TW202221135A - Production of alpha-1,3 glycosylated form of fuc-a1,2-gal-r - Google Patents

Abstract

The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered cells. The present invention describes a cell metabolically engineered for production of an alpha-1,3 glycosylated form of fucose-alpha1,2-galactose-R (Fuc-a1,2-Gal-R). Furthermore, the present invention provides a method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R by a cell as well as the purification of said alpha-1,3 glycosylated form Fuc-a1,2-Gal-R from the cultivation.

Description

Production of α-1,3 glycated form of Fuc-a1,2-Gal-R

The present invention belongs to the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered cells. The present invention describes metabolically engineered cells for the production of the alpha-1,3 glycosylated form of fucose-alpha1,2-galactose-R (Fuc-a1,2-Gal-R). Also, the present invention provides a method for producing α-1,3 glycosylated form of Fuc-a1,2-Gal-R by cells and purifying the α-1,3 glycosylated form of Fuc-a1,2- Gal-R's method.

Oligosaccharides are usually present in proteins and lipids in sugar-coupled form and are involved in many important phenomena such as differentiation, development and biorecognition related to the development and progression of fertilization, embryogenesis, inflammation, metastasis and host pathogen attachment process. Oligosaccharides can also exist as unconjugated glycans in body fluids and breast milk, where they also regulate important developmental and immune processes (Bode, Early Hum. Dev. 1-4 (2015); Reily et al., Nat. Rev. . Nephrol. 15, 346-366 (2019); Varki, Glycobiology 27, 3-49 (2017)). Fucose-α1,2-galactose-R (Fuc-a1,2-Gal-R) has been identified in several types of oligosaccharides in glycoconjugated forms to proteins and lipids. It contains the disaccharide fucose-α1,2-galactose epitope linked to other glycans, glycoproteins or glycolipids. The fucose-α1,2-galactose epitope is frequently reported to be involved in neuronal morphology, neuronal development, learning and memory (Kalovidouris et al., J. Am. Chem. . Soc. 127(5), 1340-1341 (2005); Tosh et al., Sci. Rep. 9, 18806 (2019)). Fuc-a1,2-Gal is also part of the most abundant oligosaccharide in breast milk, 2'-fucosyllactose (2'FL, Fuc-a1,2-Gal-b1,4-Glc). Human milk oligosaccharides (HMO), especially 2'FL, have multiple functions including prebiotic, immune, gut and cognitive benefits (Reverri et al., Nutrients 10(10), 1346 (2018)). Fuc-al,2-Gal also forms the H antigen, which is a substructure of the A and B blood group antigens. The α-1,3 glycosylated form of the fucose-α1,2-galactose epitope is part of the histo-blood group ABH carbohydrate epitope (also known as histo-blood group antigens, HBGA). HBGA is a complex carbohydrate found on the surface of many cell types, including epithelial enterocytes and as a free oligosaccharide in biological fluids such as saliva and milk (Marionneau et al., Biochimie 83, 565-573 (2001) ). The Fuc-a1,2-Gal group is also present in lacto-N-fucose pentasaccharide I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4 -Glc), a highly abundant oligosaccharide present in human milk. LNFP-I represents an important immunomodulatory agent, which can avoid severe infectious diarrhea in parenting infants by inhibiting the attachment of pathogenic bacteria such as Escherichia coli (EPEC, UPEC) and viruses. LNFP-I is also associated with binding to pathogen toxins, growth inhibition of group B Streptococci and selective stimulation of bifidobacterial communities (Derya et al., J. Biotechnol. 318, 31-38 ( 2020); Gotoh et al., Sci. Rep. 8, 13958 (2018); Lin et al. J. Biol. Chem. 292, 11243-11249 (2017); Sotgiu et al., Int. J. Biomed. Sci 2(2), 114-120 (2006)). LNFP-I can be further modified with terminal galactose or N-acetylgalactosamine groups to produce B antigen (Gal-a1,3-(Fuc-a1,2)-Gal-beta) or A antigen (GalNAc- Oligosaccharide structure of a1,3-(Fuc-a1,2)-Gal-beta). These oligosaccharide structures are of great scientific and commercial interest, but their availability is limited because their production relies on chemical or chemical enzymatic synthesis, or purification from natural sources such as animal milk. Chemical synthesis methods are time-consuming, labor-intensive, and difficult to scale up due to the large number of steps involved. Enzymatic methods have advantages over chemical synthesis, but the stereospecificity and regioselectivity of essential enzymes remains a formidable challenge.

It is an object of the present invention to provide tools and methods by means of which an efficient, time and cost effective manner, and if desired, a continuous process to produce the α-1,3 saccharified form of Fuc-a1,2 can be achieved -Gal-R.

This and other objects are achieved according to the present invention by providing cells, methods and novel forms of glycosyltransferases for the production of Fuc-a1,2-Gal-R in the α-1,3 glycosylated form , wherein the cells are genetically modified to produce the α-1,3 glycosylated form of Fuc-a1,2-Gal-R.

Surprisingly, it has now been found that the α-1,3 glycosylated form of Fuc-a1,2-Gal-R can be produced by a single cell. The present invention provides a cell and method for producing the α-1,3 glycosylated form of Fuc-a1,2-Gal-R. The method includes providing the ability to synthesize Fuc-a1,2-Gal-R, the expression of alpha-1,3-glycosyltransferase (alpha-1,3-glycosyltransferas) and the ability to synthesize as alpha-1,3-glycosyl transferase donor nucleotide-sugar step and culture the cells under conditions that allow the production of the α-1,3 glycosylated form of Fuc-a1,2-Gal-R. The present invention also provides methods for isolating the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R. Furthermore, the present invention provides a metabolically engineered cell to produce the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R.

definition

The terms used in this specification to describe the invention and its various embodiments are not to be understood solely in their normally defined meanings, and should include structures, materials or acts outside the scope of their normally defined meanings through specific definitions in this specification. Thus, if an element can be understood in the context of this specification to include more than one meaning, use of that element in the scope of the claim is to be understood as being generic to all possible meanings supported by the specification and the term itself.

The various embodiments and aspects of the invention disclosed herein are to be read not only in the order and context specifically described in this specification, but also in any order and in any combination. Whenever the context requires, all words in the singular shall be deemed to contain the plural and vice versa. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental procedures of cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and the hybridization procedures described herein are those well known and routinely employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, purification steps are performed according to the manufacturer's instructions.

Embodiments of the present invention are disclosed in this specification, and although specific terms are used, the terms are used for descriptive nature only, not for limitation, and the scope of the present invention is described in the following claims. It should be understood that the described embodiments are described for illustrative purposes only and should not be construed as limiting the invention. It is obvious to those with ordinary knowledge in the technical field to which the present invention pertains that other embodiments, improvements, details and uses are consistent with the text and spirit of the present invention and are within the scope of the present invention, and are only limited by the scope of the patent application. The scope of the invention is to be construed in terms of patent law including the doctrine of egalitarianism. In the following claims, reference signs are provided to indicate the steps of the claims for ease of description only and are not intended to imply a particular order in which the steps are performed.

In this document and the scope of its claims, the verb "comprise" and its conjugations are used in a non-limiting manner to mean the items included after this word, but do not exclude that no specific mention is made and items. Throughout this application, the verb "comprises" may be replaced by "consisting of" or "consisting essentially of" and vice versa. In addition, the verb "consisting of" may be replaced by "consisting essentially of", "consisting essentially of" means that the composition as defined herein may include additional ingredients than those specifically specified, The additional ingredients do not alter the unique characteristics of the present invention. Furthermore, reference to an element with the indefinite article "a (a or an)" does not preclude the presence of more than one element, unless the context clearly states that there is only one or only one of the elements. Therefore, the indefinite article "a (a or an)" generally means "at least one".

Throughout this application, unless expressly stated otherwise, the features of "synthesize", "synthesized" and "synthesis" are respectively associated with the features "produce", "produced" and Used interchangeably for "production".

Each of the embodiments identified herein may be combined unless otherwise stated. All publications, patents and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. Priority applications, including EP20190208, EP20190198 and EP20190199, are also incorporated herein by reference in their entirety as if expressly and individually indicated that such priority applications were incorporated by reference.

According to the present invention, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA as a mixture of single- and double-stranded regions or single-, double-, and triple-stranded regions, single- and double-stranded RNA, and as single- and double-stranded regions RNA of a mixture of double-stranded regions, comprising hybrid molecules of DNA and RNA (which may be single-stranded, or more typically double- or triple-stranded regions, or a mixture of single- and double-stranded regions). Furthermore, "polynucleotide" as used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chains in these regions can be from the same molecule or from different molecules. These regions may include all of one or more molecules, but more typically only involve regions of some molecules. One of the molecules of the triple helix region is usually an oligonucleotide. As used herein, the term "polynucleotide" also includes DNA or RNA containing one or more modified bases as described above. Thus, DNA or RNA having a backbone modified for stability or other reasons is a "polynucleotide" according to the present invention. Furthermore, DNA or RNA comprising unusual bases (eg, inosine) or modified bases (eg, tritylated bases) should be understood to be encompassed by the term "polynucleotide". It will be appreciated that various 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 forms of polynucleotides, as well as chemical forms of DNA and RNA that are characteristic of viruses and cells, including, for example, simple and complex cells. The term "polynucleotide" also includes short polynucleotides commonly referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to short chains, commonly referred to as peptides, oligopeptides and oligomers, and to long chains, commonly referred to as proteins. Polypeptides may 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, as well as polypeptides modified by chemical modification techniques. Such modifications are well described in basic textbooks and more detailed monographs, as well as in the multi-volume research literature, and are well known to those skilled in the art. The same type of modification may be present at several sites in a given polypeptide to the same degree or to different degrees. Furthermore, a given polypeptide can contain many types of modifications. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amination, covalent attachment of flavin, covalent attachment of heme moieties, nucleotides or Covalent attachment of nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulphide bond formation, Demethylation, formation of covalent cross-links, formation of pyroglutamate, methylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation carboxylation, myristolyation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid linkage, sulfation, gamma-carboxylation of glutamic acid residues , hydroxylation and ADP-ribosylation, selenoylation, addition of transfer RNA-mediated amino acids to proteins, such as arginylation and ubiquitination. Polypeptides can be branched or cyclic with or without branching. Cyclic, branched and branched cyclic polypeptides can be formed by post-translational natural processes, and can also be made by entirely synthetic methods.

As used herein, the term "polynucleotide encoding a polypeptide" includes a polynucleotide comprising a sequence encoding a polypeptide of the present invention. The term also includes polynucleotides that include a single contiguous region or discrete regions (eg, integrated phage or insert sequences or separated by editing) encoding a polypeptide and may also include encoding and/or Additional regions of non-coding sequences.

"Isolated" means altered "by artificial means" from its natural state, that is, if present in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide that occurs naturally in an organism is not "isolated," but the same polynucleotide or polypeptide that is separated from coexisting materials in its natural state is "isolated," as the term is used herein used in. Likewise, the term "synthetic" sequence as used herein refers to any sequence produced synthetically rather than directly isolated from a natural source. The term "synthetic" as used herein refers to any synthetically produced sequence and not isolated directly from a natural source.

"recombinant" or "transgenic" or "metabolically engineered" or "genetically modified" as used herein with reference to a cell or host cell The words are used interchangeably and refer to a cell replicating a heterologous nucleic acid or expressing a heterologous nucleic acid (ie, a sequence foreign to the cell, or foreign to the location or environment in the cell sequence) encoded peptide or protein. Such cells are described as being transformed with at least one heterologous or exogenous gene, or described as being transformed by introducing at least one heterologous or exogenous gene. Metabolically engineered or recombinant or transgenic cells may contain genes that are not present in the cell's native (non-recombinant) form. Recombinant cells can also contain genes that are present in the cell's native form, wherein these genes have been modified and artificially reintroduced into the cell. These terms also include cells that contain nucleic acid endogenous to the cell, which has been modified, or whose expression or activity has been modified without removing the nucleic acid from the cell, including through genetic substitution Modifications obtained, substitution of promoters, site-specific mutations and related techniques. Thus, "recombinant polypeptides" are produced by recombinant cells. As used herein, a "heterologous sequence" or "heterologous nucleic acid" is derived from a source that is foreign to a particular cell (eg, from a different species), or, if derived from the same source, from Its original form or position in the gene body is modified. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, modified from its original form or location in the gene body. Heterologous sequences can be stably introduced, eg, by transfection, transformation, conjugation or transduction, into the genome of a host microbial cell, wherein techniques depending on the cell and the sequence to be introduced can be applied. Various techniques are known to those of ordinary skill in the art to which this invention pertains, and are disclosed in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). A "mutant" cell or microorganism as used in the context of the present invention refers to a genetically modified cell or microorganism.

The term "endogenous" in the context of the present invention refers to any polynucleotide, polypeptide or protein sequence that is a natural part of the cell and is present in its natural location in the cell's chromosome. The term "exogenous" refers to any polynucleotide, polypeptide or protein sequence which is derived from outside the cell under study and which is not a native part of the cell, or which is not present in the cell's chromosomes or plastids natural location.

The term "heterologous" when used in reference to a polynucleotide, gene, nucleic acid, polypeptide or enzyme refers to a polynucleotide, gene, nucleic acid, polypeptide from or derived from a source other than the host species or enzymes. In contrast, "homologous" polynucleotides, genes, nucleic acids, polypeptides or enzymes as used herein refers to polynucleotides, genes, nucleic acids, polypeptides or enzymes derived from a species of host organism. When referring to gene regulatory sequences or helper nucleic acid sequences for maintaining or manipulating gene sequences (e.g., promoters, 5' untranslated regions, 3' untranslated regions, poly A additional sequences, intron sequences, splice site (splice site, ribosome binding site, internal ribosomal entry sequence, gene body homology region, recombination site, etc.), "heterologous" refers to the regulatory sequence or auxiliary sequence and in the construct, gene The genes in the somatic, chromosomal or episome juxtaposed with regulatory or helper nucleic acid sequences are not naturally associated. Thus, a promoter operably linked to a gene to which it is not operably linked in its native state (ie, in the gene body of a non-GM organism) is referred to herein as a "heterologous promoter" , even though the promoter may be derived from the same species (or in some cases, the same organism) as the gene to which it is linked.

The term "modified activity" of a protein or enzyme refers to a change in the activity of a protein or enzyme compared to the wild-type activity (ie, native activity) of the protein or enzyme. The modified activity can be a disrupted, attenuated, reduced or delayed activity of the protein or enzyme compared to the wild-type activity of the protein or enzyme, but can also be the activity of the protein or enzyme compared to the wild-type activity of the protein or enzyme. The accelerated or enhanced activity of a protein or enzyme. The modified activity of a protein or enzyme is achieved by modifying the expression of the protein or enzyme or by expressing a modified (ie, mutant) protein or enzyme. The modified activity of the enzyme is more related to the modification in the apparent Michaelis constant Km and/or the apparent maximum rate (Vmax) of the enzyme.

The term "modified expression" of a gene refers to a change in the amount of expression of the gene compared to wild type at any stage of the production of the encoded protein. The modified expression is a lower or higher expression level compared to the wild type, wherein the term "higher expression level" is also defined as "overexpression" of the gene with respect to the endogenous gene , or expression in terms of heterologous genes not present in wild-type strains. Lower expression levels or reduced expression levels are achieved by techniques commonly known to the skilled artisan, such as the use of siRNA, CrispR, CrispRi, riboswitches, recombination-mediated genetic engineering, recombineering , homologous recombination, ssDNA-induced mutagenesis (mutagenesis), RNAi, miRNA, asRNA, mutant genes, knock-out genes, transposon-induced mutagenesis, etc. Alter a gene in a way that is statistically significantly "unlikely") or completely incapable (e.g., knocking out the gene) to produce a functional end product. As used herein, the term "riboswitch" is defined as a portion of messenger RNA that folds into an intricate structure that blocks expression by interfering with translation. Binding to effector molecules results in a conformational change that regulates post-transcriptional expression.

Altering the gene of interest in a manner that reduces the amount of expression is obtained by altering the transcription unit, promoter, untranslated region, ribosome binding site, Shine Dalgarno sequence, or transcription termination as described above. sequence (terminator) to obtain lower performance. For example, one or more base pairs in the promoter sequence can be mutated or the promoter sequence can be completely changed to a constitutive promoter with a lower expression strength than the wild type or an inducible promoter that regulates the amount of expression promoter or a repressible promoter that regulates the amount of expression.

Overexpression or expression is achieved by techniques commonly known to the skilled artisan, such as the use of artificial transcription factors, de novo synthetic design of promoter sequences, engineering of RNA switches, introduction or re-introduction of expression modules in euchromatin, or the use of high replication A number of plastids in which the gene is part of an "expression cassette" regarding the presence therein of promoter sequences, untranslated region sequences, coding sequences, and optionally transcription termination sequences Any sequence that results in the performance of a functionally active protein. The expression is constitutive or regulated.

The term "constitutive expression" is defined as an expression that is not regulated by transcription factors other than subunits of RNA polymerase (eg, bacterial sigma factors) under specific growth conditions. Non-limiting examples of these transcription factors are CRP, Lacl, ArcA, Cra and IclR in E. coli. These transfer factors bind to specific sequences and can block or enhance performance under specific growth conditions. RNA polymerase binds to specific sequences to initiate transcription, such as sigma factors by prokaryotic hosts.

The term "regulated expression" is defined as expression under specific growth conditions that is regulated by transcription factors other than subunits of RNA polymerase (eg, bacterial sigma factors). Examples of these transcription factors are described above. Common expression regulation is achieved through inducers or repressors, such as, but not limited to, IPTG, arabinose, rhamnose, fucose, allolactose Either by adjusting pH, or by adjusting temperature or carbon depletion, or by substrate, product or chemical inhibition.

The term "expression via a natural elicitor" is defined as the facultative or regulatory expression of a gene that is expressed only under the host's natural conditions (eg, an organism in parturition, or during lactation), with respect to environmental changes (eg, including hormonal , heat, cold, pH changes, light, oxidative stress or osmotic stress/signals), or depending on the location of the developmental stage or the cell cycle of the host cell, but not limited to apoptosis or cell self-regulation Addicted (autophagy).

The term "chemically inducible expression" is defined as a facultative or regulatory expression of a gene that is expressed only upon treatment with a chemical inducer or repressor, including but not limited to alcohols (e.g., ethanol, methanol), carbohydrates (eg, glucose, galactose, glycerol, lactose, arabinose, rhamnose, fucose, allo-lactose), metal ions (eg, aluminum, copper, zinc) , nitrogen, phosphate, isopropyl-β-D-thiogalactoside (Isopropyl β-D-1-thiogalactopyranoside, IPTG), acetate, formate or xylene.

The term "control sequences" refers to sequences recognized by host cell transcription and translation systems that enable the transcription and translation of polynucleotide sequences into polypeptides. Thus, such DNA sequences are necessary for expression of the operably linked coding sequence in a particular host cell or organism. Such control sequences can be, but are not limited to, promoter sequences, ribosome binding sequences, ShineDalgarno sequences, Kozak sequences, transcription terminator sequences. For example, control sequences suitable for use in prokaryotes include promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers. The DNA of the presequence or secretory leader is operably linked to the DNA of the polypeptide if the DNA of the presequence or secretory leader is expressed as a preprotein involved in polypeptide secretion; if the promoter or the enhancer affects the transcription of the sequence, then the promoter or enhancer is operably linked to the coding sequence; or if the ribosome binding site affects the transcription of the sequence, then the ribosome binding site is operably linked to the coding sequence; Alternatively, the ribosome binding site is operably linked to the coding sequence if the location of the ribosome binding site facilitates translation. The control sequences may also utilize external chemicals such as, but not limited to, IPTG, arabinose, lactose, allolactose, rhamnose, or fucose via an inducible promoter or via induction or inhibition of transcription of the polynucleotide or genetic circuits that are translated into polypeptides are additionally controlled.

In general, "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.

The term "wild type" refers to a genotypic or phenotypic condition that is commonly known to occur in nature.

As used herein, the term "modified expression of a protein" refers to: i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein as compared to a wild-type (ie, native) protein , or iii) expression and/or over-expression of a variant protein with higher activity.

As used herein, the term "mammary cells" generally refers to mammary epithelial cells, mammary epithelial luminal cells, or mammalian epithelial alveolar cells, or any combination of the foregoing. As used herein, the term "mammary-like cells" generally refers to cells that have a phenotype/genotype similar (or substantially similar) to natural mammary cells but derived from sources other than mammary cells. Such mammary-like cells can be engineered to remove at least one unwanted genetic component, and/or include at least one predetermined genetic construct typical of mammary gland cells. Non-limiting examples of mammary-like cells may include mammary-like epithelial cells, mammary-like luminal cells, non-mammary cells exhibiting one or more characteristics of cells of the mammary cell lineage, or any combination of the foregoing. More non-limiting examples of mammary-like cells may include cells with a phenotype similar (or substantially similar) to natural mammary cells. Cells having a phenotype or exhibiting at least one characteristic similar (or substantially similar) to native mammary cells or mammary epithelial cells can include cells that exhibit at least one milk component naturally or are engineered to express at least one milk component (eg, derived from a mammary cell line or a non-mammary cell line).

As used herein, "non-mammary cells" can generally include any cell of a non-mammary cell lineage. In the context of the present invention, a non-mammary gland cell can be any mammalian cell that can be engineered to express at least one milk component. Non-limiting examples of such non-mammary cells include hepatocytes, blood cells, kidney cells, umbilical cord blood cells, epithelial cells, epidermal cells, muscle cells, fibroblasts, interstitial cells, or any combination of the foregoing. In some paradigms, molecular biology and genome editing techniques can be designed to simultaneously eliminate, silence or attenuate a wide variety of genes.

In this application, unless expressly stated otherwise, the expressions "capable of...<verb>" and "capable to...<verb>" are preferably replaced by the active voice of the verb, and vice versa The same is true. For example, the expression "expressible" is preferably replaced with "expressive", and vice versa, that is, "expressible" is preferably replaced with "expressible".

As used herein, a "variant" is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains the necessary properties. A typical variant of a polynucleotide or polypeptide differs from the nucleotide sequence of another reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. As discussed below, nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptides encoded by the reference sequences . A typical variant of a polypeptide differs from another reference polypeptide in its amino acid sequence. In general, limited differences result in the sequence of the reference polypeptide and the variant being very similar overall and identical in many regions. A variant can differ from a reference polypeptide by any combination of one or more substitutions, additions, deletions. A substituted or inserted amino acid residue may or may not be an amino acid residue encoded by the genetic code. Variants of a polynucleotide or polypeptide may be naturally occurring, such as allelic variants, or may be known non-naturally occurring variants. Non-naturally occurring variants of polynucleotides or polypeptides can be produced using mutational techniques, direct synthesis, and other recombinant methods known to those of ordinary skill in the art to which this invention pertains.

As used herein, the term "derivative" of a polypeptide is one that may contain deletions, additions or substitutions of amino acid residues in the amino acid sequence of the polypeptide, but which result in silent changes resulting in Polypeptides of functionally equivalent polypeptides. Amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphiphilic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; planar neutral amino acids include Glycine, serine, threonine, cysteine, tyrosine, aspartic acid, and glutamic acid; positively charged (basic) amino acids include arginine, lysine Acids and histidines; negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In the context of the present invention, a derived polypeptide as used herein refers to a polypeptide capable of exhibiting substantially similar in vitro and/or in vivo activity as the original polypeptide, As judged by any of a number of criteria, including but not limited to enzymatic activity, various modifications can be made during or after translation. Furthermore, non-classical amino acids or chemical amino acid analogs can be substituted or added to the original polypeptide sequence.

In some embodiments, the present invention contemplates generating functional variants by modifying the structure of enzymes as used in the present invention. Variants can be made by amino acid substitution, deletion, addition, or a combination of the foregoing. For example, replacement of leucine with isoleucine or valine alone, aspartic acid with glutamic acid alone, threonine with serine alone, or a structurally related amino acid may be reasonably expected Similar substitutions to amino acids (eg, conservative mutations) do not have a significant effect on the biological activity of the resulting molecule. Conservative substitutions are those that take place within a family of amino acids related to their side chains. Whether changes in the amino acid sequence of the polypeptides of the invention result in functional homology can be readily determined by assessing the ability of the variant polypeptide to generate a response in cells in a manner similar to the wild-type polypeptide thing.

The term "functional homologues" as used herein describes those molecules that share sequence similarity (in other words, homology) and also share at least one functional characteristic such as biochemical activity (Altenhoff et al. , PLoS Comput. Biol. 8 (2012) e1002514). Functional homologues generally produce similar, but not necessarily the same, degree for the same characteristics. Functionally homologous polypeptides have the same characteristics, wherein one homolog produces a quantitative measure of 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 the original 100% of the quantitative measurement produced by the molecule. Thus, when the molecule has enzymatic activity, the functional homologue will have the above percentage of enzymatic activity compared to the original enzyme. If the molecule is a DNA binding molecule (eg, a polypeptide), the homologue will have the above-mentioned percentage binding affinity, as measured by the weight of the bound molecule compared to the original molecule.

The functional homologue and the reference polypeptide may be naturally occurring polypeptides, and the sequence similarity may be the result of convergent or divergent evolutionary events. Functional homologs are sometimes referred to as orthologs, where "heterolog" refers to a homologous gene or protein that is functionally equivalent to a reference gene or protein in another species.

Heterologous proteins are homologous genes in different species that originate from the vertical descent of a single gene from the last common ancestor, where this gene and its main function are conserved. Homologous genes are genes of two species inherited from a common ancestor.

When the term "heterologous" is used for amino acid or nucleotide/nucleic acid sequences from a given species, it refers to amino acid or nucleotide/nucleic acid sequences from different species. It will be appreciated that two sequences are heterologous to each other when they are derived from a common ancestor through linear descent and/or are closely related in sequence and biological function. Heterologs typically share a high degree of sequence similarity, but may not (and generally do not) share 100% sequence similarity.

A paralogous gene is a homologous gene derived from the phenomenon of gene duplication. Homologous genes usually belong to the same species, but this is not a requirement. Homologs can be divided into in-paralogs (in-paralogs, which appear after a speciation event) and out-paralogs (out-paralogs, which appear before a speciation event). homologous pair). An exoparalog between species is a pair of homologs that existed between two organisms by duplication prior to speciation. Among species, ectoparalogs within species are paired homologs that exist in the same organism, but the replication event occurs after speciation. Homologs generally have the same or similar function.

Functional homologues can be identified by alignment analysis of nucleotide and polypeptide sequences. For example, performing a query against a database of nucleotide or polypeptide sequences can identify homologues, polypeptides of interest such as biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis or membrane transport proteins. Sequence analysis may involve BLAST, reciprocal BLAST using the amino acid sequences of biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane transporters as reference sequences, respectively, of non-redundant databases (reciprocal BLAST) or PSI-BLAST analysis. In some cases, the amino acid sequence is deduced from the nucleotide sequence. Generally, polypeptides with sequence similarity greater than 40% in the database are suitable candidates for further evaluation as biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane transport proteins, respectively. Amino acid sequence similarity allows conservative amino acid substitutions such as one hydrophobic residue for another hydrophobic residue, or one polar residue for another polar residue, or one acidic residue for another acidic residue base, or substitution of one basic residue for another, etc. Preferably, conservative substitutions refer to combinations such as glycine with alanine and vice versa; valine, isoleucine, leucine with methionine, and vice versa; Combinations where aspartic acid is replaced by glutamic acid and vice versa; combinations where asparagine is replaced by glutamine and vice versa; combinations where serine is replaced by threonine and vice versa; lysine Combinations where arginine is substituted with arginine, and vice versa; combinations where cysteine is substituted with methionine, and vice versa; combinations where phenylalanine and tyrosine are substituted with tryptophan, and vice versa. Such candidates can be manually inspected to narrow down the number of candidates for further evaluation, if desired. Manual inspection can be performed by selecting candidates that appear to have domains present in productivity-regulating polypeptides, eg, conserved functional domains.

In terms of polynucleotides, a "fragment" refers to a clone or any portion of a polynucleotide molecule, particularly a portion of a polynucleotide that retains functional characteristics available to a full-length polynucleotide molecule. Useful fragments include oligonucleotides and polynucleotides, which are useful in hybridization or amplification techniques or the regulation of replication, transcription or translation. "Polynucleotide fragment" refers to any subsequence of a polynucleotide sequence identification number (or Genbank NO.), typically including or as a result of providing at least about 9 of the polynucleotide sequence identification number (or Genbank NO.) herein. , 10, 11, 12 contiguous nucleotides, eg, at least about 30 nucleotides or at least about 50 nucleotides of any polynucleotide sequence. Exemplary fragments may additionally or alternatively include regions comprising, consisting essentially of, or consisting of, a region encoding a conserved family domain of a polypeptide. Exemplary fragments may additionally or alternatively include fragments comprising conserved domains of polypeptides. Therefore, a fragment of a polynucleotide sequence identification number (or Genbank NO.) preferably refers to a nucleotide sequence comprising or consisting of said sequence identification number (or Genbank NO.), wherein no more than 200, 150 , 100, 50 or 25 contiguous nucleotide deletions (missing), preferably no more than 50 contiguous nucleotide deletions, and the above-mentioned nucleotide sequence retains the functional characteristics (eg, activity) available to the full-length polynucleotide molecule , the available functional characteristics can be assessed using routine experimental methods of the general knowledgeable. Alternatively, a fragment of a polynucleotide SEQ ID NO: (or Genbank NO.) preferably refers to a nucleotide sequence comprising or consisting of some contiguous nucleotides from said SEQ ID NO: (or Genbank NO.), wherein some of said consecutive nucleotides are at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5% , 99.0%, 99.5%, 100.0%, preferably at least 80.0%, more preferably at least 87%, more preferably at least 90.0%, more preferably at least 95.0%, more preferably at least 97.0%, and retain full-length polynucleosides Functional characteristics (eg, activity) available to the acid molecule. Thus, a fragment of a polynucleotide SEQ ID NO: (or Genbank NO.) preferably refers to a nucleotide sequence comprising or consisting of said SEQ ID NO: (or Genbank NO.), in which some contiguous nucleotides Deletion, and the content of deletion is not more than 50.0%, 40.0%, 30.0% of the full length of the SEQ ID NO: (or Genbank NO.), preferably not more than the full length of the SEQ ID NO: (or Genbank NO.) 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5 %, more preferably not more than 15.0% of the full length of the SEQ ID NO. (or Genbank NO.), more preferably not more than 10.0% of the full length of the SEQ ID NO: (or Genbank NO.), more preferably not more than 10.0% More than 5.0% of the full length of the SEQ ID NO: (or Genbank NO.), preferably no more than 2.5% of the full length of the SEQ ID NO: (or Genbank NO.), and wherein the fragment remains a full-length polynucleotide The available functional characteristics (eg, activity) of the molecule can be assessed using routine experimental methods of the general knowledgeable person.

Fragments may additionally or alternatively include subsequences of polypeptide and protein molecules, or subsequences of polypeptides. In certain instances, a fragment or domain is a subsequence of a polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner, or preferably to a similar extent, as the intact polypeptide. As defined herein, a "subsequence of a polypeptide" refers to a sequence of contiguous amino acid residues derived from a polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain, such as a DNA binding site or domain, which is associated with a DNA promoter region, activation domain, or functional domain for protein-protein interactions. domains bind and can initiate transcription. Fragments can range in size from as few as 3 amino acid residues to the full length of a complete polypeptide, eg, at least about 20 amino acid residues in length, eg, at least about 30 amino acid residues in length. Therefore, a polypeptide sequence identification number (or UniProt ID or Genbank NO.) preferably refers to a polypeptide sequence comprising or consisting of said polypeptide sequence identification number (or UniProt ID or Genbank NO.), wherein no more than 80, 60, 50, 40, 30, 20 or 15 consecutive amino acid residues are deleted, preferably no more than 40 amino acid residues are deleted, and it is substantially the same as the complete polypeptide Perform at least one biological function of the intact polypeptide in the same manner as above, or preferably to a similar extent, and the biological function can be assessed using routine experimental methods of the ordinary knowledgeable person. Alternatively, a fragment of a Polypeptide Sequence Identification Number (or UniProt ID or Genbank NO.) refers to a number of contiguous amino acids comprising or derived from the Polypeptide Sequence Identification Number (or UniProt ID or Genbank NO.) A polypeptide sequence consisting of residues, and wherein some of the consecutive amino acid residues are at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0% , 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 100.0%, preferably at least 80.0%, more preferably at least 87.0%, more preferably at least 90.0% %, more preferably at least 95.0%, more preferably at least 97.0%, and it performs at least one biological function of the intact polypeptide in substantially the same manner or preferably to a similar extent as the intact polypeptide, using common knowledge Routine experimental methods to assess biological function. Therefore, a fragment of a polypeptide sequence identification number (or UniProt ID or Genbank NO.) preferably refers to a polypeptide comprising or consisting of said polypeptide sequence identification number (or UniProt ID or Genbank NO.) sequence, wherein some consecutive amino acid residues are deleted, and the content of the deletion is not more than 50.0%, 40.0%, 30.0% of the full length of the polypeptide sequence identification number, preferably not more than the sequence recognition number No. (or UniProt ID or Genbank NO.) 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5% of the full length , 2.0%, 1.5%, 1.0%, 0.5%, more preferably not more than 15% of the full length of the polypeptide sequence identification number (or UniProt ID or Genbank NO.), more preferably not more than the 10% of the full length of the peptide sequence identification number (or UniProt ID or Genbank NO.), more preferably not more than 5% of the full length of the polypeptide sequence identification number (or UniProt ID or Genbank NO.), the best No more than 2.5% of the full length of the polypeptide SEQ ID NO: (or UniProt ID or Genbank NO.), and which performs substantially the same or preferably to a similar extent as the intact polypeptide At least one biological function that can be assessed using routine laboratory methods of ordinary knowledge.

In the present application, the sequence identification number or UniProt ID or GenBank NO. can be used to indicate the sequence of the polypeptide. Therefore, unless expressly stated otherwise, the terms "polypeptide sequence identification number" and "polypeptide UniProt ID" and "polypeptide GenBank NO." are used interchangeably.

Preferably, a fragment of a polypeptide is a functional fragment having at least one property or activity of the polypeptide from which the fragment is derived, to a similar extent, for example, a functional fragment may comprise a functional domain or a conserved domain of the polypeptide . It should be understood that a polypeptide or fragment thereof may have conservative amino acid substitutions that have no substantial effect on the activity of the polypeptide. Conservative substitutions can be one hydrophobic amino acid for another hydrophobic amino acid, or one polar amino acid for another polar amino acid, or one acidic amino acid for another acidic amino acid, or one A basic amino acid replaces another basic amino acid, etc. Preferably, conservative substitutions refer to combinations such as glycine with alanine and vice versa; valine, isoleucine, leucine with methionine, and vice versa; Combinations where aspartic acid is replaced by glutamic acid and vice versa; combinations where asparagine is replaced by glutamine and vice versa; combinations where serine is replaced by threonine and vice versa; lysine Combinations where arginine is substituted with arginine, and vice versa; combinations where cysteine is substituted with methionine, and vice versa; combinations where phenylalanine and tyrosine are substituted with tryptophan, and vice versa.

For example, by Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427-D432), an IPR (InterPro domain) (Mitchell et al., Nucleic Acids Res. 47 (2019) D351-D360 ), a protein fingerprint domain (PRINTS) (Attwood et al., Nucleic Acids Res. 31 (2003) 400-402), a SUBFAM domain (Gough et al., J. Mol. Biol. 313 (2001) 903-919), a TIGRFAM domain (Selengut et al., Nucleic Acids Res. 35 (2007) D260-D264), a Conserved Domain Database (CDD) designation (https://www.ncbi.nlm.nih.gov/cdd) (Lu et al., Nucleic Acids Res. 48 (2020) D265-D268), a PTHR domain (http://www.pantherdb.org ) (Mi et al., Nucleic Acids. Res. 41 (2013) D377-D386; Thomas et al., Genome Research 13 (2003) 2129-2141) or a PATRIC identifier or PATRIC DB global family domain (global family domain) (https://www.patricbrc.org/) (Davis et al., Nucleic Acids Res. 48(D1) (2020) D606-D612). It should be understood in the art that, for the databases used here, Pfam 32.0 (released Sept 2018), CDD v3.17 (released 3rd April 2019), eggnogdb 4.5.1 (released Sept 2016), InterPro 75.0 (released 4th July 2019), TCDB (released 17th June 2019) and PATRIC 3.6.9 (released March 2020) are specified to represent domains, and the content of each database is fixed and will not change in each release. When the content of a particular database changes, this particular database receives a new release version with a new release date. All release versions of each database, their corresponding release dates, and the specific content noted are available and known to those of ordinary skill in the art to which this invention pertains.

Comprehensive sources of protein sequence and annotation data can provide protein or polypeptide sequence information and functional information, such as Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1) , D480-D489). UniProt includes a professional and rich protein database, called UniProt Knowledgebase (UniProt Knowledgebase, UniProtKB) and UniProt Reference Cluster (UniRef) and UniProt Archive (UniParc). The UniProt Identification Number (UniProt ID) is unique to each protein in the database. The UniProt ID used here is the UniProt ID from the May 5, 2021 UniProt database release. Proteins without UniProt IDs were used in this department using the May 5, 2021 edition of the NIH Gene Sequence Database (https://www.ncbi.nlm.nih.gov/genbank/) (Nucleic Acids Res. 2013, 41( D1), D36-D42) of the individual GenBank accession numbers (GenBank No.) presented.

The terms "identical" or "percent similarity" or "% similarity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences, when used with Amino acid residues or nucleotides that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence as measured by sequence comparison algorithms or by visual inspection. For sequence comparison, one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence similarity of the test sequence relative to the reference sequence according to the specified program parameters. The percent similarity can be calculated globally over the full-length sequence of the reference sequence, resulting in an overall percent similarity score. Alternatively, the percent similarity can be calculated over a partial sequence of the reference sequence, resulting in a partial percent similarity score. Using the full length of the reference sequence in the local sequence alignment yields an overall percent similarity score between the test and reference sequences.

Different algorithms can be used to determine percent similarity, such as BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25: 17, 3389 -402), Clustal Omega method (Sievers et al., 2011, Mol. Syst. Biol. 7:539), MatGAT method (Campanella et al., 2003, BMC Bioinformatics, 4:29) or EMBOSS Needle.

The BLAST (Basic Tool for Local Alignment Search) method of alignment is an algorithm provided by the National Center for Biotechnology Information, which uses preset parameters to compare sequences. The program compares nucleotide or protein sequences to sequence databases and calculates statistical significance. PSI-BLAST (Basic Tool for Position-Specific Iterative Local Alignment Search) derives a position-specific scoring matrix from multiple sequence alignments of sequences detected using protein-protein BLAST (BLASTp) above a given score threshold -specific scoring matrix, PSSM) or overview. The BLAST method can be used for pairwise or multiple sequence alignment. Pairwise sequence alignment is used to identify similar regions that may indicate functional, structural and/or evolutionary relationships between two biological sequences (protein or nucleic acid). The web interface of BLAST is located at: https://blast.ncbi.nlm.nih.gov/Blast.cgi.

Clustal Omega (Clustal W) is a multiple sequence alignment program that uses seeded guided tree and HMM profile-profile techniques to generate alignments between three or more sequences. It produces biologically meaningful multiple sequence alignments of different sequences. The web interface of Clustal W is located at: https://www.ebi.ac.uk/Tools/msa/clustalo/. The preset parameters for the percent similarity between multiple sequence alignments and protein sequences using the Clustal W method are: enable de-alignment of input sequences: FALSE; enable mbed-like clustering guide-tree: TRUE; Enable mbed-like clustering iterations (mbed-like clustering iteration): TRUE; (combined with bootstrap tree/HMM) number of iterations: preset(0); max bootstrap tree iterations: preset[-1]; max HMM iterations [-1]; order: aligned.

MatGAT (Matrix Global Alignment Tool) is a computer application that generates a similarity/identity matrix of DNA or protein sequences without pre-aligning the data. The program uses the Myers and Miller global alignment algorithm to perform a series of pairwise alignments, calculates similarity and similarity, and puts the results into a distance matrix. The user can specify which type of alignment matrix (eg, BLOSUM50, BLOSUM62, and PAM250) is used to view protein sequences.

The EMBOSS Needle (https://galaxy-iuc.github.io/emboss-5.0-docs/needle.html) uses the Needleman-Wunsch ensemble alignment algorithm to find the optimal alignment of two sequences when their full length is considered ( including gaps). The dynamic programming method ensures the best alignment by exploring all possible alignments and selecting the best alignment. The Needleman-Wunsch algorithm is a member of a class of algorithms that can compute optimal scores and alignment results in an order of mn steps (where m and n are the lengths of the two sequences). The gap open penalty (default 10.0) is the fraction of when a gap is generated. The default values assume you are using the EBLOSUM62 matrix for protein sequences. A gap extension (default 0.5) penalty is added to the standard gap penalty for each base or residue in the gap. This is how long gaps are punished.

As used herein, a polypeptide having an amino acid sequence having at least 80% sequence similarity with respect to the full-length sequence of a reference polypeptide sequence can be understood to be the amino acid of this sequence to the reference polypeptide sequence The full length of the sequence has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.50%, 92.00%, 92.50%, 93.00 %, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence similarity. In this application, unless expressly indicated otherwise, a polypeptide includes/consists of/has a full-length amino acid sequence relative to a reference polypeptide sequence, usually indicated by a SEQ ID NO: or UniProt ID or Genbank NO., having It consists of amino acid sequences with at least 80.0% sequence similarity, preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0% relative to the full-length reference sequence , 98.0% or 99.0% sequence similarity, more preferably at least 85.0% sequence similarity, more preferably at least 90.0% sequence similarity, and most preferably at least 95.0% sequence similarity. Additionally, unless expressly indicated otherwise, a polynucleotide sequence includes/has/is at least 80.0% sequence similar to the full-length nucleotide sequence of a reference polynucleotide sequence, usually as indicated by a SEQ ID NO: or Genbank NO. It consists of amino acid sequences of high degrees, preferably at least 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, or 99.0% relative to the full-length reference sequence. % sequence similarity, more preferably at least 85.0% sequence similarity, more preferably at least 90.0% sequence similarity, and most preferably at least 95.0% sequence similarity.

For the purpose of the present invention, MatGAT2.01 (Campanella et al., 2003, BMC Bioinformatics 4:29) was used to determine percent similarity. Preset parameters for the following proteins were used: (1) gap cost presence: 12 and extension: 2; (2) the matrix used was BLOSUM65. In a preferred embodiment, the sequence similarity is calculated from the full-length sequence or a portion thereof for a given sequence identification number (ie, a reference sequence). A portion thereof preferably refers to at least 50%, 60%, 70%, 80%, 90% or 95% of the complete reference sequence.

As used herein, the term "glycosyltransferase" refers to an enzyme capable of catalyzing the transfer of sugar moieties from an activated donor molecule to a specific acceptor molecule, forming glycosidic bonds.

The terms "α-1,3-glycosyltransferase", "a1,3-glycosyltransferase", "α1,3-glycosyltransferase", "a1,3-glycosyltransferase" refer to an enzyme capable of catalyzing An enzyme that transfers a sugar group from an activated donor molecule to an alpha-1,3 glycosidic linkage in a specific acceptor molecule. The term "α-1,3-glycosyltransferase" as used herein refers to α-1,3-galactosyltransferase and α-1,3-N-acetylgalactosyltransferase.

