α-1,3-Fucosyltransferase for the production of 3-fucosyllactose and inversion of lactose

Abstract

The present invention relates to a method for producing 3-fucosyllactose (3-FL) and a novel fucosyltransferase, more specifically a novel lactose-binding α-1,3-fucosyltransferase polypeptide, and their use. In addition, the present invention provides a method for producing 3-fucosyllactose (3-FL) using a novel lactose-binding α-1,3-fucosyltransferase.

Description

Technical Field

[0001] The present invention relates to a method for producing 3-fucosyllactose (3-FL) and a newly identified fucosyltransferase, more specifically a newly identified lactose-binding α-1,3-fucosyltransferase polypeptide, and their uses. In addition, the present invention provides a method for producing 3-fucosyllactose (3-FL) using a newly identified lactose-binding α-1,3-fucosyltransferase. Technical Background

[0002] More than 80 compounds belonging to the human milk oligosaccharide (HMO) family have now been structurally characterized. These HMOs represent a class of complex oligosaccharides that function as prebiotics. Furthermore, structural homology of HMOs to epithelial epitopes suggests a protective effect against bacterial pathogens. In the infant gastrointestinal tract, HMOs selectively nourish the growth of selected bacterial strains and, therefore, induce the development of a unique intestinal microbiota in breastfed infants.

[0003] Some of these human milk oligosaccharides require the presence of specific fucosylated structures that are likely to exhibit specific biological activities. The production of these fucosylated oligosaccharides requires the action of fucosyltransferases. These fucosyltransferases belong to the glycosyltransferase family and are widely expressed in vertebrates, invertebrates, plants, fungi, yeast and bacteria. They catalyze the transfer of fucose residues from donors (usually guanosine diphosphate-fucose (GDP-fucose)) to acceptors, including oligosaccharides, sugars (proteins) and sugars (lipids). Therefore, fucosylated acceptor substrates are involved in a variety of biological and pathological processes.

[0004] Several fucosyltransferases have been identified, for example in the bacteria Helicobacter pylori, Escherichia coli, Salmonella enterica, mammals, Caenorhabditis elegans and Schistosoma mansoni, and in plants.

[0005] Fucosyltransferases are classified based on the site of fucose addition into, for example, α-1,2, α-1,3, α-1,4, and O-fucosyltransferases.

[0006] Several α-1,3-fucosyltransferases have been described in the art. WO 1998 / 055630 describes a bacterial α-1,3-fucosyltransferase gene from Helicobacter pylori that can be used to produce oligosaccharides such as Lewis X, Lewis Y and sialyl Lewis X. WO 2016 / 040531 describes several α-1,3-fucosyltransferases for the production of fucosylated oligosaccharides. Here, the α-1,3-fucosyltransferases are described as having 25% to 100% sequence identity to the Bacteroides nordii CafC enzyme. However, in Table 1 of that document, the authors clearly show that more than half (7 / 12) of the enzymes they tested (many of which had >25% sequence identity to CafC) were unable to produce 3-fucosyllactose using lactose as an acceptor substrate. This shows that obviously not all putative fucosyltransferases have lactose-binding fucosyltransferase activity. WO2012 / 049083 describes some new α-1,3-fucosyltransferases and their use in the production of fucosylated products. Huang et al. 2017 compared various exogenous α-1,3-fucosyltransferase candidates and a series of E. coli host strains and demonstrated that Helicobacter pylori futA using E. coli BL21 (DE3) as the host strain produced the highest titer of 3-fucosyllactose (one of the human milk oligosaccharides).

[0007] Generally, α-1,3-fucosyltransferase (also called 3-fucosyltransferase) is known to have a low affinity for lactose. 3-Fucosyltransferase is required for the production of HMO 3-fucosyllactose. Low affinity has a negative impact on the productivity of 3-fucosyllactose. In order to improve conversion and productivity, a transferase with sufficient lactose affinity, preferably higher lactose affinity, is required.

[0008] It is therefore an object of the present invention to provide tools and methods by which 3-fucosyllactose can be produced or synthesized in an efficient, time- and cost-effective manner and yields similar or higher quantities of the desired product compared to prior art methods. Summary of the invention

[0009] Surprisingly, it has now been found that the newly identified lactose binding α-1,3-fucosyltransferases of the present invention provide transferases with similar or improved lactose binding and / or transferase properties compared to currently known lactose binding α-1,3-fucosyltransferases.

[0010] Therefore, the present invention provides a method for producing 3-fucosyllactose (3FL) using a newly identified lactose-binding α-1,3-fucosyltransferase. 3FL can be obtained by reacting lactose in the presence of an α-1,3-fucosyltransferase that is capable of catalyzing the formation of 3-fucosyllactose oligosaccharides from lactose and GDP-fucose. Alternatively, it can also be obtained from a microorganism that produces an α-1,3-fucosyltransferase according to the present invention.

[0011] definition

[0012] The words used in this specification to describe the present invention and its various embodiments should be understood not only in their conventionally defined meanings, but also in their special definitions in this specification to include structures, materials or behaviors beyond the meaning of the conventionally defined meanings. Therefore, if an element can be understood to include more than one meaning in the context of this specification, its use in the claims must be understood to be universal to all possible meanings supported by the specification and the word itself.

[0013] The various embodiments of the present invention disclosed herein and the aspects of the embodiments should not only be understood in the order and context specifically described in this specification, but also include any order and any combination thereof. When the context requires, all words used in the singular should be deemed to include the plural, and vice versa. Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings as those of ordinary skill in the art to which the present invention belongs. In general, the terms used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, nucleic acid chemistry and hybridization described herein are well known in the art and commonly used. Standard techniques are used for the synthesis of nucleic acids and peptides. Typically, enzymatic reactions and purification steps are carried out according to the manufacturer's instructions.

[0014] In the drawings and description, embodiments of the present invention have been disclosed, and although specific terms are used, these terms are used only in a descriptive sense and not for the purpose of limitation, and the scope of the present invention is set forth in the appended claims. It must be understood that the embodiments shown are set forth for illustrative purposes only and should not be considered to limit the present invention. It is obvious to those skilled in the art that modifications, other embodiments, improvements, details and uses can be made consistent with the text and spirit of the present disclosure and within the scope of the present disclosure, and the present disclosure is only limited by the claims according to the interpretation of patent law, including the doctrine of equivalents. In the subsequent claims, reference characters for specifying claim steps are provided only for ease of description and are not intended to imply any particular order of performing the steps.

[0015] According to the present invention, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA."Polynucleotide" includes but is not limited to single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions or single-stranded, double-stranded and triple-stranded regions, single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA (which can be single-stranded, or more typically double-stranded or triple-stranded regions, or a mixture of single-stranded and double-stranded regions).In addition, "polynucleotide" used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.The chains in these regions can come from the same molecule or from different molecules.These regions can include all one or more molecules, but more typically only relate to the regions of some molecules.One of the molecules in the triple helix region is typically an oligonucleotide.As used herein, the term "polynucleotide" also includes DNA or RNA containing one or more modified bases as described above.Therefore, DNA or RNA with a main chain modified for stability or other reasons is "polynucleotide" according to the present invention. In addition, DNA or RNA containing unusual bases (e.g., inosine) or modified bases (e.g., triacylated bases) are understood to be encompassed in the term "polynucleotide". It should be understood that a variety of modifications have been made to DNA and RNA for many useful purposes known to those skilled in the art. The term "polynucleotide" as used herein includes such chemically, enzymatically or metabolically modified polynucleotide forms, as well as chemical forms of DNA and RNA that are unique to viruses and cells (including, for example, simple and complex cells). The term "polynucleotide" also includes short polynucleotides commonly referred to as oligonucleotides.

[0016] "Polypeptide" refers to any peptide or protein containing two or more amino acids connected to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains (commonly referred to as peptides, oligopeptides and oligomers) and long chains (commonly referred to as proteins). Polypeptides can contain amino acids other than the 20 gene-encoded amino acids. "Polypeptide" includes polypeptides modified by natural processes (such as processing and other post-translational modifications) and polypeptides modified by chemical modification techniques. Such modifications are fully described in basic textbooks and more detailed monographs and in multi-volume research literature, and are well known to those skilled in the art. The same type of modification can exist at several sites in a given polypeptide to the same degree or to different degrees. In addition, a given polypeptide can contain many types of modifications. Modifications can occur at any position of a polypeptide, including the peptide backbone, amino acid side chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfidation, γ-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, transfer RNA-mediated addition of amino acids to proteins (such as arginylation) and ubiquitination. Polypeptides may be branched or cyclic with or without branches. Circular, branched, and branched circular polypeptides can be formed by post-translational natural processes and can also be made entirely synthetically.

[0017] "Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," as the term is used herein. Similarly, the term "synthetic" sequence as used herein refers to any sequence that is synthetically produced and not directly isolated from a natural source. The term "synthetic" as used herein refers to any sequence that is synthetically generated and not directly isolated from a natural source.

[0018] "Recombinant" means genetically engineered DNA that is prepared by transplanting or splicing a gene from one species into a cell of a host organism of a different species. This DNA becomes part of the host's genetic makeup and is replicated. A "mutant" cell or microorganism used in the context of the present invention refers to a cell or microorganism that is genetically engineered or has an altered genetic makeup.

[0019] In the context of the present invention, the term "cell genetically modified for producing 3-fucosyllactose" refers to a cell of a microorganism that has been genetically manipulated to contain at least one of: i) a recombinant gene encoding an α1,3 fucosyltransferase necessary for the synthesis of said 3-fucosyllactose, ii) a biosynthetic pathway for producing GDP-fucose suitable for transfer to lactose by said fucosyltransferase, and / or iii) a biosynthetic pathway for producing lactose or a mechanism for internalization of lactose from the culture medium into the cell, where lactose is fucosylated to produce 3-fucosyllactose.

[0020] The term "a nucleic acid sequence encoding an enzyme for 3-fucosyllactose synthesis" relates to a nucleic acid sequence encoding an enzyme essential for the 3-fucosyllactose synthesis pathway, such as an enzyme capable of transferring the fucose moiety of a GDP-fucose donor substrate to the 3-hydroxyl group of the galactose moiety of lactose to produce 3-fucosyllactose.

[0021] In the context of the present disclosure, the term "endogenous" refers to any polynucleotide, polypeptide, or protein sequence that is a natural part of a cell and is present in its natural location in a cell chromosome. The term "exogenous" refers to any polynucleotide, polypeptide, or protein sequence that originates from outside the cell being studied and is not a natural part of the cell, or is not present in its natural location in a cell chromosome or plasmid.

[0022] The term "heterologous" refers to polynucleotides, genes, nucleic acids, polypeptides or enzymes from or derived from the source outside the host species. On the contrary, "homologous" polynucleotides, genes, nucleic acids, polypeptides or enzymes are used herein to represent polynucleotides, genes, nucleic acids, polypeptides or enzymes derived from host organism species. When it comes to the gene regulatory sequence or auxiliary nucleic acid sequence for maintaining or manipulating a gene sequence (for example, promoter, 5' non-translated region, 3' non-translated region, poly A additional sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genome homology region, recombination site etc.), "heterologous" refers to that regulatory sequence or auxiliary sequence are not naturally associated with the gene in parallel with regulatory or auxiliary nucleic acid sequence in construct, genome, chromosome or episome. Therefore, the promoter that is operably connected to the gene that is not operably connected to in its natural state (i.e. in the genome of non-genetically engineered organisms) is referred to as "heterologous promoter" in this article, even if the promoter can be derived from the same species (or in some cases, same organism) as the gene connected thereto.

[0023] As used herein, the term "polynucleotide encoding a polypeptide" includes polynucleotides comprising a sequence encoding a polypeptide of the present invention, in particular an α-1,3-fucosyltransferase having an amino acid sequence as shown in SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 in the attached sequence listing. For clarity, polynucleotides encoding polypeptides of SEQ ID NO 18, 24 and 26 are also polynucleotides encompassed by this definition, but the polynucleotide of SEQ ID NO 18 is a prior art α-1,3-fucosyltransferase used as a reference, and the polynucleotides of SEQ ID NO 24 and 26 are α-1,3-fucosyltransferases that do not function with lactose as an acceptor. The term also includes polynucleotides comprising a single continuous region encoding the polypeptide or discontinuous regions (e.g., separated by integrated phage or insertion sequences or editing) and additional regions that may also contain coding and / or non-coding sequences.

[0024] The term "variant" as used herein is a polynucleotide or polypeptide that is different from a reference polynucleotide or polypeptide, respectively, but retains basic properties. Typical variants of polynucleotides differ from another reference polynucleotide in nucleotide sequence. Changes in the nucleotide sequence of a variant may or may not change the amino acid sequence of a polypeptide encoded by a reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in a polypeptide encoded by a reference sequence, as discussed below. Typical variants of a polypeptide differ from another reference polypeptide in amino acid sequence. Typically, the difference is limited so that the sequences of the reference polypeptide and the variant are closely similar overall and identical in many regions. Variants and reference polypeptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring, such as allelic variants, or they may be variants that are not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art. In some embodiments, the present invention contemplates preparing functional variants by modifying the structure of the membrane protein used in the present invention. Variants can be produced by amino acid substitution, deletion, addition or a combination thereof. For example, it is reasonable to expect that similar replacements of amino acids with isoleucine or valine for leucine alone, glutamic acid alone for aspartic acid, serine alone for threonine, or with structurally related amino acids (e.g., conservative mutations) will not have a significant impact on the biological activity of the resulting molecule. Conservative replacements are replacements that occur within the amino acid family associated with its side chain. By assessing the ability of variant polypeptides to produce responses in cells in a manner similar to that of the wild-type polypeptide, in the case of the present invention, better yields, productivity and / or growth rates are provided than cells without variants, it is easy to determine whether changes in the amino acid sequence of a polypeptide of the present disclosure result in functional homologues.

[0025] As used herein, the term "functional homolog" describes those molecules that have sequence similarity and also share at least one functional characteristic, such as biochemical activity. Functional homologs typically produce similar, but not necessarily identical, degrees of the same characteristic. Functionally homologous proteins have the same characteristic, wherein one homolog produces a quantitative measurement that is at least 10% of the other; more typically, at least 20%, between about 30% and about 40%; for example, between about 50% and about 60%; between about 70% and about 80%; or between about 90% and about 95%; between about 98% and about 100%, or more than 100% of the quantitative measurement produced by the original molecule. Thus, when the molecule has enzymatic activity, the functional homolog will have the above percentage of enzymatic activity compared to the original enzyme. If the molecule is a DNA binding molecule (e.g., a polypeptide), the homolog will have the above percentage of binding affinity, measured by the weight of the bound molecule compared to the original molecule.

[0026] Functional homologs and reference polypeptides may be naturally occurring polypeptides, and sequence similarities may be due to convergent or divergent evolutionary events. Functional homologs are sometimes referred to as orthologs, where "ortholog" refers to a homologous gene or protein in another species that is functionally equivalent to a reference gene or protein.

[0027] Functional homologues can be identified by nucleotide and peptide sequence comparison analysis.For example, the database of nucleotide or peptide sequence is performed to query and can identify the homologue of biomass regulating polypeptide.Sequence analysis can relate to the BLAST, reverse BLAST or PSI-BLAST analysis of the non-redundant database using the amino acid sequence of biomass regulating polypeptide as reference sequence.In some cases, the amino acid sequence is derived from the nucleotide sequence.Usually, those polypeptides with sequence identity greater than 40% in the database are further evaluated as the suitability of biomass regulating polypeptide.Amino acid sequence similarity allows conservative amino acid replacement, for example a hydrophobic residue replaces another hydrophobic residue or a polar residue replaces another polar residue.If necessary, this type of candidate can be carried out artificial inspection, to reduce the quantity of candidate to be further evaluated.Artificial inspection can be performed by selecting those candidate that seem to have the domain (for example, conservative functional domain) existing in productivity regulating polypeptide.

[0028] "Fragment", with respect to polynucleotides, refers to any portion of a clone or polynucleotide molecule, particularly a portion of a polynucleotide that retains useful functional characteristics. Useful fragments include oligonucleotides and polynucleotides that can be used for hybridization or amplification techniques or regulation of replication, transcription or translation. "Polynucleotide fragment" refers to any subsequence of a polynucleotide, typically having at least about 9 consecutive nucleotides, such as at least about 30 nucleotides or at least about 50 nucleotides, of any sequence provided herein. Exemplary fragments may additionally or alternatively include regions comprising, essentially consisting of, or consisting of conserved family domains encoding polypeptides. Exemplary fragments may additionally or alternatively include fragments comprising conserved domains of polypeptides.

[0029] Fragment can additionally or alternatively include subsequences of polypeptide and protein molecules, or subsequences of polypeptide.In some cases, fragment or domain is a subsequence of polypeptide, and it performs at least one biological function of complete polypeptide in the substantially same mode or similar degree as complete polypeptide.For example, polypeptide fragment can comprise recognizable structural motif or functional domain, for example DNA binding site or domain, it combines with DNA promoter region, activation domain or domain for protein-protein interaction, and can start transcription.The size of fragment can vary from as little as 3 amino acid residues to the total length of complete polypeptide, for example, length is at least about 20 amino acid residues, for example, length is at least about 30 amino acid residues.Preferably, fragment is at least one property of polypeptide with derived fragment or active functional fragment, for example, fragment can include functional domain or conserved domain of polypeptide.For example, can specify to characterize domain by Pfam or conserved domain database (CDD).

[0030] As used herein, the terms "alpha-1,3-fucosyltransferase", "alpha 1,3-fucosyltransferase", "3-fucosyltransferase", "α-1,3-fucosyltransferase", "α1,3 fucosyltransferase", "3 fucosyltransferase", "3-FT" or "3FT" are used interchangeably and refer to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate GDP-L-fucose to an acceptor molecule lactose in an α-1,3-linkage. A polynucleotide encoding "α-1,3-fucosyltransferase" or any of the above terms refers to a polynucleotide encoding such a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate GDP-L-fucose to an acceptor molecule lactose in an α-1,3-linkage.

[0031] As used herein, the terms "3-fucosyllactose", "alpha-1,3-fucosyllactose", "alpha1,3 fucosyllactose", "α-1,3-fucosyllactose", "α1,3 fucosyllactose", "Galβ-4(Fucα1-3)Glc", "3FL" or "3-FL" are used interchangeably and refer to the product obtained by the transfer of fucose residues from GDP-L-fucose to lactose in an α-1,3-linkage by α-1,3-fucosyltransferase.

[0032] "Oligosaccharide" as the term is used herein and as generally understood in the art, refers to a sugar polymer containing a small number (usually 3 to 10) of simple sugars (ie, monosaccharides).

[0033] The term "purified" refers to a material that is substantially or essentially free of components that interfere with the activity of a biomolecule. For cells, carbohydrates, nucleic acids, and polypeptides, the term "purified" refers to a material that is substantially or essentially free of components that normally accompany the material when the material is present in its native state. Typically, the purified carbohydrates, oligosaccharides, proteins, or nucleic acids of the present invention are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, as measured by band intensity on a silver-stained gel or other method of determining purity. Purity or homogeneity can be indicated by many methods known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization when stained. For some purposes, high resolution is required, and HPLC or similar purification methods are used. For oligosaccharides, such as 3-fucosyllactose, purity can be determined using methods such as, but not limited to, thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis, or mass spectrometry.

[0034] The term "identical" or percentage "identity" or % "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical or have a specific percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence using a sequence comparison algorithm or visual inspection. For sequence comparison, a sequence is used as a reference sequence to which a test sequence is compared. When a sequence comparison algorithm is used, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence according to the specified program parameters. BLAST and PSI-BLAST can be used to determine the identity percentage (Altschul et al., 1990, J Mol Biol 215:3,403-410; Altschul et al., 1997, Nucleic Acids Res 25:17,3389-402). For the purposes of the present invention, percent identity was determined using MatGAT 2.01 (Campanella et al., 2003, BMC Bioinformatics 4:29). MatGAT uses the Myers and Miller global alignment algorithm for pairwise alignments. The following default parameters for proteins were used: (1) Gap penalty presence: 12 and extension: 2; (2) the matrix used was BLOSUM50.

[0035] The term "control sequence" refers to a sequence recognized by the host cell transcription and translation system, allowing the polynucleotide sequence to be transcribed and translated into a polypeptide. Therefore, this DNA sequence is necessary for expressing an operably connected coding sequence in a specific host cell or organism. This control sequence can be, but is not limited to, a promoter sequence, a ribosome binding sequence, a ShineDalgarno sequence, a Kozak sequence, a transcription terminator sequence. For example, a control sequence suitable for prokaryotes includes a promoter, an optional operator sequence, and a ribosome binding site. It is known that eukaryotic cells utilize promoters, polyadenylation signals, and enhancers. If the DNA of a presequence or a secretable leader is expressed as a preprotein involved in the secretion of a polypeptide, the DNA of the presequence or the secretable leader is operably connected to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a sequence, the promoter or enhancer is operably connected to a coding sequence; or if a ribosome binding site affects the transcription of a sequence, the ribosome binding site is operably connected to a coding sequence; or if the positioning of the ribosome binding site is convenient for translation, the ribosome binding site is operably connected to a coding sequence. The control sequences may also be additionally controlled using external chemicals such as, but not limited to, IPTG, arabinose, lactose, allolactose, rhamnose or fucose via inducible promoters or via genetic circuits that induce or repress transcription or translation of the polynucleotide into a polypeptide.

[0036] The term "end of fermentation" as used in the present invention refers to the time when the fermentation is harvested for product purification.

[0037] Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretable leader, contiguous and in reading frame. However, enhancers need not be contiguous. DETAILED DESCRIPTION OF THE INVENTION

[0039] According to a first embodiment, the present invention provides a method for producing α-1,3-fucosyllactose. The method comprises the following steps:

[0040] a) providing a polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate, wherein the polypeptide comprises

[0041] i) an amino acid sequence encoding a conserved GDP-fucose binding domain [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R] (SEQ ID NO 33);

[0042] ii) an amino acid sequence encoding a conserved [K / D][L / K / M]XXX[F / Y] domain (SEQ ID NO 34), and

[0043] iii) wherein if the domain of ii) is equal to DM[A / S]VSF (SEQ ID NO 36), then additionally in the N-terminal region the conserved motif [N / H]XDPAXLD (SEQ ID NO 35) is present;

[0044] wherein X can be any different amino acid; and

[0045] wherein the C-terminus of the polypeptide has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain;

[0046] b) contacting the polypeptide having α-1,3-fucosyltransferase activity in step a) with a mixture comprising GDP-fucose as a donor substrate and lactose as an acceptor substrate, under conditions wherein the polypeptide catalyzes the transfer of a fucose residue from a donor substrate to an acceptor substrate, thereby producing α-1,3-fucosyllactose.

[0047] Relative to currently known α-1,3-fucosyltransferases, these newly identified polypeptides comprising two of the above domains (or all of SEQ ID NOs 33 to 36, as the case may be) provide an alternative α-1,3-fucosyltransferase having the ability to use lactose as an acceptor substrate. Polypeptides comprising two of the above domains (or all of SEQ ID NOs 33 to 36, as the case may be) provide transferases having similar or higher lactose binding and / or similar or higher transferase properties compared to currently known α-1,3-fucosyltransferases.

[0048] In a first preferred embodiment of the present invention, the polypeptide for use in the present invention comprises two of the domains having SEQ ID NOs 33 to 34 or 36 (or all of SEQ ID NOs 33 to 36, as the case may be) and wherein said SEQ ID NO 33 is a conserved domain having an amino acid sequence YXTEK (SEQ ID NO: 37), wherein X can be any different amino acid.

[0049] In a second preferred embodiment of the present invention, the polypeptide for use in the present invention comprises two of the domains having SEQ ID NOs 33 to 34 or 36 (or all of SEQ ID NOs 33 to 36, as the case may be) and wherein said SEQ ID NO 34 is a conserved domain having an amino acid sequence of [K / D]LX[I / L / M]G[F / Y] (SEQ ID NO: 38), [K / D][L / K]xL[S / G][F / Y] (SEQ ID NO: 39) or [K / D]LXLG[F / Y] (SEQ ID NO: 40), wherein X can be any different amino acid.

[0050] Another advantage of using some of the newly identified polypeptides having the ability to use lactose as an acceptor substrate and having α-1,3-fucosyltransferase activity and having a newly identified domain is that at the end of the reaction or fermentation, 3-fucosyllactose is produced at a higher purity than that obtained using the reference prior art polypeptide having SEQ ID NO 18, due to the better conversion ability of the newly identified 3-fucosyltransferase, lactose can be used for 3FL production. More specifically, the ratio of lactose concentration to 3-fucosyllactose concentration is less than 1:5, preferably less than 1:10, more preferably less than 1 / 20, preferably less than 1:40. In another preferred embodiment, the 3-fucosyllactose purity is 80% or more at the end of the fermentation.

