene will occur. . The DNA sequences that “follow” the gene in the DNA polymer molecule and provide the transcription termination signal in the mRNA are called transcription “terminator” sequences.

A wide variety of expression systems can be used to produce the polypeptides of the invention. Such systems include, among others, chromosomal, episomal, and viral vectors, such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and vectors. derived from combinations thereof, such as vectors derived from genetic elements of plasmids and bacteriophages, such as cosmids and phagemids. Expression system constructs may contain control regions that regulate as well as induce expression. In general, in this regard, any system or vector suitable for maintaining, increasing the number or expression of polynucleotides and for synthesizing the polypeptide in a host cell can be used for expression. The appropriate DNA sequence can be inserted into the expression system by any of a variety of well known and routine methods, such as those described in Sambrook et al., supra. Accordingly, a nucleic acid molecule suitable for expressing a heterologous fucosyltransferase in a genetically modified cell is selected from the group consisting of plasmids, phagemids, cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs).

According to a fourth aspect, fucosyltransferases are provided that are capable of transferring a fucose residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is lactotetraose.

Fucosyltransferases are of bacterial origin. Fucosyltransferases can be used to synthesize fucosylated oligosaccharides, preferably complex fucosylated lactose-based HMOs, LNT and/or LNnT or other oligosaccharides. The fucosylated oligosaccharide is lactofucopentaose.

Fucosyltransferases are capable of transferring a fucose residue from a donor substrate, preferably GDP-fucose, to an acceptor molecule. In a preferred embodiment, the acceptor molecule is lactotetraose, preferably LNT and/or LNnT.

In an additional and/or alternative embodiment, the fucosyltransferase is selected from the group consisting of polypeptides represented by any of SEQ ID NO: 16-30, functional variants of polypeptides represented by any of SEQ ID NO: 16-30, functional fragments of polypeptides represented by any of SEQ ID NO: 16-30, and functional variants of functional polypeptide fragments represented by any of SEQ ID NO: 16-30.

Fucosyltransferases, which are represented by any of the sequences of SEQ ID NO: 16-30, have not been described as fucosyltransferases capable of transferring a fucose residue from a donor substrate to lactotetraose as an acceptor substrate. New fucosyltransferases, which are represented by any of the sequences with SEQ ID NO: 16-27, have not previously even been described as fucosyltransferases themselves, i.e. despite their acceptor molecules.

Fucosyltransferases described earlier in this application can be used to produce complex fucosylated oligosaccharides either by whole cell fermentation, for example, as described earlier in this application, or by in vitro biocatalysis. Therefore, the fifth aspect provides the use of at least one of the fucosyltransferases capable of transferring a fucose residue from a donor substrate to an acceptor molecule, said acceptor molecule being lactotetraose, to produce a fucosylated oligosaccharide.

In an additional and/or alternative embodiment, the fucosyltransferases previously described herein are used to synthesize complex fucosylated HMOs by in vitro biocatalysis.

Use of at least one of the new fucosyltransferases in an in vitro biocatalysis method involves adding a suitable donor substrate containing a fucose moiety, preferably GDP-fucose, and a suitable acceptor molecule to at least one of the new fucosyltransferases in a solvent and under conditions suitable for transfer from the participation of fucosyltransferase of a fucose residue from a donor substrate to an acceptor molecule, resulting in the synthesis of a fucosylated oligosaccharide. Preferably, a suitable acceptor molecule is lactotetraose, more preferably LNT or LNnT. When lactotetraose is used as an acceptor molecule, the reaction product involving fucosyltransferase in the in vitro biocatalysis reaction is lactofucopentaose. Said lactofucopentaose is preferably one of the pentasaccharides selected from the group consisting of lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-neofucopentaose III, lacto-N-fucopentaose V and lacto-N-neofucopentaose V.

According to the sixth aspect, a method is provided for producing fucosylated oligosaccharides, preferably fucosylated human milk oligosaccharides, more preferably complex human milk oligosaccharides, most preferably lactofucopentaose, by in vitro biocatalysis, which uses a fucosyltransferase, wherein said fucosyltransferase is capable of transferring a fucose residue from a donor substrate to an acceptor substrate molecule.

In one embodiment of in vitro biocatalysis, the method includes the steps

a) providing a fucosyltransferase capable of transferring a fucose residue from a donor substrate to an acceptor molecule into the reaction mixture;

b) contacting said fucosyltransferase with a donor substrate containing a fucose residue and an acceptor molecule, said acceptor molecule being lactotetraose, to synthesize a fucosylated oligosaccharide.

This embodiment provides for the provision of fucosyltransferase into the reaction mixture. The term "fucosyltransferase" as used herein also includes functional variants, functional fragments of said fucosyltransferase and functional variant fucosyltransferase fragments, wherein "functional" means that said variants and fragments may have fucosyltransferase activity as previously described herein .

The method of this embodiment further comprises reacting the components of the mixture under conditions suitable to effect the transfer of a fucose moiety from a donor substrate, preferably GDP-fucose, to an acceptor moiety of an acceptor molecule, wherein said acceptor molecule is preferably lactotetraose.

In a further embodiment, the in vitro biocatalysis method further comprises subsequent purification and/or subsequent isolation of the fucosylated acceptor molecule from the reaction mixture.

The seventh aspect provides fucosylated oligosaccharides produced by whole cell fermentation or in vitro biocatalysis as previously described herein.