"α-1,3-galactosyltransferase", also known as "a-1,3-galactosyltransferase", "a1,3-galactosyltransferase", is a catalytic galactose residue A glycosyltransferase that transfers from UDP-galactose (UDP-Gal) to a specific acceptor molecule with an alpha-1,3 linkage.

α-1,3-N-acetylgalactosamine transferase, also known as "a-1,3-N-acetylgalactosamine transferase", "a1,3 N-acetylgalactosamine transferase" "a1,3-N-acetylgalactosamine transferase" is an enzyme that catalyzes the transfer of N-acetylgalactosamine (GalNAc) from UDP-GalNAc to a specific acceptor molecule with α-1,3 linkage. Glycosyltransferase.

In the present invention, polypeptide sequences are used to mean fragments of the alpha-1,3-glycosyltransferases used in the present invention, which are common to those alpha-1,3-glycosyltransferases. Such polypeptide segments are written as sequences of amino acids in one-letter codes. If an amino acid at a specific position in such a polypeptide segment can be several amino acids, the specific position will have the amino acid code X. Unless otherwise mentioned herein, the letter "X" refers to any possible amino acid. The term [FHMQT] in a sequence refers to F, H, M, Q or T as possible amino acids at that particular position. The term [ACG] in a sequence refers to A, C or G as possible amino acids at that particular position. The term [ACIL] in a sequence refers to A, C, I or L as possible amino acids at that particular position. The term [AG] in a sequence refers to A or G as a possible amino acid at that particular position.

As used herein, the term "nucleotide sugar" or "activated sugar" refers to the activated form of a monosaccharide. Examples of activated monosaccharides include UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine Mannosamine (UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2 ,6-dideoxy-L-arabino-4-hexulose (UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose), UDP-2-acetamido-2,6 -Dideoxy-L-lyxo-4-hexulose (UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose), UDP-N-acetamido-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucose Amines (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-6-deoxytalosamine (UDP- L-PneNAc or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N- Acetyl-L-isorhamnose (UDP-L-QuiNAc or UDP-2-acetylamino-2,6-dideoxy-L-glucose), GDP-L-isorhamnose (GDP-L- quinovose), CMP-N-acetylneuraminic acid (N-acetylneuraminic acid, CMP-Neu5Ac), CMP-N-glycolylneuraminic acid (N-glycolylneuraminic acid, CMP-Neu5Gc), CMP-Neu4Ac, CMP-Neu5Ac9N _3. CMP-Neu4,5Ac ₂ , CMP-Neu5,7Ac ₂ , CMP-Neu5,9Ac ₂ , CMP-Neu5,7(8,9)Ac ₂ , UDP-galacturonate (UDP-galacturonate), UDP - UDP-glucuronate, GDP-rhamnose, or GDP-xylose. Nucleotide sugars act as sugar donors in glycosylation reactions. These reactions are catalyzed by glycosyltransferases.

The term "oligosaccharide" as used herein and generally understood in the art refers to a sugar polymer containing a small amount, usually three to twenty simple sugars, i.e. monosaccharides . The monosaccharides used herein are reducing sugars. Oligosaccharides can be reducing or non-reducing sugars and have reducing and non-reducing ends. A reducing sugar is any sugar that is capable of reducing another compound and is itself oxidized, ie, the carbonyl carbon of the sugar is oxidized to a carboxyl group. The oligosaccharides used in the present invention may be of a linear structure or may include branches. A linkage (eg, glycosidic linkage, galactosidic linkage, glycosidic linkage, etc.) between two sugar units can be represented, for example, as 1,4, 1->4 or ( 1-4). For example, the terms "Gal-b1,4-Glc", "b-Gal-(1->4)-Glc", "Galbeta1-4-Glc" have the same meaning as "Gal-b(1-4)-Glc" The meaning of , that is, a β-glycosidic bond connects the carbon-1 of galactose (Gal) to the carbon-4 of glucose (Glc). Each monosaccharide can be in a cyclic form (eg, the furanose form of pyranose). The linkages between individual monosaccharide units may include α1->2, α1->3, α1->4, α1->6, α2->1, α2->3, α2->4, α2->6 , β1->2, β1->3, β1->4, β1->6, β2->1, β2->3, β2->4 and β2->6. Oligosaccharides may contain both α- and β-glycosidic bonds, or only β-glycosidic bonds.

The term "monosaccharide" as used herein refers to sugars that cannot be broken down into simpler sugars by hydrolysis, are classified as aldose or ketose, and each molecule contains a or multiple hydroxyl groups. Monosaccharides are sugars that contain only one simple sugar. Examples of monosaccharides include hexose, D-glucopyranose, D-galactofuranose, D-galactopyranose, L-galactopyranose, D- Mannoguaranose, D-isoguaranose (D-allopyranose), L-altropyranose (L-altropyranose), D-gulopyranose (D-gulopyranose), L-aiduranose (L-idopyranose), D-talopyranose (D-talopyranose), D-ribofuranose, D-ribofuranose, D-arabinofuraanose, D-arabinofuraanose, L-arabinofuraanose , L-arabinopyranose, D-xylopyranose, D-lyxopyranose, D-erythrofuranose, D-threthrofuranose (D-threofuranose), heptose, L-glycerol-D-mannanoheptose (LDmanHep), D-glycerol-D-mannanoheptose (DDmanHep), 6-deoxy-L-aldrophanan Sugar, 6-deoxy-D-guroguaranose, 6-deoxy-D-taloranose, 6-deoxy-D-galactopyranose, 6-deoxy-L-galactopyranose Mannanose, 6-Deoxy-D-Mannoranose, 6-Deoxy-L-Mannoranose, 6-Deoxy-D-Gurogranose, 2-Deoxy-D-Arabinose Sugar, 2-deoxy-D-erythropentose, 2,6-dideoxy-D-arabinohexanose, 3,6-dideoxy-D-arabinohexanose, 3,6- Dideoxy-L-arabinohexanose, 3,6-dideoxy-D-xylopyranose, 3,6-dideoxy-D-core Hexanose, 2,6-dideoxy-D-nucleohexanose, 3,6-dideoxy-L-xylohexanose, 2-amino-2-deoxy-D-glucose Quaranose, 2-amino-2-deoxy-D-galactanose, 2-amino-2-deoxy-D-mannanose, 2-amino-2-deoxy-D -Isoguananose, 2-amino-2-deoxy-L-aldroranose, 2-amino-2-deoxy-D-gulopyranose, 2-amino-2-destoranose Oxy-L-iduranose, 2-amino-2-deoxy-D-tarotranose, 2-acetamido-2-deoxy-D-glucoranose, 2-ethyl Acetamino-2-deoxy-D-galactopyranose, 2-acetamido-2-deoxy-D-mannanose, 2-acetamido-2-deoxy-D- Isofranose, 2-acetamido-2-deoxy-L-aldroranose, 2-acetamido-2-deoxy-D-gulopyranose, 2-acetamide Alkyl-2-deoxy-L-iduranose, 2-acetamido-2-deoxy-D-tarotranose, 2-acetamido-2,6-dideoxy- D -Galactanose, 2-acetamido-2,6-dideoxy-L-galactopyranose, 2-acetamido-2,6-dideoxy-L-mannan Sugar, 2-acetamido-2,6-dideoxy-D-glucopyranose, 2-acetamido-2,6-dideoxy-L-aldroranose, 2-ethyl Amino-2,6-dideoxy-D-tarotranose, D-glucopyanuronic acid, D-galactofuranoic acid, D-mannanose uronic acid, D-isoguaranuronic acid, L-aldroguaranuronic acid, D-guloguaranuronic acid, L-guloguaranuronic acid, L-iduoguaranuronic acid acid, D-taroturonanuronic acid, sialic acid, 5-amino-3,5-dideoxy-D-glycerol-D-galacto-non-2-ketonic acid (5 -Amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid), 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-non- -2-Kulonic acid, 5-Glycolylamido-3,5-dideoxy-D-glycerol-D-galacto-non-2-ketonic acid (5-Glycolylamido-3,5-dideoxy-D -glycero-D-galacto-non-2-ulosonic acid), erythritol, arabitol, xylitol, ribitol, glucitol, galactitol, mannitol, D-nucleo-hexyl alcohol -2-Hex-2-ulopyranose (D-ribo-Hex-2-ulopyranose), D-Arabino-Hex-2-Furanose (D-Fructofuranose), D-Arabino-Hex-2-Ganulose , D-wood-hexyl-2-guranulose, L-lysu-hexyl-2-guranulose, D-lysu-hexyl-2-guranulose, D-threo-pentane-2- D-threo-pent-2-ulopyranose, D-alzo-hept-2-guaranose, 3-C-(hydroxymethyl)-D-erythrofuranose, 2,4, 6-Tri-deoxy-2,4-diamino-D-glucoranose, 6-deoxy-3-O-methyl-D-glucose, 3-O-methyl-D-rhamnose ( 3-O-mehtyl-rhamnose), 2,6-dideoxy-3-methyl-D-ribohexose, 2-amino-3-O-[(R)-1-carboxyethyl]-2- Deoxy-D-glucopyranose (2-Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose), 2-acetamido-3-O-[( R)-Carboxyethyl]-2-deoxy-D-glucoranose, 2-ethanolamido-3-O-[(R)-1-carboxyethyl]-2-deoxy-D- Grape guaranose (2-Glycolylamido-3-O-[(R)- 1-carboxyethyl]-2-deoxy-D-glucopyranose), 3-Deoxy-D-lyxo-hept-2-guaranulonic acid (3-Deoxy-D-lyxo-hept-2-ulopyranosaric acid), 3-Deoxy-D-manno-octanoic-2-guaranulonic acid, 3-deoxy-D-glycerol-D-galacto-non-2-guaranulonic acid, 5,7-diamino -3,5,7,9-Tetradeoxy-L-glycerol-L-mannose-non-2-guaranulonic acid, 5,7-diamino-3,5,7,9-tetradeoxy -L-glycerol-L-alzo-non-2-guaranulonic acid, 5,7-diamino-3,5,7,9-tetradeoxy-D-glycerol-D-galacto-non- -2-guaranulonic acid, 5,7-diamino-3,5,7,9-tetradeoxy-D-glycerol-D-talo-non-2-guaranulonic acid, glucose, Galactose, N-acetylglucosamine, glucosamine, mannose, xylose, N-acetylmannosamine, N-acetylneuraminic acid, N-glycolylneuraminic acid , sialic acid, N-acetylgalactosamine, galactosamine, fucose, rhamnose, glucuronic acid, gluconic acid, fructose and polyols.

The term polyol refers to alcohols containing multiple hydroxyl groups. For example, glycerol, sorbitol or mannitol.

As used, the term "disaccharide" refers to a sugar consisting of two monosaccharide units. Examples of disaccharides include lactose (Gal-b1,4-Glc), lacto-N-disaccharide (Gal-b1,3-GlcNAc), N-acetyllactosamine (Gal-b1,4-GlcNAc), LacDiNAc ( GalNAc-b1,4-GlcNAc), N-acetylgalactosamine (GalNAc-b1,4-Glc).

Preferably, the oligosaccharides described herein contain monosaccharides selected from the list used above. Examples of oligosaccharides include, but are not limited to, Lewis-type antigenic oligosaccharides, mammalian milk oligosaccharides, and human milk oligosaccharides.

As used herein, "mammalian milk oligosaccharide" (MMO) refers to oligosaccharides such as, but not limited to, 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose Glycosyllactose, 2',3-bisfucosyllactose, 2',2-bisfucosyllactose, 3,4-bisfucosyllactose, 6'-sialyllactose, 3'-sialyllactose Acid lactose, 3,6-disialylactose, 6,6'-disialylactose, 8,3-disialylactose, 3,6-disialylactosyl-N-saccharide, lactosyl bismuth alginose, lactosyl-N-fucosyl, lactosyl-N-sulfanose, lactosyl-N-fucosyl II, lactosyl-N-fucopentose I, lactosyl-N-fucosyl Pentose III, lactosyl-N-fucopentose V, lactosyl-N-fucopentose VI, sialyllosyl-N-fucosyl c, sialyllosyl-N-fucosyl b, sialic acid Lactosyl-N-difucosyl a, lactosyl-N-bisfucose hexose I, lactosyl-N-bisfucose hexose II, lactosyl-N-hexose, lactosyl-N-neohexose Sugar, p-lactosyl-N-hexose, monofucosyl monosialyllacto-N-neotetraose c (monofucosylmonosialyllacto-N-neotetraose c), monofucosyl p-lactose-N-hexose (monofucosyl para-lacto-N-hexaose), monofucosyllacto-N-hexaose III, isomeric fucosylated lacto-N -hexaose III), isomeric fucosylated lacto-N-hexaose I (isomeric fucosylated lacto-N-hexaose I), sialyllosyl-N-hexose, sialyllosyl-N-neohexose II. Difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose Glycosyllactose-N-hexose a (difucosyllacto-N-hexaose a), difucosyllacto-N-hexose c (difucosyllacto-N-hexaose c), galactosyllacto-N-hexaose c, galactosyllactosan, fucoid Glycosylated oligosaccharides, neutral oligosaccharides and/or sialic acid oligosaccharides.

Mammalian milk oligosaccharides or MMOs include oligosaccharides present in milk at any stage of lactation, including human colostrum (ie, human milk oligosaccharides or HMOs) and mammalian colostrum, including But not limited to cattle ( Bos Taurus ), sheep ( Ovis aries ), goats ( Capra aegagrus hircus ), Bactrian camels ( Camelus bactrianus ), horses ( Equest ferus caballus ), pigs ( Sus scropha ), dogs ( Canis lupus familiaris ), Ezo Brown Bear ( Ursus arctos yesoensis ), Polar Bear ( Ursus maritimus ), Japanese Black Bear ( Ursus thibetanus japonicus ), Striped Skunk ( Mephitis mephitis ), Seal ( Cystophora cristata ), Asian Elephant ( Elephas maximus ), African Elephant ( Loxodonta africana ) , Giant Anteater ( Myrmecophaga tridactyla ), Common Bottlenose Dolphin ( Tursiops truncates ), Northern Whale ( Balaenoptera acutorostrata ), Tama Wallaby ( Macropus eugenii ), Red Kangaroo ( Macropus rufus ). Common brush-tailed possum ( Trichosurus Vulpecula ), koala ( Phascolarctos cinereus ), eastern quoll ( Dasyurus viverrinus ), platypus ( Ornithorhynchus anatinus ). Human milk oligosaccharides (HMOs), also known as identical human milk oligosaccharides, are chemically identical to human milk oligosaccharides in human milk, but are produced by biotechnology (e.g., using cell-free systems or including bacteria, fungi, etc.) , yeast, plant, animal or protozoa cells and organisms, preferably genetically modified cells and organisms). The same human milk oligosaccharide is marketed as HiMO.

The term "Lewis-type antigen" as used herein includes the following oligosaccharides: H1 antigen, which is Fuca1-2Galβ1-3GlcNAc, or 2'FLNB for short; Lewisa, the trisaccharide Galβ1-3[Fucα1-4]GlcNAc , or 4-FLNB for short; Lewisb, namely tetraose Fucα1-2Galβ1-3[Fucα1-4]GlcNAc, or simply DiF-LNB; sialyl Lewisa, namely 5-acetylneuramido-(2-3)-galactosyl -(1-3)-(fucopyranosyl-(1-4))-N-acetylneuraminyl-(2-3)-galactosyl-(1-3)-(fucopyranosyl -(1-4))-N-acetylglucosamine), or Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAc for short; H2 antigen, namely Fucα1-2Galβ1-4GlcNAc, or 2'fucosyl-N-acetyl -Lactosamine, referred to as 2'FLacNAc; Lewisx, the trisaccharide Galβ1-4[Fucα1-3]GlcNAc, or 3-fucosyl-N-acetyl-lactosamine (3-Fucosyl-N-acetyl- lactosamine), referred to as 3-FLacNAc; Lewisy, tetose Fuca1-2Galβ1-4[Fucα1-3]GlcNAc, and sialic acid Lewisx, 5-acetylneuramido-(2-3)-galactosyl-(1 -4)-(fucopyranosyl-(1-3))-N-acetylneuraminyl-(2-3)-galactosyl-(1-4)-(fucopyranosyl-(1 -3))-N-acetylglucosamine), or Neu5Acα2-3Galβ1-4[Fucα1-3]GlcNAc for short.

The term "Fuc-a1,2-Gal-R" as used herein refers to the terminal disaccharide Fuc-alpha-1,2-Gal attached to the R group. The term "Fuc-a1,2-Gal-b1,3-R" as used herein refers to the terminal disaccharide Fuc-alpha-1,2-Gal attached to the R group in a β-1,3 glycosidic linkage. The term "Fuc-a1,2-Gal-b1,3-GlcNAc-R" as used herein refers to the terminal trisaccharide Fuc-alpha-1,2-Gal-b1,3-GlcNAc attached to an R group. As used throughout this application, the "R-group" or "R" refers to a monosaccharide, disaccharide, oligosaccharide, lipid, peptide or protein, or is associated with a peptide, glycopeptide, protein, glycoprotein, lipid Or glycolipid-bound mono-, di- or oligosaccharides.

The terms "lacto-N-triose", "LN3" and "LNT II" refer to the trisaccharide GlcNAc-bl,3-Gal-bl,4-Glc. The terms "lacto-N-dose" and "LNT" refer to the oligosaccharide Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc.

Terms "lacto-N-fucopentaose I", "lacto-N-fucopentaose-I", "LNFP-I", "LNFP I", "LNF I OH type I determinant", "LNF I", "LNF1", "LNF 1" and "Blood group H antigen pentaose type 1" refer to the The oligosaccharide Fuc-a1,2-Gal-b1,3- obtained by the catalysis of the transfer of a halose residue from GDP-L-fucose to the terminal galactose residue of LNT by α-1,2-linkage GlcNAc-b1,3-Gal-b1,4-Glc, such as Miyazaki et al. (2010, Methods Enzym. 480, 511-524), Baumgärtner et al. (2015, Bioorg. Med. Chem. 23, 6799-6806 ), Zhao et al. (2016, Chem. Commun. 52, 3899-3902), Sugiyama et al. (2016, Glycobiology 26, 1235-1247) and a patent document (for example, WO19008133, WO2014018596A2).

The terms "GalNAc-LNFP-I" and "A blood group antigen hexose type I" are used interchangeably and mean GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3 -Gal-b1,4-Glc.

The term "LNFP-II" is used interchangeably with "lactosyl-N-fucosepentose II" and means Gal-b1,3-(Fuc-a1,4)-GlcNAc-b1,3-Gal-b1 , 4-Glc.

The term "LNFP-III" is used interchangeably with "lactosyl-N-fucosepentose III" and means Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1,3-Gal-b1 , 4-Glc.

The term "LNFP-V" is used interchangeably with "lactosyl-N-fucosepentose V" and means Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1, 3)-Glc.

The terms "LNFP-VI", "LNnFP V" and "lactosyl-N-neofucopentose V" are used interchangeably and mean Gal-b1,4-GlcNAc-b1,3-Gal-b1,4- (Fuc-a1,3)-Glc.

The terms "LNnFP I" and "lactosyl-N-neofucosepentose I" are used interchangeably and mean Fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4 -Glc.

The terms "LNDFH I", "lactosyl-N-bisfucohexose I", and "LDFH I" are used interchangeably and mean Fuc-a1,4-(Fuc-a1,2-Gal-b1,3 )-GlcNAc-bl,3-Gal-bl,4-Glc, which includes the Lewisb antigen Fuc-al,4-(Fuc-al,2-Gal-bl,3)-GlcNAc.

The terms "LNDFH II", "lactosyl-N-bisfucohexose II", "Lewis a-Lewis x" and "LDFH II" are used interchangeably and mean Fuc-a1,4-(Gal-b1, 3)-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3)-Glc.

The terms "LNnDFH", "lactosyl-N-neobisfucose" and "Lewis x hexose" are used interchangeably and mean Gal-b1,4-(Fuc-a1,3)-GlcNAc-b1, 3-Gal-b1,4-(Fuc-a1,3)-Glc.

The terms "alpha-tetose" and "A-tetose" are used interchangeably and mean GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc.

As used herein and as commonly understood in the art, a "fucosylated oligosaccharide" is an oligosaccharide bearing a fucose residue. Examples include 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyl lactose (diFL), lactodifucotetraose (LDFT), lactosyl-N-fucose pentose I (LNFP I), lactosyl-N-fucosyl pentose II (LNFP II), Lactosyl-N-fucopentose III (LNFP III), Lactosyl-N-fucopentose V (LNFP V), Lactosyl-N-fucopentose VI (LNFP VI) , Lactosyl-N-neofucose pentose I, Lactosyl-N-bisfucose I (LDFH I), Lactosyl-N-bisfucose II (LDFH II), Monofucose Monofucosyllacto-N-hexaose III (MFLNH III), bis-fucosyllacto-N-hexose (DFLNHa), bis-fucosyl-lactosyl-N-neohexose.

Terms used in the present invention "alpha-1,2-fucosyltransferase", "alpha 1,2-fucosyltransferase", "2-fucosyltransferase", "α-1,2-fucosyltransferase" "2-fucosyltransferase", "α 1,2-fucosyltransferase", "2-fucosyltransferase", "2-FT" or "2FT" are used interchangeably to mean catalytic rock Fucose is transferred from the donor GDP-L-fucose to the glycosyltransferase in the acceptor molecule with an α-1,2-linkage. Terms used in the present invention "2'-fucosyllactose", "2'-fucosyllactose", "alpha-1,2-fucosyllactose", "alpha 1,2-fucose" "α-1,2-fucosyllactose", "α-1,2-fucosyllactose", "Galβ-4(Fucα1-2)Glc", "2FL" or "2'FL" "Used interchangeably, refers to the transfer of fucose residues from GDP-L-fucose to lactose with α-1,2-bonds catalyzed by alpha-1,2-fucosyltransferase. product obtained. Terms used in the present invention "bisfucosyllactose", "di-fucosyllactose", "lactobisfucotose", "2',3-bisfucosyllactose", "2', 3 Bifucosyllactose", "α-2', 3-fucosyllactose", "α 2', 3-fucosyllactose", "Fucα1-2Galβ 1-4(Fucα1-3)Glc" , "DFLac", "2',3diFL", "DFL", "DiFL" or "diFL" are used interchangeably.

As used herein, the "fucosylation pathway" is composed of enzymes and their respective genes, mannose-6-phosphate sugar isomerase, phosphomannose mutase, mannose-1- Phosphoguanosyltransferase, GDP mannose 4,6-dehydratase, GDP-L-fucose synthesis pathway and/or recycling pathway L-fucose kinase/GDP-L-fucose pyrophosphorylase ( GDP-L-fucose synthase and/or the salvage pathway L-fucokinase/GDP-fucose pyrophosphorylase), binding to fucose leading to α1,2, α1,3, α1,4 or α1,6 fucosylated oligosaccharides base transferase.

As used herein, "galactosylation pathway" is a biochemical pathway composed of enzymes and their respective genes, galactose-1-epimerase, galactokinase, glucokinase, galacto- Lactose-1-phosphate uridine transferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridine transferase and/or phosphoglucomutase, with 2 , 3, 4, and 6 hydroxyl groups to form alpha or beta bound galactose galactosyltransferase binding.

As used herein, the "N-acetylglucosamine carbohydrate pathway" is a biochemical pathway composed of enzymes and their respective genes, L-glutamate-D-fructose-6 - Phosphoaminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase , N-acetylglucosamine-1-phosphate uridine transferase, glucosamine-1-phosphate acetyltransferase and/or glucosamine-1-phosphate acetyltransferase, and oligosaccharides leading to 3, 4, Glycosyltransferase binding to the 6 hydroxyl group to form an alpha or beta bound N-acetylglucosamine.

The term "glycopeptide" as used herein refers to a peptide containing one or more glycosyl groups, which are monosaccharides, disaccharides, oligosaccharides, polysaccharides and/or glycans, covalently linked to on the side chains of amino acid residues in peptides. Glycopeptides include natural glycopeptide antibiotics, such as glycosylated non-ribosomal peptides produced by various soil actinomycetes, which interact with the growth of peptidoglycan on the outer surface of the cytoplasmic membrane. -D-alanine (acyl-D-alanyl-D-alanine, D-Ala-D-Ala) terminal binding against Gram-positive bacteria, and synthetic glycopeptide antibiotics. The common core of natural glycopeptides is composed of a cyclic peptide consisting of 7 amino acids to which 2 sugars are bound. Examples of glycopeptides include vancomycin, teicoplanin, oritavancin, chloroeremomycin, telavancin, and dalbavancin ).

The terms "glycoprotein" and "glycopolypeptide" are used interchangeably and refer to monosaccharides, disaccharides, oligosaccharides, polysaccharides, and/or polysaccharides containing side chains of amino acid residues covalently attached to a polypeptide or a polypeptide of one or more sugar groups of a glycan.

As used herein, the term "glycolipid" refers to any glycolipid generally known in the art. Glycolipids (GLs) can be subdivided into simple glycolipids (SGLs) and complex (CGLs) glycolipids. Simple GLs, sometimes referred to as saccharolipids, are two-component (glycosyl and lipid) GLs in which the sugar and lipid molecules are directly linked to each other. Examples of SGLs include glycosylated fatty acids, fatty alcohols, carotenoids, hopanoids, sterols, or paraconic acid. SGL produced by bacteria can be divided into rhamnolipid, glucolipid, trehalolipid, other glycosylated (without trehalose) mycolate, oligosaccharide lipid containing trehalose, sugar Sylated fatty alcohols, glycosylated macrolides (macro-lactone) and macro-lactam (macro-lactam), glycosyl macrolides (glycomacrodiolides) (glycosylated macrolides (glycosylated macrocyclic dilactones), glycosyl carotenoids and glyco-terpenoids, and glycosylated hopanes/sterols. However, complex glycolipids (CGLs) are more heterogeneous in structure, as they contain other residues such as glycerol (glycoglycerolipids), peptides ( glycopeptides), acylated-sphingosines (glycosphingolipids) or other residues (lipopolysaccharides, phenolic glycolipids, nucleoside lipids) .

As used herein, "membrane transporter" refers to a protein that is part of or interacts with the cell membrane and controls the flow of molecules and information in and out of the cell. Thus, membrane proteins are involved in transport, either for import into the cell or export to the outside of the cell.

Such membrane transport proteins can be transport proteins (porters), P-P bond hydrolysis-driven transport proteins, β-barrel porins, auxiliary transport proteins, putative transport proteins and Phosphotransfer-driven group translocators are defined by the Saier Laboratory Bioinformatics Group, which operates and curates the Transporter Taxonomy Database at www.tcdb.org, which provides membrane transporters Functional and phylogenetic classification of . The Transporter Classification Database details a comprehensive IUBMB-approved classification system for membrane transporters, known as the Transporter Classification (TC) system. TCDB taxonomy searches as described herein are defined according to the version published by TCDB.org on June 17, 2019.

Porter is the common name for uniporter, symporter, and antiporter, which utilize a carrier-mediated process (Saier et al., Nucleic Acids Res . 44 (2016) D372-D379). They are electrochemical potential-driven transport proteins, also known as secondary carrier-type facilitators. When membrane transporters utilize carrier-mediated processes to catalyze unidirectional transport of secondary carriers or permeation of a single species to facilitate diffusion or transport in membrane potential-dependent processes (if the solute is charged); when two or more When multiple species are transported in opposite directions by secondary carriers in a tightly coupled process, not coupled to direct energy forms other than chemical energy; and/or when two or more species are transported in a tightly coupled process Intermediate and secondary carriers transported together in the same direction are not coupled to direct energy forms other than chemical energy, and are included in this classification by (Forrest et al., Biochim. Biophys. Acta 1807 (2011) 167-188) among. These systems are usually stereospecific. Solutes: Solute back transport is a feature of secondary carriers. The dynamic association of transport proteins and enzymes produces functional membrane transport metabolons that transport channel substrates normally obtained outside the cell directly into their cellular metabolism (Moraes and Reithmeier, Biochim. Biophys. Acta 1818 (2012) , 2687-2706). Solutes transported by this transport protein system include, but are not limited to, cations, organic anions, inorganic anions, nucleosides, amino acids, polyols, phosphorylated glycolytic intermediates, osmolytes, chelated irons (siderophores).

If membrane transporters hydrolyze the diphosphate bonds of inorganic pyrophosphate, ATP or another nucleoside triphosphate to drive active uptake and/or extrusion of solutes, membrane transporters are involved in the hydrolysis of PP bonds. A class of kinesin transporters (Saier et al. , Nucleic Acids Res. 44 (2016) D372-D379). Membrane proteins may or may not be temporarily phosphorylated, but substrates are not. Substances transported by PP-bonded hydrolysis-driven transport protein classes include, but are not limited to, cations, heavy metals, beta-glucans, UDP-glucose, lipopolysaccharides, teichoic acids.

β-Barrel porins membrane transporter proteins form transmembrane pores that normally allow solutes to traverse the membrane without requiring energy. The transmembrane portion of these proteins consists entirely of β-strands that form a β-barrel (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). These porin-type proteins are present in the outer membranes of Gram-negative bacteria, mitochondria, plastids, and possibly acid-fast Gram-positive bacteria. Solutes transported by these β-barrel porins include, but are not limited to, nucleosides, raffinose, glucose, β-glucosides, oligosaccharides.

Auxiliary transport proteins are defined as proteins that facilitate transport across one or more biological membranes, but are not themselves directly involved in the transport process. These membrane transporters always act in conjunction with one or more established transport systems such as but not limited to outer membrane factors (OMFs), polysaccharide porters (PST porters), ATP-binding cassettes (ATP-binding cassette, ABC) transport protein. They may provide functions related to energy-coupled transport, play structural roles in complex formation, exert biological or stabilizing functions, or perform regulatory functions (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379 ). Examples of helper transport proteins include, but are not limited to, the family of polysaccharide copolymerases involved in the transport of polysaccharides, the family of membrane fusion proteins involved in the transport of bacteriocins and chemical toxins.

Families encompassed by putative transport proteins were classified elsewhere when a member's transport function was established, or were removed from the transport protein classification system when the proposed transport function was denied. These families include one or more members that have been suggested to have a transport function, but the evidence for this function is incomplete (Saier et al., Nucleic Acids Res. 44 (2016) D372-D379). As released on June 17, 2019, examples of putative transport proteins classified into this group under the TCDB system include, but are not limited to, copper transport proteins.

Phosphotransfer-driven group translocators are also known as bacterial phosphoenolpyruvate:PEP-dependent phosphotransferase-driven group translocators of the sugar phosphotransferase system (PTS). The reaction product derived from extracellular sugar is cytoplasmic sugar-phosphate. The enzymatic components that catalyze the phosphorylation of sugars are superimposed on the transport process in a tightly coupled process. The PTS system is involved in many different aspects, including regulation and chemotaxis, biofilm formation and pathogenesis (Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93; Saier, J. Mol. Microbiol. Biotechnol. 25 (2015) 73-78). As released on June 17, 2019, the family of membrane transporters classified under the TCDB system among the phosphate transfer driver translocators includes gluco-glucoside, fructose-mannitol, lactose-N, N'-diacetylchitobiose-beta-glucoside (lactose-N, N'-diacetylchitobiose-beta-glucoside), glucose alcohol, galactitol, mannose-fructose-sorbose and ascorbic acid transport related PTS system.

The main facilitator superfamily (MFS) is a superfamily of membrane transporters that catalyze unidirectional transport, solute:cation (H ⁺ , but a few Na ⁺ ) co-transport and/or solute:H ⁺ or solute : solute reverse transport. Most transport proteins are 400-600 amino acid residues in length, with 12, 14, or occasionally 24, as defined by the Transporter Protein Taxonomy Database run by the Bioinformatics Group in the Saier Laboratory (www.tcdb.org). Transmembrane α-helical spanners (TMS).

As used herein, "sugar efflux transporter (SET)" refers to a membrane protein of the SET family, and the membrane protein of the SET family is a protein with the InterPRO domain IPR004750 and/or belonging to the eggNOGv4.5 family ENOG410XTE9. protein. Can be identified using the online tool at https://www.ebi.ac.uk/interpro/ or using the standalone version of InterProScan (https://www.ebi.ac.uk/interpro/download.html) with preset values interPRO domain. A stand-alone version of eggNOG-mapperv1 (http://eggnogdb.embl.de/#/app/home) can be used to identify heterologous families in eggNOGv4.5.

As used herein, "siderophore" refers to secondary metabolites of various microorganisms that are primarily iron-specific chelators. These molecules are classified as catecholate, hydroxamate, carboxylate and mixed types. Chelaterin is usually synthesized by the nonribosomal peptide synthetase (NRPS)-dependent pathway or the NRPS independent pathway (NIS). The most important precursor in the NRPS-dependent chelatin biosynthesis pathway is chorismate. 2,3-DHBA can be formed from chorismate using a three-step reaction catalyzed by isochorismate synthase, isochorismate and 2,3-dihydroxybenzoate-2,3-dehydrogenase. When ornithine is used as the precursor of chelatin, biosynthesis depends on the hydroxylation of ornithine catalyzed by L-ornithine N5-monooxygenase. In the NIS pathway, an important step in the biosynthesis of chelatin is N(6)-hydroxylysine synthase (N(6)-hydroxylysine synthase).

Transport protein required for the export of chelatin outside the cell. To date, four membrane protein superfamilies have been identified in the process: Major Promoter Superfamily (MFS), Multidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily (MOP) , resistance, nodulation and cell division superfamily (the resistance, nodulation and cell division superfamily, RND) and ABC superfamily. In general, genes involved in chelatin export are clustered with chelatin genes. As used herein, the term "chelatin export protein" refers to a transport protein required for the export of chelatin to the outside of the cell.

The ATP-binding cassette (ABC) superfamily contains both uptake and efflux transport systems, and members of these two groups are generally loosely clustered. ATP hydrolysis without protein phosphorylation provides energy for transport. There are dozens of families in the ABC superfamily, and families are usually associated with substrate specificity. Members are classified according to class 3.A.1 as defined by the Transporter Taxonomy Database run by the Bioinformatics Group of the Saier Laboratory, located at www.tcdb.org, and provides functional and phylogenetic classification of membrane transporters.

The term "enabled efflux" refers to the transport activity of introduced solutes across the cell membrane and/or cell wall. Said transport can be achieved by introducing and/or increasing the expression level of the transport protein described in the present invention. The term "enhanced efflux" refers to improved solute transport activity across the cell membrane and/or cell wall. The transport of solutes across the cell membrane and/or cell wall can be enhanced by introducing and/or increasing the expression of the membrane transporters described in the present invention. "Expression" of a membrane transporter is defined as "overexpression" of the gene encoding the membrane transporter in the case where the gene is an endogenous gene, or in the case where the gene encoding the membrane transporter is a heterologous gene "Expression" under the heterologous gene is not present in the wild-type strain or cell.

The term "purified" refers to a material that is substantially free of components that interfere with the activity of a biomolecule. With respect to cells, carbohydrates, nucleic acids, polypeptides, peptides, glycoproteins, glycopeptides, lipids, and glycolipids, the term "purified" means substantially or substantially free from what normally accompanies in its natural state The material of the components of the material. In general, the purified carbohydrates, oligosaccharides, peptides, glycopeptides, proteins, glycoproteins, lipids, glycolipids or nucleic acids of the present invention are at least about 50%, 55%, 60%, 65%, 70% pure , 75%, 80% or 85%, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, using silver stained gels band intensity or other methods to determine purity. Purity or homogeneity can be demonstrated using a number of methods known in the art, eg, polyacrylamide gel electrophoresis of protein or nucleic acid samples followed by staining for visualization. For some purposes, high resolution and the use of HPLC or similar purification methods are required. For oligosaccharides, purity can be determined using, but not limited to, thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis, or mass spectrometry.

The term "culture" refers to the medium in which the cells are grown or fermented, the cells themselves, and the alpha-1,3-glycosylated oligosaccharides produced by the cells of the invention in whole broth, i.e., inside the cells (intracellularly) ) and extracellular (extracellularly).

As used herein, the term "precursor" refers to substances that are taken up and/or synthesized by cells for the production of specific oligosaccharides. In this sense, a precursor may be an acceptor as defined herein, but may also be another substance-metabolite, which is first modified intracellularly as part of the biochemical synthesis pathway of the oligosaccharide. Examples of such precursors include recipients as defined herein, and/or glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, dihydroxyacetone, glucosamine , N-acetylglucosamine, mannosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, phosphorylated sugars such as but not limited to glucose-1-phosphate, galactose-1- Phosphoric acid, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, mannose-6-phosphate, mannose-1-phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, Dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-1-phosphate, N-Acetyl-neuraminic acid-9-phosphate and/or nucleotide-activated sugars as defined herein, eg UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, CMP-sialic acid , GDP-mannose, GDP-4-dehydro-6-deoxy-α-D-mannose, GDP-fucose.

As used herein, the term "acceptor" refers to a monosaccharide, disaccharide or oligosaccharide, protein, glycoprotein, peptide, glycopeptide, lipid or glycolipid that can be modified by a glycosyltransferase. Examples of such recipients include glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, lactose-N-disaccharide (LNB), lacto-N-triose, lactose -N-tetrasaccharide (LNT), lacto-N-neotetraose (LNnT), N-acetyl-lactosamine (LacNAc), lacto-N-pentose (LNP), lacto-N-neopentose, p-lacto-N-pentose, p-lacto-N-neopentose, p-lacto-N-neopentose I, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), p-lacto- N-neohexaose (pLNnH), p-lacto-N-hexaose (pLNH), lacto-N-heptose, lacto-N-neoheptose, p-lacto-N-neoheptose, p-lacto-N-heptose Sugar, lacto-N-octaose (LNO), lacto-N-octaose, isolacto-N-octaose, p-lacto-N-octaose, isolacto-N-octaose, new lacto-N- Neo-octa sugar, p-lacto-N-octa sugar, isolacto-N-nona sugar, neo-lacto-N-nona sugar, lacto-N-nona sugar, lacto-N-deca sugar, isolacto-N-deca sugar , neolacto-N-deca, lacto-N-neodeca, galactosyl lactose, with 1, 2, 3, 4, 5 or more N-acetyllactosamine units and/or 1, 2, 3 , 4, 5 or more lactose-N-disaccharide units extended, and oligosaccharides containing 1 or more N-acetyllactosamine units and or 1 or more lacto-N-disaccharide units , or become an oligosaccharide intermediate (intermediate into oligosaccharide), its fucosylated and sialylated forms, peptides, polypeptides, lipids, sphingolipids, cerebrosides, ceramide lipids, phosphatidylinositol Lipids and glycosylated forms of peptides, polypeptides, lipids, sphingolipids, cerebrosides, ceramide lipids, phosphatidylinositol.

Detailed Description of the Invention

According to a first aspect, the present invention provides a method of producing an α-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the α-1,3 glycosylation occurs on fucose-α - Terminal "fucose-α1,2-galactose"-group of 1,2-galactose R (Fuc-a1,2-Gal-R). The method includes the following steps: i) Provide the ability to synthesize Fuc-a1,2-Gal-R, express an alpha-1,3-glycosyltransferase (alpha-1,3-glycosyltransferase), and synthesize the alpha-1,3-sugar A nucleotide-sugar capable cell that is a donor of syltransferase, and ii) After allowing synthesis of the Fuc-a1,2-Gal-R, expression of the α-1,3-glycosyltransferase, synthesis of the nucleotide-sugar and synthesis of the α-1,3 glycosylated form of Fuc-a1 , the cells were cultured under the condition of 2-Gal-R, iii) Preferably the alpha-1,3 glycated form of Fuc-a1,2-Gal-R is isolated from the culture.

In one embodiment, the present invention provides a method of producing a mixture of Fuc-a1,2-Gal-R in the alpha-1,3 glycosylated form. The method includes the following steps: i) Provide a supply capable of synthesizing at least two different Fuc-a1,2-Gal-Rs, expressing an α-1,3-glycosyltransferase and having the ability to synthesize the α-1,3-glycosyltransferase a nucleotide-sugar-capable cell, preferably a single cell, and ii) After allowing synthesis of the at least two different Fuc-a1,2-Gal-Rs, expression of the α-1,3-glycosyltransferase, synthesis of the nucleotide-sugar and synthesis of the α-1,3 glycosylation The cells are cultured under the conditions of each of the forms of Fuc-a1,2-Gal-R, iii) Preferably, each Fuc-a1,2-Gal-R in the α-1,3 glycated form is isolated from the culture.

According to the present invention, the mixture comprises or consists of at least two different "α-1,3 glycated forms of Fuc-a1,2-Gal-R", preferably at least three different "α-1,3 glycated forms of Fuc-a1,2-Gal-R" Fuc"-a1,2-Gal-R', more preferably at least four different "α-1,3 glycosylated forms of Fuc-a1,2-Gal-R". Preferably, the at least two, more preferably at least three, even more preferably at least four different Fuc-a1,2-Gal-Rs are synthesized by the cells. In further and/or alternative embodiments, a mixture of alpha-1,3 glycosylated forms of Fuc-a1,2-Gal-R may be obtained by a method as disclosed herein, wherein the cells express a variety of alpha-1,3 - Glycosyltransferases (preferably α-1,3-galactosyltransferase and α-1,3-N-acetylgalactosaminyltransferase). In further and/or alternative embodiments, mixtures of alpha-1,3 glycated forms of Fuc-a1,2-Gal-R may be obtained by methods as disclosed herein, wherein a variety of different recipients as disclosed herein are provided .

In a second aspect, the present invention provides metabolically engineered cells for producing the alpha-1,3 glycated form of Fuc-al,2-Gal-R as described herein. In the context of the present invention, the alpha-1,3 glycosylated form of Fuc-al,2-Gal-R described herein preferably does not occur in the wild-type progenitor of said cell.

Provide a metabolically engineered cell, preferably a single cell, which has the ability to synthesize Fuc-a1,2-Gal-R, which expresses α-1,3-glycosyltransferase and which has the ability to synthesize the α-1,2-Gal-R, A nucleotide-sugar capacity of a donor of 3-glycosyltransferase.

According to the present invention, the method for producing the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R may utilize non-metabolically engineered cells or may utilize metabolically engineered cells as disclosed herein.

In the context of the present invention, it should be understood that the Fuc-a1,2-Gal-R form of the Fuc-a1,2-Gal-R, nucleotide-sugar and α-1,3 glycosylated forms are preferably in intracellular synthesis. The skilled artisan will further appreciate that a portion or substantially all of the synthetic α-1,3 glycated form of Fuc-a1,2-Gal-R remains intracellularly and/or is excreted passively or by active transport to Extracellular.

Throughout the application, unless expressly stated otherwise, according to the present invention, "genetically modified cell" or "metabolically engineered cell" preferably means genetically modified or metabolically engineered, respectively, to produce alpha-1, Cells with 3-glycosylated form of Fuc-a1,2-Gal-R.

In the context of the present invention, Fuc-a1,2-Gal-R (or derivative structures thereof, as described herein) of the term "α-1,3 glycated form" preferably means that a sugar moiety (eg a monosaccharide) is passed through α-1,3-glycosidic bond with the described "Fuc-a1,2-Gal"-group of fucose-α-1,2-galactose-R (Fuc-a1,2-Gal-R) Galactose residue binding, i.e., the sugar moiety is not directly linked to another residue contained in Fuc-a1,2-Gal-R, such as a1,2-linked fucose or any other residue contained in the R moiety Residues.