[0051] According to the present invention, the method of producing α-1,3-fucosyllactose can be carried out in a cell-free system or a system containing cells. The substrates GDP-fucose and lactose are reacted with the α-1,3-fucosyltransferase polypeptide for a sufficient time and under sufficient conditions to form an enzyme product. These conditions will vary depending on the amount and purity of the substrate and enzyme, and whether the system is a cell-free system or a cell-based system. Those skilled in the art will readily adjust these variables.

[0052] In a cell-free system, polypeptides according to the invention, acceptor substrates, donor substrates, and optionally other reaction mixture components (including other glycosyltransferases and auxiliary enzymes) are combined by mixing in an aqueous reaction medium to perform an enzymatic reaction. The enzyme can be freely available in solution, or can be bound or immobilized on a support (e.g., a polymer), and the substrate can be added to the support. For example, the support can be packaged in a column.

[0053] The cell-containing system or cell-based system for synthesizing 3-fucosyllactose described herein may include a genetically modified host cell. According to one aspect of the invention, a polypeptide having α-1,3-fucosyltransferase activity is produced by a cell that produces the polypeptide (e.g., a host cell as described herein). According to another aspect of the invention, GDP-fucose and / or lactose are provided by cells that produce the GDP-fucose and / or lactose. The cell may be a host cell that also produces α-1,3-fucosyltransferase. Alternatively, the cell may be another cell other than the host cell that produces α-1,3-fucosyltransferase, in which case the technician will discuss cell coupling. This cell that produces GDP-fucose may express an enzyme that converts, for example, fucose to be added to the host cell into GDP-fucose. The enzyme can be, for example, a bifunctional fucokinase / fucose-1-phosphate guanylyltransferase, such as Fkp from Bacteroides fragilis, or a combination of a single fucokinase and a single fucose-1-phosphate guanylyltransferase, as are known from several species, including Homo sapiens, Sus scrofa, and Rattus norvegicus.

[0054] In another embodiment, the present invention relates to a method for producing α-1,3-fucosyllactose, comprising the following steps:

[0055] i) providing a cell genetically modified for producing α-1,3-fucosyllactose, the cell comprising at least one nucleic acid sequence encoding an enzyme for α-1,3-fucosyllactose synthesis, the cell comprising expression of a polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate, wherein the polypeptide comprises:

[0056] a) an amino acid sequence encoding a conserved GDP-fucose binding domain [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R] (SEQ ID NO 33);

[0057] b) an amino acid sequence encoding a conserved [K / D][L / K / M]XXX[F / Y] domain (SEQ ID NO 34), and

[0058] c) wherein if the domain of b) is equal to DM[A / S]VSF (SEQ ID NO 36), then the conserved motif [N / H]XDPAXLD (SEQ ID NO 35) is present in the N-terminal region;

[0059] wherein X can be any different amino acid; and

[0060] wherein the C-terminus of the polypeptide has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain, and

[0061] ii) culturing the cells in a culture medium under conditions that allow for the production of α-1,3-fucosyllactose, thereby producing 3-FL.

[0062] In another embodiment, the present invention relates to a method for producing α-1,3-fucosyllactose, the method comprising the following steps:

[0063] a) providing a host cell expressing said polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate as defined herein;

[0064] b) growing the host cell under suitable nutritional conditions that allow the production of α-1,3-fucosyllactose and the expression of a polypeptide having α-1,3-fucosyltransferase activity;

[0065] c) simultaneously or after step b), providing a donor substrate GDP-fucose and an acceptor substrate lactose, so that the α-1,3-fucosyltransferase polypeptide catalyzes the transfer of fucose residues from GDP-fucose to lactose, thereby producing α-1,3-fucosyllactose.

[0066] The produced 3FL is then optionally isolated from the host cells and / or the medium in which they were grown.

[0067] According to yet another embodiment, the production of 3-fucosyllactose in the methods described herein is performed by heterologous or homologous (over)expression of a polynucleotide encoding an α-1,3-fucosyltransferase by a cell.

[0068] In the methods of the invention described herein, the host cell can be transformed or transfected to express the exogenous polypeptide described herein and having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate. Therefore, the present invention relates to a method for producing α-1,3-fucosyllactose using a host cell, comprising the following steps:

[0069] a) growing a host cell transformed or transfected to express an exogenous polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate, wherein the polypeptide is as described herein; and

[0070] b) simultaneously with or after step a), providing a donor substrate GDP-fucose and an acceptor substrate lactose, wherein the α-1,3-fucosyltransferase polypeptide catalyzes the transfer of fucose residues from the donor substrate to the acceptor substrate, thereby producing α-1,3-fucosyllactose.

[0071] Preferably, the exogenous polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate as used herein produces 3FL having a ratio of lactose concentration to 3FL concentration of less than 1:5 at the end of fermentation.

[0072] The ratio of lactose concentration to 3FL concentration can be less than 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38 , 1:39, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000.

[0073] In a preferred embodiment, a ratio of lactose concentration to 3FL concentration of less than 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80 :80, 1:85, 1:90, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180; 1:190, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, resulting in a final lactose concentration of less than 25 g / L, 20 g / L, 15 g / L, 10 g / L, 9 g / L, 8 g / L, 7 g / L, 6 g / L, 5 g / L, 4 g / L, 3 g / L, 2 g / L, 1 g / L, 0.5 g / Lg / L, 0.25 g / L, 0.1 g / L or 0 g / L.

[0074] In another embodiment, a ratio of lactose concentration to 3FL concentration of less than 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:45 is obtained during the production process. , 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, wherein the lactose concentration is fed under substrate limiting conditions, wherein substrate limitation is defined as the concentration in the bioreactor that determines the conversion rate of the substrate.

[0075] In another embodiment, a ratio of lactose concentration to 3FL concentration of less than 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38 is obtained during the production process. , 1:39, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, wherein lactose is formed in the cell under rate-limiting conditions.

[0076] In another embodiment, the purity of 3-fucosyllactose in the broth based on the sum of (lactose and 3FL) in the broth is greater than about 80%, for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95,5%, 96%, 96,5%, 97%, 97,5%, 98%, 98,5%, 99%, 99,5%, 99,6%, 99,7%, 99,8%, 99,9%. As used herein, 3-fucosyllactose purity is defined as the ratio of 3FL concentration to the sum of 3FL concentration and lactose concentration ([3FL] / ([3FL]+[lactose]).

[0077] According to the invention, GDP-fucose and / or lactose may be fed to the host cell in a fermentation medium or an aqueous medium. Alternatively, GDP-fucose and / or lactose may be provided by enzymes simultaneously expressed in the host cell or by the metabolism of the host cell. Thus, the host cell will also produce an α-1,3-fucosyltransferase after GDP-fucose and / or lactose. In another embodiment, GDP-fucose and / or lactose may be produced by a cell that is another cell other than the host cell producing the α-1,3-fucosyltransferase, in which case the skilled person will talk about cell coupling. Such a cell producing GDP-fucose may express an enzyme that converts, for example, fucose to be added to the host cell into GDP-fucose. Such an enzyme may, for example, be a bifunctional fucokinase / fucose-1-phosphate guanylyltransferase, such as Fkp of Bacteroides fragilis, or a combination of a single fucokinase and a single fucose-1-phosphate guanylyltransferase, as known from several species, including Homo sapiens, Sus suis and Rattus norvegicus.

[0078] According to yet another embodiment, the production of α-1,3-fucosyllactose is performed by a host cell as described herein, comprising heterologous or homologous (over) expression of a polynucleotide encoding an α-1,3-fucosyltransferase.

[0079] In another aspect, the present invention provides a method for producing α-1,3-fucosyllactose as described herein, wherein the method further comprises the step of isolating α-1,3-fucosyllactose from the host cell or the culture medium in which it is grown.

[0080] As used herein, the term "isolating" means harvesting, collecting or recovering α-1,3-fucosyllactose produced by α-1,3-fucosyltransferase according to the present invention from a reaction mixture and / or from cells producing α-1,3-fucosyltransferase.

[0081] If α-1,3-fucosyllactose is produced by cells or fermentation, 3-FL can be separated from the aqueous medium in which the mixture is prepared by conventional means. If α-1,3-fucosyllactose is still present in the cells that produce α-1,3-fucosyllactose, conventional methods for releasing or extracting α-1,3-fucosyllactose from the cells can be used, such as disrupting the cells using high pH, ​​heat shock, ultrasound, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis, etc. The culture medium, reaction mixture and / or cell extract are collectively and individually referred to as a mixture containing 3-FL, which can then be further used to separate 3-FL. This preferably involves clarifying the mixture containing 3-FL to remove suspended particles and contaminants, in particular cells, cell components, insoluble metabolites and debris produced by culturing genetically modified cells and / or performing enzymatic reactions. In this step, the mixture containing 3-FL can be clarified in a conventional manner. Preferably, the mixture containing 3-FL is clarified by centrifugation, flocculation, decantation and / or filtration. The second step of separating 3-FL from a mixture containing 3-FL preferably involves removing substantially all proteins as well as peptides, amino acids, RNA and DNA from the mixture containing 3-FL (preferably after it has been clarified) and any endotoxins and glycolipids that may interfere with subsequent separation steps. In this step, proteins and related impurities can be removed from the mixture containing 3-FL in a conventional manner. Preferably, proteins, salts, by-products, colors and other related impurities are removed from the mixture containing 3-FL by ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography (for example, but not limited to cation exchange, anion exchange, mixed bed ion exchange), hydrophobic interaction chromatography and / or gel filtration (i.e., size exclusion chromatography), in particular by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography. In addition to size exclusion chromatography, proteins and related impurities are retained by the chromatographic medium or selected membrane, while 3-FL is retained in the mixture containing 3-FL.

[0082] 3-FL is further isolated from the reaction mixture and / or culture medium and / or cells by further purification steps of evaporation, lyophilization, crystallization, precipitation and / or drying, spray drying or without further purification steps.

[0083] In a further aspect, the present invention also provides further purification of α-1,3-fucosyllactose. The further purification of the α-1,3-fucosyllactose can be accomplished, for example, by using (activated) charcoal or carbon, nanofiltration, ultrafiltration or ion exchange to remove any remaining DNA, protein, LPS, endotoxin or other impurities. Alcohols such as ethanol and aqueous alcohol mixtures can also be used. Another purification step is to crystallize, evaporate or precipitate the product. Another purification step is to dry, spray dry or freeze dry the α-1,3-fucosyllactose.

[0084] The isolated and preferably also purified 3-FL can be used as a supplement in infant formula and for the treatment of various diseases in newborn infants.

[0085] Another aspect of the present invention provides a method, wherein the polypeptide and also preferably 3-FL are produced in and / or by fungi, yeast, bacteria, insects, animals and plants expression systems or cells as described herein. The expression system or cell is selected from a list including bacteria, yeast or fungi, or refers to a plant or animal cell. The latter bacteria preferably belong to the Proteobacteria phylum or Firmicutes phylum or Cyanobacteria phylum or Deinococcus-Thermus phylum. The latter bacteria belonging to the Proteobacteria phylum preferably belong to the Enterobacteriaceae family, preferably to the Escherichia coli species. The latter bacteria preferably relate to any strain belonging to the Escherichia coli species, such as, but not limited to, Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to a cultured Escherichia coli strain, referred to as an Escherichia coli K12 strain, which is very suitable for laboratory environments and, unlike wild-type strains, has lost the ability to reproduce in the intestine. Well-known examples of E. coli K12 strains are K12 wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Therefore, the present invention particularly relates to E. coli host cells or strains mutated and / or transformed as described above, wherein the E. coli strain is a K12 strain. More specifically, the E. coli K12 strain is E. coli MG1655. The latter bacterium belonging to Firmicutes preferably belongs to Bacilli, preferably to Lactobacilliales, whose members are such as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacillales, whose members are such as from Bacillus, such as Bacillus subtilis or B. amyloliquefaciens. The latter bacterium belonging to Actinobacteria preferably belongs to Corynebacteriaceae, whose members are Corynebacterium glutamicum or C. afermentans, or to Streptomycetaceae, whose members are Streptomyces griseus or S. fradiae.The latter yeast preferably belongs to the phylum Ascomycota or Basidiomycota or Deuteromycota or Zygomycete. The latter yeast preferably belongs to the genus Saccharomyces, Pichia, Komagataella, Hansunella, Kluyveromyces, Yarrowia or Starmerella. The latter fungi preferably belong to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus.

[0086] According to another aspect of the present invention, the polynucleotide encoding the polypeptide having α-1,3-fucosyltransferase activity is suitable for the codon usage of the corresponding cell or expression system.

[0087] In another preferred embodiment, the method of the present invention uses a culture medium for growing a host cell or microorganism comprising an α-1,3-fucosyltransferase of the present invention, wherein the lactose concentration in the culture medium is in the range of 50 to 150 g / L. The lactose concentration in the culture medium may be 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, 105 g / L, 110 g / L, 115 g / L, 120 g / L, 125 g / L, 130 g / L, 135 g / L, 140 g / L, 145 g / L or 150 g / L.

[0088] In another preferred embodiment, the method of the present invention produces a final concentration of 3-fucosyllactose ranging from 70 g / L to 200 g / L. Such a 3-FL concentration is 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, 105 g / L, 110 g / L, 115 g / L, 120 g / L, 125 g / L, 130 g / L, 135 g / L, 140 g / L, 145 g / L, 145 g / L, 150 g / L, 155 g / L, 160 g / L, 165 g / L, 170 g / L, 175 g / L, 180 g / L, 185 g / L, 190 g / L, 195 g / L or 200 g / L. Higher lactose concentrations in the culture medium may provide even higher final concentrations of 3-FL obtained in the production process.

[0089] In another preferred embodiment, the method of the present invention produces a final concentration of 3FL ranging from 70 g / L to 200 g / L, as described above, and wherein the purity of 3FL in the broth is 80% or more. The purity of 3FL according to the present invention is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%.

[0090] In the methods of the present invention as described herein, the polypeptide having alpha-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate comprises:

[0091] a) an amino acid sequence encoding a conserved GDP-fucose binding domain [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R] (SEQ ID NO 33);

[0092] b) an amino acid sequence encoding a conserved [K / D][L / K / M]XXX[F / Y] domain (SEQ ID NO 34), and

[0093] c) wherein if the domain of b) is equal to DM[A / S]VSF (SEQ ID NO 36), then additionally in the N-terminal region the conserved motif [N / H]XDPAXLD (SEQ ID NO 35) is present;

[0094] wherein X can be any different amino acid; and

[0095] The C-terminus of the polypeptide has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain.

[0096] Within the scope of the present invention, the polypeptide is demonstrated to have lactose-binding α-1,3-fucosyltransferase activity and preferably has a better lactose conversion efficiency than the currently known α-1,3-fucosyltransferases.

[0097] In a preferred embodiment of the present invention, the polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate comprises an amino acid sequence selected from the group consisting of:

[0098] i) any one of SEQ ID NO 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 in the attached sequence listing;

[0099] ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO 2, 20 or 22;

[0100] iii) an amino acid sequence having 80% or greater sequence identity to the full-length amino acid sequence of any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32;

[0101] iv) a fragment of the amino acid sequence shown in SEQ ID NO 2, 20 or 22, wherein the fragment comprises at least 45 consecutive amino acids thereof;

[0102] v) a fragment of the amino acid sequence shown in any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32, wherein the fragment comprises at least 10 consecutive amino acids thereof and has lactose-binding α-1,3-fucosyltransferase activity.

[0103] Optionally, the polypeptide is further modified by an N-terminal and / or C-terminal amino acid extension.

[0104] The amino acid sequence of the polypeptide used herein may be a sequence selected from SEQ ID NO 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 in the attached sequence listing. The amino acid sequence may also be an amino acid sequence having greater than about 87% sequence identity, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity to the full-length amino acid sequence of any one of SEQ ID NO 2, 20 or 22. The amino acid sequence can also be an amino acid sequence having greater than about 80% sequence identity, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity to the full-length amino acid sequence of any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32.

[0105] In addition, within the scope of the present invention, the amino acid sequence may be a fragment of the amino acid sequence shown in any one of SEQ ID NOs 2, 20 or 22, wherein the fragment comprises at least 45 consecutive amino acids thereof; alternatively, the amino acid sequence may be a fragment of the amino acid sequence shown in any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32, wherein the fragment comprises at least 10 consecutive amino acids thereof and has lactose-binding α-1,3-fucosyltransferase activity.

[0106] Further included within the scope of the present invention are α-1,3-fucosyltransferase polypeptides as described herein, optionally further modified by N-terminal and / or C-terminal amino acid extensions. Such amino acid extensions are understood to be the addition of polypeptide sequences at the N-terminus and / or C-terminus of the polypeptide. For example, a polypeptide sequence may be fused to an α-1,3-fucosyltransferase polypeptide to achieve additional enzymatic activity. Such amino acid extensions may be specific tags and / or HQ-tags; extensions of up to 20 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids; such extensions may also be 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more amino acids in length. The optional N-terminal and / or C-terminal amino acid extension can also be a tag for purification, a tag for increasing the solubility of the polypeptide, a tag or amino acid extension for metabolite formation, a tag for protein metabolomics, a tag for substrate binding, another polypeptide with the same or different function in gene fusion, such as, but not limited to, a polypeptide encoding GDP-fucose synthase, a galactosyltransferase, a fucosyltransferase, a bifunctional fucokinase / fucose-1-phosphate guanylyltransferase or a fucose-1-phosphate guanylyltransferase, wherein the other polypeptide is optionally fused to the α-1,3-fucosyltransferase polypeptide via a peptide linker. For example, the α-1,3-fucosyltransferase polypeptide as described herein optionally includes one or more exogenous affinity tags, such as purification or substrate binding tags, such as a 6His tag sequence, a GST tag, an HQ tag, an HA tag sequence, multiple 6His tag sequences, multiple GST tags, multiple HA tag sequences, a SNAP tag, a SUMOstar tag. Other examples include a proteolytic cleavage site, a retention site, a cleavage site, a polyhistidine tag, biotin, avidin, a BiTag sequence, an S tag, an enterokinase site, a thrombin site, an antibody or antibody domain, an antibody fragment, an antigen, a receptor, a receptor domain, a receptor fragment, a ligand, a dye, an acceptor, a quencher, or a combination thereof.

[0107] In addition, α-1,3-fucosyltransferase polypeptides may include proteins or polypeptides that represent functionally equivalent polypeptides. Such equivalent α-1,3-fucosyltransferase polypeptides may comprise deletions, additions or substitutions of amino acid residues within the amino acid sequence encoded by the α-1,3-fucosyltransferase polynucleotides described herein, but which result in silent changes, thereby producing functionally equivalent α-1,3-fucosyltransferases. Amino acid substitutions may be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; planar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In the context of the present invention, "functional equivalent" as used herein refers to a polypeptide capable of exhibiting substantially similar in vivo activity to the lactose binding α-1,3-fucosyltransferase polypeptide of the present invention, as judged by any of a number of criteria including but not limited to enzyme activity.

[0108] Included within the scope of the invention are α-1,3-fucosyltransferase proteins, polypeptides and derivatives (including fragments) that are differentially modified during or after translation. In addition, non-classical amino acids or chemical amino acid analogs may be introduced as substitutions or additions into the α-1,3-fucosyltransferase polypeptide sequence.

[0109] α-1,3-fucosyltransferase polypeptides can be produced by expression of polynucleotides produced by recombinant DNA technology using techniques known in the art. Methods well known to those skilled in the art can be used to construct expression vectors comprising α-1,3-fucosyltransferase coding sequences and appropriate transcription and / or translation control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo genetic recombination. For example, see Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989 and updated annually). Alternatively, α-1,3-fucosyltransferase polypeptides can be produced by direct synthesis, by extracting cells that produce the polypeptide in nature or in a cell-free and / or in vitro system.

[0110] The suitability of the newly identified α-1,3-fucosyltransferases with the ability to bind lactose for producing 3-fucosyllactose and preferably producing such 3FL with a purity of 80% or more is very surprising, and therefore, their use represents an excellent tool to easily, efficiently and cost-effectively produce 3-fucosyllactose.

[0111] Polynucleotides encoding α-1,3-fucosyltransferase polypeptides can be produced via recombinant DNA techniques using techniques known in the art. Methods well known to those skilled in the art can be used to construct expression vectors comprising α-1,3-fucosyltransferase coding sequences and appropriate transcriptional and / or translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. For example, see Sambrook et al. (2001) Molecular Cloning: alaboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989 and updated annually).

[0112] According to another aspect of the present invention, a vector is provided, comprising a polynucleotide encoding a polypeptide having α-1,3-fucosyltransferase activity as described herein, wherein the polynucleotide is operably linked to a control sequence recognized by a host cell transformed with the vector. In a particularly preferred embodiment, the vector is an expression vector, and according to another aspect of the present invention, the vector may be in the form of a plasmid, a cosmid, a phage, a liposome or a virus.

[0113] Thus, the polynucleotide according to the invention may, for example, be contained in a vector that is stably transformed / transfected into a host cell. In the vector, the polynucleotide of the invention is controlled by a promoter. The promoter may, for example, be an inducible promoter, so that the expression of the gene / polynucleotide can be specifically targeted, and, if desired, the gene can be overexpressed in this way. The promoter may also be a constitutive promoter.

[0114] A variety of expression systems can be used for producing polypeptides of the present invention. These vectors especially include chromosomes, episomes and virus-derived vectors, for example, derived from bacterial plasmids, phages, transposons, yeast episomes, insertion elements, yeast chromosome elements, viral vectors, and vectors derived from their combinations, for example, those derived from plasmids and bacterial phage genetic elements, such as clay and phagemids. These vectors can include selection markers, such as but not limited to antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA justice / antisense markers. The expression system construct may include the control region for regulation and production of expression. Generally speaking, any system or vector suitable for maintaining, breeding or expressing polynucleotides and / or expressing polypeptides in a host can be used for expression in this regard. Suitable DNA sequence dna can be inserted into the expression system by any of various known and conventional techniques, for example, those described in Sambrook et al., see above.

[0115] For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the present invention. The introduction of polynucleotides into host cells can be accomplished by the methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986) and Sambrook et al., 1989, supra.

[0116] According to another aspect of the present invention, a host cell is provided, which comprises the vector as described above.

[0117] According to another aspect, the present invention provides a genetically modified host cell for producing alpha-1,3-fucosyllactose, wherein the host cell comprises at least one nucleic acid sequence encoding an enzyme for 3-fucosyllactose synthesis and wherein the cell comprises expression of a polypeptide having alpha-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate. The polypeptide is as described herein.

[0118] As used herein, the term "host cell" is now defined as a cell that has been transformed or transfected or is capable of being transformed or transfected by an exogenous polynucleotide sequence, and therefore comprises at least one sequence that does not naturally occur in said host cell.

[0119] A variety of host-expression vector systems can be used to express the α-1,3-fucosyltransferase polynucleotides of the present invention. Such host-expression systems represent vehicles that can produce and subsequently purify the target coding sequence, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, display the α-1,3-fucosyltransferase gene product of the present invention in situ.

[0120] According to another aspect of the present invention, a host cell for producing 3-fucosyllactose is provided, wherein the host cell comprises a sequence consisting of a polynucleotide encoding a polypeptide having lactose-binding α-1,3-fucosyltransferase activity, as described herein, wherein the sequence is a sequence exogenous to the host cell and wherein the sequence is integrated into the genome of the host cell. The polynucleotide is operably linked to a control sequence recognized by the host cell.

[0121] According to an alternative aspect of the present invention, there is provided a host cell for producing 3-fucosyllactose, wherein the host cell comprises a vector comprising the polynucleotide described herein, wherein the polynucleotide is operably linked to a control sequence recognized by the host cell transformed with the vector.

[0122] In another aspect, the present invention also provides a method for producing α-1,3-fucosyllactose, comprising the steps of: a) providing the cells described herein, and b) culturing the cells in a culture medium under conditions allowing production of α-1,3-fucosyltransferase.

[0123] Preferably, the α-1,3-fucosyltransferase is isolated from a culture as described herein. Preferably, it can also be purified as described herein.

[0124] In another aspect, the present invention provides use of the cell of the present invention for producing 3-fucosyllactose.

[0125] According to another aspect of the present invention, there is provided a microorganism expressing the α-1,3-fucosyltransferase described herein and preferably encoded by the polynucleotide described herein.