In one embodiment, the fucosylated oligosaccharide is a human milk oligosaccharide. The individual oligosaccharides in human milk are 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, 3-fucosyl-3'-sialyllactose, 3-fucosyl-6'-sialyllactose, lacto-N-fucopentaose I, lacto-N- neofucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose I, lacto-N-neofucopentaose V, lacto-N-difucosylhexaose I, lacto-N-difucosylhexaose II, fucosyl-lacto-N-sialylpentaose b, fucosyl-lacto -N-sialylpentaose c, lacto-N-neodifucohexaose I, disialyl-lacto-N-fucopentaose V and fucosyl-p-lacto-N-hexaose IV. The structures of individual human milk oligosaccharides are shown in Table 2.

In one embodiment, the fucosylated oligosaccharide, which can be produced using a fucosyltransferase described earlier in this application, is selected from the group consisting of pentasaccharides, preferably selected from the group consisting of lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-neofucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V.

Although the lactofucopentaoses mentioned in the previous paragraph are products of a direct reaction of the enzymatic action of fucosyltransferase described earlier in this application, these lactofucopentaoses can be subjected to further processing. For example, these lactofucopentaoses can be proposed as acceptor molecules for other glycosyltransferases, so the resulting hexaoses, for example, hexaoses from Table 2, and heptaoses can also be considered as fucosylated oligosaccharides, which can be obtained using the fucosyltransferases described in this application. In addition, it can be envisaged that fucosyltransferases can also be used in the preparation of trisaccharides and/or tetrasaccharides, such as those listed in Table 2.

According to the eighth aspect, the use of a fucosylated oligosaccharide obtained by whole cell fermentation or in vitro biocatalysis as previously described in this application for the preparation of a food composition is proposed. Said food composition contains at least one fucosylated oligosaccharide obtained by the method previously described in this application.

Thus, according to the ninth aspect, there is provided a food composition containing at least one fucosylated oligosaccharide obtained by the method previously described in this application. At least one fucosylated oligosaccharide is lactofucopentaose. The at least one lactofucopentaose in the food composition is selected from the group consisting of lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-neofucopentaose III, lacto-N-fucopentaose V and lacto-N-neofucopentaose V.

In a further embodiment, the nutritional composition is selected from the group consisting of medicinal compositions, compositions for infants and nutritional supplements.

The food composition may be in liquid form or in solid form, including, but not limited to, powders, granules, flakes and pellets.

The present invention will be described in relation to specific embodiments and with reference to drawings, however, the present invention is not limited to them, but only to the claims. In addition, the terms “first,” “second,” and the like in the specification and claims are used to distinguish like elements and not necessarily to describe a sequence either in time, space, ranking, or any other manner of description. It should be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described in this application may operate in sequences other than those set forth or illustrated herein.

It should be noted that the term "comprising" used in the claims should not be interpreted as a term limited to the following means; it does not exclude other elements or stages. Thus, it should be interpreted as a term specifying the presence of specified features, integers, stages or components that are mentioned, but not excluding the presence or addition of one or more other features, integers, stages or components or groups thereof. Thus, the scope of the expression "device comprising means A and B" should not be limited to devices consisting only of components A and B. For the purposes of the present invention, this means that the only relevant components of a given device are A and B.

Reference throughout this description to “one of the embodiments” or “any embodiment” means that at least one embodiment of the present invention includes a particular feature, a particular structure, or a particular characteristic property described in conjunction with that embodiment. Thus, occurrences at various places throughout this specification of the phrases “in one of the embodiments” or “in an embodiment” do not necessarily refer to, but may all refer to, the same embodiment. Moreover, specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as will be apparent to one of ordinary skill in the art from this description.

Likewise, it will be apparent that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, drawing, or description thereof for the purpose of streamlining the description and aiding in the understanding of one or more than one of the various aspects of the invention. This manner of description, however, should not be interpreted as reflecting the intention that the claimed invention requires more features than those expressly stated in each claim. Rather, as reflected by the following claims, aspects of the invention are not embodied in all of the features of a single embodiment described above. Thus, the claims set forth after the detailed description are hereby expressly incorporated into the detailed description, with each claim standing on its own as a separate embodiment of the invention.

In addition, although some embodiments described herein include some but not other features included in other embodiments, combinations of features of various embodiments are intended to be within the scope of the invention and form different embodiments, as will be appreciated by those skilled in the art. field of technology. For example, in the following claims, any of the claimed embodiments can be used in any combination.

In addition, some of the embodiments are described herein as a method or combination of elements of a method that can be implemented using a computer system processor or other means of performing a function. Thus, a processor with the necessary instructions for executing such method or method element constitutes means for implementing the method or method element. In addition, the device embodiment element described herein is an example of a means for performing a function performed by the element for the purpose of carrying out the invention.

Numerous specific details are set forth in the specification and drawings provided herein. However, it will be appreciated that embodiments of the invention may be practiced without these specific details. In other cases, well-known methods, structures and methods are not shown in detail so as not to obscure the understanding of this invention.

The present invention will now be described by way of detailed description of several embodiments of the invention. It will be appreciated that other embodiments of the invention may be made in accordance with the knowledge of those skilled in the art without departing from the true spirit or technical doctrine of the invention, and the invention is limited only by the terms of the appended claims.

Example 1: Identification of fucosyltransferases that fucosylate lacto-N-tetraose and lacto-N-neotetraose

The GenBank database was searched for putative fucosyltransferases encoded in the genome of various bacterial species. This approach identified one hundred and twenty-five putative fucosyltransferases that had not previously been annotated as fucosyltransferases.