In a preferred embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is (i)Gal-a1,3-(Fuc-a1,2)-Gal-R, more Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-R is preferred, Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R is more preferred , even better Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R, even better Gal-a1,3-(Fuc-a1,2) -Gal-b1,3-GlcNAc-b1,3-Gal-R, preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc; or (ii) GalNAc-a1,3-(Fuc-a1,2)-Gal-R, preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-R , more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, even more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1, 3-GlcNAc-b1,3-R, even more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-R, most preferably GalNAc-a1 ,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc.

In other words, in a preferred embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is α-1,3 galactose or α-1,3 GalNAc modified form of Fuc -a1,2-Gal-R, wherein the galactose or GlcNAc is bound to fucose-α-1,2-galactose-R with an α-1,3-glycosidic bond (Fuc-a1,2-Gal- R) the galactose residue of the "Fuc-a1,2-Gal" group.

In a more preferred embodiment, the Fuc-a1,2-Gal-R of the α-1,3 glycosylated form is Gal-a1,3-(Fuc-a1,2)-Gal-R, preferably Gal -a1,3-(Fuc-a1,2)-Gal-b1,3-R, more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, even More preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R, even more preferably Gal-a1,3-(Fuc-a1,2) -Gal-b1,3-GlcNAc-b1,3-Gal-R, most preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1 , 4-Glc. In other words, in a more preferred embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is α-1,3 galactose-modified Fuc-a1,2-Gal-R.

In another more preferred embodiment, the Fuc-a1,2-Gal-R of the α-1,3 glycosylated form is GalNAc-a1,3-(Fuc-a1,2)-Gal-R, preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-R, more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, Even more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R, even more preferably GalNAc-a1,3-(Fuc-a1,2 )-Gal-b1,3-GlcNAc-b1,3-Gal-R, most preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal- b1,4-Glc. In other words, in a more preferred embodiment, the Fuc-a1,2-Gal-R of the α-1,3 glycosylated form is 1,3GalNAc modified Fuc-a1,2-Gal-R.

Throughout the application, unless expressly stated otherwise, the Fuc-a1,2-Gal-R is preferably Fuc-a1,2-Gal-b1,3-R, more preferably the Fuc-a1, 2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-R, even more preferably, said Fuc-a1,2-Gal-R is lacto-N-fucose pentasaccharide I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc).

In one embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is a structure of the histo blood group antigen (HBGA) system. In a preferred embodiment, the Fuc-a1,2-Gal-R of the α-1,3 glycosylated form is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc- b1,3-R. In a more preferred embodiment, the Fuc-a1,2-Gal-R of the α-1,3 glycosylated form is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc- b1,3-Gal-b1,4-Glc. In an alternative preferred embodiment, the Fuc-a1,2-Gal-R of the α-1,3 glycosylated form is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc -b1,3-R. In a more preferred embodiment, the Fuc-a1,2-Gal-R of the α-1,3 glycosylated form is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc- b1,3-Gal-b1,4-Glc.

In another embodiment, the α-1,3 glycated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, wherein glucose can be Fucosylated (preferably a1,3-fucosylated) as necessary. Preferably, Fuc-a1,2-Gal-R in the α-1,3 glycosylated form is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc.

In another embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1, 3)-Glc.

In another embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is the α-tetrasaccharide GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glcthe (alpha-tetrasaccharide GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc).

In another embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is an α-1,3 GalNAc or an α-1,3 galactose-modified Fuc-a1,2-Gal - GlcNAc, wherein the galactose in Fuc-a1,2-Gal-GlcNA is bound to GlcNAc via a β-1,3 or β-1,4 bond. In a preferred embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is GalNAc-a1,3-(Fuc-a1,2)-Gal-GlcNAc, wherein galactose is via β- 1,3 or β-1,4 bonds bind to GlcNAc. In a more preferred embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc. In another more preferred embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc. In a preferred embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is a Gal-a1,3-(Fuc-a1,2)-Gal-GlcNAc, wherein galactose is via β -1,3 or β-1,4 bonds bind to GlcNAc. In a more preferred embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc. In another more preferred embodiment, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc.

Within the scope of the present invention, the expression "permissive conditions" should be understood as conditions related to physical or chemical parameters, including but not limited to temperature, pH, pressure, osmotic pressure and product/precursor/acceptor concentrations.

In certain embodiments, such conditions may include a temperature range of 30+/-20 degrees Celsius, a pH range of 7+/-3.

According to a preferred embodiment of the present invention, cells are modified with one or more expression modules. Such expression modules are also known as transcriptional units and include polynucleotides that express recombinant genes including the coding gene sequence and appropriate transcriptional and/or translational control signals operably linked to the coding gene. The control signals include promoter sequences, untranslated regions, ribosome binding sites and terminator sequences. The expression module may include one expression unit of a single recombinant gene, but may also include expression units of more recombinant genes, or may be organized into an operon structure to integrate two or more recombinant genes expressed. The polynucleotides can be produced using recombinant DNA techniques using techniques well known in the art. Methods of constructing expression modules are well known to those of ordinary skill in the art to which the present invention pertains, and include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. See eg Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989 and updated annually) technology described in.

The expression of each of the expression modules may be constitutive or produced by natural or chemical inducers. As used herein, constitutive expression is understood to mean the expression of genes that are continuously transcribed in an organism. Expression produced by a natural inducer should be understood as a tropic or regulatory expression of a gene that is only found in certain natural conditions of the host (eg, the organism is in labor, or during lactation) Performance, as a response to environmental changes (such as, including but not limited to, hormonal, heat, cold, pH changes, light, oxidative or osmotic stress/signals), or depending on the developmental stage of the cell or position in the cell cycle, including But not limited to apoptosis and autophagy. Expression by chemical inducers is to be understood as a facultative or regulatory expression of a gene that is only externally sensed by an inducible promoter or by a genetic circuit that induces or inhibits the transcription or translation of said polynucleotide into a polypeptide Chemicals such as IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose.

The expression module can be integrated into the genome of the cell, or can be presented to the cell in the form of a vector. The vector may be in the form of plastids, cosmids, bacteriosomes, liposomes or viruses, and is stably transformed/transfected into the metabolically engineered cells. Such vectors include, among others, chromosomal, episomal, and virus-derived vectors, such as those derived from bacterial plastids, bacteriophages, yeast episomes, insertion units, yeast chromosomal units and vectors derived from combinations of the foregoing, such as vectors derived from plastid and phagemid gene units, such as cohesoplasts and phagemids. These vectors may contain selection markers such as, but not limited to, antibiotic markers, auxotrophic markers, toxin-antitoxin markers, or RNA forward/anti-sense markers. A performance system construct may include control regions that regulate and cause performance. In general, any system or vector suitable for maintaining, propagating or expressing polynucleotides and/or expressing polypeptides in a host can be used for expression in this regard. Suitable DNA sequences can be inserted into the expression system by any of a variety of well-known conventional techniques, such as those described in Sambrook et al. For recombinant production, cells can be genetically engineered to incorporate an expression system or portion thereof or a polynucleotide of the invention. Polynucleotides can be introduced into cells using a number of methods described in standard laboratory practice manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986), and Sambrook et al., supra. 1989.

As used herein, an expression module includes polynucleotides for expression of at least one recombinant gene. The recombinant gene is involved in the expression of polypeptides that play a role in the synthesis of LNFP-I or LNFP-I in the α-1,3 glycosylated form; or the recombinant gene and the cell are not involved in α-1,3 Glycated forms of LNFP-1 or other pathways for the synthesis of LNFP-1 are associated. The recombinant gene encodes an endogenous protein with a modified expression or activity, preferably, the endogenous protein is overexpressed; or the recombinant gene encodes a heterologous protein, and the heterologous protein is modified in the modification. It is introduced and expressed, preferably overexpressed, heterologously in the cell. An endogenous protein can have a modified expression in a cell that also expresses the heterologous protein.

In one embodiment of the method and/or cell according to the invention, the cell has the ability to synthesize lacto-N-fucosepentaose I (LNFP-I). LNFP-I is a fucosylated pentasaccharide derived from lacto-N-tetraose (LNT) modified by a fucose group a1,2-linked to its terminal galactose residue. In a preferred embodiment, the cell is capable of synthesizing LNT and expresses an α-1,2-fucosyltransferase that transfers fucose residues from a GDP fucose donor to a recipient LNT to LNFP-I is produced. In a more preferred embodiment, the cells have the ability to synthesize LNT, express an α-1,2-fucosyltransferase using LNT as an α-1,2-fucosylation acceptor, and have the ability to synthesize LNT as The GDP-fucose capacity of the donor of the alpha-1,2-fucosyltransferase.

LNT can be produced in cells by overexpression of the galactoside β-1,3-N-acetylglucosamine transferase gene and the N-acetylglucosamine β-1,3-galactosyltransferase gene, They transfer GlcNAc residues from UDP-GlcNAc to lactose to form LN3 and Gal residues from UDP-Gal to LN3 to form LNT, respectively. Preferably, the cells do not have an activated galactosidase, such as lacZ, which degrades lactose into glucose and galactose. The lactose required by the galactoside β-1,3-N-acetylglucosaminyltransferase can be synthesized in culture or by metabolism of cells. The UDP-GlcNAc and UDP-Gal required by the enzyme can be provided by the enzyme expressed in the cell or by the metabolism of the cell.

Cells that use lactose as a recipient in the saccharification reaction preferably have transporters for uptake of lactose from culture. More preferably, the cells are optimized for lactose uptake. The optimization may be the overexpression of a lactose transporter such as the lactose permease from Escherichia coli or Kluyveromyces lactis.

In preferred embodiments of the methods and/or cells of the invention, the cells are resistant to lactose killing when grown in an environment where lactose is combined with one or more other carbon sources. The term "lactose killing" refers to the arrest of cell growth in a medium containing lactose and another carbon source. In a preferred embodiment, as described in WO 2016/075243, the cell line is genetically modified to retain at least 50% of the lactose influx without undergoing lactose killing, even at high lactose concentrations. The genetic modification includes expression and/or overexpression of exogenous and/or endogenous lactose transporter genes through heterologous promoters that do not result in a lactose-killing phenotype, and/or modification of the codon usage of the lactose transporter protein Preference is given to producing an altered expression of the lactose transporter that does not contribute to the lactose killing phenotype. The contents of WO 2016/075243 are hereby incorporated by reference in this regard. In the context of the present invention, lactose is preferably taken up by cells as a result of this disclosure, wherein the lactose is further glycosylated to synthesize MMOs, preferably HMOs, as a result of the disclosed glycosyltransferases.

Alternatively, lactose-producing cells can be obtained by expressing β-1,4-galactosyltransferase and UDP-glucose 4-eprimerase. More preferably, the cells are modified to increase lactose production. The modification may be any one or more selected from the group consisting of overexpression of β-1,4-galactosyltransferase, overexpression of UDP-glucose 4-epimerase.

Cells that produce UDP-GlcNAc can express enzymes that convert, for example, GlcNAc to be added to the cell to UDP-GlcNAc. These enzymes can be N-acetyl-D-glucosamine kinase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine from several species including Homo sapiens, Escherichia coli Mutase and N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase. Preferably, the cells are modified to produce UDP-GlcNAc. More preferably, the cells are modified to enhance the production of UDP-GlcNAc. The modification may be selected from the group consisting of knockout of N-acetylglucosamine-6-phosphate deacetylase, overexpression of L-glutamic acid-D-fructose-6-phosphate aminotransferase, phosphoglucosamine Overexpression of amine mutase, and any one or more of the group of overexpression of N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase.

Cells that produce UDP-Gal can express an enzyme that converts, for example, UDP-glucose to UDP-Gal. This enzyme can be, for example, the UDP-glucose 4-epimerase GalE, as known from several species including Homo sapiens, Escherichia coli and brown rat. Preferably, the cells are modified to produce UDP-Gal. More preferably, the cells are modified to enhance UDP-Gal production. The modification may be selected from the group consisting of knockout of a gene encoding bifunctional 5'-nucleotidase/UDP-glycohydrolase, knockout of a gene encoding galactose-1-phosphate uridine transferase, and UDP-glucose 4- Any one or more of an overrepresented group of epimerase-encoding genes.

GDP-fucose can be provided by enzymes expressed in cells or by metabolism of cells. Such GDP-fucose-producing cells may express an enzyme that converts, for example, fucose to be added to the cell to GDP-fucose. This enzyme can be, for example, a bifunctional fucose kinase/fucose-1-phosphate guanosine transferase, such as Fkp from Bacteroides fragilis , or a fucose kinase alone combined with a Combinations of individual fucose-1-phosphate guanylyltransferases, as they are known from several species including Homo sapiens, pigs (Sus scrofa) and brown rats.

Preferably, the cells are modified to produce GDP fucose. More preferably, the cells are modified to increase GDP fucose production. The modification may be selected from the group consisting of knockout of a gene encoding UDP-glucose:undecylallyl-phosphoglucose-1-phosphotransferase, overexpression of a gene encoding GDP-L-fucose synthase, Overexpression of the gene encoding GDP-mannose 4,6-dehydratase, overexpression of the gene encoding mannose-1-phosphate citrulline transferase, overexpression of the gene encoding phosphomannase and mannose-6-phosphate Any one or more of an overexpressed group of genes encoding isomerases.

α-1,2-fucosyltransferase that transfers fucose residues from GDP-fucose to intracellularly synthesized LNT is an α-1,2-fucosyltransferase that accepts LNT The terminal galactose residues act as acceptors for fucosylation. The alpha-1,2-fucosyltransferase can use other acceptors in addition to LNT for fucosylation. Such additional recipients may include, but are not limited to, monosaccharides, disaccharides, and oligosaccharides, such as galactose, glucose, N-acetylglucosamine (GlcNAc), lactose, lactulose, lacto-N-biosaccharide (LNB) ), N-acetylgalactosamine (LacNAc), 3'-fucose (3'FL), lacto-N-trisaccharide (LN3) and lacto-N-neotetraose (LNnT). The alpha-1,2-fucosyltransferase may be, for example, the alpha-1,2-fucosyltransferase of Helicobacter pylori exemplified herein.

In a preferred embodiment of the method and/or cell of the present invention, the α-1,2-fucosyltransferase is selected from the group consisting of a polypeptide (UniProt ID) from Brachyspira pilosicoli A0A2N5RQ26), a polypeptide from Dysgonomonas mossii (UniProt ID F8X274), a polypeptide from Dechlorosoma suillum (UniProt ID G8QLF4), a polypeptide from Desulfovibrio alaskensis (UniProt ID Q316B5) and a polypeptide from Polaribacter vadi ( UniProt ID A0A1B8TNT0).

In an alternative preferred embodiment, the α-1,2-fucosyltransferase is derived from a hair-like short Polypeptide from Treponema (UniProt ID A0A2N5RQ26), Polypeptide from Dysgonomonas mossii (UniProt ID F8X274), Polypeptide from Dechlorosoma suillum (UniProt ID G8QLF4), Polypeptide from Desulfovibrio alaskensis (UniProt ID Q316B5) and Functional fragment of any of the polypeptides from P. vadi (UniProt ID A0A1B8TNTO).

In alternative preferred embodiments, the α-1,2-fucosyltransferase is a polypeptide from Brachyspira trichomes (UniProt ID A0A2N5RQ26), a polypeptide from Dysgonomonas mossii (UniProt ID F8X274) , a functional homologue, variant or any of a polypeptide from Dechlorosoma suillum (UniProt ID G8QLF4), a polypeptide from Desulfovibrio alaskensis (UniProt ID Q316B5) and a polypeptide from P. vadi (UniProt ID A0A1B8TNT0) Derivatives with α-1,2-fucosyltransferase activity on the terminal galactose residues of LNT.

In an alternative preferred embodiment, the α-1,2-fucosyltransferase is a polypeptide comprising an amino acid sequence, or consisting of an amino acid sequence, the amino acid sequence With a polypeptide from Brachyspira trichomes (UniProt ID A0A2N5RQ26), a polypeptide from D. mossii (UniProt ID F8X274), a polypeptide from D. suillum (UniProt ID G8QLF4), a polypeptide from D. alaskensis ( The polypeptide of UniProt ID Q316B5) has at least 80% sequence similarity to the full-length amino acid sequence of any of the polypeptides from P. vadi (UniProt ID A0A1B8TNTO) and has a terminal galactose residue for LNT Alpha-1,2-fucosyltransferase activity.

In another preferred embodiment of the method and/or cell of the present invention, the cell expresses an α-1,2-fucosyltransferase, which preferably uses LNT as α-1,2-fucose Sylated acceptors other than other acceptors such as galactose, glucose, N-acetylglucosamine (GlcNAc), lactose, lactulose, lactose-N-disaccharide (LNB), N-acetyllactosamine (LacNAc) ), 3'-fucosyllactose (3'FL), lactose-N-triose (LN3) and lacto-N-neotetraose (LNnT). In a more preferred embodiment, at least 50% of the fucosylated compounds obtained in the mixture by the α-1,2-fucosyltransferase expressed in the cell are derived from LNT α-1,2- Fucosylation. In other words, at least 50% of the fucosylated compounds obtained in the mixture by the alpha-1,2-fucosyltransferase expressed in the cells are fucosylated LNTs. At least 50% of the fucosylated compound is in the mixture, it should be understood as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84% %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95,50 %, 96.00%, 96,50%, 97.00%, 97,50%, 98.00%, 98,50%, 99.00%, 99,50%, 99,60%, 99,70%, 99,80%, 99 , 90% and 100% of the fucosylated compounds in the mixture were fucosylated LNT. Preferably, at least 60%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably At least 95% of the fucosylated compounds obtained in the mixture by expression in cells are fucosylated LNTs.

In a more preferred embodiment, the α-1,2-fucosyltransferase uses only LNT as a recipient for α-1,2-fucosylation. The term "solely" means only. In other words, the α-1,2-fucosyltransferase only accepts LNT as an acceptor for fucosylation in the α-1,2-bond of the terminal galactose residue of the LNT, but not other recipients.

According to one embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase having the ability to produce the α-1,3-glycosylated form of Fuc-a1,2-Gal-R is An α-1,3-galactosyltransferase, which is a glycosyltransferase with a terminal "fucose-a1" that transfers galactose residues from UDP-Gal to Fuc-a1,2-Gal-R ,2-galactose" group, wherein said R includes a monosaccharide, disaccharide, oligosaccharide, peptide, glycopeptide, protein, glycoprotein, lipid or glycolipid, as hereinbefore defined.

In one embodiment of the method and/or cell of the present invention, the α-1,3-galactosyltransferase has a PFAM PF03414 domain and includes a motif with SEQ ID NO: 01 YX[FHMQT]XAXX[ACG][ACG], where X can be any amino acid residue.

In an alternative embodiment, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and includes the motif YXQXCXX[ACG][ACG] with SEQ ID NO: 02, where X can be any amine acid residues.

In an alternative embodiment, the α-1,3-galactosyltransferase has a PFAM PF03414 domain and includes a PFAM PF03414 domain according to SEQ ID NO: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or A polypeptide sequence of any one of 37.

In an alternative embodiment, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and is SEQ ID NO: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 Any one of a functional homolog (functional homolog), variant (variant) or derivative (derivative), with and with the sequence identification number: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, The full-length of any of the alpha-1,3-galactosyltransferase polypeptides of 36 or 37 has at least 80% overall sequence similarity and is identical to fucose-al,2-galactose-R (fucose- The terminal "fucose-a1,2-galactose"-group of a1,2-galactose-R, Fuc-a1,2-Gal-R) has a-1,3-galactosyltransferase activity.

In an alternative embodiment, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and is SEQ ID NO: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 A functional fragment that has a- 1,3-galactosyltransferase activity.

In a preferred alternative embodiment, the α-1,3-galactosyltransferase has a PFAM PF03414 domain and includes a SEQ ID NO: 03, 04, 05, 06, 07, 08, 09, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or an oligopeptide sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues of any of 37, and for The terminal "fucose-a1,2-galactose"-group of Fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R) has an a-1,3-galactosyl group transferase activity.

In an alternative embodiment, the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain and includes or consists of a polypeptide that includes or consists of an amino acid sequence. Composition, this amino acid sequence and sequence identification number: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, The full-length amino acid sequence of any of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 has at least 80% sequence similarity, and Has a-1,3-galactose to the terminal "fucose-a1,2-galactose"-group of Fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R) Syltransferase activity.

According to another embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase having Fuc-a1,2-Gal-R that produces the α-1,3-glycosylated form is a α-1,3-N-Acetylgalactosamine transferase, which is a glycosyltransferase with the ability to transfer N-acetylgalactosamine residues from UDP-GalNAc to Fuc-a1,2-Gal- the ability of a terminal "fucose-a1,2-galactose" group of R, wherein said R comprises a monosaccharide, disaccharide, oligosaccharide, peptide, glycopeptide, protein, glycoprotein, lipid or glycolipid, as defined hereinbefore.

In one embodiment of the method and/or cell of the present invention, the α-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain and includes motif YX with SEQ ID NO: 38 [ACIL]XGXX[ACG][ACG], where X can be any amino acid residue.

In an alternative embodiment, the alpha-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain and includes motif YX[AG]XAXX[ACG][ with SEQ ID NO: 39 ACG], where X can be any amino acid residue.

In an alternative embodiment, the alpha-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain and includes, eg, SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 A polypeptide sequence of any one of , 97, 98, 99, 100, 101 or 102.

In an alternative embodiment, the α-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain and is SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, A functional homologue, variant or derivative of any one of 97, 98, 99, 100, 101 or 102, having and having SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 , 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 At least 80% of the full length of any of the α-1,3-N-acetylgalactosyltransferase (α-1,3-N-acetylgalactosyltransferase) polypeptides of 98, 99, 100, 101 or 102 Overall sequence similarity with a-1 to the terminal "fucose-al,2-galactose" group of Fucose-al,2-galactose-R (Fuc-al,2-Gal-R) , 3-N-acetylgalactosyltransferase activity.

In an alternative embodiment, the α-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain and is SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, One of the functional fragments of 97, 98, 99, 100, 101 or 102, and the terminal "fucose-a1" of Fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R) ,2-galactose" group has a-1,3-N-acetylgalactosyltransferase activity.

In an alternative embodiment, the alpha-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain and includes the α-1,3-N-acetylgalactosamine transferase from SEQ ID NO: 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99, 100, 101 or 102 at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acids An oligopeptide sequence of residues with a terminal "fucose-al,2-galactose" group for the fucose-al,2-galactose-R (Fuc-al,2-Gal-R) a-1,3-N-Acetylgalactosyltransferase activity.

In an alternative embodiment, the α-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain and comprises or consists of a polypeptide comprising or consisting of It consists of an amino acid sequence, the amino acid sequence and the sequence identification number: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, The full-length amine of any of 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, or 102 The amino acid sequence has at least 80% sequence similarity and is "fucose-a1,2-galactose" to the end of Fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R) The group has a-1,3-N-acetylgalactosyltransferase activity.

As used herein, the PFAM PF03414 domain refers to the PF03414 domain present in the Pfam 32.0 database published in September 2018 and present in the glycosyltransferase 6 (GT6) family. Both the α-1,3-galactosyltransferase and the α-1,3-N-acetylgalactosyltransferase belong to the GT6 family.

Overall sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably using default parameters and preferably using the sequence of the mature protein (i.e. regardless of secretion signal or transit peptide). Sequence similarity is generally higher when only conserved domains or motifs are considered compared to overall sequence similarity.

from with serial identification numbers: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, at least 8, 9, 10, 11, 12, 13, 14, 15 of any of the polypeptides of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 , 16, 17, 18, 19, 20 consecutive amino acid residues and have a-1,3-half to the terminal "fucose-a1,2-galactose" group of Fuc-a1,2-Gal-R Oligopeptide sequences for lactosyltransferase activity are understood to be derived from those provided herein with SEQ ID NOs: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 of any of the polypeptides Any of the oligopeptide sequences of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 up to the total number of contiguous amino acid residues, preferably wherein if present , the oligopeptide does not completely overlap the PFAM domain, more preferably wherein, if present, the oligopeptide does not overlap the PFAM domain, and the terminal "fucose- The a1,2-galactose" group has a-1,3-galactosyltransferase activity.

from with serial identification numbers: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, At least 8, 9, 10, 11, 12 of any of the polypeptides of 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 , 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues and have a to the terminal "fucose-a1,2-galactose" group of Fuc-a1,2-Gal-R - Oligopeptide sequences with 1,3-N-acetylgalactosyltransferase activity are to be understood as provided herein from those having SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 up to the total number of contiguous amino acid residues of any of , 99, 100, 101 or 102 of the polypeptides any of the oligopeptide sequences, preferably wherein, if present, the oligopeptide does not completely overlap the PFAM domain, more preferably wherein, if present, the oligopeptide does not overlap the PFAM domain, and The terminal "fucose-al,2-galactose" group of Fuc-a1,2-Gal-R has a-1,3-N-acetylgalactosyltransferase activity.

In a preferred embodiment of the method and/or cell of the present invention, the cell expresses an α-1,3-glycosyltransferase having the ability to modify intracellularly synthesized Fuc-a1,2-Gal-R, thereby The alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is formed as disclosed hereinbefore. Preferably, the cells are capable of synthesizing nucleotide-sugars as donors of the alpha-1,3-glycosyltransferase.

In a more preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-galactosyltransferase described herein, having the The ability of lactose residues to transfer from UDP-Gal to the terminal "fucose-α1,2-galactose" of Fuc-a1,2-Gal-R, as described here, the nucleotide-sugar is UDP -Gal, resulting in alpha-1,3 galactose-modified Fuc-a1,2-Gal-R as described herein.

In another preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-N-acetylgalactosamine described herein Transferase with the ability to transfer GalNAc residues from UDP-GalNAc to the terminal "fucose-a1,2-galactose" of Fuc-a1, as described herein, the nucleotide-sugar is UDP- GalNAc, resulting in alpha-1,3GalNAc modified Fuc-a1,2-Gal-R as described herein.

In a more preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-galactosyltransferase described herein, The ability of a lactose residue to transfer from UDP-Gal to the terminal "fucose-α1,2-galactose" of Fuc-a1,2-Gal-b1,3-R, as described herein, the nucleotide - The sugar is UDP-Gal, resulting in alpha-1,3 galactose modified Fuc-a1,2-Gal-b1,3-R as described herein.

In another preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-N-acetylgalactosamine described herein Transferases with the ability to transfer GalNAc residues from UDP-GalNAc to the terminal "fucose-a1,2-galactose" of Fuc-a1,2-Gal-b1,3-R, as described herein, The nucleotide-sugar is UDP-GalNAc, resulting in a-1,3 GalNAc modified Fuc-a1,2-Gal-b1,3-R as described herein.

In a more preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-galactosyltransferase described herein, The ability of lactose residues to transfer from UDP-Gal to the terminal "fucose-α1,2-galactose" of Fuc-a1,2-Gal-b1,3-GlcNAc-R, as described here, the core The phospho-sugar is UDP-Gal, resulting in alpha-1,3 galactose-modified Fuc-a1,2-Gal-b1,3-GlcNAc-R as described herein.

In another preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-N-acetylgalactosamine described herein Transferases with the ability to transfer GalNAc residues from UDP-GalNAc to the terminal "fucose-a1,2-galactose" of Fuc-a1,2-Gal-b1,3-GlcNAc-R, as described herein As stated, the nucleotide-sugar is UDP-GalNAc, resulting in a-1,3GalNAc modified Fuc-a1,2-Gal-b1,3-GlcNAc-R as described herein.

In a more preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-galactosyltransferase described herein, Ability to transfer lactose residues from UDP-Gal to the terminal "fucose-α1,2-galactose" of Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R, as described here , the nucleotide-sugar is UDP-Gal, resulting in alpha-1,3 galactose-modified Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R as described herein.

In another preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-N-acetylgalactosamine described herein A transferase with the ability to transfer GalNAc residues from UDP-GalNAc to the terminal "fucose-a1,2-galactose" of Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R, As described herein, the nucleotide-sugar is UDP-GalNAc, resulting in α-1,3GalNAc modified Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3- as described herein R.

In a more preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-galactosyltransferase described herein, The ability of lactose residues to transfer from UDP-Gal to the terminal "fucose-α1,2-galactose" of Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R, as described here Said, said nucleotide-sugar is UDP-Gal, resulting in alpha-1,3 galactose modified Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal as described herein -R.

In another preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-N-acetylgalactosamine described herein Transferase with terminal "fucose-a1,2-galactose" that transfers GalNAc residues from UDP-GalNAc to Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R The ability, as described herein, the nucleotide-sugar is UDP-GalNAc, resulting in α-1,3GalNAc modified Fuc-a1,2-Gal-b1,3-GlcNAc-b1, as described herein, 3-Gal-R.

In a more preferred embodiment of the method and/or cell of the present invention, the cell expresses an α-1,3-glycosyltransferase, which is capable of modifying intracellularly synthesized LNFP-I to an α-1,3 glycosylated form The LNFP-I. In another additional embodiment of the method and/or cell of the present invention, the cell is capable of synthesizing nucleotide-sugars that are donors of said alpha-1,3-glycosyltransferase.

In a more preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-galactosyltransferase described herein, The ability of lactose residues to transfer from UDP-Gal to the terminal "fucose-α1,2-galactose" group of LNFP-1, the nucleotide-sugar being UDP-Gal and the α-1,3 Glycated form 5 lacto-N-fucose pentasaccharide I (LNFP-I) is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4 -Glc (Gal-a1,3-LNFP-I).

In a more preferred embodiment of the method and/or cell of the present invention, the α-1,3-glycosyltransferase is the α-1,3-N-acetylgalactosaminyltransferase described herein Enzyme with the ability to transfer GalNAc residues from UDP-GalNAc to the terminal "fucose-α1,2-galactose" group of LNFP-I, the nucleotide-sugar being UDP-GalNAc and the α - The lacto-N-fucose pentasaccharide I (LNFP-I) in the 1,3 glycated form is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal -b1,4-Glc (GalNAc-a1,3-LNFP-I).

In a still further embodiment of the methods and/or cells of the invention, the cells are modified in the expression or activity of at least one glycosyltransferase, including a galactosyltransferase (eg, beta-1 ,3-galactosyltransferase, β-1,4-galactosyltransferase, α-1,3-galactosyltransferase, α-1,4-galactosyltransferase), N-acetyltransferase Galactosyltransferase, fucosyltransferase (eg 2-fucosyltransferase, α-1,3/1,4-fucosyltransferase, α-1,6-fucosyltransferase transferase), N-acetylglucosaminyltransferase, mannosyltransferase, N-acetylmannosyltransferase, glucosyltransferase. In a preferred embodiment, the glycosyltransferase comprises an α-1 having α-1,3-galactosyltransferase activity to a terminal "fucose-α1,2-galactose-R" group ,3-galactosyltransferase and α-1,3 with α-1,3-N-acetylgalactosyltransferase activity to the terminal "fucose-α1,2-galactose-R" group -N-Acetylgalactosyltransferase, as described herein.

In one embodiment, the glycosyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably the endogenous glycosyltransferase is overexpressed; alternatively, the glycosyltransferase is a heterologous protein that is introduced heterologously and expressed, preferably overexpressed, in the cell. The endogenous glycosyltransferase can have a modified expression in a cell that also expresses a heterologous glycosyltransferase.

In one embodiment of the method of the invention, the culture is fed for the synthesis of fucose-al,2-galactose-R and/or alpha-1,3 glycated form of fucose-al,2-galactose Lactose-R precursor. Precursors that can be supplied to the culture for the synthesis of fucose-a1,2-galactose-R and/or α-1,3 glycated forms of fucose-a1,2-galactose-R include lactose, Lactose-N-triose (LN3, LNT II), fucose, glucose, galactose, GlcNAc, GDP-fucose, UDP-galactose and UDP-GlcNAc or any other precursor as defined herein.

In one embodiment of the method of the present invention, the culture is supplied with a precursor for the synthesis of LNFP-1 and/or the alpha-1,3 glycosylated form of LNFP-1. Precursors that can be supplied for culturing to synthesize LNFP-I and/or α-1,3 glycated forms of LNFP-I include lactose, lactose-N-triose (LN3, LNT II), fucose, glucose and Galactose.

In one embodiment of the method and/or cell according to the invention, the cell expresses a membrane transporter or a polypeptide having transport activity whereby the compound is transported across the outer membrane of the cell wall. In a preferred embodiment of the method and/or cell of the invention, the cell expresses more than one membrane transporter or polypeptide with transport activity, whereby the compound is transported across the outer membrane of the cell wall. In a more preferred embodiment of the method/or cell of the present invention, the cell line is modified with respect to the expression or activity of said membrane transporter or polypeptide having transport activity. The membrane transporter or polypeptide with transport activity is an endogenous protein with modified expression or activity of the cell, preferably the endogenous membrane transporter or polypeptide with transport activity is overexpressed Alternatively, the endogenous membrane transporter or polypeptide with transport activity is a heterologous protein introduced into the cell and expressed in the cell, preferably overexpressed. The endogenous membrane transporter or polypeptide with transport activity may have a modified expression in cells that also express the heterologous membrane transporter or polypeptide with transport activity.

In another embodiment of the method/or cell of the present invention, the membrane transporter or polypeptide having transport activity is selected from a list comprising the following: transporter, P-P linkage hydrolysis-driven transporter , b-barrel porin (b-barrel porin), auxiliary transport protein, putative transport protein (putative transport protein) and phosphotransfer-driven group translocator (phosphotransfer-driven group translocator). In a more preferred embodiment of the method/or cell of the present invention, the porter includes MFS transporter, sugar efflux transporter and siderophore exporter. In another preferred embodiment of the method/or cell of the present invention, the P-P bond hydrolysis-driven transport protein includes ABC transport protein and chelated iron export protein.

In another preferred embodiment of the method and/or cell of the present invention, the membrane transporter or polypeptide having transport activity controls the α-1,3 glycosylated form of fucose- Flow of α1,2-galactose-R on the outer membrane of the cell wall. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, membrane transporters or polypeptides having transport activity control the flow of one or more precursors on the outer membrane of the cell wall, said precursors was used for the described production of fucose-α1,2-galactose-R in the α-1,3 glycosylated form. In alternative and/or additional preferred embodiments of the methods and/or cells of the present invention, membrane transporters or polypeptides having transport activity control the flow of one or more recipients on the outer membrane of the cell wall, so The recipient was used for the described production of the α-1,3 glycosylated form of fucose-α1,2-galactose-R.

In another preferred embodiment of the method/or cell of the present invention, the cell expresses a membrane transporter belonging to the MFS transporter family, such as MdfA polypeptides from the MdfA family of multidrug transporters from species including Escherichia coli ( UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23) and Yokenella regensburgei (UniProt ID) G9Z5F4). In another more preferred embodiment of the method/or cell of the invention, the cell expresses a membrane transporter belonging to the sugar efflux transporter family, such as SetA polypeptides from the SetA family of species including Escherichia coli (UniProt ID P31675 ), Citrobacter koseri (UniProt ID A0A078LM16), Klebsiella pneumoniae (UniProt ID A0A0C4MGS7). In another preferred embodiment of the method/or cell of the present invention, the cell expresses a membrane transporter belonging to the siderophore exporter family, such as E. coli entS (UniProt ID P24077) and E. coli iceT (UniProt ID A0A024L207). In another more preferred embodiment of the method/or cell of the present invention, the cell expresses a membrane transporter belonging to the ABC transporter family, such as oppF from Escherichia coli (UniProt ID P77737), lactic acid diacetate from Lactococcus lactis subsp. ImrA (UniProt ID A0A1V0NEL4) of varietal ( Lactococcus lactis subsp. lactis bv. Diacetylactis ) and Blon_2475 (UniProt ID B7GPD4) of Bifidobacterium longum subsp. Infantis . In a more preferred embodiment of the method/or cell of the present invention, the cell expresses a membrane transporter selected from a list comprising: LacY or lac12 permease, fucose transporter, glucose transporter, galactose Transporters, transporters of nucleotide-activated sugars, such as transporters of UDP-GlcNAc, UDP-Gal and/or GDP-Fuc, MdfA protein from E. coli (UniProt ID P0AEY8), MdfA protein from Cronobacter (UniProt ID A0A2T7ANQ9), MdfA protein from Citrobacter johnsonii (UniProt ID D4BC23), MdfA protein from Yorkia spp. (Z5F protein from SetZ5F ID G9). SetA protein from E. coli (UniProt ID P31675), SetA protein from Citrobacter cruzi (UniProt ID A0A078LM16), SetA protein from Klebsiella pneumoniae (UniProt ID A0A0C4MGS7), entS protein from Escherichia coli (UniProt ID P24077), iceT protein from Escherichia coli (UniProt ID A0A024L207), oppF protein from Escherichia coli (UniProt ID P77737), lmrA protein from Lactococcus lactis subsp. diacetate lactis (UniProt ID A0A1VONEL4) and Blon_2475 from Bifidobacterium longum infantum subsp. (UniProt ID B7GPD4) . Preferably, the cells are transformed to include at least one nucleic acid sequence encoding a protein selected from the group consisting of lactose transporters such as LacY or lac12 permease, fucose transporter, glucose transporter, galactose transporter, nuclear Glycoside-activated sugar transporters, such as UDP-GlcNAc, UDP-GalNAc and/or GDP-Fuc transporters. Thus, the transporter internalizes upon addition of a precursor and/or acceptor for the synthesis of fucose-α1,2-galactose-R in the α-1,3 glycosylated form of the present invention By.

In additional and/or alternative embodiments of the methods and/or cells according to the invention, the cells are genetically modified to export the α-1,3 glycosylated form of fucose-α1,2 of the invention through the membrane -Galactose-R. For example, this transporter is a membrane transporter that belongs to the siderophore exporter family, major facilitator superfamily (MFS), ATP-binding cassette (ABC) transporter protein family or sugar efflux transporter family.

In further embodiments of the methods and/or cells of the invention, the cells preferably comprise multiple copies of the same coding DNA sequence encoding a protein. In the context of the present invention, the protein may be a glycosyltransferase, a membrane transporter or any other protein disclosed herein. Throughout this application, the feature "plurality" means at least 2, preferably at least 3, more preferably at least 4, even more preferably at least 5.

In another embodiment of the methods and/or cells of the present invention, the cell includes modifications for reducing acetate production. The modification may be any one or more selected from the group including overexpression of acetyl-CoA synthase, complete or partial knockout or less functional pyruvate dehydrogenase and complete or partial knockout or functional Poor lactate dehydrogenase.

In a further embodiment of the method and/or cell of the present invention, the cell line is modified with the expression or activity of at least one acetyl-coenzyme A synthetase (acs), such as from Escherichia coli, Saccharomyces cerevisiae , human or mouse ( M. musculus ) acs. In preferred embodiments, the acetyl-CoA synthase is an endogenous protein of the cell with modified expression or activity, preferably the endogenous acetyl-CoA synthase is overexpressed; Alternatively, the acetyl-CoA synthetase is a heterologous protein introduced into and expressed in the cell, preferably overexpressed. The endogenous acetyl-CoA synthetase can have a modified expression in a cell that also expresses a heterologous acetyl-CoA synthase. In a more preferred embodiment, the cell line is modified for the expression or activity of the acetyl-CoA synthase acs (UniProt ID P27550) from E. coli. In another and/or additional preferred embodiment, the cell line is modified with a functional homologue, variant or derivative of the acetyl-CoA synthase acs (UniProt ID P27550) from Escherichia coli, which is relative to that from The polypeptide of E. coli (UniProt ID P27550) has at least 80% overall sequence similarity and has acetyl-CoA synthase activity.

In a further alternative and/or additional embodiment of the methods and/or cells of the invention, the cell line is modified for the expression or activity of at least one pyruvate dehydrogenase, for example from Escherichia coli, yeast, brown mouse ( R. norvegicus ) pyruvate dehydrogenase. In a preferred embodiment, the cell line is modified to have at least a portion or loss of pyruvate dehydrogenase activity by methods generally known to those of ordinary skill in the art to cause at least one protein to have less function or loss of pyruvate dehydrogenase activity. Completely deleted or mutated gene encoding pyruvate dehydrogenase. In a more preferred embodiment, the gene encoding poxB is completely deleted from the cell, resulting in a lack of pyruvate dehydrogenase activity in the cell.

In a further alternative and/or additional embodiment of the methods and/or cells of the invention, the cell line is modified for the expression or activity of at least one lactate dehydrogenase, eg from E. coli, yeast, R. norvegicus ) of lactate dehydrogenase. In a preferred embodiment, the cell line is modified to have at least a partial or complete loss of at least one protein by methods generally known to those of ordinary skill in the art to cause at least one protein to have less function or loss of lactate dehydrogenase activity Knocked out or mutated gene encoding lactate dehydrogenase. In a more preferred embodiment, the gene encoding ldhA is completely deleted from the cell, resulting in a lack of lactate dehydrogenase activity in the cell.

According to another preferred embodiment of the method and/or cell of the present invention, the cell comprises a reduced or reduced expression and/or disrupted, attenuated, reduced or delayed expression of any one or more of the following proteins as compared to an unmodified precursor cell activity, the one or more proteins include: β-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deacetylase Aminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose: undecyl isopentenyl-phosphoglucose 1-phosphotransferase (UDP -glucose:undecaprenyl-phosphate glucose-1-phosphate transferase), L-fucose kinase, L-fucose isomerase (L-fucose isomerase), N-acetylneuraminic acid lyase, N-acetylene Mannosamine Kinase, N-Acetylmannosamine-6-Phosphate 2-Epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, Galactose-1-Phosphate Uridine Transferase, Glucose- ATP-dependent 6-phosphofructokinase isozyme 1 (ATP-dependent 6-phosphofructokinase isozyme 1), ATP-dependent 6-phosphofructokinase isozyme 2 , glucose-6-phosphate isomerase, aerobic respiration control protein, transcription inhibitor protein IclR, lon protease, glucose-specific translocating phosphotransferase (glucose-specific translocating phosphotransferase) enzyme IIBC to form ptsG, glucose-specific translocating phosphate Transferase (phosphotransferase, PTS) enzyme IIBC composed of malX, enzyme IIA ^Glc , β-glucoside-specific PTS enzyme II, fructose-specific PTS polyphosphoryl transfer proteins FruA and FruB, alcohol dehydrogenase, aldehyde dehydrogenase, Pyruvate-formate lyase (pyruvate-formate lyase), acetate kinase, phosphoacyltransferase, phosphoacetyltransferase and pyruvate decarboxylase.

According to another preferred embodiment of the method and/or cell of the present invention, the cell has the ability to produce phosphoenolpyruvate (PEP). In another preferred embodiment of the method and/or cell of the present invention, the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP).