[0126] The term microorganism or organism or cell or host cell used herein refers to a microorganism selected from a list including bacteria, yeast or fungi, or refers to a plant or animal cell. The latter bacterium preferably belongs to the Proteobacteria phylum or the Firmicutes phylum or the Cyanobacteria phylum or the Deinococcus-Thermus phylum. The latter bacterium belonging to the Proteobacteria phylum preferably belongs to the Enterobacteriaceae family, preferably to the Escherichia coli species. The latter bacterium preferably relates to any strain belonging to the Escherichia coli species, such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to a cultivated Escherichia coli strain, referred to as the Escherichia coli K12 strain, which is very suitable for laboratory environments, and unlike wild-type strains, it has lost the ability to reproduce in the intestine. Well-known examples of E. coli K12 strains are K12 wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Therefore, the present invention particularly relates to E. coli host cells or strains mutated and / or transformed as described above, wherein the E. coli strain is a K12 strain. More preferably, the E. coli K12 strain is E. coli MG1655. The latter bacterium belonging to Firmicutes preferably belongs to Bacilli, preferably to Lactobacilliales, whose members are such as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacillales, whose members are such as from Bacillus, such as Bacillus subtilis or B. amyloliquefaciens. The latter bacterium belongs to Actinobacteria, preferably to Corynebacteriaceae, whose members are Corynebacterium glutamicum or C. afermentans, or to Streptomycetaceae, whose members are Streptomyces griseus or S. fradiae. The latter yeasts preferably belong to the phylum Ascomycota or Basidiomycota or Deuteromycota or Zygomycete.The latter yeast preferably belongs to the genus Saccharomyces, Pichia, Komagataella, Hansunella, Kluyveromyces, Yarrowia or Starmerella. The latter fungi preferably belong to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus.

[0127] According to another aspect of the present invention, the polynucleotide encoding the polypeptide having lactose-binding α-1,3-fucosyltransferase activity is suitable for the codon usage of the corresponding host cell.

[0128] Another aspect of the present invention provides the use of the polypeptide described herein for producing α-1,3-fucosyllactose. Another aspect of the present invention provides the use of the polynucleotide described herein or the vector described herein for producing α-1,3-fucosyllactose.

[0129] According to another embodiment, a hitherto unknown lactose-binding α-1,3-fucosyltransferase is provided. The present invention provides an isolated and / or synthetic polypeptide having lactose-binding α-1,3-fucosyltransferase activity, wherein the polypeptide comprises:

[0130] - an amino acid sequence encoding a conserved GDP-fucose binding domain [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R] (SEQ ID NO 33) and a conserved [K / D][L / K / M]XXX[F / Y] domain (SEQ ID NO 34), wherein if the domain is equal to DM[A / S]VSF (SEQ ID NO 36), then in the N-terminal region there is additionally a conserved motif [N / H]XDPAXLD (SEQ ID NO 35), wherein X can be any different amino acid, and the C-terminus of the amino acid sequence has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain.

[0131] Preferably, the polypeptide is selected from the group consisting of:

[0132] i) SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 in the attached sequence listing;

[0133] ii) an amino acid sequence having 87% or greater sequence identity to the full length of SEQ ID NO 2, 20 or 22;

[0134] iii) comprising an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any one of SEQ ID NO 6, 8, 10, 12, 14, 16, 28, 30 or 32;

[0135] iv) a fragment of the sequence shown in any one of SEQ ID NOs 2, 20 or 22, wherein the fragment comprises at least 45 consecutive amino acids thereof;

[0136] v) a fragment of the amino acid sequence shown in any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32, wherein the fragment comprises at least 10 consecutive amino acids thereof.

[0137] Optionally, the polypeptide is further modified by an N-terminal and / or C-terminal amino acid extension.

[0138] Within the scope of the present invention, an isolated and / or synthetic polypeptide has lactose binding α-1,3-fucosyltransferase activity. Such a polypeptide comprises an amino acid sequence encoding a conserved GDP-fucose binding domain [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R] (SEQ ID NO 33) and a conserved [K / D][L / K / M]XXX[F / Y] domain (SEQ ID NO 34), wherein if the domain is equal to DM[A / S]VSF (SEQ ID NO 36), then in addition there is a conserved motif [N / H]XDPAXLD (SEQ ID NO 35), wherein X can be any different amino acid and the C-terminus of the amino acid sequence has less than or equal to 100 amino acids starting from the first amino acid of the conserved GDP-fucose binding domain defined above, for example 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 amino acids.

[0139] Also included within the scope of the present invention are α1,3-fucosyltransferase polypeptides as described herein, optionally further modified by N-terminal and / or C-terminal amino acid extensions.

[0140] It has surprisingly been found that the newly identified lactose binding α-1,3-fucosyltransferase can be used to carry out a non-naturally occurring reaction. Furthermore, it has been found that the above identified α-1,3-fucosyltransferase is able to use lactose as a substrate, has similar or higher lactose binding properties than currently known α-1,3-fucosyltransferases, and is able to produce 3-fucosyllactose.

[0141] Until today, as shown in Table 1, the newly identified fucosyltransferase of the present invention has not been described to have lactose-binding α-1,3-fucosyltransferase activity.

[0142] Table 1:

[0143]

[0144] As shown in Table 2, newly identified α-1,3-fucosyltransferases having the ability to bind lactose and used to produce 3-fucosyllactose were also found, all of which have the same special feature, namely, having an amino acid sequence comprising a conserved GDP-fucose binding domain [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R] (SEQ ID NO 33), wherein x can be any different amino acid and wherein the C-terminus of the amino acid sequence has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain. This is in contrast to known lactose-binding 3-fucosyltransferases, which have a C-terminus longer than 100 amino acids starting from the first amino acid of the GDP-fucose binding domain defined above, such as those described in WO2012 / 049083.

[0145] Table 2:

[0146]

[0147] In addition, it was found that the polypeptide sequences of SEQ ID NO 6, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14 and SEQ ID NO 16 share the domain PENXXXXXXXTEK (SEQ ID NO 37), wherein X can be any different amino acid, such as Figure 1All alignments were performed using MAFFT v7.307 and visualization using Jalview 2.10.

[0148] Furthermore, it was found that, in addition to the conserved GDP-fucose binding domain [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R] (SEQ ID NO 33), the newly identified polypeptides all share a common conserved motif [K / D][L / K / M]XXX[F / Y] (SEQ ID NO 34), wherein X can be any different amino acid, and a conserved amino acid region [FW]W that is important for lactose binding, e.g. Fig.11 As shown, the domain is boxed.

[0149] Furthermore, we note that when this common feature is DM[A / S]VSF (SEQ ID NO 36), additionally a conserved [N / H]XDPAXLD (SEQ ID NO 35) motif is required in the N-terminal domain of the protein, where X can be any different amino acid, in order for the enzyme to have α-1,3-fucosyltransferase activity towards lactose as an acceptor substrate, e.g. Fig.12 As shown in the alignment of , wherein the domain is boxed. As exemplified in Example 14, polypeptides having SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 20 and SEQ ID NO 22 contain two consensus motifs and appear to have α-1,3-fucosyltransferase activity towards lactose as an acceptor substrate, whereas polypeptides having SEQ ID NO 24 and SEQ ID NO 26 do not contain the N-terminal [NH]XDPAXLD motif (where X can be any different amino acid (SEQ ID NO 35)) and do show such activity.

[0150] In addition, it was found that the polypeptide sequences of SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 28, SEQ ID NO 30 and SEQ ID NO 32 share the structural domains K[IV]F[FL]XGEN (SEQ ID NO 41) and RFPLW (SEQ ID NO 42), wherein x can be any different amino acid, such as Fig.13 , where the domain is boxed.

[0151] In a second embodiment, the present invention also relates to an isolated and / or synthetic polynucleotide encoding a polypeptide having lactose-binding α-1,3-fucosyltransferase activity as described above.

[0152] Within the scope of the present invention, the polynucleotide may be an allelic variant of a polynucleotide encoding any one of the amino acid sequences shown in SEQ ID NOs 2, 6, 8, 10, 12, 14, 16, 20, 22, 28, 30, 32.

[0153] Therefore, the present invention also relates to an isolated and / or synthetic polynucleotide, which encodes a polypeptide having α-1,3-fucosyltransferase activity and comprises a sequence selected from the group consisting of: a) SEQ ID NO 1, 5, 7, 9, 11, 13, 15, 19, 21, 27, 29, 31 in the attached sequence listing; b) a nucleic acid sequence complementary to SEQ ID NO 1, 5, 7, 9, 11, 13, 15, 19, 21, 27, 29, 31; c) a nucleic acid sequence having 80% or higher sequence identity with SEQ ID NO 1, 5, 7, 9, 11, 13, 15, 19, 21, 27, 29, 31.

[0154] Therefore, the present invention also relates to 3-fucosyllactose obtained by the method according to the present invention, and the use of the polynucleotides, vectors, host cells, microorganisms or polypeptides as described above for producing 3-fucosyllactose. α-1,3-fucosyllactose can be used as a food additive, a prebiotic, a symbiotic, for supplementing infant food, adult food or feed, or as a therapeutic or pharmaceutically active compound. By these new methods, α-1,3-fucosyllactose can be easily and efficiently provided without the need for complex, time-consuming and costly synthesis processes.

[0155] Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings as those of ordinary skill in the art to which the invention belongs. In general, the terms used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry described in the context are well known and commonly used in the art. Standard techniques are used for the synthesis of nucleic acids and peptides. Typically, enzymatic reactions and purification steps are carried out according to the manufacturer's instructions.

[0156] Further advantages emerge from the detailed description, examples and figures.

[0157] It goes without saying that the features mentioned above and those still to be explained below can be used not only in the respectively specified combination but also in other combinations or alone, without departing from the scope of the present invention.

[0158] The present invention relates to the following specific embodiments:

[0159] 1. A method for producing α-1,3-fucosyllactose, the method comprising the following steps:

[0160] a) providing a polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate, wherein the polypeptide comprises

[0161] i) an amino acid sequence encoding a conserved GDP-fucose binding domain [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R] (SEQ ID NO 33);

[0162] ii) an amino acid sequence encoding a conserved [K / D][L / K / M]XXX[F / Y] domain (SEQ ID NO 34), and

[0163] iii) wherein if the domain of ii) is equal to DM[A / S]VSF (SEQ ID NO 36), then additionally in the N-terminal region the conserved motif [N / H]XDPAXLD (SEQ ID NO 35) is present;

[0164] wherein X can be any different amino acid; and

[0165] wherein the C-terminus of the polypeptide has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain;

[0166] b) contacting the polypeptide having α-1,3-fucosyltransferase activity of step a) with a mixture comprising GDP-fucose as a donor substrate and lactose as an acceptor substrate under conditions wherein the polypeptide catalyzes the transfer of a fucose residue from a donor substrate to an acceptor substrate,

[0167] Thus, α-1,3-fucosyllactose is produced.

[0168] c) optionally isolating the α-1,3-fucosyllactose.

[0169] 2. The method according to embodiment 1, wherein the polypeptide is provided in a cell-free system.

[0170] 3. The method according to embodiment 1, wherein the polypeptide is produced by a cell comprising a polynucleotide encoding the polypeptide.

[0171] 4. The method according to any one of embodiments 1 or 3, wherein the GDP-fucose and / or lactose is provided by cells that produce the GDP-fucose and / or lactose.

[0172] 5. The method according to any one of embodiments 1, 3 or 4, comprising the following steps:

[0173] i) providing a cell genetically modified to produce α-1,3-fucosyllactose, said cell comprising at least one nucleic acid sequence encoding an enzyme for the synthesis of α-1,3-fucosyllactose,

[0174] The cell comprises expression of the polypeptide having alpha-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate,

[0175] ii) culturing the cells in a culture medium under conditions that allow production of α-1,3-fucosyllactose,

[0176] iii) preferably isolating α-1,3-fucosyllactose from the culture.

[0177] 6. The method according to embodiment 3, comprising the following steps:

[0178] a) providing a host cell expressing the polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate;

[0179] b) growing the host cell under suitable nutritional conditions that allow the production of α-1,3-fucosyllactose and the expression of the polypeptide having α-1,3-fucosyltransferase activity;

[0180] c) simultaneously with or after step b), providing a donor substrate GDP-fucose and an acceptor substrate lactose, such that the α-1,3-fucosyltransferase polypeptide catalyzes the transfer of fucose residues from GDP-fucose to lactose, thereby producing α-1,3-fucosyllactose;

[0181] d) optionally isolating the α-1,3-fucosyllactose from the host cell or the culture medium in which it is grown.

[0182] 7. The method according to any one of embodiments 5 or 6, wherein the host cell is transformed or transfected to express an exogenous polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate.

[0183] 8. The method according to any one of embodiments 3 to 7, characterized in that GDP-fucose and / or lactose are provided by enzymes simultaneously expressed in the host cell or by metabolism of the host cell.

[0184] 9. The method according to any one of embodiments 1 to 8, further comprising purification of α-1,3-fucosyllactose.

[0185] 10. The method according to any one of the preceding embodiments, wherein the polypeptide is selected from the group consisting of:

[0186] i) any one of SEQ ID NO 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 in the attached sequence listing;

[0187] ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO 2, 20 or 22;

[0188] iii) an amino acid sequence having 80% or greater sequence identity to the full-length amino acid sequence of any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32;

[0189] iv) a fragment of the amino acid sequence shown in SEQ ID NO 2, 20 or 22, wherein the fragment comprises at least 45 consecutive amino acids thereof;

[0190] v) a fragment of the amino acid sequence shown in any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32, wherein the fragment comprises at least 10 consecutive amino acids thereof and has lactose-binding α-1,3-fucosyltransferase activity;

[0191] Optionally, the polypeptide is further modified by an N-terminal and / or C-terminal amino acid extension.

[0192] 11. The method for producing 3-fucosyllactose according to any one of the preceding embodiments, further comprising at least one of the following steps:

[0193] i) adding a lactose feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g lactose per initial reactor volume to the culture medium, preferably in a continuous manner and preferably such that the final volume of the culture medium is not more than 3 times, preferably not more than 2 times, more preferably less than 2 times the volume of the culture medium before the addition of the lactose feed;

[0194] ii) adding a lactose feed to the culture medium via a feed solution in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, and 5 days;

[0195] iii) adding lactose feed to the culture medium in a continuous manner via a feed solution during 1 day, 2 days, 3 days, 4 days, 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably 550 g / L, most preferably 600 g / L; and wherein the pH of the solution is preferably set at 3 to 7 and wherein the temperature of the feed solution is preferably maintained at 20°C to 80°C;

[0196] iv) the method results in a 3-fucosyllactose concentration in the final volume of the culture medium of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L.

[0197] 12. A host cell genetically modified to produce α-1,3-fucosyllactose, wherein the host cell comprises at least one nucleic acid sequence encoding an enzyme involved in the synthesis of α-1,3-fucosyl sugar; the cell comprises expressing a polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate, wherein the polypeptide comprises:

[0198] i) an amino acid sequence encoding a conserved GDP-fucose binding domain [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R] (SEQ ID NO 33);

[0199] ii) an amino acid sequence encoding a conserved [K / D][L / K / M]XXX[F / Y] domain (SEQ ID NO 34), and

[0200] iii) wherein if the domain of ii) is equal to DM[A / S]VSF (SEQ ID NO 36), then additionally in the N-terminal region the conserved motif [N / H]XDPAXLD (SEQ ID NO 35) is present;

[0201] wherein X can be any different amino acid; and

[0202] The C-terminus of the polypeptide has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain.

[0203] 13. A cell according to embodiment 12, wherein the host cell comprises: i) a sequence comprising a polynucleotide encoding the polypeptide having lactose-binding α-1,3-fucosyltransferase activity, wherein the sequence is an exogenous sequence to the host cell, and wherein the sequence is integrated into the genome of the host cell, or ii) a vector comprising the polynucleotide encoding the polypeptide, wherein the polynucleotide is operably linked to a control sequence recognized by a host cell transfected with the vector.

[0204] 14. The cell according to any one of embodiments 12 or 13, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:

[0205] i) any one of SEQ ID NO 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 in the attached sequence listing;

[0206] ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO 2, 20 or 22;

[0207] iii) an amino acid sequence having 80% or greater sequence identity to the full-length amino acid sequence of any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32;

[0208] iv) a fragment of the amino acid sequence shown in SEQ ID NO 2, 20 or 22, wherein the fragment comprises at least 45 consecutive amino acids thereof;

[0209] v) a fragment of the amino acid sequence shown in any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32, wherein the fragment comprises at least 10 consecutive amino acids thereof and has lactose-binding α-1,3-fucosyltransferase activity;

[0210] Optionally, the polypeptide is further modified by an N-terminal and / or C-terminal amino acid extension.

[0211] 15. The method according to any one of embodiments 3 to 11 or the cell according to any one of embodiments 12, 13 or 14, wherein the cell is selected from the group consisting of a microorganism, a plant or an animal cell, preferably the microorganism is a bacterium, a fungus or a yeast, preferably the plant is rice, cotton, rapeseed, soybean, corn or a cereal plant, preferably the animal is an insect, a fish, a bird or a non-human mammal; preferably the cell is an E. coli cell.

[0212] 16. A host cell according to any one of embodiments 12 to 15, characterised in that the host cell is a bacterium, preferably a cell of an Escherichia coli strain, more preferably a K12 strain of an Escherichia coli strain, even more preferably the Escherichia coli K12 strain is Escherichia coli MG1655.

[0213] 17. The host cell according to any one of embodiments 12 to 15, characterized in that the host cell is a yeast cell.

[0214] 18. The host cell according to any one of embodiments 12 to 17, characterized in that the polynucleotide encoding the polypeptide having lactose-binding α-1,2-fucosyltransferase activity is adapted to the codon usage of the corresponding host cell.

[0215] 19. A method for producing α-1,3-fucosyllactose, comprising the following steps:

[0216] a) providing a cell according to any one of embodiments 12 to 18,

[0217] b) culturing the cells in a culture medium under conditions that allow production of α-1,3-fucosyltransferase,

[0218] c) Preferably, the α-1,3-fucosyltransferase is isolated from the culture.

[0219] 20. Use of the host cell according to any one of embodiments 12 to 18 for the production of α-1,3-fucosyllactose.

[0220] 21. Use of the polypeptide as described in any one of embodiments 1 or 11 for producing α-1,3-fucosyllactose.

[0221] 22. A microorganism heterologously expressing a lactose-binding α-1,3-fucosyltransferase polypeptide, wherein the polypeptide comprises:

[0222] i) an amino acid sequence encoding a conserved GDP-fucose binding domain [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R] (SEQ ID NO 33);

[0223] ii) an amino acid sequence encoding a conserved [K / D][L / K / M]XXX[F / Y] domain (SEQ ID NO 34), and

[0224] iii) wherein if the domain of ii) is equal to DM[A / S]VSF (SEQ ID NO 36), then additionally in the N-terminal region the conserved motif [N / H]XDPAXLD (SEQ ID NO 35) is present;

[0225] wherein X can be any different amino acid; and

[0226] The C-terminus of the polypeptide has less than or equal to 100 amino acids starting from the first amino acid of the GDP-fucose binding domain.

[0227] 23. The microorganism according to embodiment 22, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:

[0228] i) any one of SEQ ID NO 6, 2, 4, 8, 10, 12, 14, 16, 20, 22, 28, 30 or 32 in the attached sequence listing;

[0229] ii) an amino acid sequence having 87% or greater sequence identity with the full-length amino acid sequence of SEQ ID NO 2, 20 or 22;

[0230] iii) an amino acid sequence having 80% or greater sequence identity to the full-length amino acid sequence of any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32;

[0231] iv) a fragment of the amino acid sequence shown in SEQ ID NO 2, 20 or 22, wherein the fragment comprises at least 45 consecutive amino acids thereof;

[0232] v) a fragment of the amino acid sequence shown in any one of SEQ ID NOs 6, 8, 10, 12, 14, 16, 28, 30 or 32, wherein the fragment comprises at least 10 consecutive amino acids thereof and has lactose-binding α-1,3-fucosyltransferase activity;

[0233] Optionally, the polypeptide is further modified by an N-terminal and / or C-terminal amino acid extension.

[0234] 24. Use of the microorganism according to embodiment 22 or 23 for producing α-1,3-fucosyllactose.

[0235] 25. The method according to any one of embodiments 1 to 11, 15 or 19, further comprising the step of isolating the α-1,3-fucosyllactose from the host cell or the culture medium in which it is grown.

[0236] 26. The method according to any one of embodiments 1 to 11, 15, 19 or 25, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and / or gel filtration, ligand exchange chromatography.

[0237] 27. The method of any one of embodiments 1 to 11, 15, 19, 25 or 26, further comprising purification of α-1,3-fucosyllactose.

[0238] 28. The method of embodiment 27, wherein the purification of the α-1,3-fucosyllactose comprises at least one of the following steps: using activated charcoal or carbon, using charcoal, nanofiltration, ultrafiltration or ion exchange, using alcohol, using an aqueous alcohol mixture, crystallization, evaporation, precipitation, drying, spray drying or freeze drying.

[0239] 29. The method of any one of embodiments 1 to 11, 15, 19, 25 to 28, wherein the polypeptide is produced in a fungal, yeast, bacterial, insect, animal or plant expression system.

[0240] 30. The method according to embodiment 29, wherein the host cell is a bacterium, preferably a cell of an E. coli strain, more preferably a K12 strain of E. coli, even more preferably the E. coli K12 strain is E. coli MG1655.

[0241] 31. The method of embodiment 29, wherein the host cell is a yeast cell.

[0242] 32. The method of any one of embodiments 1 to 11, 15, 19, 25 to 31, wherein the lactose concentration in the culture medium is in the range of 50 to 150 g / L.

[0243] 33. The method of any one of embodiments 1 to 11, 15, 19, 25 to 32, wherein the final concentration of 3-fucosyllactose is in the range of 70 g / L to 200 g / L.

[0244] 34. The method of any one of embodiments 1 to 11, 15, 19, 25 to 33, wherein the production results in a ratio of the concentration of lactose to the concentration of 3-fucosyllactose of less than 1:5 at the end of the fermentation.

[0245] 35. The method of any one of embodiments 1 to 11, 15, 19, 25 to 34, wherein the production results in a 3-fucosyllactose purity of 80% or more at the end of the fermentation.

[0246] 36. A method for producing α-1,3-fucosyllactose, the method comprising the following steps:

[0247] a) providing a polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate,

[0248] b) contacting the polypeptide having α-1,3-fucosyltransferase activity of step a) with a mixture comprising GDP-fucose as a donor substrate and lactose as an acceptor substrate under conditions wherein the polypeptide catalyzes the transfer of a fucose residue from a donor substrate to an acceptor substrate,

[0249] Thus, α-1,3-fucosyllactose is produced.

[0250] c) wherein the catalysis results in a ratio of lactose concentration to 3-fucosyllactose concentration of less than 1:5 at the end of the fermentation,

[0251] d) optionally isolating the α-1,3-fucosyllactose.

[0252] 37. A method for producing α-1,3-fucosyllactose, the method comprising the following steps:

[0253] a) providing a polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate,

[0254] b) contacting the polypeptide having α-1,3-fucosyltransferase activity of step a) with a mixture comprising GDP-fucose as a donor substrate and lactose as an acceptor substrate under conditions wherein the polypeptide catalyzes the transfer of a fucose residue from a donor substrate to an acceptor substrate,

[0255] Thus, α-1,3-fucosyllactose is produced.

[0256] c) wherein said catalysis results in a 3-fucosyllactose purity of 80% or more at the end of the fermentation,

[0257] d) optionally isolating the α-1,3-fucosyllactose.

[0258] 38. A method for producing 3-fucosyllactose, comprising at least one of the following steps:

[0259] i) adding a lactose feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g lactose per initial reactor volume to the culture medium, preferably in a continuous manner and preferably such that the final volume of the culture medium is not more than 3 times, preferably not more than 2 times, more preferably less than 2 times the volume of the culture medium before the addition of the lactose feed;

[0260] ii) adding a lactose feed to the culture medium via a feed solution in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, and 5 days;

[0261] iii) adding lactose feed to the culture medium in a continuous manner via a feed solution during 1 day, 2 days, 3 days, 4 days, 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably 550 g / L, most preferably 600 g / L; and wherein the pH of the solution is preferably set at 3 to 7 and wherein the temperature of the feed solution is preferably maintained at 20°C to 80°C;

[0262] The method results in a 3-fucosyllactose concentration in the final volume of the culture medium of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L, and preferably the ratio of lactose concentration to 3FL concentration in the final volume of the culture is lower than 1:5, more preferably 1:10, even more preferably 1:20, most preferably 1:40.

[0263] 39. A method for producing 3-fucosyllactose, comprising at least one of the following steps:

[0264] i) adding a lactose feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g lactose per initial reactor volume to the culture medium, preferably in a continuous manner and preferably such that the final volume of the culture medium is not more than 3 times, preferably not more than 2 times, more preferably less than 2 times the volume of the culture medium before the addition of the lactose feed;

[0265] ii) adding a lactose feed to the culture medium via a feed solution in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, and 5 days;

[0266] iii) adding lactose feed to the culture medium in a continuous manner via a feed solution during 1 day, 2 days, 3 days, 4 days, 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably 550 g / L, most preferably 600 g / L; and wherein the pH of the solution is preferably set at 3 to 7 and wherein the temperature of the feed solution is preferably maintained at 20°C to 80°C;

[0267] The method results in a 3-fucosyllactose concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the culture medium, and preferably a 3FL purity of 80% or more in the final volume of the culture.