Genes encoding putative fucosyltransferases (fucT) were codon optimized for expression in E. coli and were purchased from GenScript Cooperation (Piscataway, USA). The fucosyltransferase genes were subcloned through sequence-independent ligase-free cloning (SLIC) into the pINT-malE-zeo vector downstream of the malE gene, which encodes maltose-binding protein (MBP) and allows the synthesis of an MBP fusion protein (Fig. 1), using primers 2700 and 2702 for amplification of fucT genes and primers 2701 and 2703 for amplification of pINT-malE-zeo (the oligonucleotide primers used are shown in Table 3).

fucT genes were expressed in E. coli ER 2508 (New England Biolabs, Ipswich, USA). The genes encoding the alpha-1,2-fucosyltransferases wbgL from E. coli O126 and fucT2Hp from Helicobacter pylori were also codon optimized for expression in E. coli and were also purchased from the GenScript Cooperation and cloned using the sites for Ncol and BamH1 in pACYC vector (Novagen, Merck, Darmstadt, Germany) under the transcriptional control of the T7 promoter. WbgL and fucT2Hp were amplified using oligonucleotides 141 and 142 and 143 and 144, respectively. The vector, as well as PCR (polymerase chain reaction) amplification products, were digested using Ncol and BamH1 restriction endonucleases and ligated. E. coli transformants were selected on chloramphenicol. WbgL and fucT2HP were expressed in E. coli BL21(DE3)ΔlacZ (bacterial strains used in this work are listed in Table 4).

E. coli strain ER 2508, containing the pINT-malE-fucT-zeo plasmids, was grown in 2YT (2X tryptone yeast extract) medium (Sambrook et al., 1989, supra) containing ampicillin (100 μg/ml) and zeocin (40 μg/ml) to an optical density at 600 nm (OD _{600 nm} ) of 0.3, after which expression of the malE-fucT genes was induced by adding anhydrotetracycline (200 μg/ml). E. coli strain BL21(DE3) ΔlacZ, carrying pACYC-wbgL or pACYC-fucT2Hp, was grown in 2YT nutrient medium containing chloramphenicol (34 μg/ml). After reaching an OD _{600 nm} of 0.3, expression was induced with 0.3 mM IPTG. After 16 h of incubation at 30°C, cells were collected by centrifugation.

Cells were opened mechanically using glass beads. Cell-free extracts of expressing clones containing the same concentration of protein were incubated for 22 h at 37°C in 100 μl of reagent mixture for fucosyltransferase activity assays. These assay reagent mixtures contained 5 mM GDP-L-fucose, 5 mM LNT or LNnT, and 1 mM ATP in 20 mM Tris/HCI, pH 7.4, with 200 mM NaCI.

The formation of fucosylated LNT or LNnT was determined by thin layer chromatography (TLC) using TLC silica gel plates (Silica Gel 60 F ₂₅₄ (Merck KGaA, Darmstadt, Germany)). A mixture of butanol:acetone:acetic acid: _H2O (35/35/7/23 (v/v/v/v)) was used as the mobile phase. To detect the separated substances, TLC plates were impregnated with a thymol-based reagent (0.5 g of thymol dissolved in 95 ml of ethanol with the addition of 5 ml of sulfuric acid) and heated.

In addition, product formation was monitored and its identification was performed using mass spectrometry. Mass spectrometry analysis was performed using multiple reaction monitoring (MRM) mode using a liquid chromatography-triple-quadrupole MS (LC-MS 8050 from Shimadzu) detection system (Shimadzu Corporation, Kyoto , Japan). Quadrupole 1 (Q1) selects and analyzes parent ions, fragmentation occurs in a collision cell using argon as a collision initiating dissociation gas (CID gas), and selects fragmented ions in quadrupole 3 (Q3). Table 5 shows selected transitions and collision energies (CE) for intermediates and final product metabolites. Chromatographic separation of lactose, LNT-II, LNT and LNFP variants after diluting the culture supernatant, particle-free biocatalytic reaction mixture or crude extract, respectively 1:50 or 1:100 in H ₂ O (LC/MS grade), performed on an XBridge Amide HPLC column (3.5 μm; 2.1 × 50 mm (Waters, USA)). Prior to LC/MS analyses, samples were prepared using filtration (0.22 μm pore size) and purification by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex). The HPLC system consists of a Nexera X2 SIL-30AC _MP autosampler from Shimadzu, operating at 8°C, an LC-20AD pump from Shimadzu, and a column thermostat STO-20AC from Shimadzu, which operated at 35°C (Shimadzu Corporation, Kyoto, Japan) . The mobile phase consisted of acetonitrile:H ₂ O with 0.1% (v/v) ammonium hydroxide. A 1 μL sample was injected into the instrument; chromatographic separation was carried out for 5.00 min at a flow rate of 300 μL/min. All metabolites were analyzed using MRM in negative electrospray ionization (ESI) mode. A unit resolution mass spectrometer was used for this work. The collision energy and the specified bias (pre bias) for Q1 and Q3 were optimized individually for each analyte. Quantification methods were performed using commercially available standards (Carbosynth, Compton, UK and Elicityl, Crolles, France).

LNFP variants based on fucosylation of the lacto-N-tetraose (LNT) intermediate can be identified using chromatographic separation (2.4 min for LNFP-I and 2.5 min for LNFP-V). The slightly different retention times are due to differences in the type of LNT fucosylation (alpha-1,2-fucosylation of galactose in the case of LNFP-I; alpha-1,4-fucosylation of N-acetylglucosamine in the case of LNFP-II and alpha-1,3-fucosylation of glucose in case of LNFP-V). In addition, they can be identified based on their corresponding transition pattern (see Table 5), based on specific fragmentation patterns associated with the position of the fucosylation site.

LNnFP variants based on differences in fucosylation position (alpha-1,2-fucosylation of galactose in the case of LNnFP-I, alpha-1,3-fucosylation of N-acetylglucosamine in the case of LNnFP-III and glucose in the case of LNnFP-V) of the intermediate lacto- N-neotetraoses (LNnTs) can be identified using chromatographic separation (Figure 2a). In addition, they can be identified based on their corresponding transition pattern (see Table 5), based on specific fragmentation patterns associated with the position of the fucosylation site.