In a preferred embodiment and as a method of enhancing the production and supply of PEP, one or more PEP-dependent sugar transport phosphotransferase systems are disrupted, such as but not limited to: 1) N-acetyl-D-glucosamine Npi- Phosphotransferase (EC 2.7.1.193), encoded by the nagE gene (or cluster nagABCD) of species such as E. coli or Bacillus, 2) ManXYZ, which encodes the import of exogenous six-carbon sugars (mannose, glucose, glucosamine, Fructose, 2-deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and the enzyme II Man complex that releases phosphate to the cytoplasm (mannose PTS permease, protein-Npi-phosphohistidine- D-mannose phosphotransferase (protein-Npi-phosphohistidine-D-mannose phosphotransferase), 3) a glucose-specific PTS transporter protein (eg encoded by PtsG/Crr) that takes up glucose and forms glucose-6 in the cytoplasm - Phosphate, 4) sucrose-specific transport proteins, which take up sucrose and form sucrose-6-phosphate in the cytoplasm, 5) fructose-specific transport proteins (encoded for example by the genes fruA and fruB and the gene fruK), which take up fructose and fructose-1-phosphate in the first step and fructose 1,6-diphosphate in the second step, 6) the lactose PTS transport protein (encoded for example by lacE in Lactococcus casei ), It takes up lactose and forms lactose-6-phosphate, 7) Galactitol-specific PTS enzymes, which take up galactitol and/or sorbitol and form galactitol-1-phosphate or sorbitol-6-phosphate, respectively , 8) Mannitol-specific PTS enzymes, which take up mannitol and/or sorbitol and form mannitol-1-phosphate or sorbitol-6-phosphate, respectively, and 9) Trehalose-specific PTS enzymes, which take up Trehalose and form trehalose-6-phosphate.

In another and/or additional preferred embodiment and as a method of enhancing PEP generation and supply, the complete PTS system is disrupted by disrupting the PtsIH/Crr pitch cluster. PtsI (enzyme I) is a cytoplasmic protein that acts as a gateway to the phosphoenolpyruvate:sugar phosphotransferase system (PTS sugar) of E. coli K-12. PtsI is one of the two sugar-nonspecific protein components of PTS sugars (PtsI and PtsH), which causes a phosphorylation transfer reaction (cascade) with sugar-specific inner membrane permease, and the phosphorylation transfer reaction results in coupled phosphorylation and a Transport of serial sugar substrates. HPr (histidine-containing protein) is one of two carbohydrate-specific proteins composed of PTS sugar. The HPr complex phosphorylase I (PtsI-P) accepts a phosphate group and then transfers to the EIIA domain of any of the many glycospecific enzymes of the PTS sugar. Crr or EIIAGlc is phosphorylated by PEP in a reaction that requires PtsH and PtsI.

In another and/or preferred embodiment, the cells are further modified to compensate for the absence of the carbon source's PTS system by introducing and/or overexpressing the corresponding permease. These are such as permease or ABC transporters, which include, but are not limited to, transporters that specifically import lactose, such as those encoded by the LacY gene from E. coli, and transporters that specifically import sucrose, such as those from the large intestine. The transport protein encoded by the cscB gene of Bacillus, the transport protein for the specific import of glucose, such as the transport protein encoded by the galP gene from Escherichia coli, the transport protein for the specific import of fructose, for example, from Streptococcus mutans The transport protein encoded by the fruI gene, or the sorbitol/mannitol ABC transport protein, such as the transport protein encoded by the cluster SmoEFGK of Rhodobacter sphaeroides , trehalose/sucrose/maltose transport protein, such as alfalfa China The transport protein encoded by the cluster ThuEFGK of Sinorhizobium meliloti , and the N-acetylglucosamine/galactose/glucose transport protein, such as the transport protein encoded by NagP of Shewanella oneidensis . Examples of combinations of PTS deletion and alternative transporter overexpression are: 1) deletion of a glucose PTS system, such as the ptsG gene, combined with the introduction and/or overexpression of a glucose permease (eg, galP or glcP), 2) deletion of a fructose PTS system, such as One or more of the fruB, fruA, fruK genes in combination with the introduction and/or overexpression of a fructose permease such as fruI, 3) deletion of the lactose PTS system in combination with the introduction and/or overexpression of a lactose permease such as LacY, and/or or 4) deletion of the sucrose PTS system in combination with the introduction and/or overexpression of a sucrose permease such as cscB.

In a more preferred embodiment, cells are modified to compensate for the absence of the carbon source PTS system by introducing and/or overexpressing glycokinases such as glucokinase (EC 2.7.1.1, EC 2.7.1.2, EC 2.7.1.63 ), galactokinase (EC 2.7.1.6) and/or fructokinase (EC 2.7.1.3, EC 2.7.1.4). Examples of combinations of PTS deletion and alternative transporter and kinase overexpression are: 1) Deletion of a glucose PTS system, such as the ptsG gene, combined with the introduction and/or overexpression of a glucose permease (eg, galP or glcP), combined with the introduction and/or overexpression Expression of glucokinase (eg, glk), and/or 2) deletion of the fructose PTS system, such as one or more of the fruB, fruA, fruK genes, in conjunction with the introduction and/or overexpression of a fructose permease, such as fruI, in conjunction with the introduction and/or Or overexpress fructokinase (eg frk or mak).

In another and/or additional preferred embodiment and as a method of enhancing PEP production and supply, cells are modified by introducing or modifying one or more of the following: phosphoenolpyruvate synthase activity (EC: 2.7.9.2, eg encoded by ppsA in E. coli), phosphoenolpyruvate carboxykinase activity (EC 4.1.1.32 or EC 4.1.1.49, eg by Corynebacterium glutamicum , respectively) PCK or encoded by pckA in E. coli), phosphoenolpyruvate carboxylase activity (EC 4.1.1.31, eg encoded by ppc in E. coli), oxaloacetate decarboxylase activity (EC 4.1.1.112, eg encoded by eda in E. coli), pyruvate kinase activity (EC 2.7.1.40, eg encoded by pykA and pykF in E. coli), pyruvate carboxylase activity (EC 6.4.1.1 , eg encoded by pyc in Bacillus subtilis), and malate dehydrogenase activity (EC 1.1.1.38 or EC 1.1.1.40 eg encoded by maeA or maeB in E. coli, respectively).

In a more preferred embodiment, the cell line is modified to overexpress a polypeptide comprising any one or more of the following: ppsA from E. coli (UniProt ID P23538 ), PCK from C. glutamicum (UniProt ). ID Q6F5A5), pcka of E. coli (UniProt ID P22259), eda of E. coli (UniProt ID P0A955), maeA of E. coli (UniProt ID P26616) and maeB of E. coli (UniProt ID P76558).

In another and/or additional preferred embodiment, the cell line is modified to express any one or more polypeptides having phosphoenolpyruvate synthase activity, phosphoenolpyruvate Carboxykinase activity, oxalate acetate decarboxylase activity or malate dehydrogenase activity.

In another and/or additional preferred embodiment and as a method of enhancing PEP production and supply, cells are modified by reducing phosphoenolpyruvate carboxylase activity and/or pyruvate kinase activity, preferably deletion Genes encoding phosphoenolpyruvate carboxylase, pyruvate carboxylase and/or pyruvate kinase.

In an exemplary embodiment, cells are modified through different adaptations, such as overexpression of phosphoenolpyruvate synthase combined with deletion of the phosphoenolpyruvate carboxylase gene; overexpression of phosphoenolpyruvate Salt synthase binding deletion of pyruvate carboxylase gene; overexpression of phosphoenolpyruvate carboxykinase binding to deletion of phosphoenolpyruvate carboxylase gene; overexpression of phosphoenolpyruvate carboxykinase binding to deletion of pyruvate carboxylate Enzyme gene; overexpressed oxalate decarboxylase in combination with deletion of pyruvate kinase gene; overexpressed in oxalate decarboxylase in combination with deletion of phosphoenolpyruvate carboxylase gene; overexpressed in malate dehydrogenase in combination with deletion of phosphate Enolpyruvate carboxylase gene, and/or malate dehydrogenase combined deletion of pyruvate carboxylase gene.

In another exemplary embodiment, cells are modified through different adaptations, such as overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase; overexpression of phosphoenolpyruvate Synthase with overexpressed oxalate decarboxylase; overexpressed phosphoenolpyruvate synthase with overexpressed malate dehydrogenase; overexpressed phosphoenolpyruvate carboxykinase with overexpressed oxalate acetate decarboxylase; overexpressed phosphoenolpyruvate carboxykinase combined with overexpressed malate dehydrogenase, overexpressed oxalate decarboxylase combined with overexpressed malate dehydrogenase; overexpressed phosphoenolpyruvate Synthase combined with overexpressed phosphoenolpyruvate carboxykinase and overexpressed oxalate decarboxylase; overexpressed phosphoenolpyruvate synthase combined with overexpressed phosphoenolpyruvate carboxykinase and overexpressed Malate dehydrogenase; overexpressed phosphoenolpyruvate synthase combined with overexpressed phosphoenolpyruvate carboxykinase, overexpressed oxalate decarboxylase, and overexpressed malate dehydrogenase; overexpressed phosphate Enolpyruvate carboxykinase in combination with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase; and/or overexpression of phosphoenolpyruvate synthase in combination with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase.

In another exemplary embodiment, cells are modified through different adaptations, such as overexpression of phosphoenolpyruvate synthase in combination with overexpression of phosphoenolpyruvate carboxykinase and in combination with a gene deficient in pyruvate kinase; Overexpressed phosphoenolpyruvate synthase combined with overexpressed oxalate decarboxylase and combined with a gene missing pyruvate kinase; overexpressed phosphoenolpyruvate synthase combined with overexpressed malate dehydrogenase and combined with deletion Genes for pyruvate kinase; overexpressing phosphoenolpyruvate carboxykinase in combination with overexpressing oxalate decarboxylase and binding genes lacking pyruvate kinase; overexpressing phosphoenolpyruvate carboxykinase in conjunction with overexpressing Malate dehydrogenase combined with a gene lacking pyruvate kinase; overexpressed oxalate decarboxylase combined with overexpressed malate dehydrogenase combined with a gene lacking pyruvate kinase; overexpressed phosphoenolpyruvate synthase Combined overexpression of oxalate decarboxylase and combined gene deletion of pyruvate kinase; overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase and combined Combines genes that lack pyruvate kinase; overexpression of phosphoenolpyruvate synthase combined with overexpression of phosphoenolpyruvate carboxykinase, overexpression of oxalate decarboxylase, and overexpression of malate dehydrogenase and Binds genes that lack pyruvate kinase; overexpresses phosphoenolpyruvate carboxykinase in combination with overexpressed oxalate decarboxylase and overexpresses malate dehydrogenase and binds genes lacking pyruvate kinase; and overexpresses phosphate Enolpyruvate synthase binds genes overexpressing oxalate acetate decarboxylase and overexpressing malate dehydrogenase and binding pyruvate kinase-deficient genes.

In another exemplary embodiment, cells are modified through different adaptations, such as overexpression of phosphoenolpyruvate synthase in combination with overexpression of phosphoenolpyruvate carboxykinase and in combination with deletion of phosphoenolpyruvate Carboxylase gene; overexpressed phosphoenolpyruvate synthase combined with overexpressed oxalate decarboxylase and combined with deletion of phosphoenolpyruvate carboxylase gene; overexpressed phosphoenolpyruvate synthase combined with overexpressed Malate dehydrogenase in combination with deletion of phosphoenolpyruvate carboxylase gene; overexpressed phosphoenolpyruvate carboxykinase in combination with overexpressed phosphoenolpyruvate in combination with deletion of phosphoenolpyruvate carboxylase gene ; Overexpressed phosphoenolpyruvate carboxykinase combined with overexpressed malate dehydrogenase combined with deletion of the phosphoenolpyruvate carboxylase gene; overexpressed oxalate decarboxylase combined with overexpressed malate dehydrogenase In combination with deletion of phosphoenolpyruvate carboxylase gene; overexpression of phosphoenolpyruvate synthase in combination with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase in combination with deletion of phosphoene Alcohol pyruvate carboxylase gene; overexpressed phosphoenolpyruvate synthase in combination with overexpressed phosphoenolpyruvate carboxykinase and overexpressed malate dehydrogenase in combination with deletion of phosphoenolpyruvate carboxylase gene ;Overexpressed phosphoenolpyruvate synthase in combination with overexpressed phosphoenolpyruvate carboxykinase, overexpressed oxalate decarboxylase and overexpressed malate dehydrogenase in combination with deletion of phosphoenolpyruvate Carboxylase genes; overexpressed phosphoenolpyruvate carboxykinase in combination with overexpressed oxalate decarboxylase and overexpressed malate dehydrogenase in combination with deletion of phosphoenolpyruvate carboxylase genes; and overexpressed phosphoenolase Enolpyruvate synthase combined with overexpressed oxalate decarboxylase and overexpressed malate dehydrogenase combined with deletion of the phosphoenolpyruvate carboxylase gene.

In another exemplary embodiment, cells are modified through different adaptations, such as overexpression of phosphoenolpyruvate synthase in combination with overexpression of phosphoenolpyruvate carboxykinase and in combination with deletion of the pyruvate carboxylase gene; Overexpressed phosphoenolpyruvate synthase combined with overexpressed oxalate decarboxylase and combined deletion of pyruvate carboxylase gene; overexpressed phosphoenolpyruvate synthase combined with overexpressed malate dehydrogenase combined with deletion Pyruvate carboxylase gene; overexpressed phosphoenolpyruvate carboxykinase in combination with overexpressed oxaloacetate decarboxylase in combination with missing pyruvate carboxylase gene; overexpressed phosphoenolpyruvate carboxykinase in conjunction with overexpressed Malate dehydrogenase in combination with deletion of pyruvate carboxylase gene; overexpression of oxalate decarboxylase in combination with overexpression of malate dehydrogenase in combination with deletion of pyruvate carboxylase gene; overexpression of phosphoenolpyruvate synthase Combines overexpressed phosphoenolpyruvate carboxykinase and overexpressed oxalate decarboxylase in conjunction with deletion of the pyruvate carboxylase gene; overexpressed phosphoenolpyruvate synthase combined with overexpressed phosphoenolpyruvate Carboxykinase and overexpressed malate dehydrogenase combined with deletion of pyruvate carboxylase gene; overexpressed phosphoenolpyruvate synthase combined with overexpressed phosphoenolpyruvate carboxykinase, overexpressed oxaloacetate dehydrogenase Carboxylase and overexpressed malate dehydrogenase in combination with deletion of the pyruvate carboxylase gene; overexpressed phosphoenolpyruvate carboxykinase in combination with overexpressed oxalate decarboxylase and overexpressed malate dehydrogenase in combination Deletion of the pyruvate carboxylase gene; and overexpression of phosphoenolpyruvate synthase in combination with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase in combination with deletion of the pyruvate carboxylase gene.

In another exemplary embodiment, cells are modified through different adaptations, such as overexpression of phosphoenolpyruvate synthase in combination with overexpression of phosphoenolpyruvate carboxykinase and in combination with a gene deficient in pyruvate kinase and Genes for phosphoenolpyruvate carboxylase; overexpressed phosphoenolpyruvate synthase combined with overexpressed oxaloacetate decarboxylase and combined genes lacking pyruvate kinase with genes for phosphoenolpyruvate carboxylase ; Overexpressed phosphoenolpyruvate synthase combined with overexpressed malate dehydrogenase and combined pyruvate kinase-deficient genes with phosphoenolpyruvate carboxylase genes; overexpressed phosphoenolpyruvate carboxykinase Combined overexpression of oxalate decarboxylase and combined gene deletion of pyruvate kinase and gene of phosphoenolpyruvate carboxylase; overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of malate dehydrogenase and Combining pyruvate kinase-deficient genes with phosphoenolpyruvate carboxylase genes; overexpressing oxalate decarboxylase in combination with overexpressing malate dehydrogenase and combining pyruvate kinase-deficient genes with phosphoenolpyruvate Genes for salt carboxylase; overexpressing phosphoenolpyruvate synthase combined with overexpressing phosphoenolpyruvate carboxykinase and overexpressing oxaloacetate decarboxylase and combining genes lacking pyruvate kinase with phosphoenol Genes for pyruvate carboxylase; overexpressed phosphoenolpyruvate synthase combined with overexpressed phosphoenolpyruvate carboxykinase and overexpressed malate dehydrogenase combined with pyruvate kinase-deficient genes with phosphoenol Genes for pyruvate carboxylase; overexpressed phosphoenolpyruvate synthase in combination with overexpressed phosphoenolpyruvate carboxykinase, overexpressed oxalate decarboxylase, and overexpressed malate dehydrogenase in combination Genes missing pyruvate kinase and genes for phosphoenolpyruvate carboxylase; overexpression of phosphoenolpyruvate carboxykinase combined with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase combined with deletion Genes for pyruvate kinase and phosphoenolpyruvate carboxylase; and overexpression of phosphoenolpyruvate synthase in combination with overexpression of oxalate decarboxylase and overexpression of malate dehydrogenase in combination with absent acetone Acid kinase gene and phosphoenolpyruvate carboxylase gene.

In another exemplary embodiment, cells are modified through different adaptations, such as overexpression of phosphoenolpyruvate synthase in combination with overexpression of phosphoenolpyruvate carboxykinase and in combination with a gene deficient in pyruvate kinase, Genes for pyruvate carboxylase and genes for phosphoenolpyruvate carboxylase; overexpressed phosphoenolpyruvate synthase combined with overexpressed oxalate decarboxylase and combined with a gene lacking pyruvate kinase, pyruvate carboxylate Enzyme genes with genes for phosphoenolpyruvate carboxylase; overexpressing phosphoenolpyruvate synthase in combination with overexpressing malate dehydrogenase and in combination with genes lacking pyruvate kinase, pyruvate carboxylase genes with phosphate Genes for enolpyruvate carboxylase; overexpressed phosphoenolpyruvate carboxykinase combined with overexpressed oxaloacetate decarboxylase and combined pyruvate kinase-deficient genes, pyruvate carboxylase genes with phosphoenol Genes for pyruvate carboxylase; overexpressed phosphoenolpyruvate carboxykinase combined with overexpressed malate dehydrogenase and combined with deficient pyruvate kinase genes, pyruvate carboxylase genes with phosphoenolpyruvate carboxylate Enzyme genes; overexpressed oxalate decarboxylase combined with overexpressed malate dehydrogenase and combined with deficient pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase genes; overexpressed Phosphoenolpyruvate synthase combined with overexpressed phosphoenolpyruvate carboxykinase and overexpressed oxaloacetate decarboxylase and combined pyruvate kinase-deficient genes, pyruvate carboxylase genes with phosphoenolacetone Genes for acid carboxylase; overexpressing phosphoenolpyruvate synthase in combination with overexpressing phosphoenolpyruvate carboxykinase and overexpressing malate dehydrogenase in combination with genes lacking pyruvate kinase, pyruvate carboxylase Genes of phosphoenolpyruvate carboxylase; overexpressed phosphoenolpyruvate synthase combined with overexpressed phosphoenolpyruvate carboxykinase, overexpressed oxaloacetate decarboxylase, and overexpressed apple Acid dehydrogenase combined with deletion of pyruvate kinase gene, pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene; overexpressed phosphoenolpyruvate carboxykinase combined with overexpressed oxaloacetate dehydrogenase Carboxylase and overexpressing malate dehydrogenase in combination with genes lacking pyruvate kinase, pyruvate carboxylase and phosphoenolpyruvate carboxylase; and overexpressing phosphoenolpyruvate synthase and overbinding Oxalate acetate decarboxylase is expressed and malate dehydrogenase is overexpressed in combination with a gene deficient in pyruvate kinase, a gene for pyruvate carboxylase, and a gene for phosphoenolpyruvate carboxylase.

According to another preferred embodiment of the method and/or cell of the present invention, the cell comprises at least partially inactivated catabolic pathways of selected monosaccharides, disaccharides or oligosaccharides involved in/ Or necessary to produce the α-1,3 glycosylated form of fucose-α1,2-galactose-R as described herein.

According to another preferred embodiment of the method and/or cell of the present invention, 90 g/L or more of the α-1,3 saccharified form of Fuc is produced in the whole broth and/or supernatant -a1,2-Gal-R, and/or wherein in the whole broth and/or supernatant, the α-1,3 glycated form of Fuc-a1,2-Gal-R is based on the α-1,3 The saccharified form of Fuc-al,2-Gal- and its precursors, measured in total in the whole broth and/or supernatant, respectively, were at least 80% pure.

According to another embodiment of the method and/or cell of the present invention, the conditions that allow the production of the α-1,3 glycosylated form of Fuc-a1,2-Gal-R include the use of conditions comprising at least one precursor and/or acceptor The medium was used to produce the α-1,3 glycosylated form of Fuc-a1,2-Gal-R. Preferably, the culture medium comprises at least one selected from the group consisting of lactose, galactose, fucose, sialic acid, GlcNAc, GalNAc, lacto-N-disaccharide (LNB), N-acetyllactosamine (LacNAc). a precursor.

According to alternative and/or additional embodiments of the methods of the present invention, the conditions permitting the production of the α-1,3 glycated form of Fuc-a1,2-Gal-R comprise the addition of at least one precursor and/or acceptor supplement to the culture medium was used to generate the alpha-1,3 glycated form of Fuc-a1,2-Gal-R.

According to an alternative embodiment of the method of the present invention, the conditions allowing the production of the glycated form of Fuc-a1,2-Gal-R in α-1,3 comprise using a culture medium to cultivate one of the cells of the present invention to produce α-1,3 A glycosylated form of Fuc-a1,2-Gal-R, wherein the medium lacks any precursor and/or recipient for the production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R , and in combination with at least one precursor and/or acceptor feed further added to the medium for production of the alpha-1,3 glycated form of Fuc-a1,2-Gal-R.

In a preferred embodiment, the method for producing an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R as described herein comprises at least one of the following steps: i) using a method comprising: at least one precursor and/or a recipient medium; ii) adding at least one precursor and/or recipient feed to the medium in a reactor with a total reactor volume in the range of 250 mL (milliliters) to 10.000 m3 (cubic meters), preferably in a continuous manner, and preferably so that the final volume of the medium is not more than three times, preferably not more than twice, more preferably less than twice the amount of time before adding the precursor (iii) Add at least one precursor and/or acceptor feed to the medium in a reactor with a total reactor volume of 250 mL (milliliters) to 10.000 m ³ (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium is no more than three times, preferably no more than twice, more preferably less than twice The volume of the culture medium prior to addition of the precursor and/or recipient feed, and wherein preferably the pH of the precursor and/or recipient feed is set between 3 and 7, and wherein more Preferably the temperature of the precursor and/or acceptor feed is maintained between 20°C and 80°C; iv) by means of a feed solution, at 1 day, 2 days, 3 days, During the course of 4 days, 5 days, at least one precursor and/or acceptor feed is added to the medium in a continuous manner; v) by means of a feed solution, on days 1, 2, 3, 4 During the course of days, 5 days, at least one precursor and/or acceptor feed is added to the medium in a continuous manner, and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained between 20°C and 80°C; the method results in at least 50 g/L, preferably at least 75 g/L in the final culture, 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 An α-1,3 glycosylated form of Fuc-a1,2-Gal-R at a concentration of g/L.

In another and/or additional preferred embodiment, the method for producing the α-1,3 glycosylated form of Fuc-a1,2-Gal-R as described herein comprises at least one of the following steps : i) using a medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reaction volume in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters); ii) add at least one precursor and/or acceptor feed to the medium in one pulse or discontinuous (pulse) , wherein the volume of the reactor is in the range of 250 mL (milliliter) to 10.000 m ³ (cubic meter), preferably so that the final volume of the medium is not more than three times, preferably not more than twice, more preferably less than twice the volume of the medium prior to adding the precursor and/or acceptor feed pulse; iii) adding at least one precursor and/or acceptor feed to the medium in a bioreactor once Pulsed or discontinuous (pulsed) mode, wherein the reactor volume is in the range of 250 mL (milliliter) to 10.000 ^m3 (cubic meter), preferably such that the final volume of the medium is not more than three times, preferably is not more than twice, more preferably less than twice the volume of the medium before adding the precursor and/or acceptor feed pulse, and wherein preferably the precursor and/or acceptor feed pulse The pH is set between 3 and 7 and wherein preferably the temperature of the precursor and/or acceptor feed pulse is maintained between 20°C and 80°C; iv) for 5 minutes , 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, by feeding the solution in a discontinuous way (pulse) adding at least one precursor and/or acceptor feed to the medium; v) at 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, During the course of 2 days, 3 days, 4 days and 5 days, at least one precursor and/or acceptor feed is added to the medium in a discontinuous (pulsed) manner by means of the feeding solution, wherein the preferred The pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained between 20°C and 80°C; 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 100 g/L in culture 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L of Fuc-a1,2-Gal-R in an α-1,3 glycated form.

In a further preferred embodiment, the method for producing the α-1,3 glycosylated form of Fuc-a1,2-Gal-R as described herein comprises at least one of the following steps: i) using a medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor The volume is in the range of 250 mL (milliliter) to 10.000 m3 (cubic meter); ii) adding a lactose feed to the medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume , wherein the reactor volume is in the range of 250 mL (milliliter) to 10.000 m3 (cubic meter), preferably in a continuous form, and preferably so that the final volume of the medium is not more than three times, preferably not more than twice, more preferably less than twice the volume of the medium before adding the lactose feed; (iii) adding to the medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams per liter of initial reactor volume Lactose, wherein the reactor volume is in the range of 250 mL (milliliters) to 10.000 m3 (cubic meters), preferably in a continuous form, and preferably such that the final volume of the medium is no greater than three times, preferably Not more than twice, more preferably less than twice the volume of the medium prior to adding the lactose feed, and wherein preferably the pH of the lactose feed is set between 3 and 7, and wherein more Preferably the temperature of the lactose feed is maintained between 20°C and 80°C; iv) adding a lactose feed to the medium in a continuous manner during the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution; v) adding a lactose feed to the medium in a continuous manner during the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution, and wherein the lactose feed solution is The concentration 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, better 225 g/L, better 250 g/L, better 275 g/L, better 300 g/L, better 325 g/L, better 350 g /L, better is 375 g/L, better is 400 g/L, better is 450 g/L, better is 500 g/L, still better is, 550 g/L, better is 600 g /L; and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained between 20°C and 80°C ; The method results in 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 in the final volume of the culture g/L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L of Fuc-a1,2-Gal- in an α-1,3 glycated form R.

Preferably, by starting the culture at a concentration of at least 5 mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably at a concentration of > 300 mM Lactose is added to effect the lactose feed.

In another embodiment, lactose feeding is achieved by adding a concentration of lactose to the medium such that a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM, is obtained throughout the production phase of the culture.

In further embodiments of the methods described herein, the cells are cultured for at least about 60, 80, 100, or about 120 hours or in a continuous manner.

In a preferred embodiment, the carbon source, preferably sucrose, is provided in the medium for 3 or more days, preferably up to 7 days; and/or the medium is provided in a continuous manner per liter of initial culture volume at least 100, advantageously at least 105, more advantageously at least 110, even more advantageously at least 120 grams of sucrose, such that the volume of the final volume of the medium does not exceed three times the volume of the medium before culturing, advantageously no more than twice, more advantageously twice as small.

Preferably, when carrying out the method as described herein, a second phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the medium prior to adding lactose to the medium in the second phase. first phase.

In another preferred embodiment of the method of the present invention, a first stage of exponential cell growth is provided by adding a carbon-based matrix (preferably glucose or sucrose) to a medium comprising a precursor (preferably lactose), and then is the second stage, in which only a carbon-based substrate, preferably glucose or sucrose, is added to the medium.

In another preferred embodiment of the method of the present invention, the first stage of exponential cell growth is provided by adding a carbon-based substrate (preferably glucose or sucrose) to a medium comprising a precursor (preferably lactose) , followed by a second stage in which a carbon-based substrate (preferably glucose or sucrose) and a precursor (preferably lactose) are added to the medium.

In an alternative preferred embodiment, in the method as described herein, lactose has been added with the carbon-based substrate during the first stage of exponential growth.

In one embodiment, the method as described herein preferably comprises the step of isolating the α-1,3 glycosylated form of fucose-α1,2-galactose-R as described herein.

In a preferred embodiment, the methods described herein preferably include the step of isolating the alpha-1,3 glycated form of LNFP-I.

The term "isolated from the culture" refers to the harvest, collection or recovery of the α-1,3 glycosylated form of fucose-α1,2-galactose described herein from the cells or the medium in which they are grown -R or said alpha-1,3 glycosylated form of LNFP-1.

The α-1,3 glycosylated form of fucose-α1,2-galactose-R or the α-1,3 glycosylated form of LNFP-I as described herein can be obtained from cells therein in a general manner Aqueous medium for growth was isolated. If the α-1,3 glycated form of fucose-α1,2-galactose-R or the α-1,3 glycated form of LNFP-1 is still present in the production of α-1,3 glycated form of fucose - In the cells of α1,2-galactose-R or α-1,3 glycated form of LNFP-1, the α-1,3 glycated form of fucose-α1,2-galactose can be freed or extracted using the general method -R or alpha-1,3 glycosylated form of LNFP-1 out of cells, eg using high pH, heat shock, sonication, French press, homogenization ), enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergents, hydrolysis, cell destruction of ... The culture medium and/or cell extracts together and separately can then be further used to isolate the α-1,3 glycosylated form of fucose-α1,2-galactose-R or the α-1,3 glycosylated form of LNFP -1. This preferably involves clarifying the mixture containing the α-1,3 glycated form of fucose-α1,2-galactose-R or the α-1,3 glycated form of LNFP-1 to remove suspended particles and contamination compounds, especially cells, cellular components, insoluble metabolites and debris produced by culturing genetically modified cells. In this step, the identity of LNFP-1 containing the α-1,3-glycosylated form of fucose-α1,2-galactose-R or the α-1,3-glycosylated form of LNFP-1 can be clarified in a general manner. mixture. Preferably, the mixture containing the α-1,3 glycosylated form of fucose-α1,2-galactose-R or the α-1,3 glycosylated form of LNFP-1 is prepared by centrifugation, flocculation, Decant and/or filter to clarify.

The α-1,3 glycated form of fucose-a1,2-galactose-R or the α-1,3 glycated form of LNFP-I is converted from the α-1,3 glycated form of LNFP-I containing the Another step of separating out the mixture preferably comprises separating the α-1,3 glycated form of said fucose-a-1,2-galactose-R or the α-1,3 glycated form of LNFP-I The mixture, preferably after clarification, is substantially free of all proteins, as well as peptides, amino acids, RNA and DNA and any endotoxins and glycolipids that might interfere with subsequent separation steps. In this step, the α-1,3-glycosylated form of fucose-α1,2-galactose-R or the α-1,3-glycosylated form of LNFP-1 can be obtained in a general manner. The mixture removes protein and related impurities. Preferably, the protein is removed from the mixture containing the α-1,3 glycosylated form of fucose-α1,2-galactose-R or the α-1,3 glycosylated form of LNFP-1, Salts, by-products, colors, endotoxins and other related impurities by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated carbon or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, cutting Concurrent flow high performance filtration, tangential flow ultrafiltration, electrophoresis (eg using plate polyacrylamide or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands) , including, for example, DEAE-Sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorbent matrices), ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange, internal and external ligand attachment), hydrophobic interaction chromatography and/or gel filtration (i.e. size exclusion chromatography), especially by chromatography, more especially by ion exchange layers Chromatography or Hydrophobic Interaction Chromatography or Ligand Exchange Chromatography. In addition to size exclusion chromatography, proteins and related impurities are retained by the chromatography medium or selected membrane, the α-1,3 glycosylated forms of fucose-α1, 2-galactose-R or α-1 , 3-glycosylated forms of LNFP-1 are retained in LNFP-1 containing α-1,3-glycosylated forms of fucose-α1,2-galactose-R or α-1,3-glycosylated forms in the mixture.

In a further preferred embodiment, the methods described herein also provide for further purification of the α-1,3 glycosylated form of fucose-α1,2-galactose-R or α-1,3 from the mixture Glycosylated form of LNFP-1. Further purification of the α-1,3-glycosylated form of fucose-α1,2-galactose-R or the α-1,3-glycosylated form of LNFP-I can be performed, for example, by using (activated) charcoal Or carbon, nanofiltration, ultrafiltration or ion exchange to remove any residual DNA, protein, LPS, endotoxin or other impurities. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used. Another purification step is accomplished by crystallization, evaporation or precipitation of the product. Other purification steps are drying such as spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying or vacuum The α-1,3 glycosylated form of fucose-α1,2-galactose-R or the α-1,3 glycosylated form of LNFP-I was produced by vacuum roller drying.

In exemplary embodiments, the isolation and purification of the α-1,3 glycated form of fucose-α1,2-galactose-R or the α-1,3 glycated form of LNFP-I is performed in any order including: Steps performed in the process: a) contacting the culture or its clarified form with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da, ensuring retention of the alpha-1,3 glycosylated form of LNFP-1 produced and allowing at least a portion of protein, salt species, by-products, color and other related impurities pass through, b) subjecting the retentate from step a) to a diafiltration process, using said membrane, with an aqueous solution of inorganic electrolyte, and then, if necessary, pure water to remove excess electrolyte, c) and the fucose-α1,2-galactose-R or the α-1,3 glycosylated form enriched in the α-1,3 glycosylated form, respectively, is collected in the form of a salt from the cation of the electrolyte The retentate of LNFP-I.

In an alternative exemplary embodiment, the isolation and purification of the α-1,3 glycosylated form of fucose-α1,2-galactose-R or the α-1,3 glycosylated form of LNFP-I is in a process comprising steps in any order of: two membrane filtration steps of the culture or its clarified version using different membranes, wherein - one membrane has a molecular weight cut-off between about 300 and about 500 Daltons, And - the other membrane has a molecular weight cutoff between about 600 to about 800 Daltons.

In an alternative exemplary embodiment, the isolation and purification of the α-1,3 glycosylated form of fucose-α1,2-galactose-R or the α-1,3 glycosylated form of LNFP-I is Conducted in a process comprising steps in any order including the step of treating the culture or its clarified form with a strong cation exchange resin in the H+- form and a weak anion exchange resin in the free base form.

In an alternative exemplary embodiment, the isolation and purification of the α-1,3 glycosylated form of fucose-α1,2-galactose-R or the α-1,3 glycosylated form of LNFP-1 is as follows: Do it in the following way.

Cultures comprising the produced α-1,3 glycated form of fucose-α1,2-galactose-R or α-1,3 glycated form of LNFP-1, biomass, medium components and contaminants are suitable for use in The following purification steps: i) isolating biomass from the culture, ii) cation exchanger treatment for removal of positively charged materials, iii) anion exchanger treatment for removal of negatively charged materials, iv) a nanofiltration step and/or an electrodialysis step, There is provided a purified solution comprising the resulting alpha-1,3 glycated form of LNFP-1 with a purity greater than or equal to 80%. Optionally, the purified solution is dried by a method selected from the group consisting of spray drying, freeze drying, spray freeze drying, freeze spray drying, strip drying, belt drying, vacuum strip drying, vacuum belt drying One or more drying steps from the list of drying, drum drying, drum drying, vacuum drum drying, or vacuum drum drying.

In an alternative exemplary embodiment, the isolation and purification of the α-1,3 glycosylated form of fucose-α1,2-galactose-R or the α-1,3 glycosylated form of LNFP-I is Conducted in a process comprising steps in any order of: enzymatic treatment of the culture; biomass removal from the culture; ultrafiltration; nanofiltration; and a column chromatography step. Preferably, such column chromatography is a single column or a multi-column. More preferably, the column chromatography step is simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises i) at least 4 columns, at least one of which comprises a weak or strong cation exchange resin; and/or ii) four zones I, II, III and IV; and/or iii) an eluent comprising water; and/or iv) an operating temperature of 15 to 60 degrees Celsius. Preferably, the method further comprises a spray drying step.

In one embodiment, the present invention provides the resulting α-1,3 glycosylated form of fucose-α1,2-galactose-R or α-1,3 glycosylated form of LNFP-I, which is dried to a powder by a method selected from the group consisting of spray drying, freeze drying, spray freeze drying, freeze spray drying, strip drying, belt drying, vacuum strip drying, vacuum belt drying, drum drying, drum drying, vacuum One or more drying steps from the list of drum drying or vacuum drum drying, wherein the dried powder contains <15%-wt. water, preferably <10%-wt. water, more preferably <7% -wt. water, preferably < 5% -wt. water.

Another embodiment of the present invention provides a method and a cell, wherein the α-1,3 glycosylated form of fucose-α1,2-galactose-R described herein, preferably α-1, 3 Glycosylated forms of LNFP-1 are produced in and/or by fungi, yeast, bacteria, insects, plants, animals or protocells as described herein. The cells are selected from a list comprising bacteria, yeast, or fungi, or refer to plant, animal or protozoan cells. The latter bacteria preferably belong to the phylum Proteobacteria or Firmicutes or Cyanobacteria or Deinococcus-Thermus. The latter bacteria belonging to the phylum Proteobacteria preferably belong to the family Enterobacteriaceae , preferably to the species Escherichia coli. The latter bacteria preferably belong to any strain of the Escherichia coli species, such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12 K12), Escherichia coli Nissle. More specifically, the latter term refers to cultured E. coli strains, designated E. coli K12 strains, that are well adapted to the laboratory environment and that, unlike wild-type strains, have lost the ability to survive in the gut. Well-known examples of E. coli K12 strains are K12 wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Accordingly, the present invention particularly relates to a mutated and/or transformed E. coli cell or strain as hereinbefore described, wherein the E. coli strain is a K12 strain. More preferably, the E. coli K12 strain is E. coli MG1655. The latter bacteria belonging to the phylum Firmicutes preferably belong to Bacilli, preferably Lactobacilliales, the members of which are Lactobacillus lactis , Leuconostoc mesenteroides , or Preferred are members of the order Bacillales, for example from the genus Bacillus , eg Bacillus subtilis or B. amyloliquefaciens . The latter bacteria belonging to Actinobacteria preferably belong to the family Corynebacteriaceae, whose members are Corynebacterium glutamicum or C. afermentans , or preferably belong to the family Corynebacteriaceae. Streptomycetaceae, whose members are Streptomyces griseus or S. fradiae . The latter yeasts preferably belong to the phylum Ascomycota or Basidiomycota or Deuteromycota or Zygomycetes. The latter yeast preferably belongs to the genus Saccharomyces (its members such as Saccharomyces cerevisiae , S. bayanus , S. boulardii ), Zygosaccharomyces ( Zygosaccharomyces ), Pichia ( Pichia pastoris , P. anomala , P. kluyveri ), Kermagtella ( Komagataella ), Hansenula , Kluyveromyces (whose members are Kluyveromyces lactis , K. marxianus, Kluyveromyces thermotolerant K. thermotolerans ), Debaromyces , Yarrowia (eg, Yarrowia lipolytica ), or Starmerella ( For example, Starmerella bombicola ). The latter yeast is preferably selected from Pichia pastoris , Yarrowia lipolytica , Saccharomyces cerevisiae and Kluyveromyces lactis. The latter fungi preferably belong to the genera Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus. Plant cells include cells of both flowering and non-flowering plants, as well as algal cells such as Chlamydomonas, Chlorella, and the like. Preferably, the plants are tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, corn or corn plants. The latter animal cells are preferably derived from non-human mammals (eg, cattle, buffalo, pigs, sheep, mice, rats), birds (eg, chickens, ducks, ostriches, turkeys, pheasants), fish (eg, swordfish, salmon, tuna, bass, trout, catfish), invertebrates (eg, lobster, crab, shrimp, clams, oysters, mussels, sea urchins), reptiles (eg, snakes, alligators) , turtle), amphibians (eg, frogs) or insects (eg, Drosophila, nematodes), or genetically modified cell lines derived from human cells other than embryonic stem cells. Both human and non-human mammalian cells are preferably selected from a list comprising: epithelial cells such as mammary epithelial cells, embryonic kidney cells (eg, HEK293 or HEK 293T cells), fibroblasts, COS cells, Chinese hamster ovary cells (Chinese Hamster ovary cell, CHO cell), murine myeloma cells (eg, N20, SP2/0 or YB2/0 cells), NIH-3T3 cells, non-mammalian adult stem cells or cells derived therefrom, eg as described in WO21067641 . The latter insect cells are preferably derived from Spodoptera frugiperda (eg, sf9 or sf21 cells), silkworm ( Bombyx mori ), Mamestra brassicae , Trichoplus ia ni (eg . , BTI-TN-5B1-4 cells) or Drosophila melanogaster (eg, Drosophila S2 cells). The latter protozoan cells are preferably Leishmania tarentolae cells.

According to a preferred embodiment of the method and/or cell of the present invention, the α-1,3 glycosylated form of fucose-a1,2-galactose-R is produced in and/or by a cell that is viable Gram-negative bacteria, the live Gram-negative bacteria include poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, can Attenuated or disrupted synthesis of colonic acid, core oligosaccharides, osmoregulated perplasmic glucan (OPG), glucosylglycerol, glycan, and/or trehalose.

In a more preferred embodiment of the method and/or cell of the present invention, by synthesizing any one or more of poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen (Enterobacterial Common Antigen (ECA), cellulose, colonic acid, core oligosaccharide, osmoregulated perplasmic glucan (OPG), glucosylglycerol, glycan and/ Or one or more glycosyltransferases of trehalose are mutated to provide poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, Attenuated or disrupted synthesis of colonic acid, core oligosaccharides, osmoregulated perplasmic glucan (OPG), glucosylglycerol, glycan and/or trehalose , wherein said mutation provides deletion or lower performance of any of said glycosyltransferases. Described glycosyltransferase comprises the following glycosyltransferase gene encoding: poly-N-acetyl-D-glucosamine synthase subunit, UDP-N-acetylglucosamine-undec isopentenyl-phosphate N-acetylglucosamine phosphotransferase (UDP-N-acetylglucosamine-undecaprenyl-phosphate N-acetylglucosaminephosphotransferase), Fuc4NAc (4-acetylamino-4,6-dideoxy-D-galactose) transferase, UDP -N-acetyl-D-mannosaminuronic acid transferase (UDP-N-acetyl-D-mannosaminuronic acid transferase), encoding the following glycosyltransferase gene: cellulose synthase catalytic subunit, cellulose production Synthetic protein, colanic acid biosynthesis glucuronosyltransferase, colanic acid biosynthesis galactosyltransferase, colanic acid biosynthesis fucosyltransferase, UDP-glucose: undecyliso Pentenyl-phosphate glucose 1-phosphotransferase (UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase), putative (putative) colamellar acid biosynthesis glycosyltransferase, UDP-glucuronide:LPS ( HepIII) glycosyltransferase, ADP-heptose-LPD heptosyltransferase 2 (ADP-heptose-LPS heptosyltransferase 2), ADP-heptose:LPS heptosyltransferase 1 (ADP-heptose:LPS heptosyltransferase 1) , putative ADP-heptose:LPS heptosyltransferase 4, lipopolysaccharide core biosynthesis protein, UDP-glucose:(glucosyl)LPSα-1,2-glucosyltransferase (UDP-glucose:(glucosyl)LPSα -1,2-glucosyltransferase), UDP-glucose:(glucosyl)LPSα-1,3-glucosyltransferase, UDP-D-galactose:(glucosyl)lipopolysaccharide-1,6-D-galactosyl Transferase, lipopolysaccharide glucosyltransferase I, lipopolysaccharide core heptosyltransferase 3, β-1,6-galactofuranosyltransferase (β-1,6-galactofuranosyltransferase), undecyl isopentenyl -phosphate 4-deoxy-6-formamido-L-arabinose transferase (undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose tr ansferase), lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase (lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase), bactoprenol glucosyl transferase ), putative family 2 glycosyltransferases, osmoregulated perplasmic glucan (OPG) biosynthesis protein G, osmoregulated interstitial glucan biosynthesis protein H, glucosylglycerate phosphorylase ), liver sugar synthase, 1,4-α-glucan branching enzyme (1,4-α-glucan branching enzyme), 4-α-glucanotransferase (4-α-glucanotransferase) and trehalose- 6-phosphate synthase. In an exemplary embodiment, the cell line is mutated to comprise one or more of the following glycosyltransferases: pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ, wcaL , waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA, and yaiP, where The mutation provides deletion or lower performance of any of the glycosyltransferases.