[0268] The following figures and examples will serve as further illustration and clarification of the present invention and are not intended to limit the present invention. Sequence Listing <110> IMBIOS <120> α-1,3-Fucosyltransferase for the production of 3-fucosyllactose and inversion of lactose <130> 005-PCT <160> 42 <170> PatentIn version 3.5 <210> 1 <211> 978 <212> DNA <213> Azospirillum oryzae <400> 1 atgattgatc agcgtaccag cgactttcta agcgagttcc tggcgtcaag ccaccgtgat 60 ccggcgcgtt tagatagctt cctgcttcac gggccaggcc gcggcgctcg cgcggcgaaa 120 ccgcgtctga aaattgcgtt ttttgacttt tggccggaat ttgacccggc tgcgaacttt 180 tttgtggaca ttctgagcgc gcgctttgat gtaagcgttg tggataacga tagcgactta 240 gcgattgtga gcgtgtttgg cacccgccat cgcgaagcgc gtaccgcgcg tagcatgttt 300 tttaccggcg aaaatgtgcg tccgccgtta gatggcgtgg atatgagcgt gagctttgat 360 cgcattgatg acccacgcca ctatcgcctg ccgttatatg tgatgcacgc gtgggatcat 420 aggcgtgaag gggcgacgcc gcacttttgc cagagcgtgc tgccgccggt gccgccgacg 480 cgtgaagaag cggcgaaacg taagttttgc gcgtttttat ataaaaatcc aaactgcgcg 540 aggcgcaacg actttttcca gatgttatgc gcgaggcgcc acgtggaaag cgtgggctgg 600 ctgctgaata ataccggcag cgtggtgaaa atgggctggc tgccgaaaat tcgtgtgttc 660 agccgctacc gctttgcgtt tgcgtttgaa aatgcgagcc acccaggcta cctgacggag 720 aaaattttag atgcgtttca agcgggcgcg gtgccgttat attggggcga cccaggagtg 780 ctgcgcgacg ttgcggccgg cagctttatt gatgtgagcc gctacgcgag cgatgaagaa 840 gcgtgcgacg cgattctggc cgcggatgat gattacgata cctaccgccg ctaccgcagc 900 acgccgccgt ttctgggcgc ggaagatttt tactttgatg cgtttcggct ggccgagtgg 960 attgagagcc gtctataa 978 <210> 2 <211> 325 <212> PRT <213> Azospirillum oryzae <400> 2 Met Ile Asp Gln Arg Thr Ser Asp Phe Leu Ser Glu Phe Leu Ala Ser 1 5 10 15 Ser His Arg Asp Pro Ala Arg Leu Asp Ser Phe Leu Leu His Gly Pro 20 25 30 Gly Arg Gly Ala Arg Ala Ala Lys Pro Arg Leu Lys Ile Ala Phe Phe 35 40 45 Asp Phe Trp Pro Glu Phe Asp Pro Ala Ala Asn Phe Phe Val Asp Ile 50 55 60 Leu Ser Ala Arg Phe Asp Val Ser Val Val Asp Asn Asp Ser Asp Leu 65 70 75 80 Ala Ile Val Ser Val Phe Gly Thr Arg His Arg Glu Ala Arg Thr Ala 85 90 95 Arg Ser Met Phe Phe Thr Gly Glu Asn Val Arg Pro Pro Leu Asp Gly 100 105 110 Val Asp Met Ser Val Ser Phe Asp Arg Ile Asp Asp Pro Arg His Tyr 115 120 125 Arg Leu Pro Leu Tyr Val Met His Ala Trp Asp His Arg Arg Glu Gly 130 135 140 Ala Thr Pro His Phe Cys Gln Ser Val Leu Pro Pro Val Pro Pro Thr 145 150 155 160 Arg Glu Glu Ala Ala Lys Arg Lys Phe Cys Ala Phe Leu Tyr Lys Asn 165 170 175 Pro Asn Cys Ala Arg Arg Asn Asp Phe Phe Gln Met Leu Cys Ala Arg 180 185 190 Arg His Val Glu Ser Val Gly Trp Leu Leu Asn Asn Thr Gly Ser Val 195 200 205 Val Lys Met Gly Trp Leu Pro Lys Ile Arg Val Phe Ser Arg Tyr Arg 210 215 220 Phe Ala Phe Ala Phe Glu Asn Ala Ser His Pro Gly Tyr Leu Thr Glu 225 230 235 240 Lys Ile Leu Asp Ala Phe Gln Ala Gly Ala Val Pro Leu Tyr Trp Gly 245 250 255 Asp Pro Gly Val Leu Arg Asp Val Ala Ala Gly Ser Phe Ile Asp Val 260 265 270 Ser Arg Tyr Ala Ser Asp Glu Glu Ala Cys Asp Ala Ile Leu Ala Ala 275 280 285 Asp Asp Asp Tyr Asp Thr Tyr Arg Arg Tyr Arg Ser Thr Pro Pro Phe 290 295 300 Leu Gly Ala Glu Asp Phe Tyr Phe Asp Ala Phe Arg Leu Ala Glu Trp 305 310 315 320 Ile Glu Ser Arg Leu 325 <210> 3 <211> 978 <212> DNA <213> Azospirillum lipoferum <400> 3 atgattgatc agcgtacgag cgattttctg agcgagtttc tagcgtcacc aaaccgtgat 60 ccagcggtgt tagatcggtt tctgcttcat gggccggagc gcgggggccg tgcggccaga 120 ccgcgcctga aaattgcgtt ttttgacttt tggccggaat ttgacccgag cgcgaatttt 180 tttgtagaaa ttctgagcag ccgctttgat gtgagcgtgg ttgataatga tagcgattta 240 gcgattctga gcgtgtttgg cgaacgccac cgcgaagcgc gtaccgcgcg cgcgctgttt 300 tttaccggcg aaaatgtgcg cccgccgtta gatggcgtgg atatgagcgt gagctttgat 360 cgcattgatc atccacgtca ttatcgcctg ccgttatacg tgatgcatgc gtgggatcac 420 cgtcgcgaag gggcgacccc gcatttttgc catccggtgc tgccgccggt gccgccgacg 480 cgtgaagaag cggcgaaacg taagttttgc gcgtttttat ataaaaatcc tcactgcgcg 540 cgccgcaacg atttttttca gatgctgtgc gcgcggcgcc atgtggaaag cgtgggctgg 600 ctgctgaata ataccggcag cgtggtgaaa atgggctggc tgccgaaaat tcgcgtgttt 660 gcgcgctatc gctttgcgtt tgcgtttgaa aacgcggcgc atccaggcta tctgaccgag 720 aaaattctgg atgcgtttca ggccgggacg gtaccgttat actggggcga cagcggcgtg 780 ctgcgcgacg ttgcggccgg cagctttatt gatgtgagcc gctatgcgag cgatgaagaa 840 gcgattgaag cgattctggc gattgatgat gactatgata gctatcgccg gtaccgcggc 900 acggcgccat ttttaggcac cgaggacttt tactttgacg cgtaccggct ggccgagtgg 960 attgagagcc gcctgtag 978 <210> 4 <211> 325 <212> PRT <213> Azospirillum lipoferum <400> 4 Met Ile Asp Gln Arg Thr Ser Asp Phe Leu Ser Glu Phe Leu Ala Ser 1 5 10 15 Pro Asn Arg Asp Pro Ala Val Leu Asp Arg Phe Leu Leu His Gly Pro 20 25 30 Glu Arg Gly Gly Arg Ala Ala Arg Pro Arg Leu Lys Ile Ala Phe Phe 35 40 45 Asp Phe Trp Pro Glu Phe Asp Pro Ser Ala Asn Phe Phe Val Glu Ile 50 55 60 Leu Ser Ser Arg Phe Asp Val Ser Val Val Asp Asn Asp Ser Asp Leu 65 70 75 80 Ala Ile Leu Ser Val Phe Gly Glu Arg His Arg Glu Ala Arg Thr Ala 85 90 95 Arg Ala Leu Phe Phe Thr Gly Glu Asn Val Arg Pro Pro Leu Asp Gly 100 105 110 Val Asp Met Ser Val Ser Phe Asp Arg Ile Asp His Pro Arg His Tyr 115 120 125 Arg Leu Pro Leu Tyr Val Met His Ala Trp Asp His Arg Arg Glu Gly 130 135 140 Ala Thr Pro His Phe Cys His Pro Val Leu Pro Pro Val Pro Pro Thr 145 150 155 160 Arg Glu Glu Ala Ala Lys Arg Lys Phe Cys Ala Phe Leu Tyr Lys Asn 165 170 175 Pro His Cys Ala Arg Arg Asn Asp Phe Phe Gln Met Leu Cys Ala Arg 180 185 190 Arg His Val Glu Ser Val Gly Trp Leu Leu Asn Asn Thr Gly Ser Val 195 200 205 Val Lys Met Gly Trp Leu Pro Lys Ile Arg Val Phe Ala Arg Tyr Arg 210 215 220 Phe Ala Phe Ala Phe Glu Asn Ala Ala His Pro Gly Tyr Leu Thr Glu 225 230 235 240 Lys Ile Leu Asp Ala Phe Gln Ala Gly Thr Val Pro Leu Tyr Trp Gly 245 250 255 Asp Ser Gly Val Leu Arg Asp Val Ala Ala Gly Ser Phe Ile Asp Val 260 265 270 Ser Arg Tyr Ala Ser Asp Glu Glu Ala Ile Glu Ala Ile Leu Ala Ile 275 280 285 Asp Asp Asp Tyr Asp Ser Tyr Arg Arg Tyr Arg Gly Thr Ala Pro Phe 290 295 300 Leu Gly Thr Glu Asp Phe Tyr Phe Asp Ala Tyr Arg Leu Ala Glu Trp 305 310 315 320 Ile Glu Ser Arg Leu 325 <210> 5 <211> 990 <212> DNA <213> Basel psittacipulmonis <400> 5 atgtcagtgc tgaaaaaact ggtaagaacg ctgaaaaaga aaaaagatat tcccagcgaa 60 aatcaggaag atattaagcc gcaggaattt ggacatatca aacattatca tttctggcct 120 ctgtcgaacg aaacgttttt taaccaattt gcgcaggaaa aaaaccttga tctgagccag 180 acggccctaa ttagctgctt tggggaacta tcagcgattc ctaaaatccc tgaacggtat 240 aaagtgtttt ttacggggga aaatatctat catccggatc gcatctcata ttcggatccg 300 gagctctatc gcatggtgga tctgtattta ggctttgaat accggacgga accgaagtat 360 ctacgctttc cgctgtgggt ttggtattta tgcggcctga cgaaaaaacc gcattttca 420 catgaatcaa ttgcggaatt tattcggaaa atgaccac ctgagtttcg cctgcagtca 480 tcaggaatc gcttttgcag ccatattagc agccatgata cgaacggcat tcggaaacgg 540 atgattgatt taatttacc gattgcgtca gtggattgcg ccggcaagtt tatgacaac 600 acggatgagt taaagcgaa gtttaatgat cavagatcg actactaaa acagtatcgg 660 tttaatctct gtcctgaaaa ctcagaatca gtcggtata tcacggaaaa gatttttgaa 720 tcaattgg ccgggtgcat tcaatttat tggggag tcagcagct ttttgtcga ccggatattt taaatccgga agcattatt tactacgaa aagggaaag aagcaatta 840 gcgaaacagg tgaagact ttggatatca cctaaacggt atgagagtt tgcagcaatt 900 gcccgttta agaggacgc agccgaagtg atttatacgt ggattgaaga actggaaaaa 960 cggctacgggg cattgacc aaagcctaa 990 <210> 6 <211> 329 <212> PRT <213> Basel psittacipulmonas <400> 6 Met Ser Val Leu Lys Lys Leu Val Arg Thr Leu Lys Lys Lys Lys Asp 1 5 10 15 Ile Pro Ser Glu Asn Gln Glu Asp Ile Lys Pro Gln Glu Phe Gly His 20 25 30 Ile Lys His Tyr His Phe Trp Pro Leu Ser Asn Glu Thr Phe Phe Asn 35 40 45 Gln Phe Ala Gln Glu Lys Asn Leu Asp Leu Ser Gln Thr Ala Leu Ile 50 55 60 Ser Cys Phe Gly Glu Leu Ser Ala Ile Pro Lys Ile Pro Glu Arg Tyr 65 70 75 80 Lys Val Phe Phe Thr Gly Glu Asn Ile Tyr His Pro Asp Arg Ile Ser 85 90 95 Tyr Ser Asp Pro Glu Leu Tyr Arg Met Val Asp Leu Tyr Leu Gly Phe 100 105 110 Glu Tyr Arg Thr Glu Pro Lys Tyr Leu Arg Phe Pro Leu Trp Val Trp 115 120 125 Tyr Leu Cys Gly Leu Thr Lys Lys Pro His Phe Ser His Glu Ser Ile 130 135 140 Ala Glu Phe Ile Arg Lys Met Asn Gln Pro Glu Phe Arg Leu Gln Ser 145 150 155 160 Ser Arg Asn Arg Phe Cys Ser His Ile Ser Ser His Asp Thr Asn Gly 165 170 175 Ile Arg Lys Arg Met Ile Asp Leu Ile Leu Pro Ile Ala Ser Val Asp 180 185 190 Cys Ala Gly Lys Phe Met Asn Asn Thr Asp Glu Leu Lys Ala Lys Phe 195 200 205 Asn Asp Asp Lys Ile Asp Tyr Leu Lys Gln Tyr Arg Phe Asn Leu Cys 210 215 220 Pro Glu Asn Ser Glu Ser Val Gly Tyr Ile Thr Glu Lys Ile Phe Glu 225 230 235 240 Ser Ile Met Ala Gly Cys Ile Pro Ile Tyr Trp Gly Gly Val Lys Gln 245 250 255 Leu Phe Val Glu Pro Asp Ile Leu Asn Pro Glu Ala Phe Ile Tyr Tyr 260 265 270 Glu Lys Gly Lys Glu Glu Gln Leu Ala Lys Gln Val Glu Glu Leu Trp 275 280 285 Ile Ser Pro Lys Arg Tyr Glu Glu Phe Ala Ala Ile Ala Pro Phe Lys 290 295 300 Glu Asp Ala Ala Glu Val Ile Tyr Thr Trp Ile Glu Glu Leu Glu Lys 305 310 315 320 Arg Leu Arg Ala Phe Glu Pro Lys Ala 325 <210> 7 <211> 930 <212> DNA <213> Planctopirus limnophila <400> 7 atgcaaaaaa ctttttctct acgtccagtc ccagtggatg caattcgatg gaactttgca 60 aacttttggt cgggttcga tgcattagca ttcgagcggc acctgttagg tgtctctggg 120 aaacataagt tccggattag tgaacaaaac cctcagattg tgttcgaatc ggtctttggg 180 actccaggga aggggcgcga gcgatggcca aaggcacgac aggtgtggta tacggggagaa 240 aacgtcgcac caccactgga tcagtttgat aaatgtttat cgttccatcg ggatattaag 300 gatccacggc atttgcgttg gccatactat ctactgcact tagcaagctt accaatgtct 360 ttcaatgatt tggtgaagtg ccagagctca gtgtcgactt gggcagaacg tccagggttc 420 tgtgcattta ttgcatttaa cgagggatgc cagacgcgga accggtttgt ggaaaagcta 480 agtcgatacc gtcgagtgga ttgtccaggt cgggtcttaa atatatgac gagtgagaca 540 ctgggtcagc gagggaactt gcatgggaaa attaacttcc tgaagcaata taatacgca 600 gtgtgcttcg agaatactag cacgcgagga tcagaagggt atgtgacgga aaagctggtt 660 gacgcgatgc tggcaggctg cataccatta tactggggcg accaccgggt tggggaagat 720 tttaacgaga actcgttcat taacttagga gtatacggga acgatgtgaa tgcaatggtc 780 cagcatgtga ttgaactgga ttctgacgaa cggttgcaaa ataacctgtt tcaagagcca 840 tggttgccag aaattaagtc gtcagagcac ttctctttcg aaacgagcaa ggatgcaatt 900 ctgaagttag tggcaaacgt aaataaatga 930 <210> 8 <211> 309 <212> PRT <213> Planctopirus limnophila <400> 8 Met Gln Lys Thr Phe Ser Leu Arg Pro Val Pro Val Asp Ala Ile Arg 1 5 10 15 Trp Asn Phe Ala Asn Phe Trp Ser Gly Phe Asp Ala Leu Ala Phe Glu 20 25 30 Arg His Leu Leu Gly Val Ser Gly Lys His Lys Phe Arg Ile Ser Glu 35 40 45 Gln Asn Pro Gln Ile Val Phe Glu Ser Val Phe Gly Thr Pro Gly Lys 50 55 60 Gly Arg Glu Arg Trp Pro Lys Ala Arg Gln Val Trp Tyr Thr Gly Glu 65 70 75 80 Asn Val Ala Pro Pro Leu Asp Gln Phe Asp Lys Cys Leu Ser Phe His 85 90 95 Arg Asp Ile Lys Asp Pro Arg His Leu Arg Trp Pro Tyr Tyr Leu Leu 100 105 110 His Leu Ala Ser Leu Pro Met Ser Phe Asn Asp Leu Val Lys Cys Gln 115 120 125 Ser Ser Val Ser Thr Trp Ala Glu Arg Pro Gly Phe Cys Ala Phe Ile 130 135 140 Ala Phe Asn Glu Gly Cys Gln Thr Arg Asn Arg Phe Val Glu Lys Leu 145 150 155 160 Ser Arg Tyr Arg Arg Val Asp Cys Pro Gly Arg Val Leu Asn Asn Met 165 170 175 Thr Ser Glu Thr Leu Gly Gln Arg Gly Asn Leu His Gly Lys Ile Asn 180 185 190 Phe Leu Lys Gln Tyr Lys Tyr Ala Val Cys Phe Glu Asn Thr Ser Thr 195 200 205 Arg Gly Ser Glu Gly Tyr Val Thr Glu Lys Leu Val Asp Ala Met Leu 210 215 220 Only Gly Cys Ile Pro Leu Tyr Trp Gly Asp His Arg Val Gly Glu Asp 225 230 235 240 Phe Asn Glu Asn Ser Phe Ile Asn Leu Gly Val Tyr Gly Asn Asp Val 245 250 255 Asn Ala Met Val Gln His Val Ile Glu Leu Asp Ser Asp Glu Arg Leu 260 265 270 Gln Asn Asn Leu Phe Gln Glu Pro Trp Leu Pro Glu Ile Lys Ser Ser 275 280 285 Glu His Phe Ser Phe Glu Thr Ser Lys Asp Ala Ile Leu Lys Leu Val 290,295,300 Only Asn Or Asn Lys 305 <210> 9 <211> 951 <212> DNA <213> Pedobacter glucosidilyticus <400> 9 atgaagtatt ttctgctgct ggctaaacgc cacaagaat acctgaaaga gagaaaatc 60 ttccgcaact cgactatctc tttttacac ttctgggaa tcgaggatta cacaacttt 120 180. tggctgcaga aattcatcgt agaccgtac ctgaaccca aaaacaaatc catcaacttc ttttctgtgt tcggcccgcg ctatgtcctt aaaaagcaaa aagcagcgat caatattttc 240 ttctcgggcg aaaccatgag ccgtttcaaa aataccacg actattgtct gcctgaagtt gatctggcgc tgggtttcga cgatctgcaa cacgagaagt acttccgtct gccgctgtgg 360 atcctggact ttttcgaacc gactgttgac cttgaaaaag ctaaagaaa actgaaacag 480. ctgaactact acaaaaacaa taaccgatc gtgcgtgaaa agttctgctc tctgatcgcc 540. cgtcacgacg aaaacggcat ccgtaaaaag attgtgaaca cgctgaaccc aatcgaaacg gttgactgtg caggcaaact gttcaacat actgctcgct tacagaccga attcgcgaac aacaaagtaa aatttctgga gaactacaag tttaacatct gcccggaaaa caccaaccag gaatcctaca ccaccgaaaa acttttcgaa agcttcgctg caggctgtat cccgatctac tggggttctg ctcagaacc ggaccgaac atcttcaaac cgtctagcat catctttttc 840. gatgagttca aaaacaccct gtctgaggat gttgaacgtc tacataaaga tccgaaactg tacctggact ttattagcca gaacccgttt caggacacag ctgctgaata catcatccag 900 actatctcca acctggaact caactgaaa gagatcatta accaggcata a 951 <210> 10 <211> 316 <212> PRT <213> Pedobacter glucosidilyticus <400> 10 Met Lys Tyr Phe Leu Leu Ala Lys Arg His Lys Lys Tyr Leu Lys 1 5 10 15 Glu Lys Lys Ile Phe Arg Asn Ser Thr Ile Phe Tyr Asn Phe Trp 20 25 30 Glu Ile Glu Asp Tyr Asn Asn Phe Trp Leu Gln Lys Phe Ile Val Asp 35 40 45 Arg Asn Leu Asn Pro Lys Asn Lys Ser Ile Asn Phe Ser Val Phe 50 55 60 Gly Pro Arg Tyr Val Leu Lys Gln Lys Ala Ala Ile Asn Ile Phe 65 70 75 80 Phe Ser Gly Glu Thr Met Ser Arg Phe Lys Lys Tyr His Asp Tyr Cys 85 90 95 Leu Pro Glu Val Asp Leu Ala Leu Gly Phe Asp Asp Leu Gln His Glu 100 105 110 Lys Tyr Phe Arg Leu Pro Leu Trp Ile Leu Asp Phe Phe Glu Pro Thr 115 120 125 Val Asp Leu Glu Lys Ala Lys Glu Lys Leu Lys Gln Leu Asn Tyr Tyr 130 135 140 Lys Asn Asn Lys Pro Ile Val Arg Glu Lys Phe Cys Ser Leu Ile Ala 145 150 155 160 Arg His Asp Glu Asn Gly Ile Arg Lys Lys Ile Val Asn Thr Leu Asn 165 170 175 Pro Ile Glu Thr Val Asp Cys Ala Gly Lys Leu Phe Asn Asn Thr Ala 180 185 190 Arg Leu Gln Thr Glu Phe Ala Asn Asn Lys Val Lys Phe Leu Glu Asn 195 200 205 Tyr Lys Phe Asn Ile Cys Pro Glu Asn Thr Asn Gln Glu Ser Tyr Thr 210 215 220 Thr Glu Lys Leu Phe Glu Ser Phe Ala Ala Gly Cys Ile Pro Ile Tyr 225 230 235 240 Trp Gly Ser Ala Gln Lys Pro Glu Pro Asn Ile Phe Lys Pro Ser Ser 245 250 255 Ile Ile Phe Phe Asp Glu Phe Lys Asn Thr Leu Ser Glu Asp Val Glu 260 265 270 Arg Leu His Lys Asp Pro Lys Leu Tyr Leu Asp Phe Ile Ser Gln Asn 275 280 285 Pro Phe Gln Asp Thr Ala Ala Glu Tyr Ile Ile Gln Thr Ile Ser Asn 290 295 300 Leu Glu Leu Lys Leu Lys Glu Ile Ile Asn Gln Ala 305 310 315 <210> 11 <211> 1029 <212> DNA <213> Porphyromonas catoniae <400> 11 atgccgatat atgatataaa agccatgaat accccgtcga agcaaccatt acgagaacga 60 ctgcatatga tgcgtcgacg taatcgtgtg cggaagcgtt cggttatagc cctgattaag 120 tcgcatttag atagctcacg tttcaggat tataactggt gggattctca tgcgtctacc 180 ttctggttac cacggtttat agatttgcat ctggaaccga agaagaaaat taatttattt 240 tcgtgctttc aaaatccgtt aatgctgatt cgttattata aaggtgttaa aattttttta 300 tcaggtgaaa acctgaccaa taacgaacat ttcggttttc acccgcgtat gctggatcac 360 cgaataaatg aagttgatct agcgttaggt ttcgaatttc gtaaagaccc gaaatattac 420 cgttttccgt tatggattta ccagaatgaa tttattagcc cgtctgccag cttagaggac 480 atatgtgttc tggtaggcca gataaacgac ccgtcgaccc gtcgtagcgc caagcgttct 540 cgttttattg gtcagatttc gagccatgat aagggtggca tgcggggacg gctgatagat 600 ctgttatctc cgattgggca aattgattgc gccggtaaat ttcgtcataa taccgacgaa 660 ctgttagaag tgtatggtga cgataaattc aaatatttag ccaactatcg ttttaactta 720 tgcccagaaa attcattagg tgagggctat attaccgaaa aagtgtttga tagcatacgt 780 gccggttgta ttccgattta ctggggcgcc tatttagaac caggtatttt aaacccgaaa 840 gcgattttac gttttgaaga gggcaaagag caagaatttt ataaccgggt taaagaatta 900 tgggaaaacg aggaagcgta tgaacagttc attctggaac caccgtttgt cgaaggggcc 960 gccgaacgta tttgggaaat tttgcagggt ttacgtgaac gtttagcgcc attagttgaa 1020 gaagggtaa 1029 <210> 12 <211> 342 <212> PRT <213> Porphyromonas catoniae <400> 12 Met Pro Ile Tyr Asp Ile Lys Ala Met Asn Thr Pro Ser Lys Gln Pro 1 5 10 15 Leu Arg Glu Arg Leu His Met Met Arg Arg Arg Asn Arg Val Arg Lys 20 25 30 Arg Ser Val Ile Ala Leu Ile Lys Serve His Leu Asp Serve Arg Tyr 35 40 45 Gln Asp Tyr Asn Trp Trp Asp Ser His Ala Ser Thr Phe Trp Leu Pro 50 55 60 Arg Phe Ile Asp Leu His Leu Glu Pro Lys Lys Ile Asn Leu Phe 65 70 75 80 CYS Physiology of Gln Asn Pro Met Leu Ile Arg Tyr Lys Gly Val 85 90 95 Lys Ile Phe Leu Ser Gly Glu Asn Leu Thr Asn Asn Glu His Phe Gly 100 105 110 Phe His Pro Arg Met Leu Asp His Arg Ile Asn Glu Val Asp Leu Ala 115 120 125 Leu Gly Phe Glu Phe Arg Lys Asp Pro Lys Tyr Tyr Arg Phe Pro Leu 130 135 140 Trp Tyr Gln Asn Glu Phe is Pro Ser Ala Leu Glu Asp 145 150 155 160 Ile Cys Val Leu Val Gly Gln Ile Asn Asp Pro Ser Thr Arg Arg Ser 165 170 175 Ala Lys Arg Ser Arg Phe Ile Gly Gln Ile Ser Ser His Asp Lys Gly 180 185 190 Gly Met Arg Gly Arg Leu Ile Asp Leu Leu Ser Pro Ile Gly Gln Ile 195 200 205 Asp Cys Ala Gly Lys Phe Arg His Asn Thr Asp Glu Leu Leu Glu Val 210 215 220 Tyr Gly Asp Asp Lys Phe Lys Tyr Leu Ala Asn Tyr Arg Phe Asn Leu 225 230 235 240 Cys Pro Glu Asn Ser Leu Gly Glu Gly Tyr Ile Thr Glu Lys Val Phe 245 250 255 Asp Ser Ile Arg Ala Gly Cys