LNnFP-III and LNnFP-V have only a slight difference in retention time of 0.1 min. In vitro enzymatic reaction analysis using the α-1,3-fucosyltransferase FucT109 demonstrated the presence of a mixture of LNnFP-III and LNnFP-V (see Figure 2b).

Table 6 summarizes the fucosyltransferases tested to produce fucopentaoses and the biocatalytic reaction products detected when LNT and LNnT were used as glycan substrates, respectively.

Example 2 Development of a Lacto-N-Triose II Producing E. coli Strain Escherichia coli BL21(DE3) was used to construct a lacto-N-triose II producing strain (LNT-2). Metabolic pathway engineering involved mutagenesis and deletion of certain genes, respectively, and integration of heterologous genes into the genome. The lacZ and araA genes were inactivated by mutagenesis using mismatched oligonucleotides.

Deletion of genome regions was carried out in accordance with the method of Datsenko and Warner. To prevent intracellular degradation of N-acetylglucosamine, the genes encoding N-acetylglucosamine 6-phosphate deacetylase (nagA) and glucosamine 6-phosphate deaminase (nagB) were deleted from the genome of E. coli strain BL21 (DE3). The wzxC-wcaJ genes were also deleted. The wcaJ gene encodes UDP-glucose:undecaprenylphosphate-glucose-1-phosphate transferase, which catalyzes the first step in the synthesis of colanic acid. In addition, the fucl and fucK genes encoding L-fucose isomerase and L-fuculose kinase, respectively, were deleted.

Integration of heterologous genes into the genome was performed through transposition. For transposition, either the EZ-Tn5™ transposase (Epicenter, USA) was used to integrate linear DNA fragments, or the highly active C9 mutant form of the mariner Himar1 transposase. To generate EZ-Tn5 transposomes, the gene of interest along with an antibiotic resistance marker flanked by FRT sites was amplified using primers 1119 and 1120; the resulting PCR product had mosaic ends at both ends consisting of 19 base pairs (bp) - recognition sites for the EZ-Tn5 transposase. For integration using the Himarl transposase, expression constructs of interest (operons) were similarly cloned along with an antibiotic resistance marker flanked by FRT sites into the pEcomar vector. The pEcomar vector encodes a highly active C9 mutant form of the mariner Himarl transposase under the control of the arabinose inducible promoter P _araB . The expressed fragment <P _tet- IacY-FRT-aaaW-FRT> (SEQ ID NO: 31) was integrated using the EZ-Tn5 transposase. After successful integration of the lactose importer gene LacY from E. coli K12 TG1 (accession no. ABN72583), the resistance gene was removed from streptomycin-resistant clones by the FLP recombinase (flippase) encoded by the plasmid. The strain resulting from these modifications was named strain #534. The N-acetylglucosamine glycosyltransferase IgtA gene from Neisseria meningitidis MC58 (accession no. NP_274923) was codon optimized for expression in E. coli and obtained by gene synthesis. Together with the galT gene, encoding galactose-1-phosphate uridylyltransferase from E. coli K-12 substr. MG1655 (Accession No. NP_415279), which was obtained in a similar manner by gene synthesis, was inserted by transposition of the IgtA gene (SEQ ID NO: 32) using the plasmid pEcomar-IgtA-gaIT. To enhance the de novo synthesis of UDP-N-acetylglucosamine, genes encoding L-glutamine:D-fructose-6-phosphate aminotransferase (glmS), phosphoglucosamine mutase from E. coli K-12 substr. MG1655 (glmM) and N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (glmU) from E. coli K12 substr. MG1655 (accession nos. NP_418185, NP_417643, NP_418186, respectively) were codon optimized and obtained by gene synthesis. The glmUM operon was cloned under the control of the constitutive tetracycline promoter P _tet , whereas glmS was cloned under the control of the constitutive P _T5 promoter. The transposon cassette <P _tet- g/mUM-P _T5 -glmS-FRT-dhfr-FRT> (SEQ ID NO: 33; dhfr means dihydrofolate reductase), flanked by inverted terminal repeats specifically recognized by the mariner-like element of the Himarl transposase, was inserted from pEcomar -glmUM-glmS, producing a lacto-N-triose 2-producing strain (No. 724). E. coli strain BL21(DE3) was used as the initial host strain to develop the E. coli production strain.

Example 3: Construction of an E. coli strain to screen fucosyltransferases in vivo to produce fucosylated LNT