In the method and/or cell replacement and/or additional preferred embodiments, by overexpression of genes encoding carbon storage regulatory proteins, deletion of genes encoding Na+/H+ antitransporters, and/or deletion of histidine-encoding sensing The gene for the kinase provides attenuated or disrupted synthesis of the poly-N-acetyl-glucosamine (PNAG).

Microorganisms or cells as used herein can be in monosaccharides, disaccharides, oligosaccharides, polysaccharides, polysaccharides, polyols, glycerol, including molasses, corn steep liquor, meringues, trypsin, yeast extracts, or mixtures thereof such as, for example, mixed feedstocks A complex medium, preferably a mixed monosaccharide raw material, such as hydrolyzed sucrose, is grown on the main carbon source. The term "primary" refers to the most important carbon source for the biological product of interest, biomass formation, carbon dioxide and/or by-product formation (eg acids and/or alcohols such as acetate, lactate and/or ethanol), i.e. all 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99% of the required carbon comes from the above carbon sources. In one embodiment of the present invention, the carbon source is the only carbon source of the organism, ie 100% of all required carbons come from the above-mentioned carbon source. Common major carbon sources include, but are not limited to, glucose, glycerol, fructose, maltose, lactose, arabinose, maltooligosaccharides, maltotriose, sorbitol, xylose, rhamnose, sucrose, galactose, mannose Sugar, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate and pyruvate. The term complex medium refers to a medium whose exact composition is not defined. Examples are molasses, corn steep liquor, egg whites, trypsin or yeast extracts. As used herein, a precursor as defined herein cannot be used as a carbon source to produce the α-1,3 glycosylated form of fucose-α1,2-galactose-R.

In a further preferred embodiment, the microorganisms or cells described herein use a split metabolism with a production pathway and a biomass pathway, as described in WO2012/007481, which is incorporated herein by reference. For example, the organism can be modified by altering a gene selected from the group consisting of a phosphoglucose isomerase gene, a phosphofructokinase gene, a fructose-6-phosphoronidase gene, a fructose isomerase gene, and/or a fructose:PEP phosphotransferase gene Genetically modified to accumulate fructose-6-phosphate.

In a third aspect, the present invention provides that the present invention provides a metabolically engineered cell as described herein for the production of an alpha-1,3 glycated form of fucose-a1,2-galactose-R, Preferred is the use of the alpha-1,3 glycated form of LNFP-I. In a preferred embodiment of the third aspect, the metabolically engineered cells described herein are used to produce (i) Gal-a1,3-(Fuc-a1,2)-Gal-R, preferably Gal -a1,3-(Fuc-a1,2)-Gal-b1,3-R, more preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, even more Preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-R, even more preferably Gal-a1,3-(Fuc-a1,2)-Gal- b1,3-GlcNAc-b1,3-Gal-R, preferably Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc or (ii) GalNAc-a1,3-(Fuc-a1,2)-Gal-R, preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-R, more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-R, even more preferably GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc -b1,3-R, even better GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-R, most preferably GalNAc-a1,3- (Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc. In a more preferred embodiment of the third aspect, the metabolically engineered cells described herein are used to generate the constructs of the blood group antigen (HBGA) system disclosed herein. In a more preferred embodiment of the third aspect, the metabolically engineered cells described herein are used to produce the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is Gal-a1,3-( Fuc-a1,2)-Gal-b1,4-Glc, wherein glucose can optionally be fucosylated (preferably a1,3-fucosylated) as disclosed herein. In another preferred embodiment of the third aspect, the metabolically engineered cells described herein are used to produce α-1,3GalNAc-modified or an α-1,3galactose-modified Fuc-a1,2- Gal-GlcNAc, wherein the galactose in Fuc-a1,2-Gal-GlcNAc binds to GlcNAc through a β-1,3 or β-1,4 bond, as disclosed herein.

In order to identify the α-1,3 glycosylated form of fucose-a1,2-galactose-R or the α-1,3 glycosylated form of LNFP-1 described herein, known in the art standard methods to identify monomeric building blocks (eg, monosaccharide or glycan unit composition), anomeric configuration of side chains, presence and location of substituent groups, degree of polymerization/molecular weight and linking modes such as methyl analysis, reductive cleavage, hydrolysis, gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), Electrospray Ionization-Mass Spectrometry (ESI-MS), High Performance Liquid Chromatography (HPLC) with UV or Refractive Index Detection, High Performance Anion Exchange Chromatography with Pulsed Current Detection (HPAEC-PAD), Capillary Electrophoresis (capillary electrophoresis, CE), far-infrared light/Raman spectroscopy and nuclear magnetic resonance (NMR) spectroscopic techniques. The crystal structure can be resolved using solid-state NMR, Fourier-far-infrared spectroscopy (FT-IR), and wide-angle X-ray scattering. The degree of polymerization (DP), DP distribution and polydispersity can be determined using, for example, a viscometer and high performance liquid chromatography. In order to identify the monomeric composition of saccharides, for example, acid-catalyzed hydrolysis, high performance liquid chromatography or gas-liquid chromatography (after conversion to sugar alcohol acetate) can be used. To determine the glycosidic bond, saccharides are methylated with methyl iodide and strong base in DMSO, hydrolyzed, reduced to partially methylated sugar alcohols, acetylated to methylated sugar alcohol acetates, and Analysis was performed by gas liquid chromatography (GLC/MS) coupled to mass spectrometry. To determine the sequence of oligosaccharides, partial depolymerization is performed using acids or enzymes to determine the structure. In order to identify the mutator configuration, the oligosaccharide is subjected to enzymatic analysis, i.e., exposed to an enzyme specific for a particular form of linkage, for example, beta-galactosidase or alpha-glucosidase, and Products including the resulting α-1,3 glycosylated form of fucose-al,2-galactose-R, preferably the α-1,3 glycosylated form of LNFPI, can be analyzed using NMR.

In some embodiments, an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein said R is a monosaccharide, disaccharide or oligosaccharide, produced as described herein, is Incorporated into food (such as human food or feed), dietary supplements, pharmaceutical ingredients, cosmetic ingredients or pharmaceuticals. In some embodiments, the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein said R is a monosaccharide, disaccharide or oligosaccharide, is combined with one or more suitable for food, feed, Composition of dietary supplements, pharmaceutical ingredients, cosmetic ingredients or pharmaceutical ingredients.

In some embodiments, the dietary supplement includes at least one probiotic ingredient and/or at least one prebiotic ingredient.

A "prebiotic" is a substance that promotes the growth of microorganisms beneficial to the host, particularly the gastrointestinal tract. In some embodiments, the dietary supplement provides a variety of xenobiotics, including Fuc-a1,2-Gal-R in an alpha-1,3 glycated form, wherein said R is a monosaccharide, disaccharide, or oligosaccharide, and which Produced and/or purified by the methods disclosed in this specification to promote the growth of one or more beneficial microorganisms. Examples of xenobiotic components used in dietary supplements include other xenobiotic molecules such as HMOs and plant polysaccharides such as inulin, pectin, beta-glucans and xylo-oligosaccharides. "Probiotic" products typically contain live microorganisms that replace or add to the gastrointestinal microbiota to provide benefit to the recipient. Examples of such microorganisms include Lactobacillus species (eg, L. acidophilus and L. bulgaricus), Bifidobacterium species (eg, Bifidobacterium animalis ( B. animalis), B. longum and B. infantis (eg Bi-26)) and Saccharomyces boulardii. In some embodiments, oligosaccharides produced and/or purified by the methods of the present specification are administered orally in conjunction with such microorganisms.

Examples of other ingredients of dietary supplements include disaccharides (eg, lactose), monosaccharides (eg, glucose and galactose), thickeners (eg, acacia), acidity regulators (eg, trisodium citrate), water, skim milk and flavorings.

In some embodiments, an alpha-1,3 glycated form of Fuc-a1,2-Gal-R produced as described herein, wherein said R is a monosaccharide, disaccharide or oligosaccharide, is added to human infant food (eg, infant formula). Infant formula is generally a food made to feed infants as a complete or partial replacement for human breast milk. In some embodiments, infant formula is sold in powder form and fed to infants mixed with water in a bottle or in a cup. The ingredients of infant formula are often designed to roughly mimic human breast milk. In some embodiments, the α-1,3 glycosylated form of Fuc-a1,2-Gal-R, wherein the R is a monosaccharide, disaccharide or oligosaccharide produced by the process in this specification, is included in infants and young children Formulated to provide nutritional benefits similar to those provided by oligosaccharides in human breast milk. In some embodiments, an alpha-1,3 glycated form of Fuc-al,2-Gal-R, wherein said R is a monosaccharide, disaccharide or oligosaccharide, is mixed with one or more ingredients of an infant formula. Examples of infant formula ingredients include skim milk, carbohydrate sources (eg lactose), protein sources (eg whey protein concentrate and casein), fat sources (eg vegetable oils such as palm oil, high oleic safflower oil, rapeseed oil) , coconut oil and/or sunflower oil; and fish oil), vitamins (eg, vitamins A, Bb, Bi2, C, and D), minerals (eg, potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and may include human milk oligosaccharides (HMO). For example, such HMOs can include DiFL, lactose-N-triose II, LNT, LNnT, lactose-N-fucopentose I, lactose-N-neofucopentose, lactose-N-fucopentose II , lactose-N-fucose III, lactose-N-fucose V, lactose-N-neofucose pentose V, lactose-N-difucose hexose I, lactose-N-di Fucose hexose II, 6'-galactosyllactose, 3'-galactosyllactose, lactose-N-hexose and lactose-N-neohexose.

In some embodiments, the one or more infant formula ingredients include skim milk, carbohydrate sources, protein sources, fat sources, and/or vitamins and minerals.

In some embodiments, the one or more infant formula ingredients may include lactose, whey protein concentrate, and/or high oleic safflower oil.

In some embodiments, the alpha-1,3 glycated form of Fuc-a1,2-Gal-R in infant formula, wherein said R is a monosaccharide, disaccharide or oligosaccharide at a concentration about the same as in human breast milk The oligosaccharides are generally present in the same concentration. In some embodiments the concentration of galactosylated oligosaccharides in the infant formula is about the same as the concentration of oligosaccharides typically present in human breast milk.

In some embodiments, the alpha-1,3 glycated form of Fuc-a1,2-Gal-R, wherein the R is a monosaccharide, disaccharide or oligosaccharide, is incorporated into a feed product, wherein the feed is selected from Self-include the following list: pet food, animal milk replacer, veterinary product, post-weaning feed or creep feed.

Each embodiment disclosed in the context of one aspect of the invention is also disclosed in the context of all other aspects of the invention, unless expressly stated otherwise.

Throughout this application, unless expressly stated otherwise, the article "a (a or an)" is preferably replaced with "at least two", more preferably "at least three", more preferably "at least four" , the better can be replaced with "at least five", the better can be replaced with "at least six", and the best can be replaced with "at least two".

Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature and laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry, and hybridization described above and below, as used herein are those known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. In general, purification steps are carried out according to the manufacturer's instructions.

Further advantages come from specific embodiments and examples. It goes without saying that the features mentioned above and those explained below can be used not only in the respectively indicated combination, but also in other combinations or alone, without departing from the scope of the present invention.

The present invention pertains to the following specific embodiments:

1. A method of producing an α-1,3 glycosylated form of fucose-α-1,2-galactose-R (fucose-alpha-1,2-galactose-R) by a cell, preferably a single cell. , Fuc-a1,2-Gal-R), wherein the α-1,3 saccharification occurs between fucose-α-1,2-galactose-R (Fuc-a1,2-Gal-R) terminal "fucose-a1,2-galactose"-group, wherein the method comprises the following steps: i. Provide the ability to synthesize Fuc-a1,2-Gal-R, express an alpha-1,3-glycosyltransferase (alpha-1,3-glycosyltransferase), and have the ability to synthesize the alpha-1,3-sugar A nucleotide-sugar capable cell that is a donor of syltransferase, and ii. After allowing synthesis of the Fuc-a1,2-Gal-R, expression of the α-1,3-glycosyltransferase, synthesis of the nucleotide-sugar and synthesis of the α-1,3 glycosylated form of Fuc-a1 , the cells were cultured under the condition of 2-Gal-R, iii. Preferably the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is isolated from the culture.

2. The method of embodiment 1, wherein the galactose (Gal) residue in the Fuc-a1,2-Gal-R is via a β-1,3 or a β-1,4 glycosidic bond (glycosidic bond). linkage) combined with R.

3. The method of embodiment 1 or 2, wherein the R comprises a monosaccharide (monosaccharide), a disaccharide (disaccharide), an oligosaccharide (oligosaccharide), a peptide, a protein, a glycopeptide, A glycoprotein, a lipid or a glycolipid.

4. The method of any one of embodiments 1 to 3, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-R, preferably the Fuc-a1,2 -Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-R.

5. The method of embodiment 4, wherein the N-acetylglucosamine (GlcNAc) residue in the Fuc-a1,2-Gal-b1,3-GlcNAc-R passes through a β-1, 3 or a β-1,4 glycosidic bond is combined with R.

6. The method of embodiment 4 or 5, wherein the Fuc-a1, 2-Gal-R is Fuc-a1, 2-Gal-b1, 3-GlcNAc-b1, 3-R, preferably, wherein the Fuc -a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R, more preferably, wherein Fuc-a1,2-Gal-R is milk-N- Fucopentaose I (lacto-N-fucopentaose I, LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc).

7. The method of any one of embodiments 1 to 3, wherein the Fuc-a1, 2-Gal-R is Fuc-a1, 2-Gal-b1, 4-R, preferably, wherein the Fuc-a1 , 2-Gal-R is Fuc-a1, 2-Gal-b1, 4-Glc, as required, wherein the Fuc-a1, 2-Gal-R is Fuc-a1, 2-Gal-b1, 4- (Fuc-a1,3)-Glc.

8. The method of any one of embodiments 1 to 7, wherein the α-1,3 glycosylated form of Fuc-a1,2-Gal-R is a structure of the histo blood group antigen (HBGA) system .

9. The method of any one of embodiments 1 to 8, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase (alpha-1,3-galactosyltransferase), It is characterized by the transfer of galactose (Gal) residues from UDP-galactose (UDP-Gal) to fucose-a1,2-galactose-R (Fuc-a1,2- Gal-R) is a glycosyltransferase capable of the terminal "fucose-al,2-galactose"-group.

10. The method of any one of embodiments 1 to 6, 8 or 9, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase that is a A glycosyltransferase capable of transferring a lactose (Gal) residue from UDP-Gal to the terminal "fucose-al,2-galactose"-group of LNFP-I.

11. The method of any one of embodiments 1 to 3, 7, 9 or 10, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase having A monosaccharide capable of transferring a galactose (Gal) residue from UDP-Gal to the terminal "fucose-a1,2-galactose"-group of Fuc-a1,2-Gal-b1,4-Glc Glucose residues in the Fuc-a1,2-Gal-b1,4-Glc are fucosylated, preferably α-1,3-fucosylated, if necessary.

12. The method of any one of embodiments 1 to 6 or 8 to 10, wherein the Fuc-a1,2-Gal-R of the α-1,3 glycated form is an α-1,3 glycated form of milk- N-fucopentaose I (LNFP-I), which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal -a1,3-LNFP-I), the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase, and the nucleotide-sugar is UDP-galactose (UDP- galactose, UDP-Gal).

13. The method of any one of embodiments 1 to 3, 7 or 9 to 11, wherein the Fuc-a1,2-Gal-R of the α-1,3 glycated form is of an α-1,3 glycated form Fuc-a1,2-Gal-b1,4-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, as needed, an alpha-1,3 glycated Form Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1 ,3)-Glc, the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase, and the nucleotide-sugar is UDP-galactose (UDP-Gal).

14. The method of any one of embodiments 9 to 13, wherein the α-1,3-galactosyltransferase has a PFAM PF03414 domain, and a. Include the motif with SEQ ID NO: 01 YX[FHMQT]XAXX[ACG][ACG], where X can be any amino acid residue, or b. Include the motif YXQXCXX[ACG][ACG] with SEQ ID NO: 02, where X can be any amino acid residue, or c. Include serial identification numbers such as: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, a polypeptide sequence of any of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, or d. is the serial identification number: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, A functional homolog, variant or derivative of any of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 (derivative) with and with serial identification numbers: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 of any of the alpha-1,3-galactosyltransferase polypeptides At least 80% of the overall sequence similarity of the full length, and to the end of fucose-a1,2-galactose-R The sugar-a1,2-galactose"-group has a-1,3-galactosyltransferase activity, or e. is a functional fragment, including from SEQ ID NO: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37. An oligopeptide sequence of 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues, and to fucose-a1,2-galactose-R (Fuc-a1,2 The terminal "fucose-al,2-galactose"-group of -Gal-R) has a-1,3-galactosyltransferase activity.

15. The method of any one of embodiments 1 to 8, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase (alpha-1,3-N-acetylgalactosamine transferase); 3-N-acetylgalactosaminyltransferase), which has the ability to transfer an N-acetylgalactosamine (N-acetylgalactosamine, GalNAc) residue from UDP-N-acetylgalactosamine (UDP-N-acetylgalactosamine, UDP-GalNAc) A glycosyltransferase capable of to the terminal "fucose-al,2-galactose"-group of fucose-al,2-galactose-R (Fuc-al,2-Gal-R).

16. The method of any one of embodiments 1 to 6, 8 or 15, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase, It is a terminal "fucose-a1,2-galactose" with the transfer of an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to LNFP-I "-group capacity of a glycosyltransferase.

17. The method of any one of embodiments 1 to 3, 7 or 15, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase, It is a terminal "fucose-al,2-galactose" with the transfer of an N-acetylgalactosamine (GalNAc) residue from UDP-GalNAc to Fuc-a1,2-Gal-b1,4-Glc A glycosyltransferase with the ability of a group, as required, the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated, preferably α-1 , 3-fucosylated.

18. The method of any one of embodiments 1 to 6, 8, 15 or 16, wherein the Fuc-a1,2-Gal-R of the α-1,3 glycated form is of an α-1,3 glycated form Lacto-N-fucopentaose I (LNFP-I) which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-a1,3-LNFP-I), the α-1,3-glycosyltransferase is an α-1,3-N-acetylgalactosamine transferase, and the nucleotide-sugar is UDP -N-Acetylgalactosamine (UDP-GalNAc).

19. The method of any one of embodiments 1 to 3, 7, 15 or 17, wherein the Fuc-a1,2-Gal-R of the α-1,3 glycated form is of an α-1,3 glycated form Fuc-a1,2-Gal-b1,4-Glc, which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc (alpha-tetrasaccharide or A- A-tetrasaccharide (A-tetrasaccharide), as required - Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc in α-1,3 glycosylated form, which is GalNAc-a1, 3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, the α-1,3-glycosyltransferase is an α-1,3-N-acetyl half Lactosamine transferase, and the nucleotide-sugar is UDP-N-acetylgalactosamine (UDP-GalNAc).

20. The method of any one of embodiments 15 to 19, wherein the α-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain, and a. Include the motif YX[ACIL]XGXX[ACG][ACG] with SEQ ID NO: 38, where X can be any amino acid residue, or b. Include the motif YX[AG]XAXX[ACG][ACG] with SEQ ID NO: 39, where X can be any amino acid residue, or c. Include such as serial identification numbers: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, a polypeptide sequence of any one of 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, or d. is the serial identification number: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, a functional homologue, variant or derivative of any of 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, With and with serial identification numbers: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 of the α-1,3-N-acetylgalactosyltransferase ( a-1,3-N-acetylgalactosyltransferase) polypeptides have at least 80% overall sequence similarity over the full length of any of the polypeptides and are The terminal "fucose-a1,2-galactose" group of R) has a-1,3-N-acetylgalactosyltransferase activity, or e. is a functional fragment, including from SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102. An oligopeptide sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues, and to fucose-a1,2-galactose-R The terminal terminal "fucose-al,2-galactose" group of (Fuc-a1,2-Gal-R) has a-1,3-N-acetylgalactosyltransferase activity.

21. The method of any one of embodiments 6, 10, 12, 14, 16, 18 or 20, wherein fucose is transferred from GDP-fucose to milk by the action of a glycosyltransferase. -The terminal galactose residue of lacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), the LNFP-I is synthesized in the cell , the glycosyltransferase is: a. an alpha-1,2-fucosyltransferase (alpha-1,2-fucosyltransferase), selected from including from Brachyspira pilosicoli with UniProt ID A0A2N5RQ26, Dysgonomonas mossii with UniProt ID F8X274 , a list of the polypeptides of Dechlorosoma suillum with UniProt ID G8QLF4, Desulfovibrio alaskensis with UniProt ID Q316B5 and Polaribacter vadi with UniProt ID A0A1B8TNT0, or b. Polypeptide from Brachyspira trichomes (UniProt ID A0A2N5RQ26) with α-1,2-fucosyltransferase activity on terminal galactose residues of LNT, from D. mossii (UniProt ID F8X274) Any of the polypeptides from D. suillum (UniProt ID G8QLF4), the polypeptides from D. alaskensis (UniProt ID Q316B5) and the polypeptides from P. vadi (UniProt ID A0A1B8TNT0) a functional fragment of , or c. From Brachyspira trichomes with UniProt ID A0A2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID A0A1B8TNT0 A functional homologue, variant or derivative of any of the peptides with B. trichomoniasis from UniProt ID A0A2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, respectively, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID A0A1B8TNT0 have at least 80% overall sequence similarity over the full length of either of the polypeptides, and to the end of lacto-N-saccharide (LNT) The galactose residue has alpha-1,2-fucosyltransferase activity, or d. a polypeptide comprises an amino acid sequence, or is composed of an amino acid sequence, the amino acid sequence has a polypeptide from Brachyspira trichomes (UniProt ID A0A2N5RQ26), from D. mossii Polypeptide from (UniProt ID F8X274), Polypeptide from D. suillum (UniProt ID G8QLF4), Polypeptide from D. alaskensis (UniProt ID Q316B5) and Polypeptide from P. vadi (UniProt ID A0A1B8TNT0) The full-length amino acid sequence of any of the peptides has at least 80% sequence similarity and has alpha-1,2-fucosyltransferase activity on the terminal galactose residue of the LNT.

22. The method of any one of embodiments 1 to 21, wherein the cell is modified in the expression or activity of a glycosyltransferase.

23. The method of any one of the preceding embodiments, wherein the cell expresses a membrane transporter protein or a polypeptide having transport activity, thereby transporting the compound through the outer membrane of the cell wall .

24. The method of embodiment 23, wherein the membrane transporter protein (membrane transporter protein) or the polypeptide having transport activity is selected from a list comprising a transport protein (porter), a P-P-bond-hydrolysis-driven transporter (P-P-bond-hydrolysis-driven transporter), b-barrel porins, auxiliary transport proteins, putative transport proteins and phosphate transfer-driven group translocations protein (phosphotransfer-driven group translocator), Preferably, the transporter includes MFS transporter, sugar efflux transporter and siderophore exporters, or Preferably, the P-P-bond-hydrolysis-driven transporter includes ABC transporter and chelatin exporter.

25. The method of any one of embodiments 23 or 24, wherein the membrane transporter or polypeptide with transport activity controls the α-1,3 glycosylated form of Fuc-a1,2-Gal-R and/or Flow of one or more precursors and/or acceptors on the outer membrane of the cell wall for the production of the alpha-1,3 glycated form of Fuc-a1,2-Gal-R.

26. The method of any one of embodiments 23 to 25, wherein the membrane transporter or polypeptide with transport activity provides improved improvement of the α-1,3 glycosylated form of Fuc-a1,2-Gal-R generation and/or activated and/or enhanced outflow.

27. The method of any of the preceding embodiments, wherein the cell is a metabolically engineered cell.

28. The method of embodiment 27, wherein the cell line is modified with a gene expression module, characterized in that the expression from any of the expression modules is constitutive, or by a natural inducer ( natural inducer).

29. The method of any one of embodiments 27 or 28, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein.

30. The medium of any one of embodiments 27-29, wherein the cell comprises a modification for reducing the production of acetic acid.

31. The method of any one of embodiments 27 to 29, wherein the cell comprises a lower or reduced expression and/or eliminated, impaired, reduced or delayed activity of any or more proteins , the one or more proteins include beta-galactosidase (beta-galactosidase), galactoside O-acetyltransferase (galactoside O-acetyltransferase), N-acetylglucosamine-6-phosphate deethyl N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotides Monophosphatase (ribonucleotide monophosphatase), EIICBA-Nag, UDP-glucose: undecaprenyl-phosphate glucose-1-phosphate transferase (UDP-glucose: undecaprenyl-phosphate glucose-1-phosphate transferase), L-fucus Glycokinase (L-fuculokinase), L-fucose isomerase (L-fucose isomerase), N-acetylneuraminate lyase (N-acetylneuraminate lyase), N-acetylmannosamine kinase (N- acetylmannosamine kinase), N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose -1-phosphate uridyltransferase (galactose-1-phosphate uridylyltransferase), glucose-1-phosphate adenylyltransferase (glucose-1-phosphate adenylyltransferase), glucose-1-phosphatase (glucose-1-phosphatase) , ATP-dependent 6-phosphofructokinase isozyme 1 (ATP-dependent 6-phosphofructokinase isozyme 1), ATP-dependent 6-phosphofructokinase isozyme 2 (ATP-dependent 6-phosphofructokinase isozyme 2) , glucose-6-phosphate isomerase (glucose-6-phosphate isomerase) ate isomerase), aerobic respiration control protein (aerobic respiration control protein), transcriptional repressor protein IclR (transcriptional repressor IclR), lon protease (lon protease), glucose-specific translocation phosphotransferase IIBC component ptsG (glucose-specific translocating phosphotransferase) enzyme IIBC component ptsG), glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX (glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX), enzyme IIAGlc, β-glucoside specific PTS enzyme II (beta -glucoside specific PTS enzyme II), fructose-specific PTS multiphosphoryl transfer protein FruA and FruB (fructose-specific PTS multiphosphoryl transfer protein FruA and FruB), ethanol dehydrogenase (ethanol dehydrogenase), aldehyde dehydrogenase (aldehyde dehydrogenase), Pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvate decarboxylase.

32. The method of any preceding embodiment, wherein the cell has the ability to produce phosphoenolpyruvate (PEP).

33. The method of any of the preceding embodiments, wherein the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP).

34. The method of any one of the preceding embodiments, wherein the cell comprises an at least partially inactivated catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide that participates in and/or is affected by This production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is required.

35. The method of any preceding embodiment, wherein the cell is resistant to lactose killing when grown in an environment in which lactose is combined with one or more other carbon sources.

36. The method of any one of the preceding embodiments, wherein the cell produces 90 g/L or more of the alpha-1,3 saccharified form of Fuc in whole broth and/or supernatant -a1,2-Gal-R, and/or wherein in the whole broth and/or supernatant, the α-1,3 glycated form of Fuc-a1,2-Gal-R is based on the α-1,3 The saccharified form of Fuc-al,2-Gal-R and its precursors, measured in total in the whole broth and/or supernatant, respectively, were at least 80% pure.

37. The method of any one of the preceding embodiments, wherein the cells are stably cultured in a culture medium.

38. The method of any one of the preceding embodiments, wherein the condition comprises: (i) using a medium comprising at least one precursor and/or acceptor for the production of the α-1,3 glycosylated form of Fuc-a1,2-Gal-R, and/or (ii) adding to the medium at least one precursor and/or acceptor feed for the production of the α-1,3 glycated form of Fuc-a1,2-Gal-R.

39. The method of any one of the preceding embodiments, comprising at least one of the following steps: i) using a culture medium comprising at least one precursor and/or recipient; ii) Add at least one precursor and/or acceptor feed to the medium in a reactor with a total reactor volume in the range of 250 mL (milliliters) to 10.000 m3 (cubic meters), preferably with Continuously, and preferably such that the final volume of the medium is no greater than three times, preferably no greater than two times, more preferably less than two times the volume of the medium prior to addition of the precursor and/or recipient feed volume; iii) Add at least one precursor and/or acceptor feed to the medium in a reactor with a total reactor volume in the range of 250 mL (milliliters) to 10.000 m3 (cubic meters), preferably with Continuously, and preferably such that the final volume of the medium is no greater than three times, preferably no greater than two times, more preferably less than two times the volume of the medium prior to addition of the precursor and/or recipient feed volume, and wherein preferably the pH of the precursor and/or acceptor feed is set between 3 and 7, and wherein preferably the temperature of the precursor and/or acceptor feed is maintained at between 20°C and 80°C; iv) adding at least one precursor and/or acceptor feed to the medium in a continuous manner during the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution; v) adding at least one precursor and/or acceptor feed to the medium in a continuous manner during the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution, and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained between 20°C and 80°C; The method results in 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 in the final culture L, more preferably at least 150 g/L, more preferably at least 175 g/L, more preferably at least 200 g/L of Fuc-a1,2-Gal-R in an α-1,3 glycated form.

40. The method of any one of embodiments 1 to 38, comprising at least one of the following steps: (i) using a culture medium comprising at least 50, more preferably at least 75 per liter of initial reactor volume , more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose, wherein the reactor volume is in the range of 250 mL (milliliter) to 10.000 m ³ (cubic meter); (ii) for The medium is supplemented with a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the The reactor volume is in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters), preferably in a continuous format, and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than twice the volume of the medium before adding the lactose feed; (iii) adding a lactose feed to the medium comprising at least 50 per liter of initial reactor volume, more preferably At least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose, wherein the reactor volume is in the range of 250 mL (milliliters) to 10.000 m3 (cubic meters), preferably In a continuous format, and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before the addition of the lactose feed, and wherein more Preferably the pH of the lactose feed is set between 3 and 7, and wherein preferably the temperature of the lactose feed is maintained between 20°C and 80°C; (iv) by By means of a feed solution, a lactose feed was added to the medium in a continuous manner during the course of 1 day, 2 days, 3 days, 4 days, 5 days; (v) by means of a feed solution, During the course of 1 day, 2 days, 3 days, 4 days, 5 days, a lactose feed is added to the medium in a continuous manner, and wherein the concentration of the lactose feed solution is 50 g/L, preferably 75 g/L g/L, better is 100 g/L, better is 125 g/L, better is 150 g/L, better is 175 g/L, better is 200 g/L, better is 225 g/L L, better is 250 g/L, better is 275 g/L, better is 300 g/L, better is 325 g/L, better is 350 g/L, better is 375 g/L, more preferably 400 g/L, more preferably 450 g/L, more preferably 500 g/L, still more preferably 550 g/L, most preferably 600 g/L; and preferably the feed The pH of the solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained between 20°C and 80°C; the method results in a final volume in the culture has at least 50 g/L, preferably at least 7 5 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 preferred is an alpha-1,3 glycated form of Fuc-a1,2-Gal-R at a concentration of at least 200 g/L.

41. The method of embodiment 39, wherein the lactose feed is started by culturing at a concentration of at least 5 mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM concentration, more preferably lactose is added at a concentration of >300 mM.

42. The method of any one of embodiments 39 or 40, wherein the lactose feed is achieved by adding lactose to the culture at a concentration such that at least 5 mM is obtained throughout the production stage of the culture, Lactose concentrations of 10 mM or 30 mM are preferred.

43. The method of any one of the preceding embodiments, wherein the cells are cultured for at least about 60, 80, 100, or about 120 hours or in a continuous manner.

44. The method of any one of the preceding embodiments, wherein the cell is in a medium comprising a carbon source comprising monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol, including molasses, corn steep liquor Steep liquor), peptone (peptone), tryptone (tryptone) or a complex medium of yeast extract (yeast extract); preferably, wherein the carbon source is selected from the group consisting of glucose, glycerol, fructose, sucrose, maltose, lactose , arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose , methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn steep liquor, high-fructose syrup, acetate, citrate, lactate and List of pyruvates.

45. The method of any one of the preceding embodiments, wherein the substratum comprises at least one precursor selected from the group consisting of lactose, galactose, fucose, sialic acid, GlcNAc, GalNAc, lacto-N-disaccharide ( lacto-N-biose, LNB), N-acetyllactosamine (N-acetyllactosamine, LacNAc) group.

46. The method of any one of the preceding embodiments, wherein the exponential type is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the medium comprising a precursor, preferably lactose A first stage of cell growth, followed by a second stage, in which only a one-carbon substrate, preferably glucose or sucrose, is added to the medium.

47. The method of any one of embodiments 1 to 45, wherein the first step for exponential cell growth is provided by adding a carbon substrate, preferably glucose or sucrose, to the medium comprising a precursor, preferably lactose. A stage is followed by a second stage in which a one-carbon substrate, preferably glucose or sucrose, and a precursor, preferably lactose, are added to the medium.

48. The method of any of the preceding embodiments, wherein the cell produces a charged, preferably sialylated and/or neutral disaccharide and oligosaccharide mixture comprising an alpha-1,3 saccharide Form Fuc-a1,2-Gal-R.

49. The method of any one of the preceding embodiments, wherein the cell produces a charged, preferably sialylated and/or neutral oligosaccharide mixture comprising an α-1,3 glycosylated form of Fuc-a1, 2-Gal-R.

50. A metabolically engineered cell for the production of fucose-alpha-1,2-galactose-R (fucose-alpha-1,2-galactose-R, Fuc-a1 ,2-Gal-R), wherein the α-1,3 glycation occurs at the terminal "fucose-α-1,2-galactose-R (Fuc-a1,2-Gal-R)" a1,2-galactose"-group, and in which the cell - Synthesis of Fuc-a1,2-Gal-R, with - expresses an alpha-1,3-glycosyltransferase, and -has the ability to generate a nucleotide-sugar, wherein the nucleotide-sugar is the donor of the alpha-1,3-glycosyltransferase.

51. The cell of embodiment 50, wherein the galactose (Gal) residue in the Fuc-a1,2-Gal-R is via a β-1,3 or a β-1,4 glycosidic bond (glycosidic bond) linkage) combined with R.

52. The cell of any one of embodiments 50 or 51, wherein the R comprises a monosaccharide, a disaccharide, an oligosaccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a lipid or a Glycolipids.

53. The cell of any one of embodiments 50 to 52, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-R, preferably the Fuc-a1,2 -Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-R.

54. The cell of embodiment 53, wherein the N-acetylglucosamine (GlcNAc) residue in the Fuc-a1,2-Gal-b1,3-GlcNAc-R is via a beta-1,3 or a beta -1,4 glycosidic bond to R.

55. The cell of any one of embodiments 53 or 54, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R, preferably , wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R, more preferably, wherein Fuc-a1,2-Gal-R is Lacto-N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc).

56. The cell of any one of embodiments 50 to 52, wherein the Fuc-a1, 2-Gal-R is Fuc-a1, 2-Gal-b1, 4-R, preferably, wherein the Fuc-a1 , 2-Gal-R is Fuc-a1, 2-Gal-b1, 4-Glc, as required, wherein the Fuc-a1, 2-Gal-R is Fuc-a1, 2-Gal-b1, 4- (Fuc-a1,3)-Glc.

57. The cell of any one of embodiments 50 to 56, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is a structure of the tissue blood group antigen (HBGA) system.

58. The cell of any one of embodiments 50 to 57, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase that is a ) residues transferred from UDP-galactose (UDP-Gal) to the terminal "fucose-al,2-galactose-R (Fuc-al,2-Gal-R) Galactose"-group capable of a glycosyltransferase.

59. The cell of any one of embodiments 50 to 55, 57 or 58, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase that is a A glycosyltransferase capable of transferring a lactose (Gal) residue from UDP-Gal to the terminal "fucose-al,2-galactose"-group of LNFP-I.

60. The cell of any one of embodiments 50 to 52, 56, 58 or 59, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase having A monosaccharide capable of transferring a galactose (Gal) residue from UDP-Gal to the terminal "fucose-a1,2-galactose"-group of Fuc-a1,2-Gal-b1,4-Glc Glucose residues in the Fuc-a1,2-Gal-b1,4-Glc are fucosylated, preferably α-1,3-fucosylated, if necessary.

61. The cell of any one of embodiments 50 to 55 or 57 to 59, wherein the Fuc-a1,2-Gal-R of the α-1,3 glycated form is an α-1,3 glycated form of milk- N-fucopentaose I (LNFP-I), which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal -a1,3-LNFP-I), the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase, and the nucleotide-sugar is UDP-galactose (UDP- Gal).

62. The cell of any one of embodiments 50 to 52, 56 or 58 to 60, wherein the Fuc-a1,2-Gal-R of the α-1,3 glycated form is of an α-1,3 glycated form. Fuc-a1,2-Gal-b1,4-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, as needed, an alpha-1,3 glycated Form Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1 ,3)-Glc, the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase, and the nucleotide-sugar is UDP-galactose (UDP-Gal).

63. The cell of any one of embodiments 58 to 62, wherein the α-1,3-galactosyltransferase has a PFAM PF03414 domain, and a. Include the motif with SEQ ID NO: 01 YX[FHMQT]XAXX[ACG][ACG], where X can be any amino acid residue, or b. Include the motif YXQXCXX[ACG][ACG] with SEQ ID NO: 02, where X can be any amino acid residue, or c. Include serial identification numbers such as: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, a polypeptide sequence of any of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, or d. is the serial identification number: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, A functional homologue, variant or derivative of any of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, having the same number as having a sequence identification number : 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 have at least 80% overall sequence similarity over the full length of any of the alpha-1,3-galactosyltransferase polypeptides, And to the terminal "fucose-al,2-galactose"-group of Fucose-al,2-galactose-R (Fuc-al,2-Gal-R) has a-1,3-galactose Lactosyltransferase activity, or e. is a functional fragment, including from SEQ ID NO: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37. An oligopeptide sequence of 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues, and to fucose-a1,2-galactose-R (Fuc-a1,2 The terminal "fucose-al,2-galactose"-group of -Gal-R) has a-1,3-galactosyltransferase activity.

64. The cell of any one of embodiments 50 to 57, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase having the Transfer of an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to fucose-a1,2-galactose-R (Fuc-a1,2-Gal -R) is a glycosyltransferase capable of the terminal "fucose-al,2-galactose"-group.

65. The cell of any one of embodiments 50 to 55, 57 or 64, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase, It is a terminal "fucose-a1,2-galactose" with the transfer of an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to LNFP-I "-group capacity of a glycosyltransferase.

66. The cell of any one of embodiments 50 to 52, 56 or 64, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase, It is a terminal "fucose-al,2-galactose" with the transfer of an N-acetylgalactosamine (GalNAc) residue from UDP-GalNAc to Fuc-a1,2-Gal-b1,4-Glc A glycosyltransferase with the ability of a group, as required, the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated, preferably α-1 , 3-fucosylated.

67. The cell of any one of embodiments 50 to 55, 57, 64 or 65, wherein the Fuc-a1,2-Gal-R of the α-1,3 glycated form is of an α-1,3 glycated form. Lacto-N-fucopentaose I (LNFP-I) which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-a1,3-LNFP-I), the α-1,3-glycosyltransferase is an α-1,3-N-acetylgalactosamine transferase, and the nucleotide-sugar is UDP -N-Acetylgalactosamine (UDP-GalNAc).

68. The cell of any one of embodiments 50 to 52, 56, 64 or 66, wherein the Fuc-a1,2-Gal-R of the α-1,3 glycated form is of an α-1,3 glycated form. Fuc-a1,2-Gal-b1,4-Glc, which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc (α-sugar or A-sugar), depending on Requires an α-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc, which is GalNAc-a1,3-(Fuc-a1,2)- Gal-b1,4-(Fuc-a1,3)-Glc, the α-1,3-glycosyltransferase is an α-1,3-N-acetylgalactosamine transferase, and the nucleotide - The sugar is UDP-N-acetylgalactosamine (UDP-GalNAc).

69. The cell of any one of embodiments 64 to 68, wherein the α-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain, and a. Include the motif YX[ACIL]XGXX[ACG][ACG] with SEQ ID NO: 38, where X can be any amino acid residue, or b. Include the motif YX[AG]XAXX[ACG][ACG] with SEQ ID NO: 39, where X can be any amino acid residue, or c. Include such as serial identification numbers: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, a polypeptide sequence of any one of 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, or d. is the serial identification number: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, a functional homologue, variant or derivative of any of 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, With and with serial identification numbers: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 of the α-1,3-N-acetylgalactosyltransferase ( a-1,3-N-acetylgalactosyltransferase) polypeptides have at least 80% overall sequence similarity over the full length of any of the polypeptides and are The terminal "fucose-a1,2-galactose" group of R) has a-1,3-N-acetylgalactosyltransferase activity, or e. is a functional fragment, including from SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102. An oligopeptide sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues, and to fucose-a1,2-galactose-R The terminal terminal "fucose-al,2-galactose" group of (Fuc-a1,2-Gal-R) has a-1,3-N-acetylgalactosyltransferase activity.

70. The cell of any one of embodiments 55, 59, 61, 63, 65, 67 or 69, wherein by the action of a glycosyltransferase, by transferring fucose from GDP-fucose to milk -The terminal galactose residue of N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), the LNFP-I is synthesized in the cell, the glycosyltransferase for a. an alpha-1,2-fucosyltransferase (alpha-1,2-fucosyltransferase) selected from the group consisting of from Brachyspira trichomes with UniProt ID A0A2N5RQ26, Dysgonomonas mossii with UniProt ID F8X274, with UniProt ID A list of the polypeptides of Dechlorosoma suillum with G8QLF4, Desulfovibrio alaskensis with UniProt ID Q316B5 and Polaribacter vadi with UniProt ID A0A1B8TNT0, or b. Polypeptide from Brachyspira trichomes (UniProt ID A0A2N5RQ26) with α-1,2-fucosyltransferase activity on terminal galactose residues of LNT, from D. mossii (UniProt ID F8X274) Any of the polypeptides from D. suillum (UniProt ID G8QLF4), the polypeptides from D. alaskensis (UniProt ID Q316B5) and the polypeptides from P. vadi (UniProt ID A0A1B8TNT0) a functional fragment of , or c. From Brachyspira trichomes with UniProt ID A0A2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID A0A1B8TNT0 A functional homologue, variant or derivative of any of the peptides with B. trichomoniasis from UniProt ID A0A2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, respectively, D. alaskensis with UniProt ID Q316B5 and P. vadi with UniProt ID A0A1B8TNT0 have at least 80% overall sequence similarity over the full length of either of the polypeptides, and to the end of lacto-N-saccharide (LNT) The galactose residue has alpha-1,2-fucosyltransferase activity, or d. a polypeptide comprises an amino acid sequence, or is composed of an amino acid sequence, the amino acid sequence has a polypeptide from Brachyspira trichomes (UniProt ID A0A2N5RQ26), from D. mossii Polypeptide from (UniProt ID F8X274), Polypeptide from D. suillum (UniProt ID G8QLF4), Polypeptide from D. alaskensis (UniProt ID Q316B5) and Polypeptide from P. vadi (UniProt ID A0A1B8TNT0) The full-length amino acid sequence of any of the peptides has at least 80% sequence similarity and has alpha-1,2-fucosyltransferase activity on the terminal galactose residue of the LNT.