Ile Pro Ile Tyr Trp Gly Ala Tyr Leu 260 265 270 Glu Pro Gly Ile Leu Asn Pro Lys Ala Ile Leu Arg Phe Glu Glu Gly 275 280 285 Lys Glu Gln Glu Phe Tyr Asn Arg Val Lys Glu Leu Trp Glu Asn Glu 290 295 300 Glu Ala Tyr Glu Gln Phe Ile Leu Glu Pro Pro Phe Val Glu Gly Ala 305 310 315 320 Ala Glu Arg Ile Trp Glu Ile Leu Gln Gly Leu Arg Glu Arg Leu Ala 325 330 335 Pro Leu Val Glu Glu Gly 340 <210> 13 <211> 972 <212> DNA <213> Porphyromonas sp. <400> 13 atgaacgcgg tcgagcgagt acggaatata cttaattatt gtattaacga agtccaaatg 60 taccggcagt gtcccaattc aaaatactat aatttctggc cctgtgatta taataataat 120 tggtttaacc atttcgtaga acaccgaggc ttagctaaag aacggcaccg gcttaacttt 180 ttctcggtct ttggtaaccc tctactgccg cggattatac cggggaagaa agtgttcttc 240 actggggaga atcttgcaga taactcaata cactcaatag ggcgagcttt caaaaagacc 300 tttccggtat atgatctggt acttgggttc gactatgaag tagaggatag ccgggtgaat 360 tatatgcggt ttccattatg gatagccttc ctgatagatc cgaccgccga ttatcagaaa 420 ataaaggaaa cgattgaacg gattaacgac ccgtcaacgc ggcttaacgc gagccgggat 480 cgtttcgcct gccttgttgc cagccacgat aaaactggta tacggcagaa attatatgat 540 gtccttatgc cgatagcgtc agtaacttgc ccaggacggt tccagaataa tacgaacgag 600 cttcacgatt tatatgcaaa cgacaagcgg gaatatttaa aactgtttaa atttaacgta 660 tgtccagaaa attcatcgac tccgggttat ataactgaaa agttattcga ttcgttcgca 720 tcaggttgta ttcccatata cttcggtggg ggaactgagg aaatagagcc cgatattgta 780 aaccaaggag cgttcatacg gtactgggat gatgggcgaa tggactggat ggacacggta 840 cgggaacttt gggaatcgcc gtcagcatac cgggccgttg ccgagatacc accgttcaaa 900 gaacaagcag cagatgtaat ttatgcctat atggaaaacc ttcacgacaa acttgcggca 960 atagtgcggt ga 972 <210> 14 <211> 323 <212> PRT <213> Porphyromonas species <400> 14 Met Asn Ala Val Glu Arg Val Arg Asn Ile Leu Asn Tyr Cys Ile Asn 1 5 10 15 Glu Val Gln Met Tyr Arg Gln Cys Pro Asn Ser Lys Tyr Tyr Asn Phe 20 25 30 Trp Pro Cys Asp Tyr Asn Asn Asn Trp Phe Asn His Phe Val Glu His 35 40 45 Arg Gly Leu Ala Lys Glu Arg His Arg Leu Asn Phe Phe Ser Val Phe 50 55 60 Gly Asn Pro Leu Leu Pro Arg Ile Ile Pro Gly Lys Lys Val Phe Phe 65 70 75 80 Thr Gly Glu Asn Leu Ala Asp Asn Ser Ile His Ser Ile Gly Arg Ala 85 90 95 Phe Lys Lys Thr Phe Pro Val Tyr Asp Leu Val Leu Gly Phe Asp Tyr 100 105 110 Glu Val Glu Asp Ser Arg Val Asn Tyr Met Arg Phe Pro Leu Trp Ile 115 120 125 Ala Phe Leu Ile Asp Pro Thr Ala Asp Tyr Gln Lys Ile Lys Glu Thr 130 135 140 Ile Glu Arg Ile Asn Asp Pro Ser Thr Arg Leu Asn Ala Ser Arg Asp 145 150 155 160 Arg Phe Ala Cys Leu Val Ala Ser His Asp Lys Thr Gly Ile Arg Gln 165 170 175 Lys Leu Tyr Asp Val Leu Met Pro Ile Ala Ser Val Thr Cys Pro Gly 180 185 190 Arg Phe Gln Asn Asn Thr Asn Glu Leu His Asp Leu Tyr Ala Asn Asp 195 200 205 Lys Arg Glu Tyr Leu Lys Leu Phe Lys Phe Asn Val Cys Pro Glu Asn 210 215 220 Ser Ser Thr Pro Gly Tyr Ile Thr Glu Lys Leu Phe Asp Ser Phe Ala 225 230 235 240 Ser Gly Cys Ile Pro Ile Tyr Phe Gly Gly Gly Thr Glu Glu Ile Glu 245 250 255 Pro Asp Ile Val Asn Gln Gly Ala Phe Ile Arg Tyr Trp Asp Asp Gly 260 265 270 Arg Met Asp Trp Met Asp Thr Val Arg Glu Leu Trp Glu Ser Pro Ser 275 280 285 Ala Tyr Arg Ala Val Ala Glu Ile Pro Pro Phe Lys Glu Gln Ala Ala 290 295 300 Asp Val Ile Tyr Ala Tyr Met Glu Asn Leu His Asp Lys Leu Ala Ala 305 310 315 320 Ile Val Arg <210> 15 <211> 951 <212> DNA <213> Selenomonas infelix <400> 15 atgttaatgc gtgcactcag aaaaatgaag cgatggggac gtgtggcgtt tgattacacg 60 aatactacga aggatggagc tgtttgctat cataattggt ggccgtgtaa ttatgaggaa 120 gagtggtttc atcgttttgt tgtacaaaat attggaacag aacgttgcta tcatttcttt 180 tctgtatttg gtccacgtat tgcgttgacg ctgccaacac cgaataaagt ttttttctgt 240 ggtgaaaatg tgcataacgc agagtggccc tataaaagct atcaagatca tgcacttgga 300 gatgtcaagc tggctctcgg atatgatgat atacaggatg aacgatatat tcgatttcct 360 ctgtggttgc tctatatgtt cgatcctgtt gttgaccgat atgccatccg tgagcgaatt 420 gaagaaatca atcatgcaga gaatacaaga aaatatgaat gtgtattgat ttccagacac 480 gataagtgga atatgcgtgg tccaatttat gatgcattga aagatcattt ggctatttcc 540 tgtgctggga aatggaagca aaacactgat gaactgtgga cggtttacaa tgatgataaa 600 ccacgctatc taaaagagtt taagtttaat atctgcccgg agaatttga cacgccgtat 660 tatgttacag agaagctgtt tgaagccttt cggagtggaa caattcctat tttgcaggc 720 ggaggcgatc atccggagcc ggaaattgtg aatcgaagcg cactactcct ttgggagcga 780 ggacaaagtg atcatagtgc cttggtacag gaagttatac ggctcgcacg cgatgagata 840 tactatgata aatttgtaca tcaggttcgt ttgcttccgt atacggaaga gtttgtttat 900 gaacagtttt catcgctgaa agagcggttg ttgcagataa gacgagggtg a 951 <210> 16 <211> 316 <212> PRT <213> Selenomonas infelix <400> 16 Met Leu Met Arg Ala Leu Arg Lys Met Lys Arg Trp Gly Arg Val Ala 1 5 10 15 Phe Asp Tyr Thr Asn Thr Thr Lys Asp Gly Ala Val Cys Tyr His Asn 20 25 30 Trp Trp Pro Cys Asn Tyr Glu Glu Glu Trp Phe His Arg Phe Val Val 35 40 45 Gln Asn Ile Gly Thr Glu Arg Cys Tyr His Phe Phe Ser Val Phe Gly 50 55 60 Pro Arg Ile Ala Leu Thr Leu Pro Thr Pro Asn Lys Val Phe Phe Cys 65 70 75 80 Gly Glu Asn Val His Asn Ala Glu Trp Pro Tyr Lys Ser Tyr Gln Asp It should be noted that "损伤新月形单胞菌" is translated as "Selenomonas infelix" here. You may need to double-check if this is the most accurate translation according to the specific context.85 90 95 His Ala Leu Gly Asp Val Lys Leu Ala Leu Gly Tyr Asp Asp Ile Gln 100 105 110 Asp Glu Arg Tyr Ile Arg Phe Pro Leu Trp Leu Leu Tyr Met Phe Asp 115 120 125 Pro Val Val Asp Arg Tyr Ala Ile Arg Glu Arg Ile Glu Glu Ile Asn 130 135 140 His Ala Glu Asn Thr Arg Lys Tyr Glu Cys Val Leu Ile Ser Arg His 145 150 155 160 Asp Lys Trp Asn Met Arg Gly Pro Ile Tyr Asp Ala Leu Lys Asp His 165 170 175 Leu Ala Ile Ser Cys Ala Gly Lys Trp Lys Gln Asn Thr Asp Glu Leu 180 185 190 Trp Thr Val Tyr Asn Asp Asp Lys Pro Arg Tyr Leu Lys Glu Phe Lys 195 200 205 Phe Asn Ile Cys Pro Glu Asn Phe Asp Thr Pro Tyr Tyr Val Thr Glu 210 215 220 Lys Leu Phe Glu Ala Phe Arg Ser Gly Thr Ile Pro Ile Tyr Ala Gly 225 230 235 240 Gly Gly Asp His Pro Glu Pro Glu Ile Val Asn Arg Ser Ala Leu Leu 245 250 255 Leu Trp Glu Arg Gly Gln Ser Asp His Ser Ala Leu Val Gln Glu Val 260 265 270 Ile Arg Leu Ala Arg Asp Glu Ile Tyr Tyr Asp Lys Phe Val His Gln 275 280 285 Val Arg Leu Leu Pro Tyr Thr Glu Glu Phe Val Tyr Glu Gln Phe Ser 290 295 300 Ser Leu Lys Glu Arg Leu Leu Gln Ile Arg Arg Gly 305 310 315 <210> 17 <211> 1437 <212> DNA <213> Helicobacter pylori <400> 17 atgtttcagc cgctgctgga tgcatatgtt gaaagcgcaa gcattgaaaa aatggcaagc 60 aaaagtccgc ctccgctgaa aattgcagtt gcaaattggt ggggtgatga agaaatcaaa 120 gagtttaaaa acagcgtcct gtactttatt ctgagccagc gttataccat taccctgcat 180 cagaatccga atgaattttc cgatctggtt tttggtaatc cgctgggtag cgcacgtaaa 240 attctgagct atcagaatgc aaaacgcgtg ttttataccg gtgaaaatga aagcccgaac 300 ttcaacctgt ttgattatgc cattggtttc gatgagctgg attttaatga tcgttatctg 360 cgtatgccgc tgtattatga tcgtctgcat cataaagcag aaagcgttaa tgataccacc 420 gcaccgtata aactgaaaga taatagcctg tacgcactga aaaaaccgag ccattgcttt 480 aaagaaaaac atccgaatct gtgtgccgtg gttaatgatg aaagcgatcc tctgaaacgt 540 ggttttgcaa gctttgttgc aagcaatccg aacgcaccga ttcgtaatgc attctatgat 600 gcactgaata gcattgaacc ggttaccggt ggtggtagcg ttcgtaatac cctgggttat 660 aatgtgaaaa acaaaaacga attcctgagc cagtataaat tcaatctgtg ctttgaaaac 720 acccagggtt atggttatgt gaccgaaaaa atcatcgatg cctatttcag ccataccatt 780 ccgatttatt ggggtagccc gagcgttgca aaagatttca atccgaaaag ctttgtgaac 840 gtgcacgact tcaaaaactt tgatgaagcc atcgattata tcaaatacct gcacacccat 900 aaaaacgcct atctggatat gctgtatgaa aatccgctga atacactgga tggtaaagcc 960 tatttttacc agaacctgag cttcaaaaaa atcctggcct ttttcaaaac catcctggaa 1020 aacgatacca tctatcacga taacccgttt atcttttgcc gtgatctgaa tgaaccgctg 1080 gttaccattg atgatctgcg tgttaattat gatgacctgc gcgtgaacta cgacgatctg 1140 cgcatcaatt atgatgatct gcgggtaaac tatgatgatc tgcgtatcaa ctacgacgac 1200 ctgcgtgtga actacgatga cctgcgggtt aattatgatg atctgcggat taattatgat 1260 gatctgcgtg tgaactatga cgatctgcgt gtgaattacg agcgtctgct gagcaaagca 1320 acccctctgc tggaactgag ccagaatacc accagtaaaa tctatcgtaa agcgtaccag 1380 aaaagcctgc ctctgctgcg tgcaattcgt cgttgggtta aaaaactggg tctgtaa 1437 <210> 18 <211> 478 <212> PRT <213> Helicobacter pylori <400> 18 Met Phe Gln Pro Leu Leu Asp Ala Tyr Val Glu Ser Ala Ser Ile Glu 1 5 10 15 Lys Met Ala Ser Lys Ser Pro Pro Pro Leu Lys Ile Ala Val Ala Asn 20 25 30 Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Asn Ser Val Leu Tyr 35 40 45 Phe Has Been Gln Arg Tyr Thr Thr Has Gln Asn Pro Asn 50 55 60 Glu Phe Ser Asp Leu Val Phe Gly Asn Pro Leu Gly Ser Ala Arg Lys 65 70 75 80 Tyr Gln Asn Is Contained With Lys Arg Val Phe Tyr Gly Glu Asn 85 90 95 Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp Glu 100 105 110 Leu Asp Phe Asn Asp Arg Tyr Leu Arg Pro Leu Tyr Tyr Asp Arg 115 120 125 Leu His His Lys Ala Glu Ser Val Asn Asp Thr Thr Ala Pro Tyr Lys 130 135 140 Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Pro Ser His Cys Phe 145 150 155 160 Lys Glu Lys His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser Asp 165 170 175 Pro Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Pro Asn Ala 180 185 190 Pro Ile Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro Val 195 200 205 Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Asn Val Lys Asn 210 215 220 Lys Asn Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu Asn 225 230 235 240 Thr Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Ile Asp Ala Tyr Phe 245 250 255 Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys Asp 260 265 270 Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Lys Asn Phe Asp 275 280 285 Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Lys Asn Ala Tyr 290 295 300 Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys Ala 305 310 315 320 Tyr Phe Tyr Gln Asn Leu Ser Phe Lys Lys Ile Leu Ala Phe Phe Lys 325 330 335 Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asp Asn Pro Phe Ile Phe 340 345 350 Cys Arg Asp Leu Asn Glu Pro Leu Val Thr Ile Asp Asp Leu Arg Val 355 360 365 Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr 370 375 380 Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr Asp Asp 385 390 395 400 Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg 405 410 415 Ile Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn 420 425 430 Tyr Glu Arg Leu Leu Ser Lys Ala Thr Pro Leu Leu Glu Leu Ser Gln 435 440 445 Asn Thr Thr Ser Lys Ile Tyr Arg Lys Ala Tyr Gln Lys Ser Leu Pro 450 455 460 Leu Leu Arg Ala Ile Arg Arg Trp Val Lys Lys Leu Gly Leu 465 470 475 <210> 19 <211> 978 <212> DNA <213> Azospirillum sp. <400> 19 atgattgatc agcggaccgg cgtgtttctg agcgaatttc ttgatacccg caacagggac 60 ccggcagtac tggatcgctt tctactgcag ggaccggatg gcggaaggcg gggagcgaaa 120 ccgaacctga aagtggcgtt ttttgacttt tggccagaat ttgaccccag cgcaaacttt 180 tttgtggaga ttctgagcgc gcgctttcag gtgagcgtgg tggaaaacga tagcgatctt 240 gcgattgtga gcgtgtttgg caccggacca cgggaaatac ggactgcgcg gagcatgttt 300 tttaccggag aaaatgtgcg gccgccgctt gatggcattg atatgagcgt gagctttgat 360 cgcattgatg acccacggca ttttcgcctg ccgctatatg tggtgcatgc gtatgatcat 420 ctgcgcgagg gagcagcacc gtatttttgc cagccagtgc tgccgccagt gccgccgact 480 cgggaagatg cggcagaacg gaagttttgc gcgtttcttt ataaaaaccc aaactgcgcg 540 cgccgcaacg atttttttca tatgcttggc gcgcggcgcc atgtggatag cgtgggctgg 600 ctgctgaaca acaccggcag cgtggtgaaa atgggatggc taccgaaaat tcgggtgttt 660 agccgctatc gctttgcgtt tgcgtttgaa aacgctagcc atccaggcta tctgaccgaa 720 aaaattctgg atgcttttca ggcgggagca gtgccgcttt attggggcga cccaggcgtg 780 ctgcgcgacg tggcagcggg cagctttatt gatgtgagca ggtatagcag cgatgaagaa 840 gcgattgaag cgattctggc gattgatgat gactatggcg cgtatcgccg ctatcgcagc 900 actccgccct ttcttggcac tgaagacttt cattttgacg cgtatcgact ggcggagtgg 960 attgagagcc gactataa 978 <210> 20 <211> 325 <212> PRT <213> Azospirillum species <400> 20 Met Ile Asp Gln Arg Thr Gly Val Phe Leu Ser Glu Phe Leu Asp Thr 1 5 10 15 Arg Asn Arg Asp Pro Ala Val Leu Asp Arg Phe Leu Leu Gln Gly Pro 20 25 30 Asp Gly Gly Arg Arg Gly Ala Lys Pro Asn Leu Lys Val Ala Phe Phe 35 40 45 Asp Phe Trp Pro Glu Phe Asp Pro Ser Ala Asn Phe Phe Val Glu Ile 50 55 60 Leu Ser Ala Arg Phe Gln Val Ser Val Val Glu Asn Asp Ser Asp Leu 65 70 75 80 Ala Ile Val Ser Val Phe Gly Thr Gly Pro Arg Glu Ile Arg Thr Ala 85 90 95 Arg Ser Met Phe Phe Thr Gly Glu Asn Val Arg Pro Pro Leu Asp Gly 100 105 110 Ile Asp Met Ser Val Ser Phe Asp Arg Ile Asp Asp Pro Arg His Phe 115 120 125 Arg Leu Pro Leu Tyr Val Val His Ala Tyr Asp His Leu Arg Glu Gly 130 135 140 Ala Ala Pro Tyr Phe Cys Gln Pro Val Leu Pro Pro Val Pro Pro Thr 145 150 155 160 Arg Glu Asp Ala Ala Glu Arg Lys Phe Cys Ala Phe Leu Tyr Lys Asn 165 170 175 Pro Asn Cys Ala Arg Arg Asn Asp Phe Phe His Met Leu Gly Ala Arg 180 185 190 Arg His Val Asp Ser Val Gly Trp Leu Leu Asn Asn Thr Gly Ser Val 195 200 205 Val Lys Met Gly Trp Leu Pro Lys Ile Arg Val Phe Ser Arg Tyr Arg 210 215 220 Phe Ala Phe Ala Phe Glu Asn Ala Ser His Pro Gly Tyr Leu Thr Glu 225 230 235 240 Lys Ile Leu Asp Ala Phe Gln Ala Gly Ala Val Pro Leu Tyr Trp Gly 245 250 255 Asp Pro Gly Val Leu Arg Asp Val Ala Ala Gly Ser Phe Ile Asp Val 260 265 270 Ser Arg Tyr Ser Ser Asp Glu Glu Ala Ile Glu Ala Ile Leu Ala Ile 275 280 285 Asp Asp Asp Tyr Gly Ala Tyr Arg Arg Tyr Arg Ser Thr Pro Pro Phe 290 295 300 Leu Gly Thr Glu Asp Phe His Phe Asp Ala Tyr Arg Leu Ala Glu Trp 305 310 315 320 Ile Glu Ser Arg Leu 325 <210> 21 <211> 978 <212> DNA <213> Azospirillum species <400> 21 atgattgaca ggcggacaag cgattttctg gcggagttcc tagcaagcgc taacaaagat 60 ccggcagtac ttgatcgatt cctactacat ggaccggacc ggggaggccg cagcgcgaaa 120 ccgcggctga aaattgcgtt ttttgacttt tggccggagt ttgacccggc agcaaatttt 180 tttgtggaaa ttctgagcgc gcgctttgat ctgagcgtgg tggataatga tagcgatcta 240 gcgattgtga gcgtgtttgg aattcgccat cgggaagctc ggactgcgcg aagcctgttt 300 tttaccggcg aaaatgtgcg gcccccgctt gatggcgtgg atatgagcgt gagctttgat 360 cgcattgatg acccacggca ttatcggctg ccgctttatg tgatgcatgc gtgggatcat 420 cggcgcgagg gagcaactcg gcatttttgc catagcgtgc tgccgccggt gcccccgact 480 cgggaagaag cagataggcg gaagttttgc gcgtttcttt ataaaaatcc aaactgcgag 540 cgccgcaacg actttttccg gatgctttgc gcgcgccgcc atgtggaaag cgtgggatgg 600 ctgctgaaca acaccggcag cgtggtgaaa atgggctggc tgccgaaaat tcgggtgttt 660 agccgctatc gctttgcgtt tgcgtttgaa aatgcgagcc atccaggcta tctgaccgaa 720 aaaattcttg atgcgtttca ggcgggcgct gtgccgcttt attgggggga cccaggcgtg 780 ctgcgggacg tagcggcggg cagctttatt gacgtgagcc gctatagcag cgatgaagaa 840 gcgattgatg cgattctggc aattgatgac gactatgata cctatcgccg ccatcgcagc 900 actgctccat ttcttggcac tgaagacttt tattttgacg cgtttcgact ggcggagtgg 960 attgagagcc gactataa 978 <210> 22 <211> 325 <212> PRT <213> Azospirillum species <400> 22 Met Ile Asp Arg Arg Thr Ser Asp Phe Leu Ala Glu Phe Leu Ala Ser 1 5 10 15 Ala Asn Lys Asp Pro Ala Val Leu Asp Arg Phe Leu Leu His Gly Pro 20 25 30 Asp Arg Gly Gly Arg Ser Ala Lys Pro Arg Leu Lys Ile Ala Phe Phe 35 40 45 Asp Phe Trp Pro Glu Phe Asp Pro Ala Ala Asn Phe Phe Val Glu Ile 50 55 60 Leu Ser Ala Arg Phe Asp Leu Ser Val Val Asp Asn Asp Ser Asp Leu 65 70 75 80 Ala Ile Val Ser Val Phe Gly Ile Arg His Arg Glu Ala Arg Thr Ala 85 90 95 Arg Ser Leu Phe Phe Thr Gly Glu Asn Val Arg Pro Pro Leu Asp Gly 100 105 110 Val Asp Met Ser Val Ser Phe Asp Arg Ile Asp Asp Pro Arg His Tyr 115 120 125 Arg Leu Pro Leu Tyr Val Met His Ala Trp Asp His Arg Arg Glu Gly 130 135 140 Ala Thr Arg His Phe Cys His Ser Val Leu Pro Pro Val Pro Pro Thr 145 150 155 160 Arg Glu Glu Ala Asp Arg Arg Lys Phe Cys Ala Phe Leu Tyr Lys Asn 165 170 175 Pro Asn Cys Glu Arg Arg Asn Asp Phe Phe Arg Met Leu Cys Ala Arg 180 185 190 Arg His Val Glu Ser Val Gly Trp Leu Leu Asn Asn Thr Gly Ser Val 195 200 205 Val Lys Met Gly Trp Leu Pro Lys Ile Arg Val Phe Ser Arg Tyr Arg 210 215 220 Phe Ala Phe Ala Phe Glu Asn Ala Ser His Pro Gly Tyr Leu Thr Glu 225 230 235 240 Lys Ile Leu Asp Ala Phe Gln Ala Gly Ala Val Pro Leu Tyr Trp Gly 245 250 255 Asp Pro Gly Val Leu Arg Asp Val Ala Ala Gly Ser Phe Ile Asp Val 260 265 270 Ser Arg Tyr Ser Ser Asp Glu Glu Ala Ile Asp Ala Ile Leu Ala Ile 275 280 285 Asp Asp Asp Tyr Asp Thr Tyr Arg Arg His Arg Ser Thr Ala Pro Phe 290 295 300 Leu Gly Thr Glu Asp Phe Tyr Phe Asp Ala Phe Arg Leu Ala Glu Trp 305 310 315 320 Ile Glu Ser Arg Leu 325 <210> 23 <211> 978 <212> DNA <213> Azospirillum brazilense <400> 23 atgcttgacc agcggacaag cgcattccta gaagagtttc tggcaaaacc aggcggcgat 60 ccggaacggc ttgatcgctt tctgctgcat ggcccatatc gcggacggcg cggcggcagg 120 ccacggctga aactggcgtt ttgatttt tggccggaat ttgatactgg caggaacttt 180 tttattgaaa ttctgagcag ccgcttcgac ctgagcgtgg tggaagatga tagcgacctt 240 gcgattgtga gcgtgtttgg cggacggcat cgcgcagcac gcagccggcg caccctgttt 300 tttaccggag aaaatgtgcg ccccccgctg gatggctttg atatggcagt gagctttgat 360 cgcgtgggcg atccgcgcca ttacgcctg ccgctttatg tgatgcatgc gtatgaacat 420 atgcgggaag gagcagtgcc gcatttttgc agcccagtgc tgccgccggt gccgccaagc 480 cgggcagcgt ttgcagaacg caacttttgc gcgtttcttt ataaaaaccc gaacggagaa 540 cgccgcaacc gcttttttcc ggcacttgat gcacggcggc gcgtggacag cgtgggctgg 600 catcttaaca acaccggcag cgtggtgaaa atgggctggc tggcaaaaat tcgcgtgttt 660 gagcgctatc gctttgcgtt tgcgtttgaa aacgcgagcc atccaggcta