The 1,3-galactosyltransferase gene wbdO from Salmonella salamae (accession no. AAV34525) was codon optimized for expression in E. coli and synthesized by GenScript Cooperation. The galE gene was amplified based on genomic DNA from E. coli K12. Both genes were inserted as a transposon <P _tet- wbdOcoP _T5 galE-FRT-cat-FRT> (SEQ ID NO: 34) into strain No. 724 via transposition using the plasmid pEcomar-wbdO-galE. The resulting strain is strain No. 753. To enhance the supply of GDP-fucose, bifunctional fucokinase/L-fucose-1-phosphate-guanyltranseferase (fkp) from Bacteroides fragilis (Accession No. AY849806), which catalyzes the conversion of L-fucose to GDP-fucose, was overexpressed in E. coli strain BL21 (DE3) . Fusion of the fkp gene (which was originally amplified based on genomic DNA from Bacteroides fragilis (ATCC 25285D)), together with the upstream P _tet promoter, to the gentamicin resistance gene flanked by lox sites was accomplished using PCR coupling with overlapping primers (SOE-PCR) and primers 1119 and 1120; the resulting transposon EZ-Tn5 <P _tet -fkp-lox-aacC1-lox> (SEQ ID NO: 35) was integrated into the E. coli strain BL21(DE3) using the EZ-Tn5™ transposase, obtaining strain No. 993. Alternatively, to perform de novo synthesis of GDP-L-fucose, the genes encoding phosphomannomutase (tapB), mannose-1-phosphate-guanosyltransferase (tapS), GDP-mannose-4,6-dehydratase (gmd) and GDP-L-fucose -synthase (wcaG) from E. coli K12 DH5α, overexpressed in E. coli strain BL21(DE3); the manCB operon was placed under the control of the constitutive P _tet promoter, the gmd, wcaG operon was transcribed, also starting from the constitutive P _T5 promoter. The transposon cassette <P _tet- manCB-P _T5 -gmd, wcaG-FRT-aacC1-FRT> (SEQ ID NO: 36), including the aacC1 gene, which confers resistance to gentamicin to the bacterial host, was flanked by inverted terminal repeats specifically recognized by mariner -like element of the Himarl transposase. The cassette was inserted into the genome of strain No. 753 from the plasmid pEcomar C9-manCB-gmd, wcaG-aacC1, obtaining strain No. 1445.

Example 4: Construction of an E. coli strain to screen fucosyltransferases in vivo to produce fucosylated LNnT

The β-1,4-galactosyltransferase IgtB gene from Neisseria meningitidis (accession no. AAF42257) was optimized for expression in E. coli and synthesized by GenScript Cooperation. Together with the tetracycline resistance gene flanked by FRT sites, it was inserted as a transposon <P _T5 -lgtB-FRT-tetA-FRT> (SEQ ID NO: 37) into strain No. 724 via transposition using the pEcomar-lgtB plasmid. The galE gene was amplified based on genomic DNA from E. coli K12 and fused by SOE-PCR to the P _tet promoter and the chloramphenicol resistance gene. Using primers that form 19 bp. mosaic ends are recognition sites for the EZ-Tn5 transposase (primers 2194 and 2235), an EZ transposon <P _tet -galE-FRT-cat-FRT> was constructed, intended for integration into the E. coli strain BL21(DE3), obtaining strain no. 1046. Alternatively, the β-1,4-galactosyltransferase 1ex1 gene from Aggregatibacter aphrophilus NJ8700 (accession number YP_003008647), obtained by synthesis and codon optimized for E. coli, was integrated into strain No. 724. The Iex1 gene was fused with the malE gene encoding maltose-binding protein (MBP), obtaining an N-terminal fusion construct of MBP with Lex1. The malE-lex1 fusion construct was co-integrated with galE (SEQ ID NO: 38) under the transcriptional control of the P _tet promoter via transposition using pEcomar-malE-lex1-galE-cat. Using the EZ fragment EZ-Tn5 <P _tet- fkp-FRT-aacC1-FRT> (see example 3), the gene encoding fucosokinase/L-fucose-1-phosphate-guanyltransferase (fkp) from Bacteroides fragilis was integrated into the chromosome strain No. 1046, obtaining strain No. 1076.

Example 5 In Vivo Screening of Fucosyltransferases Fucosylating LNT and LNnT

The medium with mineral salts used for cultivating strains for the synthesis of fucopentaoses contained: NH ₄ H ₂ PO ₄ - 7 g/l, K ₂ HPO ₄ - 7 g/l, KOH - 2 g/l, citric acid - 0. 3 g/l, MgSO ₄ × 7H ₂ O - 2 g/l and CaCl ₂ × 6H ₂ O - 0.015 g/l, together with a solution of trace elements at the rate of 1 ml/l (containing ammonium citrate - 54.4 g/ l, MnCl ₂ × 4H ₂ O - 9.8 g/l, CoCl ₂ × 6H ₂ O - 1.6 g/l, CuCl ₂ × 2H ₂ O - 1 g/l, H ₃ BO ₃ - 1.9 g/l, ZnSO ₄ × 7H ₂ O - 9 g/l, Na ₂ MoO ₄ × 2H ₂ O -1.1 g/l, Na ₂ SeO ₃ - 1.5 g/l, NiSO ₄ × 6H ₂ O - 1.5 g/l).

To produce fucosylated pentasaccharides in vivo using strains #993 or #1445 for LNT and #1076 for LNnT, pINT-malE-fucT-zeo plasmids were used as FucT substrates, respectively. Plasmid-containing strains were grown in 20 ml of mineral salts (MS) medium with 2% glucose as a carbon source, anhydrotetracycline (200 ng/ml) as an inducer of gene expression, and the antibiotics ampicillin (100 μg/ml) and zeocin (20 μg/ml). ml). Bacteria were cultured at 30°C in partitioned shake flasks to an OD _{600 nm} of 0.3, after which 3 mM lactose and 2 mM L-fucose were added for derivatization using strains #993 and #1076 and 3 mM lactose for obtaining derivatives using strain No. 1445.