71. The cell of any one of embodiments 50 to 70, wherein the cell is modified in the expression or activity of a glycosyltransferase.

72. The cell of any one of embodiments 50 to 71, wherein the cell expresses a membrane transporter or a polypeptide having transport activity to transport the compound across the outer membrane of the cell wall.

73. The cell of embodiment 72, wherein the membrane transporter or the polypeptide having transport activity is selected from a list comprising transporter, P-P-bond-hydrolysis-driven transporter, b-barrel porin, auxin transporters, putative transporters and phosphotransfer driven group translocators, Preferably, the transporter includes MFS transporter, sugar efflux transporter and siderophore exporters, or Preferably, the P-P-bond-hydrolysis-driven transporter includes ABC transporter and chelatin exporter.

74. The cell of any one of embodiments 72 or 73, wherein the membrane transporter or polypeptide with transport activity controls the α-1,3 glycosylated form of Fuc-a1,2-Gal-R and/or Flow of one or more precursors and/or acceptors on the outer membrane of the cell wall for the production of the alpha-1,3 glycated form of Fuc-a1,2-Gal-R.

75. The cell of any one of embodiments 72 to 74, wherein the membrane transporter or polypeptide with transport activity provides an improvement in the α-1,3 glycosylated form of Fuc-a1,2-Gal-R generation and/or activated and/or enhanced outflow.

76. The cell of any one of embodiments 50-75, wherein the cell line is modified with a gene expression module, characterized in that the expression from any of the expression modules is constitutive, or created by a natural inducer of.

77. The cell of any one of embodiments 50 to 76, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein.

78. The cell of any one of embodiments 50 to 77, wherein the cell comprises a modification for reducing the production of acetic acid.

79. The cell of any one of embodiments 50 to 78, wherein the cell comprises a lower or reduced expression and/or eliminated, impaired, reduced or delayed activity of any or more proteins , the one or more proteins include β-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deacetylase Enzymes, N-acetylglucosamine inhibitor protein, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecene-phosphoglucosamine-1-phosphotransferase, L-fucokinase, L -Fucose isomerase, N-acetylneuraminic acid lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC -Man, EIID-Man, ushA, galactose-1-phosphate uridylate transferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent fructose-6-phosphate activating enzyme with the same Dfase 1, ATP-dependent fructose-6-phosphate activating enzyme isoenzyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor protein IclR, lon protease, glucose-specific translocation phosphotransferase IIBC component ptsG, glucose-specific translocation phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIAGlc, β-glucoside-specific PTS enzyme II, fructose-specific PTS polyphosphate transfer proteins FruA and FruB, alcohol dehydrogenase Aldehyde dehydrogenase, pyruvate formate lyase, acetate kinase, phosphoacyltransferase, phosphoacetate transferase, pyruvate decarboxylase.

80. The cell of any one of embodiments 50 to 79, wherein the cell has the ability to produce phosphoenolpyruvate (PEP).

81. The cell of any one of embodiments 50 to 80, wherein the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP).

82. The cell of any one of embodiments 50 to 81, wherein the cell comprises an at least partially inactivated catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide that participates in and/or or required for the production of Fuc-a1,2-Gal-R in the α-1,3 glycated form.

83. The cell of any one of embodiments 50 to 82, wherein the cell is resistant to the phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon sources.

84. The cell of any one of embodiments 50 to 83, wherein the cell produces 90 g/L or more of the alpha-1,3 glycated form in whole broth and/or supernatant Fuc-a1,2-Gal-R, and/or wherein in the whole broth and/or supernatant, the Fuc-a1,2-Gal-R in the α-1,3 glycated form according to the α-1 , 3 The saccharified form of Fuc-a1,2-Gal- and its precursors, measured in total in the whole broth and/or supernatant, respectively, had a purity of at least 80%.

85. The cell of any one of embodiments 50 to 84, wherein the cell produces a charged, preferably sialylated and/or neutral disaccharide and oligosaccharide mixture comprising an alpha-1,3 glycosylated form The Fuc-a1,2-Gal-R.

86. The cell of any one of embodiments 50 to 85, wherein the cell produces a charged, preferably sialylated and/or neutral oligosaccharide mixture comprising an α-1,3 glycosylated form of Fuc- a1,2-Gal-R.

87. The method of any one of embodiments 1 to 49 or the cell of any one of embodiments 50 to 86, wherein the cell is a bacterium, a fungus, a yeast, a plant cell, an animal cell or a native animal cells (protozoan cells), - preferably, the bacteria is an Escherichia coli strain, more preferably an Escherichia coli strain, which is a K-12 strain, still more preferably, the Escherichia coli K-12 strain is a large intestine Bacillus MG1655. - preferably, the fungus belongs to a genus selected from the group consisting of Rhizopus, Dictyostelium, Penicillium, Mucor or Koji ( Aspergillus) group, - preferably, the yeast belongs to a genus selected from the group consisting of Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, the group of Hansenula, Yarrowia, Starmerella, Kluyveromyces or Debaromyces, - preferably, the plant cell is an algal cell or is derived from tobacco (tobacco), alfalfa (alfalfa), rice (rice), tomato, cotton, rapeseed (rapeseed), soybean, maize (maize) ) or corn plants, - preferably, the animal cell line is derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects, or a genetically modified cell line derived from human cells excluding embryonic stem cells, more preferably the human and non-human mammalian cells are an epithelial cell, an embryonic kidney cell, A fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell, an NIH-3T3 cell, a non-mammalian adult stem cell ( non-mammary adult stem cell) or its derivatives, more preferably the insect cell line is derived from Spodoptera frugiperda, silkworm (Bombyx mori), cabbage armyworm (Mamestra brassicae), Trichoplusia ni ) or Drosophila melanogaster, - Preferably, the protozoan cell is a Leishmania tarentolae cell.

88. The method of any one of embodiments 1 to 49 and 87, or the cell of any one of embodiments 50 to 87, wherein the cell is a bacterium, preferably an Escherichia coli strain, more preferably One cell of one Escherichia coli strain of one K-12 strain, still more preferably, the Escherichia coli K-12 strain is Escherichia coli MG1655.

89. The method of embodiment 88, or the cell of embodiment 88, wherein the cell is a live Gram-negative bacterium comprising a reduced or eliminated poly-N-acetyl-glucose Amine (poly-N-acetyl-glucosamine, PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid (colanic acid), core oligosaccharides (core oligosaccharides), osmotic regulation periplasmic glucagon Synthesis of Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, Glycan, and/or Trehalose.

90. The method of any one of embodiments 1 to 4 and 87, or the cell of any one of embodiments 50 to 87, wherein the cell is a yeast cell.

91. The method of any one of embodiments 1 to 49 and 87 to 90, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymes Enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic Hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.

92. The method of any one of embodiments 1 to 49 and 87 to 91, further comprising Fuc-a1,2-Gal-R of any one of the α-1,3 glycosylated forms from the cell, preferably from Purification of the alpha-1,3 glycosylated form of LNFP-I from the cells.

93. The method of any one of embodiments 1 to 49 and 87 to 92, wherein the purification comprises at least one of the following steps: use of activated carbon or carbon, charcoal, nanofiltration, ultrafiltration, electrophoresis ( electrophoresis), use of enzymatic treatments or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, lyophilization, spray freeze drying spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying , drum drying, roller drying, vacuum drum drying or vacuum roller drying.

94. The cell of any one of embodiments 50 to 90, or the use of the method of any one of embodiments 1 to 49 or 87 to 93, for α-1,3 glycosylated form of Fuc-a1 ,2-Gal-R, preferably the production of an alpha-1,3 glycosylated form of LNFP-I.

The present invention will be described in more detail in the examples. The following examples are intended to further illustrate and clarify the present invention, but are not intended to be limiting.

Example

Example 1 Materials and Methods Escherichia coli

culture medium

Luria Broth (LB) medium consisted of 1% trypsin (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). The minimal medium used in 96-well plates or shake flasks in culture experiments contained 2.00 g/L NH ₄ Cl, 5.00 g/L (NH ₄ ) ₂ SO ₄ , 2.993 g/L KH ₂ PO ₄ , 7.315 g/LK ₂ HPO ₄ , 8.372 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgSO ₄ .7H ₂ O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 µl/L molybdate (molybdate) solution and 1 mL/L selenium (selenium) solution. As specified in the various examples, 0.30 g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB were additionally added to the medium as precursors. The minimal medium was set to pH 7 with 1 M KOH. The vitamin solution consisted of 3.6 g/L FeCl ₂ .4H ₂ O, 5 g/L CaCl ₂ .2H ₂ O, 1.3 g/L MnCl ₂ .2H ₂ O, 0.38 g/L CuCl ₂ .2H ₂ O, 0.5 g/L L CoCl ₂ .6H ₂ O, 0.94 g/L ZnCl ₂ 0.0311 g/LH ₃ BO ₄ , 0.4 g/L Na ₂ EDTA.2H ₂ O and 1.01 g/L thiamine.HCl. The molybdate solution contained 0.967 g/L NaMoO ₄ .2H ₂ O. The selenium solution contained 42 g/L Seo2.

Minimal medium for fermentation, same composition as above, containing 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, 30 g/L sucrose or 30 g/L glycerol, 1 mL/L vitamin solution, 100 µL/L molybdate solution and 1 mL/L selenium solution. As specified in the various examples, 0.30 g/L sialic acid, 20 g/L lactose, 20 g/L LacNAc and/or 20 g/L LNB were additionally added to the medium as precursors.

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

Plasmid

pKD46 (Red helper plastid, Ampicillin resistance), pKD3 (FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (FRT-flanked chloramphenicol resistance (kan) ) gene) and pCP20 (expressing FLP recombinase activity) plastids were obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium, 2007). Plastids were maintained in host E. coli DH5alpha (F ^- , phi80d lac ZΔM15, Δ( lacZYA-argF ) U169, deoR , recA1 , endA1 , hsdR17(rk ^- , mk ⁺ ), phoA , supE44 , lambda ^- purchased from Invitrogen , thi -1, gyrA96 , rel A1).

Strains and mutations

E. coli K12 MG1655 [λ ^- , F ^- , rph-1] was obtained in March 2007 from the Coli Genetic Stock Center (US), CGSC Strain#:7740. Gene disruption, gene introduction and gene replacement were performed using techniques published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technique is based on antibiotic selection following homologous recombination by lambda Red recombinase. Subsequent catalysis by flippase recombinase ensures removal of the antibiotic selection cassette in the final production strain. Transformants carrying the Red helper plastid pKD46 were grown in 10 mL of LB medium with ampicillin (100 mg/L) and L-arabinose (10 mM) at 30°C to an OD600 nm of 0.6. Cells were made electrocompetent by washing cells first with 50 mL ice-cold water and a second with 1 mL ice-cold water. Afterwards, cells were resuspended in 50 µL of ice-cold water. After electroporation, cells were added to 1 mL of LB medium, incubated at 37°C for 1 hour, and finally plated on LB-agar containing 25 mg/L chloramphenicol or 50 mg/L kanamycin , to select for antibiotic-resistant transformants. Selected mutants were verified by PCR with primers upstream and downstream of the modified region and grown in LB agar at 42°C to eliminate helper plastids. Mutant strains were tested for ampicillin sensitivity. Linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivatives as templates. The primers used have one part of the sequence complementary to the template and another part complementary to the side of the chromosomal DNA to be recombined. For genomic knock-out, regions of homology were designed 50-nt upstream and 50-nt downstream of the start codon and stop codon of the target gene. For gene body knock-in, the transcriptional starting point (+1) must be respected. The PCR product was PCR-purified, digested with DpnI, repurified from agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0). The selected mutants were transformed with pCP20 plastids, an ampicillin- and chloramphenicol-resistant plastid, showing temperature-sensitive replication and thermal induction of FLP synthesis. Ampicillin resistant transformants were selected at 30°C, after which some colonies were purified in LB at 42°C and then tested for all antibiotic resistance and loss of FLP helper plastids. Gene knockouts and knockins were checked using control primers.

In one example of GDP-fucose production, the mutant was derived from E. coli K12 MG1655, which included a knockout of the E. coli wcaJ and thyA genes and a gene body knock-in of the constitutive transcription unit containing a Sucrose transporters such as CscB (UniProt ID EOIXR1) from Escherichia coli W, a fructose kinase such as Frk (UniProt ID Q03417) from Zymomonas mobilis and a sucrose phosphorylase (sucrose phosphorylase), eg BaSP (UniProt ID AOZZH6) derived from Bifidobacterium adolescentis . GDP-fucose production in mutant E. coli strains can be further optimized by genomically knocking out E. coli genes comprising any one or more of glgC , agp , pfkA , pfkB , pgi , arcA , iclR , pgi and lon , as described in WO2016075243 and WO2012007481. GDP-fucose production can be additionally optimized, including gene body knock-in of a mannose-6-phosphate isomerase, such as manA from E. coli (UniProt ID P00946), mannose monophosphate Phosphomannomutases such as manB from E. coli (UniProt ID P24175), mannose-1-phosphate guanylyltransferases such as manC from E. coli (UniProt ID P24174 ), a GDP-mannose 4,6-dehydratase (GDP-mannose 4,6-dehydratase), such as gmd from Escherichia coli (UniProt ID P0AC88) and a GDP-L-fucose synthase (GDP-L - fucose synthase), such as the constitutive transcription unit of fcl (UniProt ID P32055) from E. coli. GDP-fucose production can also be achieved by knocking out the E. coli fucK and fucI genes and knocking in a fucose permease such as fucP from E. coli (UniProt ID P11551) and a bifunctional rock fucose kinase/fucose-1-phosphate guanylyltransferase, such as the constitutive form of fkp (UniProt ID SUV40286.1) from Bacteroides fragilis Transcription unit to obtain. All mutant strains are additionally capable of gene body knockout of the E. coli LacZ , LacY and LacA genes and gene body knock-in of a lactose permease such as the constitutive transcription unit of E. coli LacY (UniProt ID P02920). retouch. In the next step to generate fucosylated structures as used in the present invention, mutant GDP-fucose-producing strains were treated with an enzyme containing an alpha-1,2-fucosyltransferase (alpha-1,2-fucosyltransferase) 2-fucosyltransferase), such as HpFutC (GenBank: AAD29863.1) from Helicobacter pylori ( H. pylori ), polypeptide from Brachyspira pilosicoli with UniProt ID A0A2N5RQ26, polypeptide from Dysgonomonas mossii with UniProt ID F8X274 , an expression plasmid of a constitutive transcription unit of a polypeptide from Dechlorosoma suillum with UniProt ID G8QLF4, a polypeptide from Desulfovibrio alaskensis with UniProt ID Q316B5, or a polypeptide from Polaribacter vadi with UniProt ID A0A1B8TNT0, and A selection marker, such as E. coli thyA (UniProt ID P0A884), is additionally modified by a constitutive transcription unit. The constitutive transcription unit of alpha-1,2-fucosyltransferase can also be presented to mutant E. coli strains via gene body knock-in.

Alternatively, and/or in addition, the production of GDP-fucose and/or fucosylated structures can be further optimized in mutant E. coli strains by including a membrane transporter protein, such as from MdfA from S. mogeni (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter johnsonii (UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID P0AEY8), MdfA from Yorkia rexburgh ( Genome knock-in of constitutive transcription units from UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207) or iceT from Citrobacter japonicus (UniProt ID D4B8A6).

In one example of the production of lacto-N-triose (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), the mutant was derived from E. coli K12 MG1655 , and with the knockout of the E. coli LacZ, LacY, LacA and nagB genes, and a lactose permease, such as LacY from E. coli (UniProt ID P02920), with galactoside β-1,3-N-acetyl Glucosamine transferase (galactoside beta-1,3-N-acetylglucosaminyltransferase), such as LgtA (GenBank: AAM33849.1) from N. meningitidis , is modified by gene body knock-in of the constitutive transcriptional unit. To generate lacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), either by gene body knock-in or from a N-acetylglucosamine beta-1,3-galactosyltransferase expressing plastid delivery to strains, such as from E. coli O55:H7 (UniProt ID D3QY14) A constitutive transcription unit of WbgO to further modify LN3 producing strains. In order to produce lacto-N-neotetraose (lacto- N -neotetraose, LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), a N-acetylglucosamine β-1 ,4-Galactosyltransferase (N-acetylglucosamine beta-1,4-galactosyltransferase), such as the constitutive transcription unit of LgtB (UniProt ID Q51116) from Neisseria meningitidis to modify LN3 producing strains. Lactose permease, galactoside β-1,3-N-acetylglucosamine transferase, N-acetylglucosamine β-1,3-galactosyltransferase and/or can be added as needed Multiple replication of the N-acetylglucosamine beta-1,4-galactosyltransferase gene into mutant Escherichia coli strains. Mutants can also optionally be knocked in by gene body knock-in L-glutamine-D-fructose-6-phosphate aminotransferase, such as from Escherichia coli The mutant strain glmS*54 (different from wild-type E. coli glmS protein with UniProt ID P17169, modified by A39T, R250C and G472S mutations as described by Deng et al. (Biochimie 2006, 88: 419-429), to enhance UDP-GlcNAc production.

In addition, the production of LN3, LNT and/or LNnT can include genomic knockout of any one or more of the E. coli genes of galT , ushA , ldhA and agp for further optimization in mutated E. coli. Mutant E. coli strains can also optionally be modified with UDP-glucose-4-epimerase, such as galE from E. coli (UniProt ID P09147), glucosamine monophosphate. phosphoglucosamine mutase, such as glmM (UniProt ID P31120) from Escherichia coli and N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1 - Glucosamine-1-phosphate acetyltransferase, eg gene body knock-in of the constitutive transcription unit of glmU (UniProt ID P0ACC7) from E. coli to accommodate. Mutant Escherichia coli strains can also be isolated by a phosphoglucosamine mutase, such as glmM from Escherichia coli (UniProt ID P31120), with an N-acetylglucosamine-1-phosphate uridine Transferase/glucosamine-1-phosphate acetyltransferase, such as glmU (UniProt ID P0ACC7) from Escherichia coli Knock-in-4-epimerase on demand, such as from green Genome knock-in of the constitutive transcriptional unit of WbpP (UniProt ID Q8KN66) of Pseudomonas aeruginosa to accommodate UDP-GalNAc production. In addition, the mutant strain may be an alpha-1,3-galactosyltransferase, eg SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 constitutive transcription units were modified. Alternatively and/or additionally, the mutant strain may be an alpha-1,3-N-acetylgalactosaminyl transferase, eg SEQ ID NO: 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 6, 64, 65, 66, 67, 68 , 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99, 100, 101 or 102 constitutive transcription units were modified.

Alternatively, and/or in addition, the production of LN3, LNT, LNnT and their derived oligosaccharides may involve membrane transporters such as a membrane transporter such as MdfA (UniProt ID A0A2T7ANQ9) from S. MdfA from Acidobacter (UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID POAEY8), MdfA from Yorkia rexburg (UniProt ID G90T4L) or iceT from Citrobacter japonicus (UniProt ID D4B8A6 ) Genome knock-in of constitutive transcription units for further optimization in mutant E. coli.

In one example of sialic acid production, the mutant was derived from E. coli K12 MG1655, which contains a gene body knock-in of a constitutive transcription unit containing one or more replicated monoglucosamine 6-phosphate N- glucosamine 6-phosphate N-acetyltransferase, such as GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), N-acetylglucosamine 2-epimerase, For example, AGE (UniProt ID A7LVG6) and N-acetylneuraminate synthas from Bacteroides ovatus , such as from Neisseria meningitidis (UniProt ID EONCD4) or Campylobacter jejuni (UniProt ID Q93MP9).

Alternatively, and/or additionally, sialic acid can be produced by containing a UDP-N-acetylglucosamine 2-epimerase, such as NeuC from Aspergillus (UniProt ID Q93MP8) obtained by gene body knock-in of a constitutive transcription unit, eg, from Neisseria meningitidis (UniProt ID EONCD4) or Campylobacter (UniProt ID Q93MP9) with an N-acetylneuraminic acid synthase.

Alternatively, and/or additionally, sialic acid can be produced by containing a phosphoglucosamine mutase, such as glmM (UniProt ID P31120) from E. coli, mono-N-acetylglucosamine-1-phosphate N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase, such as glmU from E. coli (UniProt ID P0ACC7), a UDP- N-acetylglucosamine 2-epimerase (UDP-N-acetylglucosamine 2-epimerase), such as NeuC (UniProt ID Q93MP8) from Aspergillus and a N-acetylneuraminic acid synthase, such as from Genome knock-in of constitutive transcriptional units of Neisseria meningitidis (UniProt ID EONCD4) or Campylobacter (UniProt ID Q93MP9) was obtained.

Alternatively, and/or additionally, sialic acid can be produced by containing a bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase ), e.g. from M. musculus (strain C57BL/6J) (UniProt ID Q91WG8), N-acylneuraminate-9-phosphate synthetase, e.g. from Pseudomonas ( Pseudomonas sp.) UW4 (UniProt ID K9NPH9) and N-acylneuraminate-9-phosphatase, e.g. from Candidatus Magnetomorum sp. HK-1 (UniProt ID KPA15328.1 ) or by gene body knock-in of constitutive transcription units from Bacteroides thetaiotaomicron (UniProt ID Q8A712).

Alternatively, and/or additionally, sialic acid can be produced by transphosphorylation containing phosphoglucosamine mutase, eg, glmM from E. coli (UniProt ID P31120), an N-acetylglucosamine-1-phosphate uridine transfer Enzymes (N-acetylglucosamine-1-phosphate uridyltransferase)/glucosamine-1-phosphate acetyltransferase (glucosamine-1-phosphate acetyltransferase), such as glmU (UniProt ID P0ACC7) from E. coli, a bifunctional UDP-GlcNAc 2 - UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase, eg from M. musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N-acetylmannosamine kinase N-acylneuraminate-9-phosphate synthetase, e.g. from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and N-acylneuraminate-9-phosphate synthetase, e.g. Genome knock-in of constitutive transcription units from Candidatus Magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or from Bacteroides polymorpha (UniProt ID Q8A712) was obtained.

Sialic acid production in mutant E. coli strains can be further knocked out with a gene body comprising any one or more E. coli genes of nagA , nagB , nagC , nagD , nagE , nanA , nanE , nanK , manX , manY , manZ such as described in WO18122225 and/or with gene body knockouts comprising any one or more of nanT , poxB , ldhA , adhE , aldB , pflA , pflC , ybiY , ackA and/or pta E. coli genes, and to include a L-glutamine-D-fructose-6-phosphate aminotransferase, such as mutant glmS*54 (different from wild-type E. coli glmS, with UniProt ID P17169, by A39T, R250C and G472S mutations, as described by Deng et al. (Biochimie 2006, 88: 419-429)), preferably a phosphatase, for example including aphA, Cof, HisB, OtsB, E. coli genes of Sure, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida , ScDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis , as described in WO1812222, combined with acetyl-CoA synthetase For example, gene body knock-in of one or more replicated constitutive transcription units from Escherichia coli acs (UniProt ID P27550) is optimized. .

For sialylated oligosaccharide production, the sialic acid producing strain is further modified to express an N-acylneuraminate cytidylyltransferase, such as the NeuA enzyme from Aspergillus (UniProt ID Q93MP7 ), NeuA enzyme (GenBank NO. AGV11798.1) from Haemophilus influenzae (GenBank NO. AGV11798.1) or NeuA enzyme (GenBank NO. AMK07891.1) from Pasteurella multocida , and showed a β-galactoside α-2,3-sialyltransferase (beta-galactoside alpha-2,3-sialyltransferase), such as PmultST3 (UniProt ID Q9CLP3) from Pasteurella PmultST3-like polypeptide (PmultST3-like polypeptide) composed of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with lactoside α-2,3-sialyltransferase activity, NmeniST3 from Neisseria meningitidis (GenBank NO. ARC07984.1) or PmultST2 (GenBank NO. AAK02592.1), a beta-galactoside alpha-2,6-sialyltransferase (beta-galactoside alpha-2,6-sialyltransferase) from Pasteurella septicemia subsp. multocida str. Pm70 -galactoside alpha-2,6-sialyltransferase), such as PdST6 from Photobacterium damselae (UniProt ID 066375) or from UniProt ID 066375 with beta-galactoside alpha-2,6-sialyltransferase activity A PdST6-like polypeptide composed of amino acid residues 108 to 497 or P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 ( UniProt ID A8QYL1) or P-JT-ISH-224-ST6-like Duosheng consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 with β-galactoside α-2,6-sialyltransferase activity Peptides, and/or alpha-2,8-sialyltransferase (alpha-2,8-sialyltransferase), for example from M. One or more copies of musculus (UniProt ID Q64689). The constitutive transcription units of N-acylneuraminate cytidylyltransferase and sialyltransferases can be delivered to mutant strains via gene knock-in or via an expression plastid. If mutant strains producing sialic acid and CMP-sialic acid are intended to make sialylated lactose structures, gene body knockout of the E. Genome knock-in of the constitutive transcription unit of P02920) additionally modified this strain.

Alternatively, and/or in addition, the production of sialic acid and/or sialylated oligosaccharides can be further optimized in mutant E. coli strains by including a membrane transporter, such as a sialic acid transporter, such as from E. coli K-12 nanT from MG1655 (UniProt ID P41036), nanT from E. coli O6:H1 (UniProt ID Q8FD59), nanT from E. coli O157:H7 (UniProt ID Q8X9G8), or nanT from E. albertii (UniProt ID P24077), or A porter, such as EntS from Escherichia coli (UniProt ID P24077), EntS from Kluyvera ascorbate (UniProt ID A0A378GQ13) or from Salmonella enterica subsp. arizonae EntS (UniProt ID A0A6Y2K4E8), MdfA from Cronobacter (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter johnsonii (UniProt ID D4BC23), MdfA from E. coli (UniProt ID P0AEY8), MdfA from Yokenella regensburgei , MdfA from Escherichia coli (UniProt ID A0A024L207), iceT from Citrobacter japonicus (UniProt ID D4B8A6), SetA from Escherichia coli (UniProt ID P31675), SetB from Escherichia coli (UniProt ID P33026) or from large intestine Bacillus SetC (UniProt ID P33026) or an ABC transporter (transporter), such as oppF (UniProt ID P77737) from Escherichia coli, lmrA from Lactococcus lactis subsp. lactis bv. Diacetylactis (UniProt ID A0A1VONEL4) Or gene body knock-in of the constitutive transcription unit of Blon_2475 (UniProt ID B7GPD4) from Bifidobacterium longum subsp. Infantis .

All mutant strains can also be optionally routed via a sucrose transporter, such as CscB from Escherichia coli W (UniProt ID EOIXR1), a fructokinase such as Frk from Z. mobilis ( Z. mobilis ). UniProt ID Q03417) with a sucrose phosphorylase, eg, gene body knock-in of the constitutive transcription unit of BaSP (UniProt ID A0ZZH6) from B. teenis , adapted for growth on sucrose.

Preferably, but not necessarily, any one or more of glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, and/or membrane transporters are N- and/or C-terminally fused to a solubility enhancer tag ( solubility enhancer tags), such as SUMO tags, MBP tags, His, FLAG, Strep-II, Halo-tag, NusA, thioredoxin, GST and/or Fh8 tags to increase their solubility (Costa et al., Front. Microbiol. 2014, https://doi.org/10.3389/fmicb.2014.00063; Fox et al., Protein Sci. 2001, 10(3), 622-630; Jia and Jeaon, Open Biol. 2016, 6: 160196).

Depending on need, to encode a chaperone such as DnaK, DnaJ, GrpE or the GroEL/ES chaperonin system (Baneyx F., Palumbo J.L. (2003) Improving Heterologous Protein Folding via Molecular Chaperone and Foldase Co-Expression. In: Vaillancourt P.E. (eds) E. coli Gene Expression Protocols. Methods in Molecular Biology™, vol 205. Humana Press) Genome knock-in of constitutive transcription units to modify mutant E. coli strains.

As desired, the mutant E. coli strains were modified to produce glycominimized E. coli strains including, including pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, Non-essential glycosyl groups of wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, wbbkgh, opjhg, glgA, glgB, malQ, otsA, and yaiP Gene body knockout of any one or more of the transferase genes.

All constitutive promoters, UTR and terminator sequences were derived from Mutalik et al. (Nat. Methods 2013, No. 10, 354-360) and Cambray et al. (Nucleic Acids Res. 2013 , 41(9), 5139-5148) described libraries. All genes were ordered synthetically on Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and codon usage was adjusted using the supplier's tools. The sequence identification numbers described in the present invention are summarized in Table 1.

All strains were stored in cryovials at -80°C (overnight LB cultures were mixed 1:1 with 70% glycerol).

Table 1. Summary of Serial Identification Numbers Described in the Present Invention serial identification number name / identifier _ species source Country of origin of digital sequence information 01 motif YX[FHMQT]XAXX[ACG][ACG] Not applicable Synthetic Not applicable 02 motif YXQXCXX[ACG][ACG] Not applicable Synthetic Not applicable 03 WbnI Escherichia coli K-12 MG1655 Synthetic USA 04 A0A379B3N6_9PAST [Pasteurella] mairii Synthetic United Kingdom 05 A0A430GH65_ACIBZ Acinetobacter bereziniae Synthetic USA 06 A0A1G6GGM3_9GAMM Acinetobacter boissieri Synthetic unknown 07 D0S6I6_ACICA Acinetobacter calcoaceticus Synthetic unknown 08 D0SG80_ACIJO Acinetobacter johnsonii Synthetic unknown 09 R5HLA7_9SPIR Brachyspira sp. CAG:484 Synthetic Humanity 10 A0A327JA01_9BACT Candidatus Melainabacteria Synthetic USA 11 A0A2D6M7J8_9ARCH Candidatus Paearchaeota Synthetic USA 12 A0A1F6ZBC2_9ARCH Candidatus Paearchaeota Synthetic USA 13 A0A1N6DQ13_9BACT Chitinophaga niabensis Synthetic unknown 14 A0A0L6XXM2_ECOLX Escherichia coli Synthetic USA 15 A0A5F2KSQ8_ECOLX Escherichia coli Synthetic USA 16 A0A377IY75_9PAST Haemophilus pittmaniae Synthetic Denmark 17 F9Q7G3_9PAST Haemophilus pittmaniae HK 85 Synthetic unknown 18 A0A268TKN7_9HELI Helicobacter sp. 11S02629-2 Synthetic Netherlands 19 A0A268U1C6_9HELI Helicobacter sp. 13S00401-1 Synthetic Netherlands 20 A0A2D8EN13_9RHOB Hyphomonas sp. Synthetic unknown twenty one G4CKK7_9NEIS Neisseria shayeganii 871 Synthetic unknown twenty two A0A448K0X4_PASAE Pasteurella aerogenes Synthetic Germany twenty three A5WE44_PSYWF Psychrobacter sp. (strain PRwf-1) Synthetic unknown twenty four A0A0Q9ZZ77_9GAMM Psychrobacter sp. P11F6 Synthetic Norway 25 U3GL52_SALER Salmonella enterica Synthetic USA 26 A0A5V0IUT3_SALER Salmonella enterica Synthetic USA 27 A0A3U0WT94_SALET Salmonella enterica I Synthetic USA 28 A0A5I2AVE0_SALET Salmonella enterica subsp. enterica serovar Ahuza Synthetic United Kingdom 29 A0A5J1JHJ9_SALET Salmonella enterica subsp. enterica serovar Kingabwa Synthetic United Kingdom 30 A0A1I6Z135_SELRU Selenomonas ruminantium Synthetic Japan 31 A0A0Q0W3X1_9DELT Smithella sp. SDB Synthetic USA 32 A0A0E3HFV4_9CAUD Synechococcus phage ACG-2014f Synthetic unknown 33 A0A0E3F846_9CAUD Synechococcus phage ACG-2014f Synthetic unknown 34 A0A0E3HAU1_9CAUD Synechococcus phage ACG-2014f Synthetic unknown 35 A0A222YW15_9CAUD Synechococcus phage Bellamy Synthetic unknown 36 A0A1D8KNT5_9CAUD Synechococcus phage S-CAM9 Synthetic unknown 37 A0A0T9L978_YERKR Yersinia kristensenii Synthetic unknown 38 motif YX[ALIC]XGXX[ACG][ACG] Not applicable Synthetic Not applicable 39 motif YX[AG]XAXX[ACG][ACG] Not applicable Synthetic Not applicable 40 BgtA Helicobacter mustelae Synthetic unknown 41 R0BXE6_9FIRM [Clostridium] bolteae 90A9 Synthetic unknown 42 A0A2N8HMV8_9BACT Akkermansia muciniphila Synthetic Netherlands 43 A0A2N8HSR4_9BACT Akkermansia muciniphila Synthetic Netherlands 44 A0A5B5WWM4_9BACT Akkermansia sp. BIOML-A61 Synthetic USA 45 R7DUA1_9BACT Akkermansia sp. CAG:344 Synthetic unknown 46 A0A139TN33_9BACT Akkermansia sp. KLE1798 Synthetic unknown 47 A0A2D8CAM3_9BACT Algoriphagus sp. Synthetic USA 48 A0A414EE60_BACOV Bacteroides ovale Synthetic unknown 49 A0A395VXC9_BACOV Bacteroides ovale Synthetic unknown 50 A0A5M5NEF9_BACOV Bacteroides ovale Synthetic unknown 51 A0A5M5BDZ6_BACOV Bacteroides ovale Synthetic unknown 52 D4WAD3_BACOV Bacteroides ovale SD CMC 3f Synthetic unknown 53 W4UP70_9BACE Bacteroides reticulotermitis JCM 10512 Synthetic unknown 54 A0A374VDT0_9BACE Bacteroides sp. OM08-11 Synthetic unknown 55 A0A414GH63_9BACE Bacteroides xylanisolvens Synthetic USA 56 A0A3A1YUS9_9PAST Bisgaard taxon 44 str. 111 Synthetic Germany 57 A0A3A1Y620_9PAST Bisgaard Taxon 44 str. B96_3 Synthetic unknown 58 A0A3A1Y6E1_9PAST Bisgaard taxon 44 str. B96_4 Synthetic Belgium 59 A0A3A1YHR3_9PAST Bisgaard taxon 44 str. EEAB3T1 Synthetic Denmark 60 A0A1F6NMG0_9BACT Candidatus Magasanikbacteria Synthetic USA 61 A0A3D4XY75_9BACT Candidatus Nomurabacteria Synthetic Canada 62 A0A1F8JKB9_9BACT Chlamydiae bacterium Synthetic USA 63 A0A1F8JQI5_9BACT Chlamydiae bacterium Synthetic USA 64 R5SZE5_9CLOT Clostridium hathewayi CAG: 224 Synthetic unknown 65 A0A1F8V813_9COXI Coxiella sp. Synthetic USA 66 M5PWK3_DESAF Desulfocurvibacter africanus PCS Synthetic USA 67 M5PWJ9_DESAF Desulfocurvibacter africanus PCS Synthetic USA 68 A0A1T4X4S8_9FIRM Gemmiger formicilis Synthetic unknown 69 A0A1T4WDI3_9FIRM Gemmiger formicilis Synthetic unknown 70 L1K4J1_GUITC Guillardia theta Synthetic unknown 71 L1JS30_GUITC Guillardia theta Synthetic unknown 72 A0A268TN00_9HELI Helicobacter sp. 11S02629-2 Synthetic Netherlands 73 A0A268U494_9HELI Helicobacter sp. 13S00401-1 Synthetic Netherlands 74 A0A1I3AV07_9FIRM Lachnospiraceae bacterium Synthetic unknown 75 A0A1M5FSV6_9GAMM Marinomonas polaris Synthetic unknown 76 A0A1H1WY84_9ACTN Marmoricola scoriae Synthetic unknown 77 A0A4Q0J8V7_9BACT Muribaculaceae bacterium Synthetic Germany 78 A0A3S0C1L8_9NEIS Neisseriaceae bacterium Synthetic USA 79 A0A1V1WBB2_9ACTN Nocardioides sp. PD653 Synthetic Japan 80 K5ZTG7_9BACT Parabacteroides goldsteinii Synthetic USA 81 S0GK24_9BACT Parabacteroides goldsteinii Synthetic USA 82 A0A0F5J8N9_9BACT Parabacteroides goldsteinii Synthetic USA 83 A0A0F5IUL0_9BACT Parabacteroides gordonii MS-1 Synthetic unknown 84 A0A0C1BYL5_9BACT Parachlamydia acanthamoebae Synthetic Germany 85 F8KUR1_PARAV Parachlamydia acanthamoebae Synthetic Germany 86 A0A2H9SLN5_9BACT Parachlamydia sp. Synthetic Germany 87 A0A1Y3N080_PIRSE Piromyces sp. Synthetic unknown 88 A0A1Y3N1S0_PIRSE Piromyces sp. Synthetic unknown 89 A0A3C1Y7L0_9PORP Porphyromonadaceae bacterium Synthetic unknown 90 Q58M87_BPPRM Prochlorococcus phage P-SSM2 Synthetic unknown 91 A0A3R5VYF4_9FIRM R. glucosophila Synthetic United Kingdom 92 F2U7J8_SALR5 Salpingoeca rosetta Synthetic unknown 93 A0A353BN36_9FIRM Subdoligranulum sp. Synthetic Humanity 94 A0A3D4DY51_9FIRM Subdoligranulum sp. Synthetic Humanity 95 A0A353BNA4_9FIRM Subdoligranulum sp. Synthetic Humanity 96 A0A1Q6SX55_9FIRM Subdoligranulum sp. Synthetic Humanity 97 A0A2V2FCK1_9FIRM Subdoligranulum variabile Synthetic Denmark 98 A0A2V2FLV2_9FIRM Subdoligranulum variabile Synthetic Denmark 99 D1PIY3_9FIRM Subdoligranulum variabile Synthetic Denmark 100 D1PRD4_9FIRM Subdoligranulum variabile Synthetic Denmark 101 D1B3X0_SULD5 Sulfurospirillum deleyianum Synthetic Germany 102 PDB: 4AYL_A Bacteroides ovale Synthetic USA

Culture conditions

Pre-incubation for 96-well microtiter plate experiments was initiated in a cryovial in 150 µL LB and incubated overnight at 37°C on an orbital shaker at 800 rpm. Use this culture as an inoculum in a 96-well square microtiter plate by adding 400 µL of minimal medium at a dilution of 400x. These final 96-well plates were then incubated at 37°C on an orbital shaker at 800 rpm for 72 hours, or shorter or longer. To measure the sugar concentration at the end of the culture experiment, remove the entire broth sample from each well by boiling the culture medium at 60°C for 15 minutes before spinning off the cells (= average of intracellular and extracellular sugar concentrations value).

Pre-culture of bioreactors starts with whole 1 mL cryovials of specific strains, inoculated in 250 mL or 500 mL of minimal medium in a 1 L or 2.5 L shake flask, and inoculated on an orbital shaker at 37°C Incubate for 24 hours at 200 rpm. A 5 L bioreactor (250 mL inoculum in 2 L batch medium) was then inoculated; this process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Cultivation conditions were set at 37°C with maximum agitation; pressure gas flow rate was dependent on strain and bioreactor. The pH was controlled at 6.8 using 0.5 MH2SO4 and ₂₀ % _{NH4OH .} Cool the exhaust gas. A 10% solution of silicone antifoaming agent was added when foaming during fermentation.

Optical density

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

Analytical analysis

Standards such as, but not limited to, sucrose, lactose, lacto-N-tricarbose II (LN3), lacto-N-tricarbose (LNT), LNFP-I were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed using in-house developed standards.

Oligosaccharides were analyzed on a Waters Acquity H-class UPLC with either an Evaporative Light Scattering Detector (ELSD) or a Refractive Index (RI) detection. A 0.7 µL volume of sample was injected onto a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm; 130 Å; 1.7 µm) and an Acquity UPLC BEH Amide VanGuard column, 130 Å, 2.1 x 5 mm. The column temperature was 50°C. The mobile phase consisted of a solution of 1/4 water and 2/4 acetonitrile to which 0.2% triethylamine was added. This method is isocratic with a flow rate of 0.130 mL/min. The drift tube temperature of the ELS detector was 50°C, the _N2 gas pressure was 50 psi, the gain was 200, and the data rate was 10 pps. The temperature of the RI detector was set to 35°C.

Sugars were also analyzed on a Waters Acquity H-class UPLC with Refractive Index (RI) detection. A 0.5 µL volume of sample was injected onto a Waters Acquity BEH Amide column (2.1 x 100 mm; 130 Å; 1.7 µm). The column temperature was 50°C. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. This method is isocratic with a flow rate of 0.260 mL/min. The temperature of the RI detector was set to 35°C.

For analysis on a mass spectrometer, a Waters Xevo TQ-MS with Electron Spray Ionisation (ESI) was used, with a desolvation temperature of 450°C, a nitrogen desolvation of 650 L/hr Gas flow and cone voltage of 20 V. For all oligosaccharides, MS was operated in negative mode in selected ion monitoring (SIM). The separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1 x 100 mm; 3 µm) at 35°C. A gradient was used where eluent A was ultrapure water with 0.1% formic acid and where eluent B was acetonitrile with 0.1% formic acid. Oligosaccharides were separated in 55 min using the following gradient: an initial increase from 2% to 12% of eluent B in 21 min, a second increase from 12% to 40% of eluent B in 11 min, at A third increase from 40% to 100% Eluent B in 5 min. As a washing step, use 100% Eluent B for 5 minutes. For column equilibration, the initial conditions of 2% Eluent B were recovered within 1 minute and held for 12 minutes.

To analyze low concentrations (below 50 mg/L) of sugars, a Dionex HPAEC system with pulsed amperometric detection (PAD) was used. A 5 µL volume of sample was injected into a Dionex CarboPac PA200 column 4 x 250 mm and a Dionex CarboPac PA200 guard column 4 x 50 mm. The column temperature was set to 30°C. A gradient was used where eluent A was deionized water, where eluent B was 200 mM sodium hydroxide, and where eluent C was 500 mM sodium acetate. Oligosaccharides were separated in 60 min while maintaining a constant ratio of 25% of eluent B using the following gradient: initial isocratic step with 75% of eluent A, 10 min, initial increase from 0 to 4 in 8 min % of eluent C, second isocratic step holding 71% of eluent A with 4% of eluent C, 6 min, increasing from 4% second to 12% elution in 2.6 min Solution C, the third isocratic step kept 63% of eluent A with 12% of eluent C for 3.4 min, and the third increased from 12% to 48% of eluent in 5 min. As a washing step, use 48% Eluent C for 3 minutes. For column equilibration, the initial conditions of 75% Eluent A and 0% Eluent C were recovered in 1 minute and held for 11 minutes. The applied flow rate was 0.5 mL/min.