tctgactgag 720 aaaattcttg atgtgtttca ggcgggagca gtgccgcttt attgggggga cccagacgtg 780 gaacgggaag tggcagcagg cagctttatt gatgtgagcc gctttgcgac tgatgaagaa 840 gcagcagaac atattctggc actggatgga gactatgatg cgtattgcgc gtatcgcgcg 900 gtggcaccat ttctgggaac tgaagaattt cattttgatg cgtatcgcct tgcggattgg 960 attgaaagcc ggctgtag 978 <210> 24 <211> 325 <212> PRT <213> Azospirillum brazilense <400> 24 Put Leu Asp Gln Arg Thr Ser Ala Phe Leu Glu Glu Phe Leu Ala Lys 1 5 10 15 Pro Gly Gly Asp Pro Glu Arg Leu Asp Arg Phe Leu Leu His Gly Pro 20 25 30 Tyr Arg Gly Arg Arg Gly Gly Arg Pro Arg Leu Lys Leu Ala Phe Tyr 35 40 45 Asp Phe Trp Pro Glu Phe Asp Thr Gly Arg Asn Phe Phe Ile Glu Ile 50 55 60 Leu Ser Ser Arg Phe Asp Leu Ser Val Val Glu Asp Asp Ser Asp Leu 65 70 75 80 Ala Ile Val Ser Val Phe Gly Gly Arg His Arg Ala Ala Arg Ser Arg 85 90 95 Arg Thr Leu Phe Phe Thr Gly Glu Asn Val Arg Pro Pro Leu Asp Gly 100 105 110 Phe Asp Met Ala Val Ser Phe Asp Arg Val Gly Asp Pro Arg His Tyr 115 120 125 Arg Leu Pro Leu Tyr Val Met His Ala Tyr Glu His Met Arg Glu Gly 130 135 140 Ala Val Pro His Phe Cys Ser Pro Val Leu Pro Pro Val Pro Pro Ser 145 150 155 160 Arg Ala Ala Phe Ala Glu Arg Asn Phe Cys Ala Phe Leu Tyr Lys Asn 165 170 175 Pro Asn Gly Glu Arg Arg Asn Arg Phe Phe Pro Ala Leu Asp Ala Arg 180 185 190 Arg Arg Val Asp Ser Val Gly Trp His Leu Asn Asn Thr Gly Ser Val 195 200 205 Val Lys Met Gly Trp Leu Ala Lys Ile Arg Val Phe Glu Arg Tyr Arg 210 215 220 Phe Ala Phe Ala Phe Glu Asn Ala Ser His Pro Gly Tyr Leu Thr Glu 225 230 235 240 Lys Ile Leu Asp Val Phe Gln Ala Gly Ala Val Pro Leu Tyr Trp Gly 245 250 255 Asp Pro Asp Val Glu Arg Glu Val Ala Ala Gly Ser Phe Ile Asp Val 260 265 270 Ser Arg Phe Ala Thr Asp Glu Glu Ala Ala Glu His Ile Leu Ala Leu 275 280 285 Asp Gly Asp Tyr Asp Ala Tyr Cys Ala Tyr Arg Ala Val Ala Pro Phe 290 295 300 Leu Gly Thr Glu Glu Phe His Phe Asp Ala Tyr Arg Leu Ala Asp Trp 305 310 315 320 Ile Glu Ser Arg Leu 325 <210> 25 <211> 912 <212> DNA <213> Azospirillum species <400> 25 atgttagatc ggtttctgct tcatgggccg gagcgcgggg gccgtgcggc cagaccgcgc 60 ctgaaaattg cgttttttga cttttggccg gaatttgacc cgagcgcgaa tttttttgta 120 gaaattctga gcagccgctt tgatgtgagc gtggttgata atgatagcga tttagcgatt 180 ctgagcgtgt ttggcgaacg ccaccgcgaa gcgcgtaccg cgcgcgcgct gttttttacc 240 ggcgaaaatg tgcgcccgcc gttagatggc gtggatatga gcgtgagctt tgatcgcatt 300 gatcatccac gtcattatcg cctgccgtta tacgtgatgc atgcgtggga tcaccgtcgc 360 gaaggggcga ccccgcattt ttgccatccg gtgctgccgc cggtgccgcc gacgcgtgaa 420 gaagcggcga aacgtaagtt ttgcgcgttt ttatataaaa atcctcactg cgcgcgccgc 480 aacgattttt ttcagatgct gtgcgcgcgg cgccatgtgg aaagcgtggg ctggctgctg 540 aataataccg gcagcgtggt gaaaatgggc tggctgccga aaattcgcgt gtttgcgcgc 600 tatcgctttg cgtttgcgtt tgaaaacgcg gcgcatccag gctatctgac cgagaaaatt 660 ctggatgcgt ttcaggccgg gacggtaccg ttatactggg gcgacagcgg cgtgctgcgc 720 gacgttgcgg ccggcagctt tattgatgtg agccgctatg cgagcgatga agaagcgatt 780 gaagcgattc tggcgattga tgatgactat gatagctatc gccggtaccg cggcacggcg 840 ccatttttag gcaccgagga cttttacttt gacgcgtacc ggctggccga gtggattgag 900 agccgcctgt ag 912 <210> 26 <211> 303 <212> PRT <213> Azospirillum species <400> 26 Met Leu Asp Arg Phe Leu Leu His Gly Pro Glu Arg Gly Gly Arg Ala 1 5 10 15 Ala Arg Pro Arg Leu Lys Ile Ala Phe Phe Asp Phe Trp Pro Glu Phe 20 25 30 Asp Pro Ser Ala Asn Phe Phe Val Glu Ile Leu Ser Ser Arg Phe Asp 35 40 45 Val Ser Val Val Asp Asn Asp Ser Asp Leu Ala Ile Leu Ser Val Phe 50 55 60 Gly Glu Arg His Arg Glu Ala Arg Thr Ala Arg Ala Leu Phe Phe Thr 65 70 75 80 Gly Glu Asn Val Arg Pro Pro Leu Asp Gly Val Asp Met Ser Val Ser 85 90 95 Phe Asp Arg Ile Asp His Pro Arg His Tyr Arg Leu Pro Leu Tyr Val 100 105 110 Met His Ala Trp Asp His Arg Arg Glu Gly Ala Thr Pro His Phe Cys 115 120 125 His Pro Val Leu Pro Pro Val Pro Pro Thr Arg Glu Glu Ala Ala Lys 130 135 140 Arg Lys Phe Cys Ala Phe Leu Tyr Lys Asn Pro His Cys Ala Arg Arg 145 150 155 160 Asn Asp Phe Phe Gln Met Leu Cys Ala Arg Arg His Val Glu Ser Val 165 170 175 Gly Trp Leu Leu Asn Asn Thr Gly Ser Val Val Lys Met Gly Trp Leu 180 185 190 Pro Lys Ile Arg Val Phe Ala Arg Tyr Arg Phe Ala Phe Ala Phe Glu 195 200 205 Asn Ala Ala His Pro Gly Tyr Leu Thr Glu Lys Ile Leu Asp Ala Phe 210 215 220 Gln Ala Gly Thr Val Pro Leu Tyr Trp Gly Asp Ser Gly Val Leu Arg 225 230 235 240 Asp Val Ala Ala Gly Ser Phe Ile Asp Val Ser Arg Tyr Ala Ser Asp 245 250 255 Glu Glu Ala Ile Glu Ala Ile Leu Ala Ile Asp Asp Asp Tyr Asp Ser 260 265 270 Tyr Arg Arg Tyr Arg Gly Thr Ala Pro Phe Leu Gly Thr Glu Asp Phe 275 280 285 Tyr Phe Asp Ala Tyr Arg Leu Ala Glu Trp Ile Glu Ser Arg Leu 290 295 300 <210> 27 <211> 1032 <212> DNA <213> Butyrivibrio sp. <400> 27 atgaatataa ttcactttta tgcaagatat ttaagagaat cacataactg gaacagagaa 60 cgtgaagtta ctcgtaacgg tgttatgact tttgctaatt ggtggagaga agacccgcac 120 aagaactggt ttgcaagatt tattgacgct ggaagcaaag accctgaacg caggattagg 180 ttctatagta tttttggacc atatagtaaa ttaaaagaag attttgatgg agctaagata 240 ttcttttccg gagagaatct tgagcagccg gtctatcaca gaatattaaa gacagatcct 300 atagaagata gaatatgggc tgacagaagg aagctgtatg gtaattatgg agcaggagat 360 gtggatcttg ctataggatt tggcaatagg gaagaggatt cacttatggg atttgaagga 420 agcaggaaga ctaaatatat ccgctttcct ttatggctta catatgtttt tgatcctgat 480 tgtactcatg atgatattaa gagaaccatt gatgagataa atgcagttcg ttccacaggc 540 aggaaggata ctctgcttct tgcatcgcat gatttctggg ggacaaggtc agatatctta 600 aagagtctag aaggtgtatg cgatgttagt attgccggta aatggcgcaa caacaccaaa 660 gaactctggg aagattataa caatgacaag aataaatatc tgtcagaatt taaatttaat 720 atatgccctg aaaatgttga tgcaccggga tatgtgacag agaagatatt tgatgcttttt 780 aaatgcggag ctattcctat atatcagggc tgtcttggta agcctgagcc ggatgtgata 840 aatacagatg cagttctttt atgggacttc gatggagata attcagatac tatatccttg 900 attaaaaaac taaattcgga taatgtatac tatgataact ttgtatctca gcccaaattc 960 aaaccggatg cggcagagta tgtggttgca tgtatggatg agctgaggcg aagctttgat 1020 cagctcatct height 1032 <210> 28 <211> 343 <212> PRT <213> Pages Communications <400> 28 Met Assn Ile Ile His Phe Tyr Ala Arg Tyr Leu Arg Glu Ser His Assn 1 5 10 15 Trp Asn Arg Glu Arg Glu Val Thr Arg Asn Gly Val Met Thr Phe Ala 20 25 30 Asn Trp Trp Arg Glu Asp Pro His Lys Asn Trp Phe Ala Arg Phe Ile 35 40 45 Asp Ala Gly Ser Lys Asp Pro Glu Arg Arg Ile Arg Phe Tyr Ser Ile 50 55 60 Phe Gly Pro Tyr Ser Lys Leu Lys Glu Asp Phe Asp Gly Ala Lys Ile 65 70 75 80 Phe Phe Ser Gly Glu Asn Leu Glu Gln Pro Val Tyr His Arg Ile Leu 85 90 95 Lys Thr Asp Pro Ile Glu Asp Arg Ile Trp Ala Asp Arg Arg Lys Leu 100 105 110 Tyr Gly Asn Tyr Gly Ala Gly Asp Val Asp Leu Ala Ile Gly Phe Gly 115 120 125 Asn Arg Glu Glu Asp Ser Leu Met Gly Phe Glu Gly Ser Arg Lys Thr 130 135 140 Lys Tyr Ile Arg Phe Pro Leu Trp Leu Thr Tyr Val Phe Asp Pro Asp 145 150 155 160 Cys Thr His Asp Asp Ile Lys Arg Thr Ile Asp Glu Ile Asn Ala Val 165 170 175 Arg Ser Thr Gly Arg Lys Asp Thr Leu Leu Leu Ala Ser His Asp Phe 180 185 190 Trp Gly Thr Arg Ser Asp Ile Leu Lys Ser Leu Glu Gly Val Cys Asp 195 200 205 Val Ser Ile Ala Gly Lys Trp Arg Asn Asn Thr Lys Glu Leu Trp Glu 210 215 220 Asp Tyr Asn Asn Asp Lys Asn Lys Tyr Leu Ser Glu Phe Lys Phe Asn 225 230 235 240 Ile Cys Pro Glu Asn Val Asp Ala Pro Gly Tyr Val Thr Glu Lys Ile 245 250 255 Phe Asp Ala Phe Lys Cys Gly Ala Ile Pro Ile Tyr Gln Gly Cys Leu 260 265 270 Gly Lys Pro Glu Pro Asp Val Ile Asn Thr Asp Ala Val Leu Leu Trp 275 280 285 Asp Phe Asp Gly Asp Asn Ser Asp Thr Ile Ser Leu Ile Lys Lys Leu 290 295 300 Asn Ser Asp Asn Val Tyr Tyr Asp Asn Phe Val Ser Gln Pro Lys Phe 305 310 315 320 Lys Pro Asp Ala Ala Glu Tyr Val Val Ala Cys Met Asp Glu Leu Arg 325 330 335 Arg Ser Phe Asp Gln Leu Ile 340 <210> 29 <211> 1059 <212> DNA <213> Porphyromonas catoniae <400> 29 atgctagccc catacaaaag ccctatcttt gtgcccatat acgacactaa ggcaatgaat 60 ccccccacca aacaaccact tagagagagg ctccacatga tgcgtaggcg caatcgtatc 120 cgaaaacgct ctgtgatagc tctcatcaaa tctcaccttg acagctcacg ctaccaggac 180 tacaactggt gggacagtca cgcctcgacc ttttggctgc cacggttcat tgacctacac 240 ctcgagccca agaagaggat caatctcttc tcttgcttcc aaaatcccct aatgctcatc 300 cgctactaca aaggggtgaa gatcttccta tcaggtgaga accttgccaa taacgagcac 360 tttggcttcc atccccgcat gctcgatcat aggatcaacg aggtggactt agccctaggc 420 tttgagttcc gcaaggatcc caagtactat cgcttccccc tttggatcta tcagaatgag 480 ttcatcagcc ccagtgctag cctagaggat atacgtgcgc tccttgagca gatcaacgat 540 ccctccaccc gtcgtagcac gggacgcagt cgcttcatcg ggcagatctc cagccacgac 600 aaaggcggaa tgcgaggacg gctcattgac ctcctgaatc ccatcggaca aatcgactgc 660 gcagggaagt tccgtcacaa caccgatgag ctcctcgagg tctacgggga tgacaagttt 720 aagtacctag ctaactaccg cttcaacctc tgcccagaga attcactagg cgaaggctac 780 atcaccgaga aggtcttcga cagcatacgc gcaggctgta tccccatcta ttggggtgct 840 tacctagagc ctggcatcct taaccccaag gctatcctac gcttcgagga agggaaggaa 900 caagagttct ataaccgagt gaaggagctg tgggagaacg aagcggccta cgagcagttt 960 atcctcgagc cccccttcgt agagggagca gcagagcgca tctgggaaat cctccagggg 1020 cttcgtgagc gccttgcccc tcttgtggag gaaggataa 1059 <210> 30 <211> 352 <212> PRT <213> Porphyromonas catoniae <400> 30 Met Leu Ala Pro Tyr Lys Ser Pro Ile Phe Val Pro Ile Tyr Asp Thr 1 5 10 15 Lys Ala Met Asn Pro Pro Thr Lys Gln Pro Leu Arg Glu Arg Leu His 20 25 30 Met Met Arg Arg Arg Asn Arg Ile Arg Lys Arg Ser Val Ile Ala Leu 35 40 45 Ile Lys Ser His Leu Asp Ser Ser Arg Tyr Gln Asp Tyr Asn Trp Trp 50 55 60 Asp Ser His Ala Ser Thr Phe Trp Leu Pro Arg Phe Ile Asp Leu His 65 70 75 80 Leu Glu Pro Lys Lys Arg Ile Asn Leu Phe Ser Cys Phe Gln Asn Pro 85 90 95 Leu Met Leu Ile Arg Tyr Tyr Lys Gly Val Lys Ile Phe Leu Ser Gly 100 105 110 Glu Asn Leu Ala Asn Asn Glu His Phe Gly Phe His Pro Arg Met Leu 115 120 125 Asp His Arg Ile Asn Glu Val Asp Leu Ala Leu Gly Phe Glu Phe Arg 130 135 140 Lys Asp Pro Lys Tyr Tyr Arg Phe Pro Leu Trp Ile Tyr Gln Asn Glu 145 150 155 160 Phe Ile Ser Pro Ser Ala Ser Leu Glu Asp Ile Arg Ala Leu Leu Glu 165 170 175 Gln Ile Asn Asp Pro Ser Thr Arg Arg Ser Thr Gly Arg Ser Arg Phe 180 185 190 Ile Gly Gln Ile Ser Ser His Asp Lys Gly Gly Met Arg Gly Arg Leu 195 200 205 Ile Asp Leu Leu Asn Pro Ile Gly Gln Ile Asp Cys Ala Gly Lys Phe 210 215 220 Arg His Asn Thr Asp Glu Leu Leu Glu Val Tyr Gly Asp Asp Lys Phe 225 230 235 240 Lys Tyr Leu Ala Asn Tyr Arg Phe Asn Leu Cys Pro Glu Asn Ser Leu 245 250 255 Gly Glu Gly Tyr Ile Thr Glu Lys Val Phe Asp Ser Ile Arg Ala Gly 260 265 270 Cys Ile Pro Ile Tyr Trp Gly Ala Tyr Leu Glu Pro Gly Ile Leu Asn 275 280 285 Pro Lys Ala Ile Leu Arg Phe Glu Glu Gly Lys Glu Gln Glu Phe Tyr 290 295 300 Asn Arg Val Lys Glu Leu Trp Glu Asn Glu Ala Ala Tyr Glu Gln Phe 305 310 315 320 Ile Leu Glu Pro Pro Phe Val Glu Gly Ala Ala Glu Arg Ile Trp Glu 325 330 335 Ile Leu Gln Gly Leu Arg Glu Arg Leu Ala Pro Leu Val Glu Glu Gly 340 345 350 <210> 31 <211> 1032 <212> DNA <213> Butyrivibrio fibrisolvens <400> 31 atgaacataa ttcactttta tgcaagatat ttaagagaat cacataattg gaacagagaa 60 cgtgaagtta ctcgtaatgg tgttatgact tttgctaact ggtggagaga agatccgcat 120 aagaactggt ttgcaagatt tattgatgct gggaataaag accctgagcg caggatcaga 180 ttctatagta ttttggacc ttatagtaaa ttgaaggaag atttgatgg agccaagata 240 ttctttccg gagaaaatct tgaacagccg gttttacaca gaatactaaa gacagatcct 300 atagaagaca ggatatgggc tgacagaaga aagctgtatg gtaattatgg agctggagaa 360 gtggatcttg ctataggttt tggtaataga gaagaggatt cacttctggg atttgaaggg 420 agcaggaaga caaaatatat ccgctttcct tttggctta catatgtctt tgatcctgac 480 tgtactcatg atgatattaa gagaaccata gatgagataa atgcagttcg ttctacaggc 540 aggaaggata ccctccttct tgcatcgcat gatttctggg ggacaaggtc agatatctta 600 aagagtcttg aaggtgtatg tgatattagt attgccggta aatggcgcaa taacaccaaa 660 gagctctggg aagattatga caatgacaag aataaatatc tgtcagaatt taaatttaac 720 atatgccctg aaaatgttga tgcaccggga tatgtaacag agaagatatt tgatgcttttt 780 aaatgcggag ctattcctat atatcagggc tgcctaggta agcctgagcc gaatgtgata 840 aatacagatg cagtacttct atgggacttc gatggagata attcagatac tatagccttg 900 attaaaaaac taaattcgga taatgtatac tatgataact ttgtatctca gcctaaattc 960 aaaccggatg cggcagagta tgtagttgca tgtatggatg agctaaggcg tagctttgac 1020 aggctgatct ga 1032 <210> 32 <211> 343 <212> PRT <213> Read more <400> 32 Met Assn Ile Ile His Phe Tyr Ala Arg Tyr Leu Arg Glu Ser His Assn 1 5 10 15 Trp Asn Arg Glu Arg Glu Val Thr Arg Asn Gly Val Met Thr Phe Ala 20 25 30 Asn Trp Trp Arg Glu Asp Pro His Lys Asn Trp Phe Ala Arg Phe Ile 35 40 45 Asp Ala Gly Asn Lys Asp Pro Glu Arg Arg Ile Arg Phe Tyr Ser Ile 50 55 60 Phe Gly Pro Tyr Ser Lys Leu Lys Glu Asp Phe Asp Gly Ala Lys Ile 65 70 75 80 Phe Phe Ser Gly Glu Asn Leu Glu Gln Pro Val Leu His Arg Ile Leu 85 90 95 Lys Thr Asp Pro Ile Glu Asp Arg Ile Trp Ala Asp Arg Arg Lys Leu 100 105 110 Tyr Gly Asn Tyr Gly Ala Gly Glu Val Asp Leu Ala Ile Gly Phe Gly 115 120 125 Asn Arg Glu Glu Asp Ser Leu Leu Gly Phe Glu Gly Ser Arg Lys Thr 130 135 140 Lys Tyr Ile Arg Phe Pro Leu Trp Leu Thr Tyr Val Phe Asp Pro Asp 145 150 155 160 Cys Thr His Asp Asp Ile Lys Arg Thr Ile Asp Glu Ile Asn Ala Val 165 170 175 Arg Ser Thr Gly Arg Lys Asp Thr Leu Leu Leu Ala Ser His Asp Phe 180 185 190 Trp Gly Thr Arg Ser Asp Ile Leu Lys Ser Leu Glu Gly Val Cys Asp 195 200 205 Ile Ser Ile Ala Gly Lys Trp Arg Asn Asn Thr Lys Glu Leu Trp Glu 210 215 220 Asp Tyr Asp Asn Asp Lys Asn Lys Tyr Leu Ser Glu Phe Lys Phe Asn 225 230 235 240 Ile Cys Pro Glu Asn Val Asp Ala Pro Gly Tyr Val Thr Glu Lys Ile 245 250 255 Phe Asp Ala Phe Lys Cys Gly Ala Ile Pro Ile Tyr Gln Gly Cys Leu 260 265 270 Gly Lys Pro Glu Pro Asn Val Ile Asn Thr Asp Ala Val Leu Leu Trp 275 280 285 Asp Phe Asp Gly Asp Asn Ser Asp Thr Ile Ala Leu Ile Lys Lys Leu 290 295 300 Asn Ser Asp Asn Val Tyr Tyr Asp Asn Phe Val Ser Gln Pro Lys Phe 305 310 315 320 Lys Pro Asp Ala Ala Glu Tyr Val Val Ala Cys Met Asp Glu Leu Arg 325 330 335 Arg Ser Phe Asp Arg Leu Ile 340 <210> 33 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Conserved GDP-fucose binding domain <220> <221> Variants <222> (1) <223> Xaa can be Tyr, Trp, Leu, His, Phe or Met <220> <221> uncertain <222> (2) <223> Xaa can be any naturally occurring amino acid <220> <221> Variants <222> (3) <223> Xaa can be Thr, Ser or Cys <220> <221> Variants <222> (4) <223> Xaa can be Glu, Gln, Asp or Asn <220> <221> Variants <222> (5) <223> Xaa can be Lys or Arg <400> 33 Xaa Xaa Xaa Xaa Xaa 1 5 <210> 34 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Conserved domain <220> <221> Variants <222> (1) <223> Xaa can be Lys or Asp <220> <221> Variants <222> (2) <223> Xaa can be Leu, Lys or Met <220> <221> uncertain <222> (3)..(5) <223> Xaa can be any naturally occurring amino acid <220> <221> Variants <222> (6) <223> Xaa can be Phe or Tyr <400> 34 Xaa Xaa Xaa Xaa Xaa Xaa 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Conserved motif <220> <221> Variants <222> (1) <223> Xaa can be Asn or His <220> <221> uncertain <222> (2) <223> Xaa can be any naturally occurring amino acid <220> <221> uncertain <222> (6) <223> Xaa can be any naturally occurring amino acid <400> 35 Xaa Xaa Asp Pro Ala Xaa Leu Asp 1 5 <210> 36 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Conserved motif <220> <221> Variants <222> (3) <223> Xaa can be Ala or Ser <400> 36 Asp Met Xaa Val Ser Phe 1 5 <210> 37 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Conserved GDP-fucose binding domain <220> <221> uncertain <222> (2) <223> Xaa can be any naturally occurring amino acid <400> 37 Tyr Xaa Thr Glu Lys 1 5 <210> 38 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Conserved domain <220> <221> Variants <222> (1) <223> Xaa can be Lys or Asp <220> <221> uncertain <222> (3) <223> Xaa can be any naturally occurring amino acid <220> <221> Variants <222> (4) <223> Xaa can be Ile, Leu or Met <220> <221> Variants <222> (6) <223> Xaa can be Phe or Tyr <400> 38 Leu ... 1 5 <210> 39 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Conserved domain <220> <221> Variants <222> (1) <223> Xaa can be Lys or Asp <220> <221> Variants <222> (2) <223> Xaa can be Leu or Lys <220> <221> uncertain <222> (3) <223> Xaa can be any naturally occurring amino acid <220> <221> Variants <222> (5) <223> Xaa can be Ser or Gly <220> <221> Variants <222> (6) <223> Xaa can be Phe or Tyr <400> 39 Xaa Xaa Xaa Leu Xaa Xaa 1 5 <210> 40 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Conserved domain <220> <221> Variants <222> (1) <223> Xaa can be Lys or Asp <220> <221> uncertain <222> (3) <223> Xaa can be any naturally occurring amino acid <220> <221> uncertain <222> (6) <223> Xaa can be Phe or Tyr <400> 40 Sorry, I'm fine. 1 5 <210> 41 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Conserved domain <220> <221> Variants <222> (2) <223> Xaa can be Ile or Val <220> <221> Variants <222> (4) <223> Xaa can be Phe or Leu <220> <221> USNURE <222> (5) <223> Xaa can be any naturally occurring amino acid <400> 41 Lys Xaa Phe Xaa Xaa Gly Glu Asn 1 5 <210> 42 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Conserved domain <400> 42 Arg Phe Pro Leu Trp 1 5 BRIEF DESCRIPTION OF THE DRAWINGS