After culture for 24 h to 72 h, cells were collected by centrifugation, washed once in saline (0.9% (wt/vol) NaCl), resuspended in saline in a volume of 150 μl to 200 μl (in depending on the size of the sediment after centrifugation) and opened mechanically using glass beads. After extraction of cellular debris, a clear supernatant was obtained. Detection of the formation of fucosylated LNT was carried out by analyzing intracellular metabolites by TLC; the results are summarized in Table 6. In order to analyze the formation of LNFP-V by FucT109 from Bacteroides fragilis NTCT 9343, which is secreted into the culture supernatant, intact cells were centrifuged and the supernatant was analyzed using TLC (Fig. 4). In FIG. Figure 4 shows the result of TLC analysis of culture supernatants of lacto-N-fucopentaose-producing E. coli strains containing pINT-malE-fucT109-zeo. Detection of LNFP-V in the supernatant after cultivation of strains expressing the product of the malE-fucT109 fusion gene is carried out by comparing the migration rate with that of a purified sugar standard. Sugar standards: line 1: lactose; line 2: LNT-2; line 3: LNT+LNFP-V; line 4: LNT. Line 5: supernatant sample after cultivation of strain No. 993, line 6: supernatant sample after cultivation of strain No. 993 with plNT-malE-fucT109-zeo, line 7: supernatant sample after cultivation of strain No. 1445, line 8: supernatant sample after cultivation of strain No. 1445 with plNT-malE-fucT109-zeo.

Example 6. Synthesis of LNFP-I by enzymatic method Integration of fucT genes into derivatives of E. coli strain BL21(DE3)

Using strain No. 993 as a host, the genes encoding fucosyltransferases FucT41 (Gramella forsetii KT0803, accession no. WP_011708479), FucT 48 (Francisella philomiragia ssp.philomiragia ATCC 25015, accession no. EET21243.1), FucT49 (Pseudogulbenkiania ferrooxidans, no. accession EEG10438.1), FucT54 (Sideroxydans lithotrophicus ES-11, accession no. ADE13114.1), FucT61 (Pseudoalteromonas haloplanktis ANT/505, accession no. EGI74693.1), FucT66 (Roseovarius nubinhibens ISM, accession no. EAP78457.1) and FucT69 (Thalassospira profundimaris WP0211, accession no. EKF09232.1). The malE-fucT fusion gene, together with its upstream P _tet promoter and zeocin resistance gene, was amplified using primers 1119 and 1120 using plasmids pINT-malE-fucT-zeo as templates; the EZ-Tn5 transposon cassette <P _tet -malE-fucT-zeo> was inserted into the E. coli strain BL21(DE3) using the EZ-Tn5™ transposase

Strains carrying the integrated malE-fucT element were grown in 96-well plates in 200 μl of MS medium containing 2% glucose and zeocin (20 μg/ml) at 30°C with shaking. After 24 h of cultivation, 50 μl of cultures were transferred into 400 μl of fresh MS medium with 2% glucose, zeocin (20 μg/ml), 3 mM lactose, and 2 mM L-fucose. After 48 h of culture, culture supernatants were analyzed by LC/MS. The results for different FucT genes are shown in Fig. 5, with the error values showing the standard deviation for five separate cultures of the same strain. The fucT61-expressing strain (no. 1197) achieved the highest LNFP-I titer compared to strains carrying other integrated elements (Fig. 5).

Construction of a fucose import pathway to produce LNFP-I

Enhanced LNFP-I production by strain 1197 was achieved through integration of the major facilitated transporter FucP from E. coli MG1655 (accession no. AIZ90162). The FucP gene, along with the upstream P _tet promoter and streptomycin resistance gene, was amplified using primers 1119 and 1120 (<P _tet -fucP-FRT-aad1-FRT>, SEQ ID NO: 39) and integrated into the chromosome via transposition using transposase EZ Тn5™. The strain containing fucT61 and fucP was named strain No. 1772.

When strains No. 1197 and No. 1772 were grown in 96-well plates in MS medium with 2% glucose as a carbon source in the presence of 3 mM lactose and 2 mM L-fucose, the LNFP-I concentration recorded in the supernatant of strain No. 1772 turned out to be two times more than that found in the supernatant of strain No. 1197.

Preparation of LNFP-I by fermentation

Pilot fermentation of strain No. 1 772 was carried out at 30°C in a 3-liter fermenter containing 1 liter of MS medium. 2% glucose and NH ₄ Cl (2.5 g/l) were added to this medium before inoculation; fermentation was carried out without the addition of antibiotics. The pH value was kept constant at 7.0 by titration with 10% ammonia solution. Cultures from shake flasks were sown into the fermenter when the OD value reached _{600 nm} . equal to 0.1. At an OD _{600 nm} of approximately 10, 1 mM lactose and 1 mM L-fucose were added; Feeding with these two substrates in doses of 1-2 mM was carried out repeatedly in accordance with the growth and production rates. When the 2% portion of glucose was exhausted, glucose was supplied for replenishment continuously from a 50% concentrated glucose solution. After 87 h of cultivation, the OD _{600 nm} value in the culture mixture reached 185. Using LC/MS analysis, it was found that the concentration of LNFP-I in the culture supernatant was 4.3 g/L. Lactose, LNT-2, and LNT were detected as sugar by-products in the supernatant (Figure 6). In the one shown in FIG. Figure 6 shows the production of LNFP-I in E. coli (strain No. 1772) during fermentation in a volume of 1 liter using glucose as a carbon source. Product concentrations were measured in the culture supernatant after fermentation for 87 hours. In addition to LNFP-I (4.3 g/L), lactose (13.11 g/L), LNT-2 (4.6 g/L) and LNT (1.1 g/l).

By-product breakdown

To facilitate separation of the desired LNFP-I product from the by-products lactose, LNT-2 and LNT, these sugars can be enzymatically digested and the resulting digestion products metabolized by E. coli strains.

β-1,4-Galactosidases, such as LacZ from E. coli, efficiently break down lactose into D-glucose and D-galactose. These two monosaccharides are metabolized by E. coli strains that express a functional gal operon.

β-N-Acetylhexosaminidase Bbhl from Bifidobacterium bifidum JCM1254 catalyzes the hydrolysis of LNT-2 to N-acetylglucosamine and lactose with high specificity and efficiency.