Example 2. Materials and Methods Saccharomyces cerevisiae

culture medium

Strains with Complete Supplement Mixture (SD CSM) or containing 6.7 g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/L L agar (Difco) (solid culture), 22 g/L glucose monohydrate or 20 g/L lactose with 0.79 g/L CSM or 0.77 g/L CSM-Ura or 0.77 g/L CSM-His (MP Biomedicals) CSM drop-out (SD CSM-Ura or SD CSM-His) for growth on Synthetic Defined yeast medium.

strain

S. cerevisiae BY4742 was created by Brachmann et al. (Yeast (1998) 14: 115-32) and is available in the Euroscarf culture collection. All mutant strains were generated via homologous recombination or plastid transformation using the method of Gietz (Yeast 11:355-360, 1995).

plastid

In the case of GDP-fucose production, yeast express plastids such as p2a_2µ_Fuc (Chan 2013, Plasmid 70, 2-17) for expressing foreign genes in Saccharomyces cerevisiae. The plastid contains an ampicillin resistance gene and a bacterial origin of replication to allow selection and maintenance in E. coli, and 2μ yeast ori and Ura3 selectable markers for selection and maintenance in yeast. The plastids further comprise a lactose permease such as LAC12 from Kluyveromyces lactis (UniProt ID P07921), a GDP-mannose 4,6-dehydratase such as gmd from Escherichia coli (UniProt ID P0AC88) and A GDP-L-fucose synthase such as the constitutive transcription unit of fcl (UniProt ID P32055) from E. coli. The yeast expression plastid p2a_2μ_Fuc2 was used as a surrogate expression plastid for the p2a_2μ_Fuc plastid containing bacterial ori, 2μ yeast ori and the Ura3 selectable marker next to the ampicillin resistance gene, a lactose permease, e.g. from Lactococcus lactis (UniProt ID P07921) LAC12, a fucose permease such as fucP from E. coli (UniProt ID P11551) and bifunctional fucose kinase/fucose-1-phosphate guanylate transfer Enzymes such as the constitutive transcription unit of fkp (UniProt ID SUV40286.1) from Bacteroides fragilis. To further produce fucosylated oligosaccharides, p2a_2µ_Fuc and its variant p2a_2µ_Fuc2 further comprise an α-1,2-fucosyltransferase, such as a component of HpFutC (GenBank: AAD29863.1) from Helicobacter pylori type transcription unit.

In an example of the production of UDP-galactose, a yeast expressed plastid derived from the pRS420-plastid series (Christianson et al., 1992, Gene 110: 119-122), which contained the HIS3 selectable marker and a glucose-4- An epimerase, such as the constitutive transcription unit of galE from E. coli (UniProt ID P09147). . To produce LN3 and LNT, the plastids are further treated with a lactose permease, such as LAC12 from Lactococcus lactis (UniProt ID P07921), galactoside β-1,3-N-acetylglucosamine transferase, such as from Lactococcus lactis (UniProt ID P07921) Constitutive formation of IgtA (GenBank: AAM33849.1) of Neisseria meningitidis with a monoN-acetylglucosamine beta-1,3-galactosyltransferase such as WbgO (UniProt ID D3QY14) from Escherichia coli O55:H7 modification of transcription units. To generate UDP-GalNAc, this plastid is additionally modified with a 4-epimerase, such as the constitutive transcription unit of WbpP (UniProt ID Q8KN66) from Pseudomonas aeruginosa, on glucosamine monophosphate mutase, such as glmM from E. coli (UniProt ID P31120) with monoN-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase, e.g. glmU from E. coli (UniProt ID P0ACC7) beside the keyed in as needed. In addition, the mutant strain may be an alpha-1,3-galactosyltransferase, eg SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 constitutive transcription units were modified. Alternatively and/or additionally, the mutant strain may be alpha-1,3-N-acetylgalactosamine transferase, eg SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 6, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, The constitutive transcription units of 101 or 102 were modified.

In one example for the production of sialic acid and CMP-sialic acid, yeast expressed plastids can be derived from the pRS420-plastid series (Christianson et al., 1992, Gene 110: 119-122), which contain the TRP1 selection marker and a L-glutamic acid-D-fructose-6-phosphate transaminase, such as mutated glmS*54 (different from wild-type E. coli glmS, with UniProt ID P17169, by A39T, R250C and G472S mutations, as in Deng et al. al. (Biochimie 2006, 88: 419-429)), monophosphatase (phosphatase), for example including aphA, Cof, HisB, OtsB, Sure, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC , YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU E. coli genes or PsMupP from Pseudomonas sputum, ScDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis, as described in WO18122225, a N-acetylglucosamine 2-epimerase, such as AGE from Bacteroides ovale (UniProt ID A7LVG6), a N-acetylneuraminic acid synthase , for example from NeuA (UniProt ID Q93MP7) from Aspergillus meningitidis (UniProt ID EONCD4) or Aspergillus (UniProt ID Q93MP9) with an N-acyl neuraminic acid cytidine transferase, for example from Aspergillus meningitidis (UniProt ID Q93MP7) , NeuA from Haemophilus influenzae (GenBank NO. AGV11798.1) or NeuA from Pasteurella septicemia (GenBank NO. AMK07891.1) of one or more replicated constitutive transcription units. Optionally, a modular transcription unit comprising one or more replications of a monoglucosamine 6-phosphate N-acetyltransferase such as GNA1 from Saccharomyces cerevisiae (UniProt ID P43577) can also be added. To produce sialylated oligosaccharides, the plastids further comprise a lactose permease, such as the constitutive transcription unit of LAC12 (UniProt ID P07921) from Lactobacillus kluyveri, and a β-galactoside α-2,3 - Sialyltransferases, such as PmultST3 from Pasteurella septicemia (UniProt ID Q9CLP3) or amino acid residues from UniProt ID Q9CLP3 with beta-galactoside alpha-2,3-sialyltransferase activity PmultST3-like polypeptide composed of bases 1 to 268, NmeniST3 from Neisseria meningitidis (GenBank NO. ARC07984.1) or from Pasteurella septicemia subsp. multocida str. Pm70 PmultST2 (GenBank NO. AAK02592.1), a β-galactoside α-2,6-sialyltransferase, such as PdST6 (UniProt ID 066375) from luminobacterium, or a β-galactoside α-2, PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375 with 6-sialyltransferase activity or from Photobacterium sp. JT-ISH- 224 of P-JT-ISH-224-ST6 (UniProt ID A8QYL1) or consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 with β-galactoside α-2,6-sialyltransferase activity P-JT-ISH-224-ST6-like polypeptide, and/or an alpha-2,8-sialyltransferase, such as one or more copies from M. musculus (UniProt ID Q64689).

Preferably, but not necessarily, any one or more of glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, and/or membrane transporters are N- and/or C-terminally fused to a SUMOstar tag (e.g. , obtained from pYSUMOstar, Life Sensors, Malvern, PA) to improve its solubility.

If desired, mutate yeast strains to encode chaperone proteins such as Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssb1, Sse1, Sse2, Ssa1, Ssa2, Ssa3, Ssa4, Ssb2, Ecm10, Ssc1, Ssq1, Ssz1, Lhs1 , Hsp82, Hsc82, Hsp78, Hsp104, Tcp1, Cct4, Cct8, Cct2, Cct3, Cct5, Cct6 or Cct7 gene body knock-in modification of constitutive transcription units (Gong et al., 2009, Mol. Syst. Biol. 5:275).

Plastids were stored in host E. coli DH5alpha (F ^- , phi80dlacZdeltaM15, delta( lacZYA-argF )U169, deoR , recA1 , endA1 , hsdR17(rk ^- , mk ⁺ ), phoA , supE44 , lambda ^- , thi -1, gyrA96 , relA 1) was purchased from Invitrogen.

Heterologous and homologous manifestations

Genes to be expressed, whether from plastids or gene bodies, are artificially synthesized by one of the following companies: DNA2.0, Gen9, IDT or Twist Bioscience. Expression can be further facilitated by optimizing codon usage for the codon usage of the expression host. Use the vendor's tools to optimize genes.

Culture conditions

Generally, yeast strains are initially grown on SD CSM plates to obtain single colonies. The plates were grown at 30°C for 2-3 days. Starting from a single colony, a preculture was grown in 5 mL overnight at 30°C with shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture in 25 mL of medium. The flasks were incubated at 30°C with orbital shaking at 200 rpm.

gene expression promoter

Genes were expressed using synthetic constitutive promoters, as described by Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012).

Example 3. Including 2'FL, DiFL, LN3, LNT, LNFP-I and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1 with modified E. coli hosts Generation of oligosaccharide mixtures of ,3-Gal-b1,4-Glc

E. coli strains modified as described in Example 1 for GDP-fucose production and growth on sucrose by β-1,3-N-β-galactoside from Neisseria meningitidis Constitutive transcription unit of glucosyltransferase LgtA (GenBank: AAM33849.1) and N-acetylglucosamine β-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from Escherichia coli O55:H7 Gene body knock-in, further suitable for LN3 and LNT production. To generate LNFP-I, the novel strain was further modified with an expression plasmid containing the constitutive transcription unit of the alpha-1,2-fucosyltransferase HpFutC (GenBank: AAD29863.1) from Helicobacter pylori. In a final step in the production of Gal-LNFP-I, the mutant strain was modified with a second compatible expression plastid containing the α-1,3-galactosyltransferase WbnI from E. coli A modular transcription unit with SEQ ID NO: 03. Novel strains were evaluated in a growth experiment for 2'FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and Gal - Generation of a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) according to Example 1 The culture conditions provided in the medium contained 30 g/L sucrose and 20 g/L lactose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 4. Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1 in fed-batch fermentation with a modified E. coli host, Production of 3-Gal-b1,4-Glc

Mutant E. coli strains as described in Example 3 were further evaluated in a fed-batch fermentation process. Bioreactor scale fed batch fermentation was performed as described in Example 1. In these examples, sucrose was used as a carbon source and lactose was added to the batch medium as a precursor. A typical broth sample was taken and oligosaccharide production was measured using UPLC as described in Example 1.

Example 5. Including 2'FL, DiFL, LN3, LNT, LNFP-I and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1 with modified E. coli hosts Generation of oligosaccharide mixtures of ,3-Gal-b1,4-Glc

E. coli strains modified for GDP-fucose production and LNFP-1 production and growth on sucrose as described in Example 3 were further modified to contain alpha-1 from H. mustelae with SEQ ID NO: 40 The second compatibility of , 3-N-acetylgalactosamine transferase BgtA with the constitutive transcription unit of the 4-epimerase WbpP (UniProt ID Q8KN66) from Pseudomonas aeruginosa expresses plastid transformation with Produced from UDP-GalNAc. Novel strains were evaluated in a growth experiment for 2'FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and GalNAc - Generation of a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) according to Example 1 The culture conditions provided in the medium contained 30 g/L sucrose and 20 g/L lactose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 6. GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1 in fed-batch fermentation with a modified E. coli host, Production of 4-Glc

Mutant E. coli strains as described in Example 5 were further evaluated in a fed-batch fermentation process. Bioreactor scale fed batch fermentation was performed as described in Example 1. In these examples, sucrose was used as a carbon source and lactose was added to the batch medium as a precursor. A typical broth sample was taken and oligosaccharide production was measured using UPLC as described in Example 1.

Example 7. Including 2'FL, DiFL, LN3, LNT, LNFP-I and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc- with modified Saccharomyces cerevisiae hosts Generation of oligosaccharide mixtures of b1,3-Gal-b1,4-Glc

Saccharomyces cerevisiae strains, as described in Example 2, are suitable for the production of GDP-fucose and LNT and express an alpha-1,2-fucosyltransferase and an alpha-1,3-galactosyltransferase Enzymes, expressed by a first yeast plastid, comprising lactose permease LAC12 (UniProt ID P07921) from Lactobacillus kluyveromyces, GDP-mannose 4,6-dehydratase gmd from Escherichia coli ( UniProt ID P0AC88), GDP-L-fucose synthase fcl from Escherichia coli (UniProt ID P32055) and al,2-fucosyltransferase HpFutC from Helicobacter pylori (GenBank: AAD29863.1) constitutive Transcription transcription unit, and expression of plastids by a second yeast comprising UDP-glucose 4-epimerase galE from Escherichia coli (UniProt ID P09147), galactoside beta-1 from Neisseria meningitidis ,3-N-acetylglucosamine transferase IgtA (GenBank: AAM33849.1), N-acetylglucosamine β-1,3-galactosyltransferase WbgO from Escherichia coli O55:H7 (UniProt ID D3QY14) Constitutive transcription transcription unit with α-1,3-galactosyltransferase WbnI from Escherichia coli with SEQ ID NO: 03. Novel strains were evaluated in a growth experiment for 2'FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and Gal - Generation of a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) according to Example 2 The culture conditions provided in SD CSM-Ura-His drop-ou medium included lactose as a precursor. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 8. Including 2'FL, DiFL, LN3, LNT, LNFP-I and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc- with modified Saccharomyces cerevisiae hosts Generation of oligosaccharide mixtures of b1,3-Gal-b1,4-Glc

Saccharomyces cerevisiae strains, as described in Example 2, are suitable for the production of GDP-fucose and LNT and express an α-1,2-fucosyltransferase and an α-1,3-N-acetylene Galactose transaminase, expressed plastid by a first yeast including lactose permease LAC12 (UniProt ID P07921) from Lactobacillus kluyveri, 4-epimerase from Pseudomonas aeruginosa WbpP (UniProt ID Q8KN66) for UDP-GalNAc production, GDP-mannose 4,6-dehydratase gmd from E. coli (UniProt ID P0AC88), GDP-L-fucose synthase fcl from E. coli (UniProt ID P32055) with the al,2-fucosyltransferase HpFutC (GenBank: AAD29863.1) constitutive transcription unit from Helicobacter pylori, and expressed plastids by a second yeast, including UDP-glucose 4-epimerase galE from Escherichia coli (UniProt ID P09147), galactoside β-1,3-N-acetylglucosamine transferase IgtA from Neisseria meningitidis (GenBank: AAM33849.1) , N-acetylglucosamine beta-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from Escherichia coli O55:H7 and alpha-1,3-galactosyltransferase from H. mustelae with SEQ ID NO: 40 Constitutive transcription transcription unit of the N-acetylgalactosamine transferase BgtA. Novel strains were evaluated in a growth experiment for 2'FL, DiFL, LN3, LNT, LNFP-I (Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) and GalNAc - Generation of a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) according to Example 2 Culture conditions are provided in , wherein SD CSM-Ura-His drop-out medium includes lactose as a precursor. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 9. GalNAc-a1,3-(Fuc-a1,2)-Gal-b1 with modified E. coli hosts expressing α-1,2-fucosyltransferase from Brachyspira trichomes, Production of 3-GlcNAc-b1,3-Gal-b1,4-Glc

E. coli strains modified for GDP-fucose production and growth on sucrose by galactoside beta-1,3-N-acetylglucose from Neisseria meningitidis as described in Example 1 Genome of the constitutive transcription unit of transaminase LgtA (GenBank: AAM33849.1) and N-acetylglucosamine β-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from Escherichia coli O55:H7 Knock-in, further applied to LN3 and LNT generation. This strain is also suitable for UDP-GalNAc production by gene body knock-in of the constitutive transcription unit of the 4-epimerase WbpP (UniProt ID Q8KN66) from Pseudomonas aeruginosa. To generate GalNAc-LNFP-I, the novel strain was further modified by a first expression plastid comprising the composition of α-1,2-fucosyltransferase with UniProt ID A0A2N5RQ26 from Brachyspira trichomes type transcription unit, and by a second compatible expression plastid, it includes an α-1,3-N-acetylgalactosamine transferase, which is selected from including from Helicobacter mustelae having SEQ ID NO: 40 BgtA, the polypeptide with SEQ ID NO: 49 from Bacteroides ovale, the polypeptide with SEQ ID NO: 74 from Lachnospiraceae bacterium , and the sequence from Roseburia inulinivorans The polypeptide of identification number: 91 and the constitutive transcription unit of the list of polypeptides with SEQ ID NO: 102 from Bacteroides ovale. The novel strain was evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/L sucrose and 20 g/L lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC. Experiments show that all strains produce GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) In the whole broth samples, as summarized in Table 2.

Table 2. Expression of mutant E. coli strains with UniProt ID A0A2N5RQ26 of α-1,2-fucosyltransferase and an α-1,3-N-acetylgalactosyltransferase from B. pilosicus Production of GalNAc-a1,3-LNFP-I in whole broth samples, when assessed in growth experiments according to culture conditions as described in Example 1, wherein the medium contained sucrose as carbon source and lactose as precursor. Expression of α-1,3-N -acetylgalactosamine transferase in Escherichia coli mutants Production of GalNAc-a1,3-LNFP-I (g/L) Serial Identification Number: 40 0.71 ± 0.07 Serial Identification Number: 49 0.36 ± 0.36 Serial Identification Number: 74 1.30 ± 0.14 Serial Identification Number: 91 1.22 ± 0.13 Serial Identification Number: 102 0.92 ± 0.08

Example 10. GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3- of E. coli hosts modified to express one of the α-1,2-fucosyltransferases from Dysgonomonas mossii Production of GlcNAc-b1,3-Gal-b1,4-Glc

E. coli strains modified as described in Example 1 for GDP-fucose production and growth on sucrose by galactoside beta-1,3-N-acetylglucose from Neisseria meningitidis Genome of the constitutive transcription unit of transaminase LgtA (GenBank: AAM33849.1) and N-acetylglucosamine β-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from Escherichia coli O55:H7 Knock-in, further applied to LN3 and LNT generation. This strain is also suitable for UDP-GalNAc production by gene body knock-in of the constitutive transcription unit of the 4-epimerase WbpP (UniProt ID Q8KN66) from Pseudomonas aeruginosa. To generate GalNAc-LNFP-I, the novel strain was further modified by a first expression plastid comprising constitutive transcription of α-1,2-fucosyltransferase with UniProt ID F8X274 from Dysgonomonas mossii unit, with a second compatible expression plasmid comprising an alpha-1,3-N-acetylgalactosamine transferase selected from the group consisting of BgtA with SEQ ID NO: 40 from Helicobacter mustelae , a constitutive transcription unit of a list of the polypeptide having SEQ ID NO: 49 from Bacteroides ovale and the list of polypeptides having SEQ ID NO: 102 from Bacteroides ovale. The novel strain was evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/L sucrose and 20 g/L lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC. Experiments show that all strains produce GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) In the whole broth samples, as summarized in Table 3.

Table 3. Expression of mutant E. coli strains with UniProt ID F8X274 of α-1,2-fucosyltransferase and an α-1,3-N-acetylgalactosyltransferase from D. mossii Production of GalNAc-a1,3-LNFP-I in whole broth samples, when assessed in growth experiments according to culture conditions as described in Example 1, wherein the medium contains sucrose as carbon source and lactose as precursor. Expression of α-1,3-N -acetylgalactosamine transferase in Escherichia coli mutants Production of GalNAc-a1,3-LNFP-I (g/L) Serial Identification Number: 40 1.07 ± 0.12 Serial Identification Number: 49 0.80 ± 0.03 Serial Identification Number: 102 0.92 ± 0.04

Example 11. GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3- of E. coli hosts modified to express one of the α-1,2-fucosyltransferases from Dechlorosoma suillum Production of GlcNAc-b1,3-Gal-b1,4-Glc

E. coli strains modified as described in Example 1 for GDP-fucose production and growth on sucrose by galactoside beta-1,3-N-acetylglucose from Neisseria meningitidis Genome of the constitutive transcription unit of transaminase LgtA (GenBank: AAM33849.1) and N-acetylglucosamine β-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from Escherichia coli O55:H7 Knock-in, further applied to LN3 and LNT generation. This strain is also suitable for UDP-GalNAc production by gene body knock-in of the constitutive transcription unit of the 4-epimerase WbpP (UniProt ID Q8KN66) from Pseudomonas aeruginosa. To generate GalNAc-LNFP-I, the novel strain was further modified by a first expressing plastid comprising constitutive transcription of α-1,2-fucosyltransferase with UniProt ID G8QLF4 from Dechlorosoma suillum unit, with a second compatible expression plasmid comprising an alpha-1,3-N-acetylgalactosamine transferase selected from the group consisting of BgtA with SEQ ID NO: 40 from Helicobacter mustelae Constitutive transcription unit with a list of polypeptides from Bacteroides ovale with SEQ ID NO: 102. The novel strain was evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/L sucrose and 20 g/L lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC. Experiments show that all strains produce GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) In the whole broth samples, as summarized in Table 4.

Table 4. Expression of α-1,2-fucosyltransferase and an α-1,3-N-acetylgalactosyltransferase from D. suillum in mutant E. coli strains with UniProt ID G8QLF4 Production of GalNAc-a1,3-LNFP-I in whole broth samples, when assessed in growth experiments according to culture conditions as described in Example 1, wherein the medium contains sucrose as carbon source and lactose as precursor. Expression of α-1,3-N -acetylgalactosamine transferase in Escherichia coli mutants Production of GalNAc-a1,3-LNFP-I (g/L) Serial Identification Number: 40 0.23 ± 0.01 Serial Identification Number: 102 0.30 ± 0.02

Example 12. GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3- of E. coli hosts modified to express one of the α-1,2-fucosyltransferases from Desulfovibrio alaskensis Production of GlcNAc-b1,3-Gal-b1,4-Glc

E. coli strains modified as described in Example 1 for GDP-fucose production and growth on sucrose by galactoside beta-1,3-N-acetylglucose from Neisseria meningitidis Genome of the constitutive transcription unit of transaminase LgtA (GenBank: AAM33849.1) and N-acetylglucosamine β-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from Escherichia coli O55:H7 Knock-in, further applied to LN3 and LNT generation. This strain is also suitable for UDP-GalNAc production by gene body knock-in of the constitutive transcription unit of the 4-epimerase WbpP (UniProt ID Q8KN66) from Pseudomonas aeruginosa. To generate GalNAc-LNFP-I, the novel strain was further modified by a first expression plastid comprising constitutive transcription of α-1,2-fucosyltransferase with UniProt ID Q316B5 from Desulfovibrio alaskensis unit, with a second compatible expression plasmid comprising an alpha-1,3-N-acetylgalactosamine transferase selected from the group consisting of BgtA with SEQ ID NO: 40 from Helicobacter mustelae Constitutive transcription unit with a list of polypeptides from Bacteroides ovale with SEQ ID NO: 102. The novel strain was evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/L sucrose and 20 g/L lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC. Experiments show that all strains produce GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) In the whole broth samples, as summarized in Table 5.

Table 5. Expression of mutant E. coli strains expressing α-1,2-fucosyltransferase with UniProt ID Q316B5 and an α-1,3-N-acetylgalactosyltransferase from D. alaskensis Production of GalNAc-a1,3-LNFP-I in whole broth samples, when assessed in growth experiments according to culture conditions as described in Example 1, wherein the medium contains sucrose as carbon source and lactose as precursor. Expression of α-1,3-N -acetylgalactosamine transferase in Escherichia coli mutants Production of GalNAc-a1,3-LNFP-I (g/L) Serial Identification Number: 40 0.02 ± 0.01 Serial Identification Number: 102 0.40 ± 0.15

Example 13. Generation of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a modified E. coli host

Escherichia coli strains modified for GDP-fucose production and growth on sucrose as described in Example 1 by β-1,3-N-β-galactoside from Neisseria meningitidis Constitutive transcription unit of glucosyltransferase LgtA (GenBank: AAM33849.1) and N-acetylglucosamine β-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from Escherichia coli O55:H7 Gene body knock-in, further suitable for LN3 and LNT production. To produce LNFP-I, the novel strain was further modified by an expression plasmid comprising constitutive transcription of the α-1,2-fucosyltransferase HpFutC (GenBank: AAD29863.1) from Helicobacter pylori unit. In a final step to generate Gal-LNFP-I, the mutant strain was modified with a second compatible expression plastid containing a plasmid selected from the group consisting of SEQ ID NOs: 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, A constitutive transcription unit of one of the lists of 34, 35, 36 and 37 alpha-1,3-galactosyltransferase. Novel strains were evaluated in a growth experiment for Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4- The production of Glc was based on the culture conditions provided in Example 1, wherein the medium contained 30 g/L sucrose and 20 g/L lactose. This strain was grown in four biological replicates in a 96-well plate. Cultivated for 72 hours Afterwards, the culture broth was collected and analyzed for sugars by UPLC.

Example 14. Generation of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a modified E. coli host

E. coli strains modified as described in Example 3 for GDP-fucose and LNFP-I production and grown on sucrose to 4-epimerase WbpP (UniProt ) from Pseudomonas aeruginosa ID Q8KN66) was further modified for UDP-GalNAc production and transformed with a second compatible expression plastid containing a plasmid selected from the group consisting of SEQ ID NOs: 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 , 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98; 99, 100, 101 and 102. A constitutive transcription unit of one of the lists of alpha-1,3-N-acetylgalactosamine transferase. Novel strains evaluated in a growth experiment 2'FL, DiFL, LN3, LNT, LNFP-I, Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal- b1,4-Glc and GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) was produced according to the culture conditions provided in Example 1, wherein the medium contained 30 g/L sucrose and 20 g/L lactose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 15. Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc and Gal-a1,3-(Fuc-a1,2)-Gal- with a modified E. coli host Production of b1,4-(Fuc-a1,3)-Glc

E. coli strains modified for GDP-fucose production and grown on sucrose as described in Example 1 were further transformed with a first expressing plastid containing a protein from Helicobacter pylori A constitutive transcription unit of α-1,2-fucosyltransferase HpFutC (GenBank: AAD29863.1), with a second compatible expression plasmid, containing a plasmid selected from the group consisting of SEQ ID NOs: 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, A constitutive transcription unit of one of the lists of 31, 32, 33, 34, 35, 36 and 37 alpha-1,3-galactosyltransferase. Novel strains evaluated in a growth experiment Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc and Gal-a1,3-(Fuc-a1,2)-Gal-b1,4- (Fuc-a1,3)-Glc was produced according to the culture conditions provided in Example 1, in which the medium contained 30 g/L sucrose and 20 g/L lactose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 16. Inclusion of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, Gal-a1,3-(Fuc-a1,2)-Gal in a modified E. coli host Generation of oligosaccharide mixtures of -b1,4-(Fuc-a1,3)-Glc and one of 3'-SL

E. coli strains modified as described in Example 15 with gene body knockout of the nagA and nagB genes and including encoding L-glutamic acid-D-fructose-6-phosphate transaminase (glmS) from E. coli *54) (different from wild-type E. coli glmS protein by A39T, R250C and G472S mutations, with UniProt ID P17169), phosphoglucosamine mutase (glmM) from E. coli (UniProt ID P31120), from E. coli N-acetylglucosamine-1-phosphate uridine transferase/glucosamine-1-phosphate acetyltransferase (glmU) (UniProt ID P0ACC7), UDP-N-acetylglucosamine 2 from Aspergillus - Epimerase (NeuC) (UniProt ID Q93MP8), N-acetylneuraminic acid synthase (NeuB) from Neisseria meningitidis (UniProt ID EONCD4), Sialic acid transporter (nanT) from Escherichia coli (UniProt ID P41036), N-acylneuraminic acid cytidine transferase from Aspergillus (UniProt ID Q93MP7) and β-galactoside α-2,3- from Pasteurella septicemia The gene body knock-in of the constitutive transcriptional unit of the gene for the sialyltransferase PmultST3 (UniProt ID Q9CLP3) was further modified. The novel strain was evaluated in a growth experiment to contain Gal-a1,3-(Fuc-a1,2)-Gal -b1,4-Glc, Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc and 3'-sialyllactose, 3 '-SL) an oligosaccharide mixture was produced according to the culture conditions provided in Example 1, in which the medium contained 30 g/L sucrose and 20 g/L lactose. This strain was grown in four in a 96-well plate Biological replicates were grown. After 72 hours of culture, culture broth was collected and analyzed for sugars by UPLC.

Example 17. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc with a modified E. coli host

E. coli strains modified for GDP-fucose production and grown on sucrose as described in Example 1, with 4-epimerase WbpP (UniProt ID Q8KN66) from Pseudomonas aeruginosa The constitutive transcription unit was further modified for UDP-GalNAc production and transformed by a first expressing plastid containing the α-1,2-fucosyltransferase HpFutC from Helicobacter pylori ( GenBank: AAD29863.1), and a second compatible expression plasmid containing SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 and 102 One of the list of integral transcription units of α-1,3-N-acetylgalactosamine transferase. The novel strain was evaluated in a growth experiment with alpha-tetrasaccharide (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc) ,2)-Gal-b1,4-Glc)) was produced according to the culture conditions provided in Example 1, in which the culture medium contained 30 g/L sucrose and 20 g/L lactose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 18. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a modified E. coli host

E. coli strains modified for GDP-fucose production and grown on sucrose as described in Example 1, were transformed with a first expressing plastid containing a A constitutive transcription unit of the α-1,2-fucosyltransferase HpFutC (GenBank: AAD29863.1), and a second compatible expression plasmid containing a gene selected from the group consisting of SEQ ID NOs: 04, 05 , 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36, and 37. A constitutive transcription unit of an alpha-1,3-galactosyltransferase. The novel strain was evaluated for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/ L sucrose and 20 g/L lacto-N-biose (lacto-N-biose, LNB, Gal-b1,3-GlcNAc). This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 19. Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a modified E. coli host

Modified E. coli strains as described in Example 1 were further transformed with genomic knockout of the nagA and nagB genes and mutant L-glutamate-D-fructose-6-phosphate from E. coli Aminase (glmS*54) (different from wild-type E. coli glmS protein by A39T, R250C and G472S mutations, with UniProt ID P17169), glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), phosphatase BsAraL (UniProt ID P94526) from Bacillus subtilis and N-acetylglucosamine beta-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from Escherichia coli O55:H7 Genome knock-in of transcription unit for production of lacto-N-disaccharide (LNB, Gal-b1,3-GlcNAc). In the final step, the novel strain is transformed with an expression plastid containing a plasmid selected from the group consisting of SEQ ID NOs: 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, One of a list of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37 A constitutive transcription unit of -1,3-galactosyltransferase. The novel strain was evaluated for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/ L sucrose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 20. Generation of an oligosaccharide mixture of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc, sialylated LNB and 6'-SL with a modified E. coli host

E. coli strains modified as described in Example 19 with N-acetylglucosamine 2-epimerase (AGE) from Bacteroides ovale (UniProt ID A7LVG6) and from Neisseria meningitidis Genome knock-in of N-acetylneuraminic acid synthase (NeuB) (UniProt ID EONCD4) was further modified and transformed with an expression plastid comprising a set of shaped expression units including Bacillus N-acylneuraminic acid cytidyltransferase (UniProt ID Q93MP7) and PdST6 (UniProt ID 066375) from Bacillus luminescens. Novel strains evaluated in a growth experiment for LNB, Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc, sialylated LNB and 6'-sialyllactose (6'-SL) production , according to the culture conditions provided in Example 1, wherein the culture medium contains 30 g/L sucrose and 20 g/L lactose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 21. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a modified E. coli host

E. coli strain modified as described in Example 1 for GDP-fucose production and growth on sucrose by 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) The constitutive transcription unit was further modified for UDP-GalNAc production and transformed by a first expressing plastid comprising the α-1,2-fucosyltransferase HpFutC from Helicobacter pylori (GenBank: AAD29863.1), and a second compatible expression plasmid containing SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 , 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 , 100, 101 and 102, a constitutive transcription unit of alpha-1,3-N-acetylgalactosamine transferase. The novel strain was evaluated for the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/ L sucrose with 20 g/L lacto-N-disaccharide (LNB, Gal-b1,3-GlcNAc). This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 22. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a modified E. coli host

E. coli strain modified as described in Example 1 for GDP-fucose production and growth on sucrose by 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) The constitutive transcriptional unit of the - Phosphotransaminase (glmS*54) (different from wild-type E. coli glmS protein by A39T, R250C and G472S mutations, with UniProt ID P17169), phosphatase BsAraL (UniProt ID P94526) from Bacillus subtilis and from E. coli Genome knock-in of the constitutive transcription unit of N-acetylglucosamine β-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) of O55:H7 for the production of lacto-N-disaccharide (LNB, Gal-b1,3-GlcNAc). In a final step, the novel strain was further transformed with an expression plastid containing a plasmid selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 , 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 A constitutive transcription unit of α-1,3-N-acetylgalactosamine transferase with one of the list of 102. The novel strain was evaluated for the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/ L sucrose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 23. Generation of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc with a modified E. coli host

E. coli strains, modified for GDP-fucose production and grown on sucrose as described in Example 1, were further transformed with a first expressing plastid comprising a strain from Helicobacter pylori The constitutive transcription unit of the α-1,2-fucosyltransferase HpFutC (GenBank: AAD29863.1), and a second compatible expression plastid, comprising the group consisting of SEQ ID NOs: 04, 05 , 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36, and 37. A constitutive transcription unit of an alpha-1,3-galactosyltransferase. The novel strain was evaluated for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/ L sucrose and 20 g/L N-acetyllactosamine (N-acetyllactosamine, LacNAc, Gal-b1,4-GlcNAc). This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 24. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc with a modified E. coli host

E. coli strains, modified for GDP-fucose production and growth on sucrose as described in Example 1, were further transformed with genomic knockouts of the nagA and nagB genes and those from E. coli Mutant L-glutamic acid-D-fructose-6-phosphate transaminase (glmS*54) (different from wild-type E. coli glmS protein by A39T, R250C and G472S mutations, with UniProt ID P17169), from Saccharomyces cerevisiae Glucosamine 6-phosphate N-acetyltransferase GNA1 from bacteria (UniProt ID P43577), phosphatase BsAraL (UniProt ID P94526) from Bacillus subtilis and N-acetylglucosamine beta-1,4 from Neisseria meningitidis - Genome knock-in of the constitutive transcription unit of the galactosyltransferase LgtB (UniProt ID Q51116) for the production of N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc). In the final step, the novel strain is transformed with an expression plastid containing a plasmid selected from the group consisting of SEQ ID NOs: 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, One of a list of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37 A constitutive transcription unit of -1,3-galactosyltransferase. The novel strain was evaluated for the production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/ L sucrose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 25. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc with a modified E. coli host

E. coli strain modified as described in Example 1 for GDP-fucose production and growth on sucrose by composition of 4-epimerase WbpP (UniProt ID Q8KN66) from Pseudomonas aeruginosa type transcription unit was further modified for UDP-GalNAc production and transformed by a first expressing plastid containing the α-1,2-fucosyltransferase HpFutC (GenBank) from Helicobacter pylori : AAD29863.1) constitutive transcription unit, and a second compatible expression plastid, comprising the group consisting of SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 A constitutive transcription unit of alpha-1,3-N-acetylgalactosamine transferase, one of the lists of 101 and 102. The novel strain was evaluated for the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/ L sucrose with 20 g/L N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc). This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 26. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc with a modified E. coli host

E. coli strain modified as described in Example 1 for GDP-fucose production and growth on sucrose by 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) The constitutive transcription unit of the Phosphotransaminase (glmS*54) (different from wild-type E. coli glmS protein by A39T, R250C and G472S mutations, with UniProt ID P17169), glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), composition of phosphatase BsAraL (UniProt ID P94526) from Bacillus subtilis and N-acetylglucosamine beta-1,4-galactosyltransferase LgtB (UniProt ID Q51116) from Neisseria meningitidis Genome knock-in of the type transcription unit for the production of N-acetyllactosamine (LacNAc, Gal-b1,4-GlcNAc). In a final step, the novel strain was further transformed with an expression plastid containing SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, A constitutive transcription unit of alpha-1,3-N-acetylgalactosamine transferase, one of the lists of 101 and 102. The novel strain was evaluated for the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/ L sucrose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 27. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc with a modified E. coli host

E. coli strains, modified for GDP-fucose production and grown on sucrose as described in Example 1, were further transformed with α-1,2-fucobacterium from Helicobacter pylori A gene body knock-in of the constitutive transcription unit of the glycosyltransferase HpFutC (GenBank: AAD29863.1), together with a first expressing plastid, comprising alpha-1,2-fucosyltransferase from Helicobacter pylori A constitutive transcription unit of the enzyme HpFutC (GenBank: AAD29863.1), and a second compatible expression plasmid comprising the group consisting of SEQ ID NOs: 04, 05, 06, 07, 08, 09, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 A constitutive transcription unit of α-1,3-galactosyltransferase with one of the list of 37. The novel strain was evaluated for Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc production in a growth experiment according to the culture conditions provided in Example 1 , in which the medium contains 30 g/L sucrose and 20 g/L lactose. This strain was grown in four biological replicates in a 96-well plate. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 28. Materials and Methods Bacillus subtilis

culture medium

Two different media were used, a rich Luria Broth (LB) and a minimal medium for shake flasks (MMsf). Minimal medium uses a mixture of trace elements.

The trace element mixture consisted of 0.735 g/L CaCl ₂ .2H ₂ O, 0.1 g/L MnCl ₂ .2H ₂ O, 0.033 g/L CuCl ₂ .2H ₂ O, 0.06 g/L CoCl ₂ .6H ₂ O, 0.17 g /L ZnCl ₂ , 0.0311 g/LH ₃ BO ₄ , 0.4 g/L Na ₂ EDTA.2H ₂ O and 0.06 g/L Na ₂ MoO ₄ . The ferric citrate (Fe-citrate) solution contained 0.135 g/L FeCl ₃ .6H ₂ O, 1 g/L sodium citrate (Na-citrate) (Hoch 1973 PMC1212887).

Luria broth (LB) medium consisted of 1% trypsin (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). Luria Broth agar (LBA) plates consisted of LB medium with concomitant addition of 12 g/L agar (Difco, Erembodegem, Belgium).

Minimal medium (MMsf) experiments for shake flasks contained 2.00 g/L (NH ₄ ) ₂ SO ₄ , 7.5 g/L KH2PO4, 17.5 g/L K ₂ HPO ₄ , 1.25 g/L sodium citrate, 0.25 g/L MgSO4.7H2O, 0.05 _g /L tryptophan, ₁₀ to 30 g/L glucose or other carbon sources, when specified in the examples, including but not limited to fructose, maltose, sucrose, glycerol and malt Trisaccharide (maltotriose), 10 ml/L trace element mixture and 10 ml/L ferric citrate solution. The medium was set to pH 7 with 1 M KOH. Depending on the experimental lactose, LNB or LacNAc can be added as precursors.

Complex media, such as LB, are sterilized by autoclaving (121°C, 21'), while minimal media are sterilized by filtration (0.22 µm Sartorius). When necessary, the medium was made selective by adding antibiotics (eg, zeocin (20 mg/L)).

Strains, plastids and mutations

Bacillus subtilis 168, available at the Bacillus Genetic Stock Center (Ohio, USA).

Plastids were constructed for gene deletion via Cre/lox as described by Yan et al. (Appl. & Environm. Microbial., Sept 2008, p5556-5562). Gene disruption is accomplished via homologous recombination with linear DNA and transformation via electroporation as described by Xue et al. (J. Microb. Meth. 34 (1999) 183-191). Liu et al. describe a method for gene knockout (Metab. Engine. 24 (2014) 61-69). This method uses 1000 bp of homology upstream and downstream of the target gene.

The integrative vectors described by Popp et al. (Sci. Rep., 2017, 7, 15158) were used as expression vectors and further used for gene body integration if necessary. Suitable promoters for expression can be obtained from the part repository (iGem): Sequence IDs: Bba_K143012, Bba_K823000, Bba_K823002 or Bba_K823003. Cloning can be performed using Gibson assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

In one example of the production of lactose-based oligosaccharides, mutant strains of B. subtilis were created to contain a gene encoding a lactose importer (eg, E. coli lacY with UniProt ID P02920). For the production of 2'FL, 3FL and diFL, alpha-1,2- and/or alpha-1,3-fucosyltransferase expression constructs were additionally added to the strains.

In one example for the production of lacto-N-tricarbon sugars (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), Bacillus subtilis strains were loaded with lactose importers (eg, with UniProt ID Escherichia coli lacY of P02920) with galactoside β-1,3-N-acetylglucosamine transaminase, such as LgtA from Neisseria meningitidis LgtA (GenBank: AAM33849.1) one gene somatic knockout of the constitutive transcription unit retouch. For LNT production, the LN3 producing strain is further modified with a constitutive transcription unit of an N-acetylglucosamine β-1,3-galactosyltransferase, such as WbgO (UniProt ID D3QY14) from E. coli O55:H7 . In order to produce lacto-N-neotetraose (lacto- N -neotetraose, LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), a N-acetylglucosamine β-1 ,4-Galactosyltransferase (N-acetylglucosamine beta-1,4-galactosyltransferase), such as the constitutive transcription unit of LgtB (UniProt ID Q51116) from Neisseria meningitidis to modify LN3 producing strains. Both N-acetylglucosamine β-1,3-galactosyltransferase and N-acetylglucosamine β-1,4-galactosyltransferase can be knocked in by gene body or delivered from an expression plastid to the strain. In order to produce fucosylated derivatives of LNFP-1 and other LNT and/or LNnT, LNT and LNnT producing strains can be further treated with α-1,2-fucosyltransferase and/or α-1,3- Fucosyltransferase expression constructs were modified.

Also, mutant strains can be α-1,3-galactosyltransferase, such as SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 of the constitutive transcription units were modified. Alternatively and/or additionally, the mutant strain may be alpha-1,3-N-acetylgalactosaminyltransferase, eg SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 6, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 , 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 101 or 102 constitutive transcription unit modifications.

For sialic acid production, the intracellular glucosamine-6-phosphate pool was enhanced by overexpressing native fructose-6-P-aminotransferase (UniProt ID POCI73) to generate mutant B. subtilis strains (pool). In addition, the enzymatic activities of the nagA, nagB and gamA genes were disrupted by gene knockout with glucosamine-6-P-aminotransferase from Saccharomyces cerevisiae (UniProt ID P43577), from Genome overexpression of N-acetylglucosamine 2-epimerase from Bacteroides ovale (UniProt ID A7LVG6) and N-acetylneuraminic acid synthase from Aspergillus ovale (UniProt ID Q93MP9) . For sialylated oligosaccharide production, the sialic acid producing strain was further modified with an expression construct including the N-acylneuraminic acid cytidine transferase NeuA (UniProt ID Q93MP7) from Aspergillus, with β-galactoside α-2,3-sialyltransferase (beta-galactoside alpha-2,3-sialyltransferase), such as PmultST3 (UniProt ID Q9CLP3) from Pasteurella PmultST3-like polypeptide (PmultST3-like polypeptide) composed of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with galactoside α-2,3-sialyltransferase activity, NmeniST3 from Neisseria meningitidis ( GenBank NO. ARC07984.1) or PmultST2 from Pasteurella septicemia subsp. multocida str. Pm70 (GenBank NO. AAK02592.1), a β-galactoside α-2,6-sialyltransferase ( beta-galactoside alpha-2,6-sialyltransferase), such as PdST6 from Photobacterium damselae (UniProt ID 066375) or from UniProt ID 066375 with beta-galactoside alpha-2,6-sialyltransferase activity A PdST6-like polypeptide consisting of amino acid residues 108 to 497 of PdST6-like polypeptide or P-JT-ISH-224-ST6 from Photobacterium sp. JT-ISH-224 (UniProt ID A8QYL1) or P-JT-ISH-224-ST6-like polyamide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 with β-galactoside α-2,6-sialyltransferase activity Peptides, and/or alpha-2,8-sialyltransferase, eg from one or more copies of M. musculus (UniProt ID Q64689).

Heterologous vs. Homologous Expression

Genes to be expressed, whether from plastids or gene bodies, are synthesized by one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

Optimizing codon usage according to the codon usage of the expression host can further facilitate expression. Use the vendor's tools to optimize genes.