[0269] Figure 1 An alignment of the polypeptide sequences of SEQ ID NO 6, SEQ ID 10, SEQ ID 12, SEQ ID 14 and SEQ ID NO 16 is shown.

[0270] Figure 2Normalized production of 3-fucosyllactose in growth experiments is shown.

[0271] Figure 3 Shown is the normalized production of 3-fucosyllactose in growth experiments with low to high amounts of lactose in the medium.

[0272] Figure 4 Shown is the normalized production of 3-fucosyllactose in growth experiments with low amounts of lactose in the medium.

[0273] Figure 5 Shown is the percentage of lactose converted to 3-FL by one of the identified lactose binding α-1,3-fucosyltransferases.

[0274] Figure 6 Shown is the percentage of lactose converted to 3-FL for different identified lactose-binding α-1,3-fucosyltransferases driven by different promoters.

[0275] Figure 7 Normalized yields of 3-fucosyllactose from further experiments are shown.

[0276] Figure 8 Shown is the normalized production of 3-fucosyllactose by a subset of identified lactose-binding α-1,3-fucosyltransferases driven by different promoters.

[0277] Fig. 9 Shown is the normalized production of 3-fucosyllactose from strains expressing H. pylori fucT (SEQ ID 18) from two different promoters.

[0278] Fig.10 Shown is the normalized production of 3-fucosyllactose for strains expressing polypeptides with the DM[AS]VSF consensus motif

[0279] Fig.11 An alignment of the polypeptide sequences of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 28, SEQ ID NO 30 and SEQ ID NO 32 is shown. The consensus motifs [Y / W / L / H / F / M]X[T / S / C][E / Q / D / A][K / R], [K / D][L / K / M]XXX[F / Y] and [FW]W are marked with boxes, wherein X can be any different amino acid.

[0280] Fig.12 An alignment of the polypeptide sequences of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24 and SEQ ID NO 26 is shown. The consensus motifs DM[A / S]VSF and [N / H]XDPAXLD are marked with boxes, where X can be any different amino acid (and unrelated motifs).

[0281] Fig.13 An alignment of the polypeptide sequences of SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 28, SEQ ID NO 30 and SEQ ID NO 32 is shown. The consensus motifs K[I / V]F[F / L]XGEN (SEQ ID NO 41) and RFPLW (SEQ ID NO 42) are marked with boxes, wherein X can be any different amino acid. Example

[0282] Example 1: Materials and Methods Escherichia coli

[0283] Culture medium

[0284] Luria broth (LB) medium is made up of 1% tryptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR.Leuven, Belgium).The medium for shaking flask experiment comprises 2.00g / L NH4Cl, 5.00g / L (NH4)2SO4, 2.993g / L KH2PO4, 7.315g / L K2HPO4, 8.372g / L MOPS, 0.5g / L NaCl, 0.5g / L MgSO4.7H2O, 14.26g / L sucrose or other carbon sources specified in the embodiment, 1ml / L vitamin solution, 100μL / L molybdate solution and 1ml / L selenium solution.The pH value of the medium is set to 7 with 1M KOH. The vitamin solution consists of 3.6g / L FeCl2.4H2O, 5g / L CaCl2.2H2O, 1.3g / L MnCl2.2H2O, 0.38g / L CuCl2.2H2O, 0.5g / L CoCl2.6H2O, 0.94g / L ZnCl2, 0.0311g / L H3BO4, 0.4g / L Na2EDTA.2H2O and 1.01g / L thiamine hydrochloride. The molybdate solution contains 0.967g / L NaMoO4.2H2O. The selenium solution contains 42g / L SeO2.

[0285] The basic medium used for fermentation contained 6.75 g / L NH4Cl, 1.25 g / L (NH4)2SO4, 2.93 g / L KH2PO4 and 7.31 g / L KH2PO4, 0.5 g / L NaCl, 0.5 g / L MgSO4.7H2O, 14.26 g / L sucrose, 1 mL / L vitamin solution, 100 μL / L molybdate solution and 1 mL / L selenium solution, and the composition was the same as above.

[0286] The complex medium was sterilized by autoclaving (121° C., 21′), and the minimal medium was sterilized by filtration (0.22 μm Sartorius). If necessary, the medium was made selective by adding antibiotics such as chloramphenicol (20 mg / L), carbenicillin (100 mg / L), spectinomycin (40 mg / L) and / or kanamycin (50 mg / L).

[0287] Plasmids

[0288] The pKD46 (Red helper plasmid, ampicillin resistance), pKD3 (containing the chloramphenicol resistance (cat) gene flanked by FRT), pKD4 (containing the kanamycin resistance (kan) gene flanked by FRT) and pCP20 (expressing FLP recombinase activity) plasmids were obtained from Professor R. Cunin (University of Brussels, Belgium, 2007).

[0289] The α-1,3-fucosyltransferase expression plasmid was constructed in the pMB1 ori vector using the Golden Gate assembly method. The gene was expressed using promoters apFAB305 ("PROM0012"), apFAB146 ("PROM0032") (both as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360)) and p14 ("PROM0016" in combination with "UTR0019") (as described by DeMey et al. (BMC Biotechnology 2007)) and UTRs Gene10-LeuAB-BCD2 ("UTR0002") (as described by Mutalik et al. (Nat. Methods 2013, No. 10, 354-360)).

[0290] Plasmids were maintained in the host Escherichia coli DH5α (F - ,phi80dlacZdeltaM15,delta(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(rk - ,mk +),phoA,supE44,lambda - ,thi-1,gyrA96,relA1).

[0291] Strains and mutations

[0292] Escherichia coli K12-MG1655[lambda - , F - , rph-1] was obtained from Coli Genetic Stock Center (US), CGSC strain #: 7740 in March 2007. Gene disruption and gene introduction were performed using the technology published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technology is based on antibiotic selection after homologous recombination by lambda Red recombinase. Subsequent catalysis by flippase recombinase ensures that the antibiotic selection cassette is removed in the final production strain.

[0293] Transformants carrying the Red helper plasmid pKD46 were cultured in 10 ml LB medium containing ampicillin (100 mg / L) and L-arabinose (10 mM) at 30°C until OD600nm was 0.6. The cells were made electrocompetent by washing with 50 ml ice-cold water for the first time and 1 ml ice-cold water for the second time. The cells were then resuspended in 50 μl of ice-cold water. The cells were then purified by using a Gene Pulser. TM Electroporation was performed with 50 μl of cells and 10-100 ng of linear double-stranded DNA product (BioRad) (600Ω, 25 μFD and 250 V).

[0294] After electroporation, the cells were added to 1 mL of LB medium and incubated at 37°C for 1 h, and finally plated on LB agar containing 25 mg / L chloramphenicol or 50 mg / L kanamycin to select antibiotic-resistant transformants. The selected mutants were verified by PCR with primers upstream and downstream of the modified region and grown in LB agar at 42°C to lose the helper plasmid. The mutants were tested for ampicillin sensitivity.

[0295] Linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivatives as templates. The primers used had a portion of sequence complementary to the template and another portion complementary to the side of the chromosomal DNA where recombination must occur. For genomic knockout, the homologous regions were designed to be 50-nt upstream and 50-nt downstream of the start codon and stop codon of the target gene. For genomic knock-in, the transcription start point (+1) must be considered. The PCR product was PCR purified, Dpnl digested, repurified from an agarose gel, and suspended in an elution buffer (5mM Tris, ph8.0).

[0296] The selected mutants (chloramphenicol or kanamycin resistance) were transformed with pCP20 plasmid, which is an ampicillin and chloramphenicol resistance plasmid that shows temperature-sensitive replication and heat-induced FLP synthesis. Ampicillin-resistant transformants were selected at 30°C, and a few colonies were then purified in LB at 42°C and then tested for all antibiotic resistance and loss of the FLP helper plasmid. Gene knockouts and knock-ins were checked with control primers (Fw / Rv-gene-out).

[0297] By knocking out the genes lacZ, lacY lacA, glgC, agp, pfkA, pfkB, pgi, arcA, iclR, wcaJ, pgi, lon and thyA, a mutant strain derived from Escherichia coli K12-MG1655 was produced. In addition, the E. coli lacY gene, the fructokinase gene (frk) from Zymomonas mobilis and the sucrose phosphorylase (SP) from Bifidobacterium adolescentis were knocked into the genome and constitutively expressed. The constitutive promoter is derived from the promoter library described by De Mey et al. (BMC Biotechnology, 2007). These genetic modifications are also described in WO2016075243 and WO2012007481.

[0298] All constructed plasmids carrying putative α-1,3-fucosyltransferase genes were evaluated in this mutant strain derived from E. coli K12 MG1655. All strains were stored in frozen vials at -80°C (overnight LB culture mixed with 70% glycerol in a 1:1 ratio). All successful lactose binding α-1,3-fucosyltransferases (SEQ ID NOs 1 to 16, 19 to 22 and 27 to 32) as well as prior art α-1,3-fucosyltransferases (SEQ ID NOs 17-18) and two non-functional α-1,3-fucosyltransferases (SEQ ID NOs 23 to 26) are provided in Table 3.

[0299] Table 3:

[0300]

[0301]

[0302] Heterologous and homologous expression

[0303] All potential α-1,3-fucosyltransferase genes for expression, either for plasmids or for genomic insertion, were synthesized at Twist Biosciences (San Francisco, USA). Expression can be further enhanced by optimizing codon usage according to the codon usage of the expression host. Genes were optimized using the supplier's tools.

[0304] Culture conditions

[0305] The pre-culture of 96-well microtiter plate experiments was started from frozen vials, in 150 μL LB and incubated overnight at 37°C on an orbital shaker at 800 rpm. The culture was used as an inoculum for 96-well square microtiter plates, diluted 400 times with 400 μL MMsf culture medium. These final 96-well culture plates were then incubated for 72 h or shorter or longer at 37°C on an orbital shaker at 800 rpm. At the end of the culture experiment, samples were collected from each well to measure the sugar concentration (extracellular sugar concentration, after the cells were centrifuged) in the broth supernatant, or the culture fluid was boiled at 90°C for 15 min (=the whole broth measurement, the mean value of the intracellular and extracellular sugar concentrations) before the cells were centrifuged.

[0306] Bioreactor precultures were started from an entire 1 mL frozen vial of the specific strain, inoculated into 250 mL or 500 mL of MMsf medium in a 1 L or 2.5 L shake flask and incubated at 37°C on an orbital shaker at 200 rpm for 24 h. The 5 L bioreactor was then inoculated (250 mL in 2 L batch medium); the process was controlled by MFCS control software (Sartorius StedimBiotech, Melsungen, Germany). Culture conditions were set at 37°C with maximum agitation; the pressure gas flow rate depended on the strain and bioreactor. The pH was controlled at 6.8 using 0.5 M H2SO4 and 20% NH4OH. The exhaust gas was cooled. A 10% silicone defoamer solution was added during the fermentation process when foaming occurred.

[0307] Optical density

[0308] The cell density of the cultures was monitored frequently by measuring the optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or Spark 10M microplate reader, Tecan, Switzerland).

[0309] Liquid chromatography

[0310] 3-fucosyllactose standard was prepared in-house. Other standards, such as but not limited to lactose, sucrose, glucose, and fructose were purchased from Sigma.

[0311] Carbohydrates were analyzed via the HPLC-RI (Waters, USA) method, where RI (refractive index) detects the change in the refractive index of the mobile phase when the sample is present. Sugars were separated in a constant flow using an X-bridge column (Waters X-bridge HPLC column, USA) and a mobile phase containing 75 ml of acetonitrile, 25 ml of ultrapure water and 0.15 ml of triethylamine. The column dimensions were 4.6 x 150 mm and the particle size was 3.5 μm. The column temperature was set at 35 ° C and the pump flow rate was 1 mL / min.

[0312] Example 2: Evaluation of different lactose binding α-1,3-fucosyltransferases spiked into E. coli

[0313] An experiment was set up to evaluate several genes encoding potential α-1,3-fucosyltransferases capable of producing 3-fucosyllactose (3-FL) from GDP-fucose and lactose. Growth experiments were performed according to the culture conditions provided in Example 1.

[0314] Figure 2 Shown is the normalized yield of 3-fucosyllactose obtained in growth experiments of strains successfully expressing various lactose-binding α-1,3-fucosyltransferases using two different promoters (PROM0012 and PROM0016) using 20 g / L lactose in the production medium. Each data point corresponds to the data for one well. The horizontal dashed line indicates the set point to which all data points were normalized.

[0315] The experiment identified the following polypeptides having lactose binding 3-fucosyltransferase activity as having similar or better lactose binding α-1,3-fucosyltransferase activity than the strain containing SEQ ID 18 having previously confirmed lactose binding α-1,3-fucosyltransferase activity: SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12 and SEQ ID NO 14. The polypeptide of SEQ ID NO 4 has 90.8% overall sequence identity with SEQ ID NO 2, thereby indicating that sequences having 87% or more sequence identity with SEQ ID NO 2 also have lactose binding α-1,3-fucosyltransferase activity.

[0316] Example 3: Incubation of lactose-binding α-1,3-fucosyltransferase in Escherichia coli in minimal medium Evaluation of the ability to produce 3-FL at low to high lactose concentrations

[0317] The gene encoding SEQ ID NO 6 (in combination with PROM0016) was evaluated for its ability to produce 3-FL in minimal medium containing different concentrations of lactose. Growth experiments were performed according to the culture conditions provided in Example 1. Strains with SEQ ID NO 6 and SEQ ID NO 18 (driven by PROM0016) were grown in multiple wells of a 96-well plate as described above. SEQ ID NO 18 has previously demonstrated α-1,3-fucosyltransferase activity on lactose.

[0318] Figure 3 The normalized yield of 3-fucosyllactose is shown, with 6 different concentrations of lactose as a precursor for 3-FL (90 g / L and its 1:2 dilution series, up to 2.8 g / L, as shown). Each data point corresponds to the data of one well. The horizontal dashed line indicates the set point to which all data points were normalized.

[0319] The experiments identified that the polypeptide of SEQ ID NO 6 had better lactose binding α-1,3-fucosyltransferase activity at all lactose concentrations compared to a strain expressing SEQ ID NO 18, a polypeptide with previously demonstrated lactose binding α-1,3-fucosyltransferase activity.

[0320] Example 4: Effect of various lactose-binding α-1,3-fucosyltransferases incorporated into Escherichia coli in a minimal culture medium Evaluation of the ability of the medium to produce 3-FL at low lactose concentrations

[0321] In a growth experiment on low lactose concentrations, the ability to produce 3-fucosyllactose from GDP-fucose and lactose was evaluated for the strains identified above that have genes encoding SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 12 and SEQ ID NO 14. The growth experiment was performed according to the culture conditions provided in Example 1.

[0322] Figure 4 Shown are the normalized production of 3-fucosyllactose for strains expressing various α-1,3-fucosyltransferases (using two different promoters PROM0012 and PROM0016) and grown in medium containing a low amount of lactose (2.8 g / L lactose). Each data point corresponds to the data for one well. The horizontal dashed line indicates the set point to which all data points were normalized.

[0323] The experiments identified that the following polypeptides having SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 12 and SEQ ID NO 14 have similar or better lactose binding α-1,3-fucosyltransferase activity when provided with low concentrations of lactose compared to a strain containing SEQ ID 18 having previously demonstrated lactose binding α-1,3-fucosyltransferase activity.

[0324] Example 5: Enzyme activity of the polypeptide of SEQ ID NO 6 incorporated into Escherichia coli against two low concentrations of lactose Evaluate

[0325] In growth experiments providing 2.8 g / L or 5.62 g / L lactose and 30 g / L sucrose, the gene encoding SEQ ID NO 6 (and in combination with PROM0016) was evaluated for its ability to convert lactose to 3-fucosyllactose in strains producing GDP-fucose. Growth experiments were performed according to the culture conditions provided in Example 1.

[0326] Figure 5 The % of lactose converted to 3-FL is shown, calculated by dividing the measured amount of 3-FL by the amount theoretically obtainable based on the lactose input concentration. Theoretically, if all lactose is converted, a value of 100% is obtained. Each data point corresponds to the data for one well.

[0327] The strain expressing the polypeptide shown in SEQ ID NO 6 was compared with the strain expressing the polypeptide shown in SEQ ID NO 18 (driven by PROM0016), which was previously confirmed to have α-1,3-fucosyltransferase activity on lactose. At two lactose concentrations, for a given amount of carbon source (30 g / L sucrose), the strain expressing the polypeptide shown in SEQ ID NO 6 was able to convert much more lactose into 3-FL than the strain expressing the polypeptide shown in SEQ ID NO 18.

[0328] Example 6: Effect of various lactose-binding α-1,3- Evaluation of fucosyltransferase enzyme activity

[0329] In growth experiments at low concentrations of lactose (2 g / L) and sucrose (7.5 g / L), the genes encoding the above identified polypeptides SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 12 and SEQ ID NO 14 were evaluated for their ability to convert lactose into 3-fucosyllactose in strains producing GDP-fucose. The growth experiments were performed according to the culture conditions provided in Example 1.

[0330] Figure 6 The % lactose converted to 3-FL is shown, calculated by dividing the measured amount of 3-FL by the amount theoretically obtainable based on the lactose input concentration. Each data point corresponds to the data for one well.

[0331] For a given amount of carbon source (7.5 g / L sucrose), strains expressing the polypeptide having SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 12 or SEQ ID NO 14 were able to convert more lactose into 3-FL than strains expressing the polypeptide having SEQ ID NO 18.

[0332] Example 7: Batch fermentation of Escherichia coli expressing various lactose-binding α-1,3-fucosyltransferases Evaluation of strains

[0333] Bioreactor scale batch fermentations were performed to evaluate strains derived from a mutant E. coli K12 MG1655 strain background as described in Example 1 expressing various α-1,3-fucosyltransferases having SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 12 and SEQ ID NO 14. Bioreactor runs were performed as described in Example 1. In these examples, sucrose was used as the carbon source. Lactose was added at 90 g / L in the batch medium as a precursor for 3-FL formation.

[0334] Figure 7Shown are the normalized yields of 3-fucosyllactose obtained in batch fermentations using strains successfully expressing various lactose-binding α-1,3-fucosyltransferases with lactose as precursor in the production medium. Each data point corresponds to the data from one fermentation run. The horizontal dashed line indicates the set point to which all data points were normalized.

[0335] The experiments showed that mutant E. coli strains expressing the lactose-binding α-1,3-fucosyltransferase gene having SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 12 or SEQ ID NO 14 produced greater amounts of 3-FL than strains expressing the polypeptide having SEQ ID NO 18.

[0336] Example 8: Evaluation of different lactose binding α-1,3-fucosyltransferases spiked into E. coli

[0337] Further experiments were performed with strains expressing enzymes having SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 14 and SEQ ID NO 16 and evaluating whether these enzymes are able to produce 3-fucosyllactose from lactose in strains producing GDP-fucose. Growth experiments were performed according to the culture conditions provided in Example 1.

[0338] Figure 8 Shown is the normalized production of 3-fucosyllactose using 20 g / L lactose in production medium using strains that successfully expressed various lactose binding α-1,3-fucosyltransferases (using three different promoters PROM0012, PROM0016 and PROM0026). Each data point corresponds to the data for one well. The horizontal dashed line indicates the set point to which all data points were normalized.

[0339] The experiment confirmed the results from Example 2 of strains expressing polypeptides having SEQ ID NO 12, SEQ ID NO 6, SEQ ID NO 12 and SEQ ID NO 14, and identified that the polypeptide having SEQ ID NO 16 also had better lactose binding α-1,3-fucosyltransferase activity than the strain containing SEQ ID 18 having previously confirmed lactose binding α-1,3-fucosyltransferase activity.

[0340] Example 9: Materials and Methods Saccharomyces cerevisiae

[0341] Culture medium

[0342] Strains were grown on synthetic defined yeast medium with a complete supplement mixture (SD CSM) or CSM drop-out (SD CSM-Ura) containing 6.7 g / L yeast nitrogen base without amino acids (YNB w / o AA, Difco), 20 g / L agar (Difco) (solid culture), 22 g / L glucose monohydrate or 20 g / L lactose, and 0.79 g / L CSM or 0.77 g / L CSM Ura (MP Biomedicals).