β-1,3-Galactosidase Bga42A from Bifidobacterium longum subsp. infantis specifically catalyzes the hydrolysis of LNT to galactose and LNT-2.

Engineering an E. coli strain to break down by-products

Escherichia coli strain BL21(DE3) #534 was used to construct a strain with cleavage ability to extract LNFP-I from the supernatant after enzymatic production without sugar by-products.

The functional galETKM operon together with its natural promoter was amplified from genomic DNA from E. coli K12 using oligonucleotides 6473 and 6474 to produce a fragment that contains 19 bp. mosaic 5' ends are recognition sites for the EZ-Tn5 transposase. The <galETKM> fragment (SEQ ID NO: 40) was integrated into the genome of the E. coli strain using the EZ-Tn5™ transposase. Clones with correctly integrated elements were selected on MacConkey agar (Difco, Sparks, USA) containing 1% galactose and appeared as red colonies after incubation for 36 h at 37°C.

The gene encoding the β-N-acetylhexosaminidase Bbhl from Bifidobacterium bifidum JCM1254 was synthesized and codon optimized for expression in E. coli. The Bbhl gene under the transcriptional control of the P _tet promoter and the gene conferring host resistance to zeocin (<P _tet -bbhl-zeo>(SEQ ID NO: 41)) were integrated from the plasmid pEcomar-bbhl-zeo using the Himarl transposase.

To ensure full β-1,4-galactosidase activity, lacZ from E. coli BL21(DE3) (accession no. AM946981) was cloned under the transcriptional control of the constitutive P _tet promoter. Together with the gentamicin resistance gene aacC1, the fragment <P _tet -lacZ-FRT-aacC1-FRT> (SEQ ID NO: 42) was amplified using primers 1119 and 1120 and integrated by transposition using EZ Tn5, resulting in strain #1369.

The β-1,3-galactosidase Bga42A genes from Bifidobacterium longum subsp.infantis were codon optimized for E. coli and produced by synthesis. The Bga42A gene under the transcriptional control of the P _tet promoter and the cat gene conferring resistance to chloramphenicol in the host (<P _tet -bga42A-cat> (SEQ ID NO: 43)) were integrated using the EZTn5 transposase. The strain expressing all three hydrolase genes is designated strain No. 1886.

Efficient breakdown of lactose and LNT-2 was achieved by adding a culture of strain #1886 to an LNFP-I-producing culture of strain #1772. To demonstrate the degradation of LNT-2 and lactose by LacZ and Bbhl, a culture of strain #1886 grown in MS medium with glucose as a carbon source was added at a volume ratio of 1:40 to a culture of strain #1772 grown in MS medium with glucose in the presence of lactose and L-fucose to obtain LNFP-I.

In FIG. Figure 7 shows the breakdown of lactose and LNT-2 by hydrolases expressed in E. coli strain No. 1886. A growing culture of E. coli strain No. 1886 was added to a culture of E. coli strain No. 1772, which produces LNFP-I as a major product and LNT and LNT-2 as by-products. After 10 h of incubation, almost no LNT-2 and lactose remained in the culture supernatant. Metabolites are labeled as follows: circles: lactose, squares: LNT-2, triangles: LNFP-I, crosses: LNT. Cleavage of LNT-2 and lactose in 10 hours passed almost to their complete disappearance, while LNFP-I and LNT were not cleaved by cells of strain No. 1886. Since strain No. 1886 contains an active galETKM operon, the monosaccharides glucose and galactose resulting from the hydrolysis of lactose are completely metabolized.

Bga42A hydrolase does not show activity outside cells. However, when cells of strain #1886 grown on MS medium with glucose as the carbon source were mechanically dissected, hydrolytic activity of Bga42A on LNT was demonstrated, as shown in FIG. 8, which shows the result of thin layer chromatography. Samples containing cell lysate from E. coli strain No. 1886 (line 1: no enzyme, lines 2-6: 100 μg protein) and 5 mM LNT in 20 mM sodium phosphate buffer, pH 7, were incubated for a period of time up to 120 minutes inclusive (line 1: 120 min, line 2: 0 min, line 3: 15 min, line 4: 30 min, line 5: 60 min, line 6: 120 min) at 30°C and then the enzyme was inactivated by heat at 95°C for 5 minutes.

In FIG. Figure 8 shows the hydrolysis of LNT and the resulting breakdown products of LNT-2 and lactose in a cell-free extract of strain #1886.

Claims (26)

1. A method for producing fucosylated oligosaccharide, including the following steps: a) providing at least one genetically modified cell that expresses a polypeptide capable of exhibiting β-1,3-N-acetylglucosaminyltransferase activity to transfer N-acetylglucosamine to lactose expresses a polypeptide capable of exhibiting β-1,3-galactosyltransferase or β- 1,4-galactosyltransferase activity leading to the formation of LNT or LN n T, and said cell is genetically modified to express a heterologous fucosyltransferase, wherein said heterologous fucosyltransferase is capable of transferring a fucose residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is lactotetraose, selected from LNT or LN n T; wherein the heterologous fucosyltransferase is selected from the group consisting of polypeptides represented by any of SEQ ID NO: 17-30, and functional fragments of polypeptides represented by any of SEQ ID NO: 17-30; b) culturing said at least one genetically modified cell in the presence of at least one carbon source and under conditions suitable for the at least one genetically modified cell to transfer a fucose residue from a donor substrate to an acceptor molecule; And c) optionally, isolating said fucosylated oligosaccharide. 2. The method according to claim 1, where the heterologous fucosyltransferase is encoded by a nucleic acid molecule containing 1) a nucleotide sequence, which is represented by any of SEQ ID NO: 2-15; 2) a nucleotide sequence having at least 80% sequence identity with one of the nucleotide sequences, which is represented by any of SEQ ID NO: 2-15, preferably over the entire length of the sequence; 3) a nucleotide sequence encoding a polypeptide having an amino acid sequence, which is represented by any of SEQ ID NO: 17-30; 4) a nucleotide sequence encoding a polypeptide having an amino acid sequence that has at least 80% identity with any of the amino acid sequences represented by any of SEQ ID NO: 17-30; 5) a nucleotide sequence encoding a functional fragment of any of the polypeptides in accordance with 3) and 4); or 6) where the nucleic acid molecule hybridizes with the complementary chain of the nucleic acid molecule in accordance with 1), 2), 3), 4) or 5) under stringent conditions. 3. A genetically modified cell to produce a fucosylated oligosaccharide, wherein said cell expresses a polypeptide capable of exhibiting β-1,3-N-acetylglucosaminyltransferase activity to transfer N-acetylglucosamine to lactose, expresses a polypeptide capable of exhibiting β-1,3-galactosyltransferase or β -1,4-galactosyltransferase activity resulting in the formation of LNT or LN n T, and said cell is genetically modified to express a heterologous fucosyltransferase, wherein said heterologous fucosyltransferase is capable of transferring a fucose residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is lactotetraose wherein said heterologous fucosyltransferase is selected from the group consisting of polypeptides represented by any of SEQ ID NO: 17-30, and functional fragments of polypeptides represented by any of SEQ ID NO: 17-30. 4. A genetically modified cell according to claim 3, where the heterologous fucosyltransferase is encoded by a nucleic acid molecule containing 1) a nucleotide sequence, which is represented by any of SEQ ID NO: 2-15; 2) a nucleotide sequence having at least 80% sequence identity with one of the nucleotide sequences represented by any of SEQ ID NOs: 2-15, preferably throughout the entire length of the sequence; 3) a nucleotide sequence encoding a polypeptide having an amino acid sequence, which is represented by any of SEQ ID NO: 17-30; 4) a nucleotide sequence encoding a polypeptide having an amino acid sequence that has at least 80% identity with any of the amino acid sequences represented by any of SEQ ID NO: 17-30; 5) a nucleotide sequence encoding a functional fragment of any of the polypeptides in accordance with 3) and 4); or 6) where the nucleic acid molecule hybridizes with the complementary chain of the nucleic acid molecule in accordance with 1), 2), 3), 4) or 5) under stringent conditions. 5. Genetically modified cell according to claim 3 or 4, where the cell is genetically modified with the possibility of having an increased ability to intracellular production of GDP-fucose compared to a given cell before it is subjected to genetic modification, wherein the cell is preferably genetically modified 1) with the ability to express a gene encoding a bifunctional fucokinase/L-fucose-1-phosphate-guanyltransferase that catalyzes the formation of GDP-fucose from L- fucose, or 2) with the ability to overexpress at least one of the genes encoding phosphomannomutase, mannose-1-phosphate-guanosyltransferase, GDP-mannose-4,6-dehydratase and GDP-L-fucose synthase. 6. Genetically modified cell according to any one of paragraphs. 3-5, wherein the cell is genetically modified to exhibit enhanced import of exogenous L-fucose across its cell membrane, wherein the cell is preferably also genetically modified to express or overexpress, relative to the progenitor cell prior to its genetic modification, one nucleotide sequence , selected from the group consisting of nucleotide sequences encoding the main facilitated transport transporter FucP from E. coli , nucleotide sequences encoding functional variants of the main facilitated transport transporter FucP from E. coli , nucleotide sequences encoding functional fragments of the main the facilitated transport transporter FucP from E. coli , and nucleotide sequences encoding functional variants of functional fragments of the major facilitated transport transporter FucP from E. coli. 7. Genetically modified cell according to any one of paragraphs. 3-6, wherein the cell is also genetically modified to prevent depletion of the intracellular supply of GDP-fucose, preferably such that the expression of the cellular gene encoding WcaJ, which catalyzes the first step of colanic acid synthesis, is disrupted or inactivated, or such that the nucleotide sequence of the gene, encoding WcaJ is changed, as a result of which the enzymatically inactive polypeptide is encoded by the altered WcaJ gene. 8. Genetically modified cell according to any one of paragraphs. 3-7, where the cell is also genetically modified so that the fucI and/or fucK genes encoding L-fucose isomerase and L-fuculose kinase, respectively, are deleted, the nucleotide sequence of the fucI and/or fucK genes is changed to irreversibly inactivate the enzymatic activity of the corresponding polypeptide(s) (s), or so that the expression of the fucI and/or fucK genes is impaired. 9. Genetically modified cell according to any one of paragraphs. 3-8, wherein the cell is also genetically modified to have an increased ability to synthesize one or more of CMP- N -acetylneuraminic acid, UDP -N -acetylglucosamine and UDP-galactose, compared to the cell before it was genetically modified, preferably that said increased ability to produce UDP- N -acetylglucosamine, UDP-galactose and, more preferably, CMP -N -acetylneuraminic acid involves overexpression of one or more genes encoding proteins having L-glutamine:D-fructose-6-phosphate aminotransferase activities, N -acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyl-transferase, phosphoglucosamine mutase, UDP-galactose-4'-epimerase, phosphoglucomutase, glucose-1-phosphate-uridylyltransferase, glucosamine-6-phosphate acetyltransferase, N -acetylglucosamine-2-epimerase, UDP -N- acetylglucosamine-2-epimerase, phosphoenolpyruvate synthetase, sialic acid synthase, sialic acid permease, CMP-sialic acid synthetase.

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