[0343] strain

[0344] Saccharomyces cerevisiae BY4742 created by Bachmann et al. (Yeast (1998) 14: 115-32) was obtained from the Euroscarf Culture Center. All mutant strains were generated by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11: 355-360, 1995). Kluyveromyces marxianus lactis was available at the LMG Culture Center (Ghent, Belgium).

[0345] Plasmids

[0346] The yeast expression plasmid p2a_2μ_sia_GFA1 (Chan 2013 (Plasmid 70 (2013) 2-17)) is used to express foreign genes in Saccharomyces cerevisiae. The plasmid contains an ampicillin resistance gene and a bacterial replication origin to allow selection and maintenance in E. coli. The plasmid also contains the 2μ yeast ori and Ura3 selection markers for selection and maintenance in yeast. Next, the plasmid can be modified to p2a_2μ_fl to contain lactose permease (e.g., LAC12 from Kluyveromyces lactis), GDP mannose 4,6-dehydratase (e.g., Gmd from E. coli), and GDP-L-fucose synthase (e.g., fcl from E. coli).

[0347] The yeast expression plasmid p2a_2μ_fl_3ft is based on p2a_2μ_ft, but is modified in such a way that it also expresses SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16 or SEQ ID NO 18. Preferably, but not necessarily, the fucosyltransferase protein is fused at the N-terminus to a SUMOstar tag (e.g., obtained from pYSUMOstar, Life Sensors, Malvern, PA) to enhance the solubility of the fucosyltransferase.

[0348] Plasmids were maintained in the host Escherichia coli DH5α (F - ,phi80dlacZdeltaM15,delta(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(rk - ,mk + ),phoA,supE44,lambda - ,thi-1,gyrA96,relA1).

[0349] Gene expression promoter

[0350] A synthetic constitutive promoter was used to express the gene as described by Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012).

[0351] Heterologous and homologous expression

[0352] The gene to be expressed, either from a plasmid or from the genome, is synthesized by one of the following companies: DNA2.0, Gen9, or IDT.

[0353] Expression can be further enhanced by optimizing codon usage for the expression host. The genes were optimized using the supplier's tools.

[0354] Culture conditions

[0355] In general, yeast strains were initially grown on SD-CSM plates to obtain single colonies. These plates were grown at 30°C for 2-3 days.

[0356] Starting from a single colony, a pre-culture was grown overnight in 5 mL at 30°C with shaking at 200 rpm. Subsequent 125 ml shake flask experiments were inoculated with 2% of this pre-culture in 25 ml of medium. These shake flasks were incubated at 30°C with orbital shaking at 200 rpm. No inducer was required because all genes were constitutively expressed.

[0357] Example 10: Production of 3-fucose in Saccharomyces cerevisiae using various lactose-binding α-1,3-fucosyltransferases Lactose

[0358] Another example provides a eukaryotic organism in the form of Saccharomyces cerevisiae for use in the present invention. Using the strains, plasmids and methods described in Example 9, a strain expressing SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16 or SEQ ID NO 18 is created.

[0359] In addition, further modifications were made to produce 3-fucosyllactose. These modifications include the addition of lactose permease, GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase. The preferred lactose permease is the KILAC12 gene from Kluyveromyces lactis (WO 2016 / 075243). The preferred GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase are gmd and fcl from Escherichia coli, respectively.

[0360] These strains are capable of growing on glucose or glycerol as a carbon source, converting the carbon source into GDP-L-fucose, absorbing lactose, and producing 3-fucosyllactose using GDP-L-fucose and lactose as substrates for an enzyme represented by SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16 or SEQ ID NO 18, with SEQ ID NO 18 being used as a reference.

[0361] Precultures of the strains were performed in 5 mL of synthetic defined medium SD-CSM (containing 22 g / L glucose) and grown at 30° C. as described in Example 9. These precultures were inoculated into 25 mL of medium in shake flasks with 10 g / L sucrose as the sole carbon source and grown at 30° C. Routine samples were collected and the production of 3-fucosyllactose was measured as described in Example 1.

[0362] Example 11: Enzymatic production of 3-fucosyllactose

[0363] Another example provides the use of an enzyme of the invention having SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14 or SEQ ID NO 16. These enzymes are produced in a cell-free expression system, such as but not limited to the PURExpress system (NEB), or in a host organism, such as but not limited to E. coli or Saccharomyces cerevisiae, after which the enzyme can be isolated and optionally further purified.

[0364] Each of the above enzyme extracts or purified enzymes is added to a reaction mixture together with GDP-fucose, lactose and a buffer component (e.g., Tris-HCl or HEPES). The reaction mixture is then incubated at a specific temperature (e.g., 37° C.) for a certain amount of time (e.g., 24 hours), during which lactose will be converted to 3-fucosyllactose by the enzyme using GDP-fucose. 3-fucosyllactose is then separated from the reaction mixture by methods known in the art. If preferred, 3-FL can be further purified. At the end of the reaction or after separation and / or purification, the production of 3-fucosyllactose is measured as described in Example 1.

[0365] Example 12: Production of 3-fucosyllactose at different lactose concentrations

[0366] Fermentation process as described in Example 1 and Example 7, wherein the lactose concentration in the culture medium is in the range of 50 to 150 g / L. In the process, the lactose is converted into 3-fucosyllactose until a very small amount of lactose remains. The final ratio of lactose to 3-fucosyllactose can be manipulated in the process by stopping the process earlier (higher lactose to 3-fucosyllactose ratio) or later (lower lactose to 3-fucosyllactose ratio). The lactose concentration in the container can be increased by feeding a high concentration of lactose solution (with or without another carbon source) to the bioreactor. The lactose feed contains a lactose concentration of 100 to 700 g / L and is maintained at a temperature such that lactose remains soluble at a pH less than or equal to 6 to avoid forming lactulose in the process, which is a standard method used in the dairy industry. The final concentration of 3-fucosyllactose achieved in this production process ranges from 70 g / L (when lower lactose concentrations are used in the process described above) to 200 g / L or more (when higher lactose concentrations are used in the process described above).

[0367] Example 13: Expression of Helicobacter pylori α-1,3-fucosyltransferase fucT (SEQ ID NO: 1) from various promoters ID 18) Evaluation

[0368] The gene encoding the Helicobacter pylori α-1,3-fucosyltransferase fucT (SEQ ID NO 18) was cloned in an expression vector under the control of the promoter PROM0012 or PROM0016, and the resulting plasmid was transformed into an E. coli mutant strain as described in Example 1. These strains were then evaluated for their ability to produce 3-FL in a growth assay. The two strains were grown in multiple wells of a 96-well plate.

[0369] Fig. 9 The normalized yield of 3-fucosyllactose produced by the strains is shown. Each data point corresponds to the data for one well. The horizontal dashed line indicates the set point to which all data points were normalized.

[0370] The experiments showed that 3-FL production in strains expressing H. pylori FucT using promoter PROM0012 was reduced to ±30% of the levels observed in similar strains expressing the fucosyltransferase from promoter PROM0016.

[0371] By extrapolating the data provided in Examples 2, 4 and 8, we can conclude that all strains containing any of SEQ ID NOs 2–16 showed significantly higher yields when the fucosyltransferases were expressed from the same promoter (PROM0012 or PROM0016) compared to the control strain with alpha 1,3-fucosyltransferase fucT (SEQ ID NO 18), with the exception that the strain with SEQ ID NO 10 showed similar yield to the control strain.

[0372] Example 14: Evaluation of 3-fucosyllactose in strains expressing polypeptides with the DM[AS]VSF consensus motif Production

[0373] In a growth experiment as described in Example 1, mutant E. coli strains containing expression constructs for any of SEQ ID NO 4, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, and SEQ ID NO 26 were evaluated for 3-FL production. Fig.12 As shown, all polypeptide sequences contain the consensus domain DM[AS]VSF (SEQ ID NO 36), but only SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 20 and SEQ ID NO 22 additionally contain the consensus motif [NH]XDPAXLD (SEQ ID NO 35) in the N-terminal region of the protein. A strain containing the Helicobacter pylori α-1,3-fucosyltransferase fucT (SEQ ID NO 18) was used as a positive control. All strains were grown in multiple wells of a 96-well plate and tested in standard medium containing 30 g / L sucrose and 20 g / L lactose.

[0374] Fig.10 The normalized yield of 3-fucosyllactose produced by the strains is shown. Each data point corresponds to the data for one well. The horizontal dashed line indicates the set point to which all data points were normalized.

[0375] The experiment showed that only strains containing polypeptides with both the consensus motifs [NH]xDPAxLD and DM[AS]VSF (i.e., SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 20, or SEQ ID NO 22) could produce 3-FL, while strains containing polypeptides with DM[AS]VSF but lacking [NH]xDPAxLD (i.e., SEQ ID NO 24 and SEQ ID NO 26) did not produce any 3-FL. Based on this data, we can conclude that the presence of the [NH]xDPAxLD (SEQ ID NO 35) consensus motif in the N-terminal region of the polypeptide with the DM[AS]VSF (SEQ ID NO 36) domain is essential for the enzyme to have lactose-binding α-1,3-fucosyltransferase activity.

[0376] Furthermore, the polypeptide of SEQ ID NO 22 has 92% global sequence identity with SEQ ID NO 2, thereby indicating that sequences having 87% or higher sequence identity with SEQ ID NO 2 also have lactose-binding α-1,3-fucosyltransferase activity.

[0377] Example 15: Expression of the polypeptide of SEQ ID NO 28, SEQ ID NO 30 or SEQ ID NO 32 in a strain Evaluate

[0378] Mutant E. coli strains containing expression constructs for SEQ ID NO 28, SEQ ID NO 30 or SEQ ID NO 32 can be evaluated for 3-FL production in a growth experiment as described in Example 1. At the end of the growth experiment, the production of 3-fucosyllactose can be observed in the culture broth.

[0379] Example 16: Evaluation of 3FL Purity at the End of Fed-Batch Fermentation

[0380] Bioreactor scale fed-batch fermentations were performed to evaluate strains derived from a mutant E. coli K12 MG1655 strain background, as described in Example 1, expressing various α-1,3-fucosyltransferases having SEQ ID NO 2, SEQ ID NO 6, and SEQ ID NO 18. Bioreactor runs were performed as described in Example 1. In these examples, sucrose was used as a carbon source. Lactose was added at 90 g / L in the batch medium as a precursor for 3-FL formation, and concentrated sucrose solution was added during the fed-batch period. For each strain, three independent fermentations were performed.

[0381] At the end of the fermentation, the broth was analyzed for the presence of lactose and 3-FL and the 3-FL purity was calculated using the formula 3FL (g / L) / (3FL (g / L) + lactose (g / L)). For the strain containing SEQ ID NO 18, an average purity of 85% was obtained, while for the strain containing SEQ ID NO 2 or 6, an average purity of more than 98% and 99%, respectively, was obtained.

[0382] Experiments have shown that mutant E. coli strains expressing a lactose-binding α-1,3-fucosyltransferase gene having SEQ ID NO 2 or SEQ ID NO 6 produce broths with higher 3-FL purity than similar strains containing SEQ ID NO 18 in bioreactor-scale fed-batch fermentations.

Claims

1. A method for producing α-1,3-fucosyllactose, the method comprising the following steps: a) providing a polypeptide having α-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate, wherein the polypeptide is selected from the group consisting of: SEQ ID NO: 6, 2 or 4, b) contacting the polypeptide having α-1,3-fucosyltransferase activity of step a) with a mixture comprising GDP-fucose as a donor substrate and lactose as an acceptor substrate under conditions wherein the polypeptide catalyzes the transfer of a fucose residue from a donor substrate to an acceptor substrate, Thus, α-1,3-fucosyllactose is produced. c) optionally isolating the α-1,3-fucosyllactose.

2. The method of claim 1, wherein the polypeptide is provided in a cell-free system.

3. The method of claim 1, wherein the polypeptide is produced by a cell comprising a polynucleotide encoding the polypeptide. 4 . The method according to claim 1 , wherein the GDP-fucose and / or lactose is provided by cells that produce the GDP-fucose and / or lactose.

5. The method according to claim 1, comprising the steps of: i) providing a cell genetically modified to produce α-1,3-fucosyllactose, said cell comprising at least one nucleic acid sequence encoding an enzyme for α-1,3-fucosyllactose synthesis, The cell comprises expression of the polypeptide having alpha-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate, ii) culturing the cells in a culture medium under conditions that permit the production of α-1,3-fucosyllactose. The method according to claim 5 , further comprising the step of isolating α-1,3-fucosyllactose from the culture.

7. The method according to claim 3, comprising the steps of: a) providing a host cell expressing the polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate; b) growing the host cell under suitable nutritional conditions that allow the production of α-1,3-fucosyllactose and the expression of the polypeptide having α-1,3-fucosyltransferase activity; c) simultaneously with or after step b), providing a donor substrate GDP-fucose and an acceptor substrate lactose, such that the α-1,3-fucosyltransferase polypeptide catalyzes the transfer of fucose residues from GDP-fucose to lactose, thereby producing α-1,3-fucosyllactose; d) optionally isolating the α-1,3-fucosyllactose from the host cell or the culture medium in which it is grown.

8. The method of claim 7, wherein the host cell is transformed or transfected to express an exogenous polypeptide having α-1,3-fucosyltransferase activity and having the ability to use lactose as an acceptor substrate.

9. The method according to any one of claims 5 or 7, characterized in that GDP-fucose and / or lactose are provided by enzymes co-expressed in the host cell or by metabolism of the host cell.

10. The method of claim 1, further comprising purification of α-1,3-fucosyllactose.

11. The method according to claim 1, further comprising at least one of the following steps: i) adding a lactose feed to the culture medium, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume; ii) adding a lactose feed to the culture medium in a continuous manner, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume; iii) adding a lactose feed to the culture medium, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume, and such that the final volume of the culture medium is no more than 3 times, or no more than 2 times, or less than 2 times the volume of the culture medium before the addition of the lactose feed; iv) adding a lactose feed to the culture medium in a continuous manner, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume, and such that the final volume of the culture medium is no more than 3 times, or no more than 2 times, or less than 2 times the volume of the culture medium before the addition of the lactose feed; v) adding a lactose feed to the culture medium via a feed solution in a continuous manner over the course of 1, 2, 3, 4 or 5 days; vi) adding a lactose feed to the culture medium in a continuous manner via a feed solution over the course of 1 day, 2 days, 3 days, 4 days or 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L or 600 g / L; vii) adding a lactose feed to the culture medium in a continuous manner via a feed solution over the course of 1 day, 2 days, 3 days, 4 days or 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L or 600 g / L; and wherein the pH of the solution is set at 3 to 7; viii) adding a lactose feed to the culture medium in a continuous manner via a feed solution over the course of 1 day, 2 days, 3 days, 4 days or 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L or 600 g / L; and wherein the pH of the solution is set at 3 to 7, and wherein the temperature of the feed solution is maintained at 20°C to 80°C; The method results in an α-1,3-fucosyllactose concentration of at least 50 g / L, at least 75 g / L, at least 90 g / L, at least 100 g / L, at least 125 g / L, at least 150 g / L, at least 150 g / L, at least 175 g / L, or at least 200 g / L in the final volume of the culture medium.

12. The method according to any one of claims 3 or 5, wherein the cell is selected from the group consisting of a microorganism, a plant cell other than a plant cell capable of developing into a complete plant, or an animal cell other than an animal cell capable of developing into a complete animal, wherein the microorganism is a bacterium or a fungus, the plant cell is derived from rice, cotton, rapeseed, soybean, corn or a cereal plant, and the animal cell is derived from an insect, a fish, a bird or a non-human mammal.

13. The method of claim 12, wherein the cell is an Escherichia coli cell or a yeast cell.

14. A host cell genetically modified to produce alpha-1,3-fucosyllactose, wherein the host cell comprises at least one nucleic acid sequence encoding an enzyme involved in the synthesis of alpha-1,3-fucosyl sugar; the cell comprises expressing a polypeptide having alpha-1,3-fucosyltransferase activity and the ability to use lactose as an acceptor substrate, wherein the polypeptide is selected from the group consisting of: SEQ ID NO: 6, 2 or 4, The cell further comprises: (1) a biosynthetic pathway for producing GDP-fucose suitable for transfer to lactose by the α-1,3-fucosyltransferase, and / or (2) a biosynthetic pathway for producing lactose or a mechanism for internalizing lactose from the culture medium into the cell where it is fucosylated to produce α-1,3-fucosyllactose, And wherein the cell is selected from the group consisting of a microorganism, a plant cell other than a plant cell capable of developing into a complete plant, or an animal cell other than an animal cell capable of developing into a complete animal, wherein the microorganism is a bacterium or a fungus, the plant cell is derived from rice, cotton, rapeseed, soybean, corn or a cereal plant, and the animal cell is derived from an insect, a fish, a bird or a non-human mammal.

15. The host cell according to claim 14, wherein the cell is an E. coli cell or a yeast cell.

16. The host cell according to any one of claims 14 or 15, comprising: i) a sequence comprising a polynucleotide encoding the polypeptide having lactose-binding α-1,3-fucosyltransferase activity, wherein the sequence is a sequence foreign to the host cell and wherein the sequence is integrated into the genome of the host cell, or ii) a vector comprising the polynucleotide encoding the polypeptide, wherein the polynucleotide is operably linked to a control sequence recognized by a host cell transfected with the vector.

17. The host cell according to claim 14, characterized in that The host cell is a bacterial cell.

18. The host cell according to claim 17, wherein the host cell is a cell of an Escherichia coli strain.

19. The host cell according to claim 18, wherein the E. coli strain is a K12 strain.

20. The host cell according to claim 19, wherein the E. coli K12 strain is E. coli MG1655.

21. The host cell according to claim 14, characterized in that The polynucleotide encoding the polypeptide having lactose-binding alpha-1,3-fucosyltransferase activity is adapted to the codon usage of the corresponding host cell.

22. A method for producing α-1,3-fucosyllactose, comprising the following steps: a) providing a host cell according to any one of claims 14 to 21, b) culturing the host cell in a culture medium under conditions that allow production of the α-1,3-fucosyltransferase.

23. The method according to claim 22, further comprising the step of isolating the α-1,3-fucosyltransferase from the culture.

24. Use of the host cell according to any one of claims 14 to 21 for producing alpha-1,3-fucosyllactose.

25. Use of the polypeptide of any one of claims 1 to 13 for producing α-1,3-fucosyllactose.

26. A microorganism heterologously expressing a lactose binding α-1,3-fucosyltransferase polypeptide, wherein the polypeptide is selected from the group consisting of: SEQ ID NO: 6, 2 or 4, The microorganism further comprises: (1) a biosynthetic pathway for producing GDP-fucose suitable for transfer to lactose by the α-1,3-fucosyltransferase, and / or (2) a biosynthetic pathway for producing lactose or a mechanism for internalizing lactose from the culture medium into the cells of the microorganism, where the lactose is fucosylated to produce α-1,3-fucosyllactose.

27. Use of the microorganism according to claim 26 for producing α-1,3-fucosyllactose.

28. The method according to any one of claims 1 or 22, further comprising the step of isolating the α-1,3-fucosyllactose from host cells or a culture medium in which they are grown.

29. The method of claim 28, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high performance filtration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and / or gel filtration, ligand exchange chromatography.

30. The method of claim 29, wherein the ultrafiltration is tangential flow ultrafiltration.

31. The method of claim 28, further comprising purification of α-1,3-fucosyllactose.

32. The method of claim 31 , wherein the purification of the α-1 ,3-fucosyllactose comprises at least one of the following steps: using activated charcoal or carbon, using charcoal, nanofiltration, ultrafiltration or ion exchange, using alcohol, using an aqueous alcohol mixture, crystallization, evaporation, precipitation or drying.

33. The method of claim 32, wherein the drying is spray drying or freeze drying.

34. The method of any one of claims 1 or 22, wherein the polypeptide is produced in a fungal, bacterial, insect, animal, and plant expression system that does not comprise: 1) insect cells capable of developing into a whole insect, 2) animal cells capable of developing into a whole animal, and 3) plant cells capable of developing into a whole plant.

35. The method of claim 34, wherein the host cell is a bacterial cell.

36. The method of claim 35, wherein the host cell is a cell of an E. coli strain.

37. The method of claim 36, wherein the E. coli strain is a K12 strain.

38. The method of claim 37, wherein the E. coli K12 strain is E. coli MG1655.

39. The method of claim 34, wherein the host cell is a yeast cell.

40. The method of any one of claims 1 or 22, wherein the lactose concentration in the culture medium is in the range of 50 to 150 g / L.

41. The method of any one of claims 1 or 22, wherein the final concentration of α-1,3-fucosyllactose is in the range of 70 g / L to 200 g / L.

42. The method of any one of claims 1 or 22, wherein the production results in a ratio of lactose concentration to alpha-1,3-fucosyllactose concentration of less than 1 :5 at the end of fermentation.

43. The method of any one of claims 1 or 22, wherein the production results in an alpha-1,3-fucosyllactose purity of 80% or more at the end of fermentation.

44. The method of claim 22, further comprising at least one of the following steps: i) adding a lactose feed to the culture medium, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume; ii) adding a lactose feed to the culture medium in a continuous manner, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume; iii) adding a lactose feed to the culture medium, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume, and such that the final volume of the culture medium is no more than 3 times, or no more than 2 times, or less than 2 times the volume of the culture medium before the addition of the lactose feed; iv) adding a lactose feed to the culture medium in a continuous manner, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume, and such that the final volume of the culture medium is no more than 3 times, or no more than 2 times, or less than 2 times the volume of the culture medium before the addition of the lactose feed; v) adding a lactose feed to the culture medium via a feed solution in a continuous manner over the course of 1, 2, 3, 4 or 5 days; vi) adding a lactose feed to the culture medium in a continuous manner via a feed solution over the course of 1 day, 2 days, 3 days, 4 days or 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L or 600 g / L; vii) adding a lactose feed to the culture medium in a continuous manner via a feed solution over the course of 1 day, 2 days, 3 days, 4 days or 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L or 600 g / L; and wherein the pH of the solution is set at 3 to 7; viii) adding a lactose feed to the culture medium in a continuous manner via a feed solution over the course of 1 day, 2 days, 3 days, 4 days or 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L or 600 g / L; and wherein the pH of the solution is set at 3 to 7, and wherein the temperature of the feed solution is maintained at 20°C to 80°C; The method results in an α-1,3-fucosyllactose concentration of at least 50 g / L, at least 75 g / L, at least 90 g / L, at least 100 g / L, at least 125 g / L, at least 150 g / L, at least 175 g / L or at least 200 g / L in the final volume of the culture medium, and optionally in a ratio of lactose concentration to α-1,3-fucosyllactose concentration in the final volume of the culture of less than 1:5, 1:10, 1:20 or 1:

40.

45. The method of claim 22, further comprising at least one of the following steps: i) adding a lactose feed to the culture medium, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume; ii) adding a lactose feed to the culture medium in a continuous manner, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume; iii) adding a lactose feed to the culture medium, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume, and such that the final volume of the culture medium is no more than 3 times, or no more than 2 times, or less than 2 times the volume of the culture medium before the addition of the lactose feed; iv) adding a lactose feed to the culture medium in a continuous manner, the lactose feed comprising at least 50 grams, at least 75 grams, at least 100 grams, at least 120 grams, or at least 150 grams of lactose per initial reactor volume, and such that the final volume of the culture medium is no more than 3 times, or no more than 2 times, or less than 2 times the volume of the culture medium before the addition of the lactose feed; v) adding a lactose feed to the culture medium via a feed solution in a continuous manner over the course of 1, 2, 3, 4 or 5 days; vi) adding a lactose feed to the culture medium in a continuous manner via a feed solution over the course of 1 day, 2 days, 3 days, 4 days or 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L or 600 g / L; vii) adding a lactose feed to the culture medium in a continuous manner via a feed solution over the course of 1 day, 2 days, 3 days, 4 days or 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L or 600 g / L; and wherein the pH of the solution is set at 3 to 7; viii) adding a lactose feed to the culture medium in a continuous manner via a feed solution over the course of 1 day, 2 days, 3 days, 4 days or 5 days, and wherein the concentration of the lactose feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L or 600 g / L; and wherein the pH of the solution is set at 3 to 7, and wherein the temperature of the feed solution is maintained at 20°C to 80°C; The method results in an α-1,3-fucosyllactose concentration of at least 50 g / L, at least 75 g / L, at least 90 g / L, at least 100 g / L, at least 125 g / L, at least 150 g / L, at least 175 g / L or at least 200 g / L in the final volume of the culture medium, and optionally an α-1,3-fucosyllactose purity of 80% or more in the final volume of the culture.

Images