Culture conditions

Pre-incubation for 96-well microtiter plate experiments starts with a cryovial or a single colony from an LB plate in 150 µL of LB and on an orbital shaker at 800 rpm at 37°C Incubate overnight. Use this culture as an inoculum in a 96-well square microtiter plate by adding 400 µL of MMsf medium at a dilution of 400x. Each strain was grown in multiple wells of a 96-well plate as biological replicates. These final 96-well plates were then incubated at 37°C on an orbital shaker at 800 rpm for 72 hours, or shorter or longer. At the end of the incubation experiment, samples were taken from each well to measure the supernatant concentration (extracellular sugar concentration, cells were spun down after 5 minutes), or by boiling the medium at 90°C for 15 minutes or Boil for 60 minutes at 60°C, then spin down the cells (= whole broth concentration, intracellular and extracellular sugar concentration, as defined here).

Again, culture dilutions were performed to measure optical density at 600 nm. The cell performance index or CPI was determined by dividing the oligosaccharide concentration by the biomass as a relative percentage compared to a reference strain. Biomass was empirically determined to be approximately 1/3 the optical density measured at 600 nm.

Example 29. Generation of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a modified Bacillus subtilis host

First, a B. subtilis strain was modified for LN3 production and growth on sucrose by gene body knockout of the nagB, glmS, and gamA genes and inclusion of the encoding lactose permease (LacY) from E. coli (UniProt ID P02920 ), native fructose-6-P-transaminase (UniProt ID P0CI73), galactoside β-1,3-N-acetylglucosamine transferase LgtA from Neisseria meningitidis (GenBank: AAM33849.1) , sucrose transporter (CscB) from Escherichia coli W (UniProt ID EOIXR1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417) and sucrose phosphorylase BaSP from Bifidobacterium adolescentis (UniProt Gene body knock-in of the constitutive transcription unit of the gene of ID A0ZZH6). In the next step, the mutant strain was further modified by means of a constitutive transcription unit comprising N-acetylglucosamine beta-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from E. coli O55:H7 Gene body knock-in to generate LNT. In a subsequent step, the LNT-producing strain is transformed with an expression plastid comprising HpFutC (GenBank: AAD29863.1) from Helicobacter pylori, Brachyspira pilosicoli Polypeptide (UniProt ID A0A2N5RQ26), Polypeptide from Dysgonomonas mossii (UniProt ID F8X274), Polypeptide from Dechlorosoma suillum (UniProt ID G8QLF4), Polypeptide from Desulfovibrio alaskensis ( UniProt ID Q316B5 ) and Polypeptide from Polaribacter List of polypeptides of vadi (UniProt ID A0A1B8TNT0) - alpha-1,2-fucosyltransferase (alpha-1,2-fucosyltransferase), and selected from the group consisting of SEQ ID NOs: 04, 05, 06, 07 , 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36 and 37. A constitutive transcription unit of an alpha-1,3-galactosyltransferase. Novel strains including LN3, LNFP-I and Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)- An oligosaccharide mixture of Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) was produced according to the culture conditions provided in Example 28. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 30. Generation of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc with a modified Bacillus subtilis host

In this example, as described in Example 29, B. subtilis strains were modified for LNT production and growth on sucrose. In the next step, the mutant LNT-producing strain was further modified with a constitutive transcription unit of the 4-epimerase WbpP (UniProt ID Q8KN66) from Pseudomonas aeruginosa for the production of UDP-GalNAc, and expressed a qualitative Body transformation, the expression plasmid includes HpFutC (GenBank: AAD29863.1) from Helicobacter pylori, a polypeptide from Brachyspira trichomes (UniProt ID A0A2N5RQ26), a polypeptide from Dysgonomonas mossii (UniProt ID F8X274), a polypeptide from Dechlorosoma suillum (UniProt ID G8QLF4), a polypeptide from Desulfovibrio alaskensis ( UniProt ID Q316B5 ) and a polypeptide from Polaribacter vadi (UniProt ID A0A1B8TNT0) List one alpha-1,2 - a fucosyltransferase, selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, One of the list of 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 and 102 alpha-1,3-N - Constitutive transcription unit of acetylgalactosamine transferase. GalNAc-a1,3-LNFP-I (GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc) was evaluated in a growth experiment with the novel strain in MMsf medium including lactose as a precursor - production of b1,3-Gal-b1,4-Glc) according to the culture conditions provided in Example 28. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 31. Including LNT, sialylated LN3, LSTa and GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1 with a modified Bacillus subtilis host , Production of an Oligosaccharide Mixture of 4-Glc

The mutant B. subtilis strain producing GalNAc-a1,3-LNFP-I as described in Example 30 was further modified by genomic knockout of the nagA gene with a second compatible expression plastid, which expressed plastids Includes TRP1 selectable marker and mutant L-glutamic acid-D-fructose-6-phosphate transaminase (glmS*54) from E. coli (different from wild-type E. coli glmS with UniProt ID P17169 by A39T , R250C and G472S mutations, as described by Deng et al. (Biochimie 2006, 88: 419-429)), monophosphatase, for example selected from the group consisting of aphA, Cof, HisB, OtsB, Sure, Yaed, YcjU, YedP, YfbT , YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU E. coli genes or from Pseudomonas sputum putida ), ScDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis , phosphatase as described in WO1812222, N-acetylglucosamine 2-epimerase from Bacteroides ovale (AGE) (UniProt ID A7LVG6), N-Acetylneuraminic acid synthase (NeuB) from Neisseria meningitidis (UniProt ID EONCD4), N-Acetylneuraminic acid cytidine transfer from Haemophilus influenzae Two replicates of the enzyme NeuA (GenBank NO. AGV11798.1) and three replicates of the PmultST3 polypeptide from Pasteurella septicemia (UniProt ID Q9CLP3) are constitutive transcription units. Novel strains including LN3, sialylated LN3, LNT, LNFP-I, 2'-FL, GalNAc-a1, 3-LNFP-I (GalNAc-a1) were evaluated in a growth experiment in MMsf medium including lactose as a precursor. ,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), 3'-SL and LSTa (Neu5Ac-a2,3-Gal-b1,3 - Generation of an oligosaccharide mixture of GlcNAc-b1,3-Gal-b1,4-Glc) according to the culture conditions provided in Example 28. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 32. Generation of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc with a modified Bacillus subtilis host

A B. subtilis strain was modified for 2'-FL production by lactose permease (LacY) (UniProt ID P02920) from E. coli and alpha-1,2 from Helicobacter pylori as described in Example 28 - Genome knock-in of the constitutive transcription unit of the fucosyltransferase HpFutC (GenBank: AAD29863.1). In the next step, the mutant strain is transformed with an expression plastid containing an expression plasmid selected from the group consisting of SEQ ID NOs: 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, One of a list of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37 - Constitutive transcription unit of 1,3-galactosyltransferase. Novel strains were evaluated for Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc production in a growth experiment on MMsf medium including lactose as a precursor, as provided in Example 28 the cultivation conditions. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 33. Generation of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc with a modified Bacillus subtilis host

A B. subtilis strain, transformed as described in Example 28, was transformed with an expression plasmid comprising the alpha-1,2-fucosyltransferase HpFutC from Helicobacter pylori (GenBank: AAD29863. 1), 4-epimerase WbpP (UniProt ID Q8KN66) from Pseudomonas aeruginosa for the production of UDP-GalNAc, and selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, Constitutive transcription unit of one of the lists of 97, 98, 99, 100, 101 and 102 alpha-1,3-N-acetylgalactosamine transferase. Novel strains were evaluated for GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc production in a growth experiment in MMsf medium including lactose as a precursor, as provided in Example 28 the cultivation conditions. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 34. Materials and Methods Corynebacterium glutamicum

culture medium

Two different media were used, a rich tryptone-yeast extract (TY) and minimal media for shake flasks. Minimal medium uses a 1000x stock trace element mix.

The trace element mixture consists of 10 g/L CaCl ₂ , 10 g/L FeSO ₄ .7H ₂ O, 10 g/L MnSO ₄ .H ₂ O, 1 g/L ZnSO ₄ .7H ₂ O, 0.2 g/L CuSO ₄ , 0.02 g/L NiCl ₂ .6H ₂ O, 0.2 g/L biotin (pH 7.0) and 0.03 g/L protocatechuic acid.

TY medium consisted of 1.6% trypsin (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). TY agar (TYA) plates consisted of TY medium with concomitant addition of 12 g/L agar (Difco, Erembodegem, Belgium).

Complex media, such as TY, are sterilized by autoclaving (121°C, 21'), while minimal media are sterilized by filtration (0.22 µm Sartorius). When necessary, the medium is made selective by the addition of antibiotics (eg, kanamycin, ampicillin).

Strains and Mutations

Corynebacterium glutamicum ATCC 13032, available from the American Type Culture Collection.

The integrated plastid vector based on Cre/loxP technology described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 Apr, 67(2):225-33) and the temperature-sensitive shuttle vector described by Okibe et al. (temperature- sensitive shuttle vectors) (Journal of Microbiological Methods 85, 2011, 155-163) were constructed for gene deletion, mutation and insertion. Suitable promoters for (heterologous) gene expression can be obtained from Yim et al. (Biotechnol. Bioeng., 2013 Nov, 110(11):2959-69). Colonization can be performed using Gibson assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

In one example of the production of lactose-based oligosaccharides, mutant strains of C. glutamicum were created to contain a gene encoding a lactose importer (eg, E. coli lacY with UniProt ID P02920). For the production of 2'FL, 3FL and diFL, alpha-1,2- and/or alpha-1,3-fucosyltransferase expression constructs were additionally added to the strains.

In an example of the production of lacto-N-tricarbon sugars (LNT-II, LN3, GlcNAc-b1,3-Gal-b1,4-Glc), C. glutamicum strains were , Escherichia coli lacY with UniProt ID P02920) with a constitutive transcription unit of galactoside β-1,3-N-acetylglucosamine transaminase, such as LgtA (GenBank: AAM33849.1) from Neisseria meningitidis A gene body knock-in modification. For LNT production, the LN3 producing strain is modified with a constitutive transcription unit of an N-acetylglucosamine beta-1,3-galactosyltransferase, such as WbgO (UniProt ID D3QY14) from E. coli O55:H7. In order to produce lacto-N-neotetraose (lacto- N -neotetraose, LNnT, Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc), a N-acetylglucosamine β-1 ,4-Galactosyltransferase (N-acetylglucosamine beta-1,4-galactosyltransferase), such as the constitutive transcription unit of LgtB (UniProt ID Q51116) from Neisseria meningitidis to modify LN3 producing strains. Both N-acetylglucosamine β-1,3-galactosyltransferase and N-acetylglucosamine β-1,4-galactosyltransferase can be knocked in by gene body or delivered from an expression plastid to the strain. To produce LNFP-1, the LNT-producing strain can be further modified with an α-1,2-fucosyltransferase expression construct.

Also, mutant strains can be α-1,3-galactosyltransferase, such as SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 of the constitutive transcription units were modified. Alternatively and/or additionally, the mutant strain may be alpha-1,3-N-acetylgalactosaminyltransferase, eg SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 6, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 , 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 101 or 102 constitutive transcription unit modifications.

For sialic acid production, intracellular glucosamine- 6-Phosphate pool. In addition, the enzymatic activities of the nagA, nagB and gamA genes were disrupted by gene knockout with glucosamine-6-P-aminotransferase from Saccharomyces cerevisiae (UniProt ID P43577), from Genome overexpression of N-acetylglucosamine 2-epimerase from Bacteroides ovale (UniProt ID A7LVG6) and N-acetylneuraminic acid synthase from Aspergillus ovale (UniProt ID Q93MP9) . For sialylated oligosaccharide production, the sialic acid producing strain was further modified with an expression construct comprising the N-acylneuraminic acid cytidyltransferase NeuA enzyme from Aspergillus (UniProt ID Q93MP7) , NeuA enzyme from Haemophilus influenzae (GenBank NO. AGV11798.1) or NeuA enzyme from Pasteurella septicemia (GenBank NO. AMK07891.1), with β-galactoside α-2,3- A sialyltransferase (beta-galactoside alpha-2,3-sialyltransferase), such as PmultST3 (UniProt ID Q9CLP3) from Pasteurella septicaemia or by alpha-2,3-sialyltransferase with β-galactoside Enzymatically active PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3, NmeniST3 from Neisseria meningitidis (GenBank NO. ARC07984.1) or from P. septicaemia PmultST2 of subsp. multocida str. Pm70 (GenBank NO. AAK02592.1), beta-galactoside alpha-2,6-sialyltransferase (beta-galactoside alpha-2,6-sialyltransferase), For example PdST6 from Photobacterium damselae (UniProt ID 066375) or consisting of amino acid residues 108 to 497 of UniProt ID 066375 with beta-galactoside alpha-2,6-sialyltransferase activity PdST6-like polypeptide (PdST6-like polypeptide) or P-JT-ISH-224-ST6 (UniProt ID A8QYL1) from Photobacterium sp. A P-JT-ISH-224-ST6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 with glycosidic α-2,6-sialyltransferase activity, and/or α-2,8- A sialyltransferase (alpha-2,8-sialyltransferase), eg from one or more copies of M. musculus (UniProt ID Q64689).

Heterologous vs. Homologous Expression

Genes to be expressed, whether from plastids or gene bodies, are synthesized by one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression can be further facilitated by optimizing codon usage for the codon usage of the expression host. Use the vendor's tools to optimize genes.

Culture conditions

Pre-cultures for 96-well microtiter plate experiments were initiated from a cryovial or a single colony from a TY plate in 150 µL of TY and incubated overnight at 37°C on an orbital shaker at 800 rpm. Use this culture as an inoculum in a 96-well square microtiter plate by adding 400 µL of MMsf medium at a dilution of 400x. Each strain was grown in multiple wells of a 96-well plate as biological replicates. These final 96-well plates were then incubated at 37°C on an orbital shaker at 800 rpm for 72 hours, or shorter or longer. At the end of the incubation experiment, samples were taken from each well to measure the supernatant concentration (extracellular sugar concentration, cells were spun down after 5 minutes), or by boiling the medium at 90°C for 15 minutes or Boil for 60 minutes at 60°C, then spin down the cells (= whole broth concentration, intracellular and extracellular sugar concentration, as defined here).

Again, culture dilutions were performed to measure optical density at 600 nm. The Cell Performance Index or CPI was determined by dividing the oligosaccharide concentration by the biomass as a relative percentage compared to a reference strain. Biomass was empirically determined to be approximately 1/3 the optical density measured at 600 nm.

Example 35. Including LN3, LNT, LNFP-I, 2'-FL and Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a modified Corynebacterium glutamicum Generation of an oligosaccharide mixture of -b1,3-Gal-b1,4-Glc

First, a strain of C. glutamicum was modified for LN3 production and growth on sucrose by gene body knockout of the ldh , cgl2645 and nagB genes and inclusion of a lactose permease (LacY) encoding from Escherichia coli (UniProt ID P02920), native fructose-6-P-transaminase (UniProt ID Q8NND3), galactoside β-1,3-N-acetylglucosamine transferase LgtA from Neisseria meningitidis (GenBank: AAM33849.1), sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), fructokinase (Frk) from Z. mobilis (UniProt ID Q03417) and sucrose phosphorylation from Bifidobacterium adolescentis Gene body knock-in of the constitutive transcriptional unit of the gene for the enzyme BaSP (UniProt ID A0ZZH6). In the next step, the mutant strain was further modified by means of a constitutive transcription unit comprising N-acetylglucosamine beta-1,3-galactosyltransferase WbgO (UniProt ID D3QY14) from E. coli O55:H7 Gene body knock-in to generate LNT. In a subsequent step, the LNT-producing strain is transformed with an expression plastid comprising HpFutC from Helicobacter pylori (GenBank: AAD29863.1) and SEQ ID NOs: 04, 05 , 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36 and 37. A constitutive transcription unit of an alpha-1,3-galactosyltransferase. Novel strains including LN3, LNT, LNFP-I, 2'-FL and Gal-a1,3-LNFP-I (Gal-a1,3-( An oligosaccharide mixture of Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc) was produced according to the culture conditions provided in Example 34. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 36. Including LN3, sialylated LN3, LNT, 3'-SL, LSTa, 2'-FL and Gal-a1,3-(Fuc-a1,2)- Generation of an Oligosaccharide Mixture of Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc

The modified Corynebacterium glutamicum as described in Example 34 was further modified by gene body knockout of the gamA and nagA genes together with the inclusion of native fructose-6-P-transaminase (UniProt ID Q8NND3) from Glucosamine-6-P-transaminase from Saccharomyces cerevisiae (UniProt ID P43577), N-acetylglucosamine 2-epimerase from Bacteroides ovale (UniProt ID A7LVG6) and Genome knock-in of the constitutive transcription unit of Bacillus N-acetylneuraminic acid synthase (UniProt ID Q93MP9). In the next step, the mutant strain is transformed with a compatible expression plastid comprising the gene encoding the NeuA enzyme (UniProt ID Q93MP7) from Aspergillus spp. and the gene encoding the enzyme from Pasteurella septicemia Constitutive transcription unit of the gene for the β-galactoside α-2,3-sialyltransferase PmultST3 (UniProt ID Q9CLP3). Novel strains including LN3, sialylated LN3, LNT, LSTa, LNFP-I, 2'-FL, 3'-SL and Gal-a1,3- were evaluated in a growth experiment in MMsf medium including lactose as a precursor Production of one oligosaccharide mixture of LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), according to Example 34 culture conditions provided in. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 37. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a modified Corynebacterium glutamicum

First, a wild-type C. glutamicum strain was modified by gene body knockout of the C. glutamicum genes ldh , cgl2645 , nagB , and glmS , along with the inclusion of a sucrose transporter (CscB) encoding a sucrose transporter from Escherichia coli W ) (UniProt ID E0IXR1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417) and sucrose phosphorylase BaSP from Bifidobacterium adolescentis (UniProt ID A0ZZH6), native fructose-6-P- Transaminase (UniProt ID Q8NND3), mutant L-glutamic acid-D-fructose-6-phosphate transaminase glmS*54 from E. coli (different from wild-type E. coli glmS protein, with UniProt ID P17169, borrowed from Mutated by A39T, R250C and G472S), glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), phosphatase BsAraL from Bacillus subtilis (UniProt ID P94526) and from Escherichia coli O55: Genome knock-in of the constitutive transcriptional unit of H7's WbgO (UniProt ID D3QY14) to generate LNB. In the next step, the mutant strain is transformed with an expression plastid comprising the α-1,2-fucosyltransferase HpFutC (GenBank: AAD29863.1) from Helicobacter pylori and selected from the group consisting of Serial ID: Serial ID: 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, Gene body of a constitutive transcription unit of the gene for alpha-1,3-galactosyltransferase, one of the lists of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37 Type in. Novel strains were evaluated for Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc production in a growth experiment in MMsf medium including lactose as a precursor, as provided in Example 34 the cultivation conditions. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 38. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc with a modified Corynebacterium glutamicum

First, a wild-type C. glutamicum strain was modified by gene somatic knockout of the C. glutamicum genes ldh , cgl2645 , nagB , and glmS , along with the inclusion of a sucrose transporter (CscB) encoding a sucrose transporter from Escherichia coli W ) (UniProt ID E0IXR1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417) and sucrose phosphorylase BaSP from Bifidobacterium adolescentis (UniProt ID A0ZZH6), WbpP from Pseudomonas aeruginosa ( UniProt ID Q8KN66), native fructose-6-P-transaminase (UniProt ID Q8NND3), mutant L-glutamate-D-fructose-6-phosphate transaminase glmS*54 from E. coli (different from wild Escherichia coli glmS protein with UniProt ID P17169 with A39T, R250C and G472S mutations), glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), phosphatase from Bacillus subtilis Genome knock-in of BsAraL (UniProt ID P94526) with the constitutive transcription unit of WbgO (UniProt ID D3QY14) from E. coli O55:H7 to generate LNB. In the next step, the mutant strain is transformed with an expression plastid comprising the α-1,2-fucosyltransferase HpFutC (GenBank: AAD29863.1) from Helicobacter pylori and selected from the group consisting of Serial identification numbers: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, Composition of the gene for alpha-1,3-N-acetylgalactosamine transferase, one of the lists of 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 and 102 Genome knock-in of type transcription unit. Novel strains were evaluated for GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc production in a growth experiment in MMsf medium including lactose as a precursor, as provided in Example 34 the cultivation conditions. After 72 hours of incubation, the culture broth was collected and analyzed for sugars by UPLC.

Example 39. Materials and Methods Chlamydomonas reinhardtii

culture medium

Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7.0). TAP medium uses 1000x stock Hutner's trace element mix. Hutner's trace element mixture includes 50 g/L Na ₂ EDTA.H ₂ O (Titriplex III), 22 g/L ZnSO ₄ .7H ₂ O, 11.4 g/LH ₃ BO ₃ , 5 g/L MnCl ₂ .4H ₂ O , 5 g/L FeSO ₄ .7H ₂ O, 1.6 g/L CoCl ₂ .6H ₂ O, 1.6 g/L CuSO ₄ .5H ₂ O and 1.1 g/L (NH ₄ ) ₆ MoO ₃ .

TAP medium contains 2.42 g/L Tris (Tris (tris(hydroxymethyl)aminomethane)), 25 mg/L salt stock solution, 0.108 g/LK ₂ HPO ₄ , 0.054 g/L KH ₂ PO ₄ with 1.0 mL/L glacial acetic acid. The salt stock solution consisted of 15 g/L _NH4CL , _{4 g} /L MgSO4.7H2O and _{2 g} /L CaCl2.2H2O. As precursors for carbohydrate synthesis, precursors such as galactose, glucose, fructose, fucose, GlcNAc, LNB and/or LacNAc can be added. The medium was sterilized by autoclaving (121°C, 21'). For stock cultures on agar slants, TAP medium containing 1% agar (purified high strength, 1000 g/cm ² ) was used.

Strains, plastids and mutations

Chlamydomonas reinhardtii wild type strain 21gr (CC-1690, wild type, mt+), 6145C (CC-1691, wild type, mt-), CC-125 (137c, wild type, mt+), CC-124 (137c, wild type) type, mt−), available from the University of Minnesota (U.S.A) Chlamydomonas Resource Center (https://www.chlamycollection.org).

Expression plastids were derived from pSI103, available from the Chlamydomonas Resource Center. Colonization can be performed using Gibson assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. Suitable promoters for (heterologous) gene expression can be derived, for example, from Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted gene modifications (such as gene knockouts or gene replacements) can be performed using Crispr-Cas technology, for example as described by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469).

Transformation was performed by electroporation as described by Wang et al. (Biosci. Rep. 2019, 39: BSR2018210). Cells were grown in liquid TAP medium under constant aeration and continuous light with a light intensity of 8000 Lx until cell density reached 1.0-2.0 x ¹⁰⁷ cells/mL. Cells were then seeded into fresh liquid TAP medium at a concentration of 1.0 x ¹⁰⁶ cells/mL and grown under continuous light for 18-20 hours until the cell density reached 4.0 x ¹⁰⁶ cells/mL. Next, cells were harvested via centrifugation at 1250 g for 5 minutes at room temperature, washed and resuspended in pre-chilled liquid TAP medium containing 60 mM sorbitol (Sigma, USA), and frozen for 10 minutes. Then, 250 µL of the cell suspension (corresponding to 5.0 x 10 ⁷ cells) was placed in a pre-chilled 0.4 cm electroporation cuvette with 100 ng of plastid DNA (400 ng/mL). Electroporation was performed using a BTX ECM830 electroporation device (1575 Ω, 50 μFD) with 6 pulses of 500 V, each with a pulse length of 4 ms and a pulse interval of 100 ms. Immediately after electroporation, place the cuvette on ice for 10 minutes. Finally, the cell suspension was transferred to a 50 ml conical centrifuge tube containing 10 mL of fresh liquid TAP medium with 60 mM sorbitol to recover overnight in dim light with slow shaking. After overnight recovery, cells were re-harvested and plated by the starch embedding method to a selective 1.5% (w/ v) On agar-TAP plates. Plates were then incubated at 23+-0.5°C under continuous illumination with a light intensity of 8000 Lx. Cells were analyzed after 5-7 days.

In one example of UDP-galactose production, C. reinhardtii cells were modified by including encoding galactokinase (KIN, UniProt ID Q9SEE5) from Arabidopsis thaliana and UDP- Transcription unit of the gene for UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1).

In one example of production of UDP-N-acetylgalactosamine, Chlamydomonas reinhardtii cells were treated with UDP-N-acetylglucosamine 4-epimerase wbpP from Pseudomonas aeruginosa serotype O6 (UniProt ID Q8KN66) a transcription unit modification.

In one example of LNB production, the C. reinhardtii cells modified to produce UDP-galactose are further to include N-acetylglucosamine beta-1,3-galactosyltransferase from E. coli O55:H7 An expression of the transcriptional unit of WbgO (UniProt ID D3QY14) was modified with a plastid. In one example of LacNAc production, the C. reinhardtii cells modified to produce UDP-galactose are further to include N-acetylglucosamine beta-1,4-galactosyltransferase LgtB from Neisseria meningitidis ( UniProt ID Q51116) of the transcription unit is modified by a presentation plastid.

In addition, the mutant Chlamydomonas reinhardtii cells can be modified with an expression vector comprising an α-1,2-fucosyltransferase, an α-1,3-fucosyltransferase, selected from Include serial identification numbers: 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37 one of the lists of alpha-1,3-galactosyltransferase and/or an alpha-1,3-N-beta Galactosamine transferase, e.g. SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, Transcription units of 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102.

In an example of CMP-sialic acid synthesis, Chlamydomonas reinhardtii cells were modified by a UDP-N-acetylglucosamine-2-epimerase/N - N-acetylmannosamine kinase, such as GNE from Homo sapiens (UniProt ID Q9Y223) or a mutant form of a human GNE polypeptide including the R263L mutation, an N-acetylneuraminic acid N-acylneuraminate-9-phosphate synthetase, such as NANS (UniProt ID Q9NR45) and N-acylneuraminate cytidylyltransferase from Homo sapiens, For example the constitutive transcription unit of CMAS (UniProt ID Q8NFW8) from Homo sapiens. In one example for the production of sialylated oligosaccharides, C. reinhardtii cells are modified with a CMP-sialic acid transporter, such as CST (UniProt ID Q61420) from mouse ( Mus musculus ), and selected from species such as A Golgi-localised sialyltransferase (Golgi-localised sialyltransferase), one of Homo sapiens, house mouse, and brown rat ( Rattus norvegicus ).

Heterologous vs. Homologous Expression

Genes to be expressed, whether from plastids or gene bodies, are synthesized by one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.

Expression can be further facilitated by optimizing codon usage for the codon usage of the expression host. Use the vendor's tools to optimize genes.

Culture conditions

Chlamydomonas reinhardtii cells were cultured on selective TAP-agar plates at 23 +/- 0.5°C under a 14/10 h light/dark cycle with a light intensity of 8000 Lx. Cells were analyzed after 5 to 7 days in culture.

For high-density culture, cells can be cultured in closed systems, such as vertical or horizontal tube photobioreactors, stirred tank photobioreactors, or flat panel photobioreactors photobioreactors), as described by Chen et al. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827).

Example 40. Production of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc in a mutant Chlamydomonas reinhardtii cell

Chlamydomonas reinhardtii cells were engineered as described in Example 39 to produce UDP-Gal by including Arabidopsis encoding galactokinase (KIN, UniProt ID Q9SEE5) and UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1) Genome knock-in of constitutive transcriptional units of genes. In the next step, the mutant strain was transformed with an expression plastid including N-acetylglucosamine β-1,4-galactosyltransferase LgtB from Neisseria meningitidis (UniProt ID Q51116 ), α-1,2-fucosyltransferase HpFutC (GenBank: AAD29863.1) from Helicobacter pylori and selected from the group consisting of SEQ ID NOs: 04, 05, 06, 07, 08, 09, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 Constitutive transcription unit of alpha-1,3-galactosyltransferase with one of the list of 37. The novel algal strains were evaluated in a culture experiment on TAP-agar plates including lactose and GlcNAc as precursors, according to the culture conditions provided in Example 39. After 5 hours of culture, cells were harvested and analyzed by UPLC for Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc production.

Example 41. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc in a mutant Chlamydomonas reinhardtii cell

Chlamydomonas reinhardtii cells were engineered as described in Example 39 to produce UDP-Gal by including Arabidopsis encoding galactokinase (KIN, UniProt ID Q9SEE5) and UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I1) Genome knock-in of constitutive transcriptional units of genes. In the next step, the mutant strain was transformed with an expression plastid including N-acetylglucosamine β-1,4-galactosyltransferase LgtB from Neisseria meningitidis (UniProt ID Q51116 ), α-1,2-fucosyltransferase HpFutC from Helicobacter pylori (GenBank: AAD29863.1), 4-epimerase WbpP from Pseudomonas aeruginosa (UniProt ID Q8KN66) for UDP -GalNAc produced, and selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, One of the lists of 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 and 102 Transcription unit of lactosamine transferase. The novel algal strains were evaluated in a culture experiment on TAP-agar plates containing lactose and GlcNAc as precursors, according to the culture conditions provided in Example 39. After 5 hours of culture, cells were harvested and analyzed by UPLC for GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-GlcNAc production.

Example 42. Materials and Methods Animal Cells

Isolation of mesenchymal stem cells from adipose tissue of different mammals

Fresh adipose tissue is obtained from slaughterhouses (eg, cattle, pigs, sheep, chickens, ducks, catfish, snakes, frogs) or liposuction (eg, after informed consent in the case of humans) and preserved in phosphate supplemented with antibiotics in buffered saline. Enzymatic digestion of adipose tissue followed by centrifugation to isolate mesenchymal stem cells. The isolated mesenchymal stem cells are transferred to cell culture flasks and grown under standard growth conditions, such as 37°C, 5% _CO . Initial media included DMEM-F12, RPMI and Alpha-MEM media (supplemented with 15% fetal bovine serum) and 1% antibiotics. After the first passage, the medium was subsequently replaced with 10% FBS (fetal bovine serum) supplemented medium. For example, Ahmad and Shakoori (2013, Stem Cell Regen Med. 9(2): 29-36), which are hereby incorporated by reference in their entirety for all purposes, describe certain variations of the methods described herein. example.

Isolation of leaf-lineage stem cells from milk

This example illustrates the isolation of leaf-line stem cells under sterile conditions from milk collected from humans or any other mammal, as described herein. An equal volume of phosphate buffered saline was added to the diluted milk and centrifuged for 20 minutes. Cell pellets were washed three times with phosphate buffered saline, then cells were seeded in cell culture flasks in DMEM-F12, RPMI and Alpha-F12 supplemented with 10% fetal bovine serum and 1% antibiotics under standard culture conditions. in MEM medium. For example, Hassiotou et al. (2012, Stem Cells. 30(10): 2164-2174), which is hereby incorporated by reference in its entirety for all purposes, describe certain variations of the methods described herein. example.

Stem Cell Differentiation Using 2D and 3D Culture Systems

Isolated mesenchymal cells can be differentiated into mammary-like epithelial and luminal cells in 2D and 3D culture systems. See, eg, Huynh et al. 1991. Exp Cell Res. 197(2): 191-199; Gibson et al. 1991, In Vitro Cell Dev Biol Anim. 27(7): 585-594; Blatchford et al. 1999; Animal Cell Technology': Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310(5 ): C348 - C356; each of which is hereby incorporated by reference in its entirety for all purposes.

For 2D cultures, isolated cells were initially seeded in culture dishes in growth medium supplemented with 10 ng/ml epithelial growth factor and 5 pg/ml insulin. At confluence, supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/ml penicillin, 100 ug/ml streptomycin) and 5 pg/ml insulin of growth medium to feed the cells for 48 hours. To induce differentiation, cells containing 5 pg/ml insulin, 1 pg/ml hydrocortisone, 0.65 ng/ml triiodothyronine, 100 nM dexamethasone and 1 pg/ml Complete growth medium with prolactin feeds cells. After 24 hours, serum was removed from the complete induction medium.

For 3D cultures, isolated cells were trypsinized, cultured for 6 days in Matrigel, hyaluronic acid, or ultra-low attachment surface culture plates, and supplemented with Growth medium with 10 ng/ml epithelial growth factor and 5 pg/ml insulin induces differentiation with lactate. When the plate is full, add growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/ml penicillin, 100 ug/ml streptomycin) and 5 pg/ml insulin Feed cells for 48 hours. To induce differentiation, cells were fed with complete growth medium containing 5 pg/ml insulin, 1 pg/ml hydrocorticosterone, 0.65 ng/ml triiodothyronine, 100 nM dexamethasone and 1 pg/ml prolactin. After 24 hours, serum was removed from the complete induction medium.

Method for making mammary gland-like cells

Mammalian cells were brought into induced pluripotency by reprogramming with viral vectors encoding Oct4, Sox2, Klf4 and c-Myc. The resulting reprogrammed cells were then cultured in Mammocult medium (available from Stem Cell Technologies) or mammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone ), heparin, corticosterone, insulin, EGF) to make it mammary-like, from which the performance of the selection of milk components can be induced. Alternatively, epigenetic remodelling is performed using a remodeling system, such as CRISPR/Cas9, to activate a selection gene of interest, such as casein, a-lactalbumin, constitutively ( constitutively on) to allow expression of their respective proteins, and/or down-regulation and/or knock-out of selected endogenous genes, as described in WO21067641, which is hereby incorporated by reference in its entirety for all purposes .

nourish

Completed growth media included high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF and 5 pg/ ml corticosterone. Complete lactation media including high glucose DMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/ml EGF, 5 pg/ml hydrocortisone with 1 pg/ml prolactin (5 ug/ml in Hyunh 1991). Cells were seeded into collagen-coated flasks at a density of 20,000 cells/cm ² in complete growth medium and allowed to adhere and expand in complete growth medium for 48 hours before switching the medium to complete lactation medium. After exposure to lactation medium, cells begin to differentiate and stop growing. Within about a week, cells begin to secrete lactation products such as milk lipids, lactose, casein and whey into the medium. The desired concentration of lactation medium can be achieved by ultrafiltration concentration or dilution. The desired salt balance of the lactation medium can be achieved by dialysis, eg, to remove unwanted metabolites from the medium. Hormones and other growth factors used can be selectively extracted by resin purification, such as the use of nickel resins to remove histidine-tagged growth factors to further reduce the level of contaminants in lactated products .

Example 43. Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3- in non-mammary adult stem cells) Preparation of GlcNAc-b1,3-Gal-b1,4-Glc)

Mesenchymal cells isolated and reprogrammed into mammary gland-like cells as described in Example 42, modified by CRISPR-CAS to overexpress GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), human semi- Lactoside alpha-1,2-fucosyltransferase FUT1 (UniProt ID P19526) and codon optimized are selected from the group consisting of SEQ ID NOs: 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37 One of the list of alpha-1,3-galactosyltransferases. Cells were seeded into collagen-coated flasks at a density of ^20,000 cells/cm in complete growth medium and allowed to adhere and expand in complete growth medium for 48 hours, then the medium was switched to complete lactation medium for approximately 7 sky. After culturing as described in Example 42, cells were subjected to UPLC to analyze Gal-a1,3-LNFP-I (Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1, 3-Gal-b1,4-Glc).

Example 44. Production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc in non-mammary adult stem cells

Mesenchymal cells isolated and reprogrammed into mammary gland-like cells as described in Example 42, modified by CRISPR-CAS to overexpress GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), human semi- Lactoside alpha-1,2-fucosyltransferase FUT1 (UniProt ID P19526) and codon optimized are selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, One of the lists of 100, 101 and 102 alpha-1,3-N-acetylgalactosamine transferase. Cells were seeded into collagen-coated flasks at a density of ^20,000 cells/cm in complete growth medium and allowed to adhere and expand in complete growth medium for 48 hours, then the medium was switched to complete lactation medium for approximately 7 sky. After culturing as described in Example 42, cells were subjected to UPLC to analyze GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc.

Example 45. Preparation of oligosaccharide mixture including one of Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, 2'-FL and 3'-SL in non-mammary adult stem cells make

Mesenchymal cells isolated and reprogrammed into mammary gland-like cells as described in Example 42, modified by CRISPR-CAS to overexpress GDP-fucose synthase GFUS from Homo sapiens (UniProt ID Q13630), human semi- Lactoside alpha-1,2-fucosyltransferase FUT1 (UniProt ID P19526), codon-optimized selected from the group consisting of SEQ ID NOs: 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37 One of the list is α-1,3-galactosyltransferase, N-acylneuraminate cytidylyltransferase from mouse (UniProt ID Q99KK2) and CMP-N from Homo sapiens - Acetylneuraminic acid-beta-1,4-galactoside alpha-2,3-sialyltransferase (CMP-N-acetylneuraminate-beta-1,4-galactoside alpha-2,3-sialyltransferase) ST3GAL3 ( UniProt ID Q11203). Cells were seeded into collagen-coated flasks at a density of ^20,000 cells/cm in complete growth medium and allowed to adhere and expand in complete growth medium for 48 hours, then the medium was switched to complete lactation medium for approximately 7 sky. After culturing as described in Example 42, cells were subjected to UPLC to analyze the production of 2'-FL, 3'-SL and Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc.

Example 46. One based on the production of GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc in a modified E. coli host Evaluation of the performance of membrane transporters

The modified E. coli hosts described in Examples 9, 10, 11 and 12 were further modified by gene body knock-in of the constitutive transcription unit of a heterologous membrane transporter selected from These include MdfA from S. mogeni (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter johnsonii (UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID POAEY8), MdfA from Yorkia rexburg List of MdfA (UniProt ID G9Z5F4), iceT from E. coli (Uni0bactrot4L A20T) and iceT from Citrobacter johnsonii (UniProt ID D4B8A6). Novel strains, each expressing one of the heterologous membrane transporters, were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/L sucrose and 20 g/L lactose . Each strain was grown in four biological replicates in a 96-well plate. After 72 hours of culture, the culture broth (ie, the extracellular and intracellular fractions) was collected along with the extracellular and intracellular fractions, respectively, and analyzed for sugars by UPLC.

Example 47. Evaluation of membrane transporter performance in modified E. coli hosts

The modified E. coli hosts described in Examples 16 and 20 were further modified by gene body knock-in of the constitutive transcription unit of a membrane transporter selected from the group consisting of S. MdfA from Citrobacter japonicus (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter japonicus (UniProt ID D4BC23), MdfA from Escherichia coli (UniProt ID P0AEY8), MdfA from Yorkia rexburg (UniProt ID G9Z5F4), from iceT from E. coli (UniProt ID A0A024L207), iceT from Citrobacter johnsonii (UniProt ID D4B8A6), nanT from E. coli O6:H1 (UniProt ID Q8FD59), nanT from E. coli O157:H7 (UniProt ID Q8X9G8 ), nanT (UniProt ID B1EFH1) from E. albertii, EntS (UniProt ID P24077) from Escherichia coli, EntS (UniProt ID A0A378GQ13) from Kluyveer ascorbate, Salmonella enterica subsp. EntS from arizonae (UniProt ID A0A6Y2K4E8), SetA from E. coli (UniProt ID P31675), SetB from E. coli (UniProt ID P33026), SetC from E. coli (UniProt ID P31436), oppF from E. coli (UniProt ID P31436) P77737), a list of lmrA from Lactococcus lactis subsp. lactis bv. Diacetylactis (UniProt ID A0A1VONEL4) and Blon_2475 from Bifidobacterium longum subsp. Infantis (UniProt ID B7GPD4). Novel strains, each expressing the heterologous membrane transporter, were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the medium contained 30 g/L sucrose and 20 g/L lactose. Each strain was grown in four biological replicates in a 96-well plate. After 72 hours of culture, the culture broth (ie, the extracellular and intracellular fractions) was collected along with the extracellular and intracellular fractions, respectively, and analyzed for sugars by UPLC.

none

none

none

                                  sequence listing
           <![CDATA[ <110> Inbiose N.V., Belgium]]>
           <![CDATA[ <120> Production of Fuc-a1,2-Gal-R in α-1,3 glycosylated form]]>
           <![CDATA[ <130> 028-TW]]>
           <![CDATA[ <160> 102 ]]>
           <![CDATA[ <170> PatentIn version 3.5]]>
           <![CDATA[ <210> 1]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> motif 1]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (2)..(2)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> VARIANT]]>
           <![CDATA[ <222> (3)..(3)]]>
           <![CDATA[ <223> Xaa can be Phe, His, Met, Gln or Thr]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (4)..(4)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (6)..(7)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> VARIANT]]>
           <![CDATA[ <222> (8)..(9)]]>
           <![CDATA[ <223> Xaa can be Ala, Cys or Gly]]>
           <![CDATA[ <400> 1]]>
          Tyr Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa
          1 5
           <![CDATA[ <210> 2]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> motif 2]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (2)..(2)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (4)..(4)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (6)..(7)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> VARIANT]]>
           <![CDATA[ <222> (8)..(9)]]>
           <![CDATA[ <223> Xaa can be Ala, Cys or Gly]]>
           <![CDATA[ <400> 2]]>
          Tyr Xaa Gln Xaa Cys Xaa Xaa Xaa Xaa
          1 5
           <![CDATA[ <210> 3]]>
           <![CDATA[ <211> 234]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli K-12 MG1655]]>
           <![CDATA[ <400> 3]]>
          Met Val Ile Asn Ile Phe Tyr Ile Cys Thr Gly Glu Tyr Lys Arg Phe
          1 5 10 15
          Phe Asp Lys Phe Tyr Leu Ser Cys Glu Asp Lys Phe Ile Pro Glu Phe
                      20 25 30
          Gly Lys Lys Tyr Tyr Val Phe Thr Asp Ser Asp Arg Ile Tyr Phe Ser
                  35 40 45
          Lys Tyr Leu Asn Val Glu Val Ile Asn Val Glu Lys Asn Cys Trp Pro
              50 55 60
          Leu Asn Thr Leu Leu Arg Phe Ser Tyr Phe Leu Lys Val Ile Asp Lys
          65 70 75 80
          Leu Gln Thr Asn Ser Tyr Thr Phe Phe Phe Asn Ala Asn Ala Val Ile
                          85 90 95
          Val Lys Glu Ile Pro Phe Ser Thr Phe Met Glu Ser Asp Leu Ile Gly
                      100 105 110
          Val Ile His Pro Gly Tyr Lys Asn Arg Ile Ser Ile Leu Tyr Pro Trp
                  115 120 125
          Glu Arg Arg Lys Asn Ala Thr Cys Tyr Leu Gly Tyr Leu Lys Lys Gly
              130 135 140
          Ile Tyr Tyr Gln Gly Cys Phe Asn Gly Gly Lys Thr Ala Ser Phe Lys
          145 150 155 160
          Arg Leu Ile Gln Ile Cys Asn Met Met Thr Met Ala Asp Leu Lys Lys
                          165 170 175
          Asn Leu Ile Ala Lys Val His Asp Glu Ser Tyr Leu Asn Tyr Tyr Tyr
                      180 185 190
          Tyr Tyr Asn Lys Pro Leu Leu Leu Ser Glu Leu Tyr Ser Trp Pro Glu
                  195 200 205
          Lys Tyr Gly Glu Asn Lys Asp Ala Lys Ile Ile Met Arg Asp Lys Glu
              210 215 220
          Arg Glu Ser Trp Tyr Gly Asn Ile Lys Lys
          225 230
           <![CDATA[ <210> 4]]>
           <![CDATA[ <211> 277]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Pasteurella mairii]]>
           <![CDATA[ <400> 4]]>
          Met Ala Lys Val Ala Ile Leu Tyr Ile Ala Thr Gly Arg Tyr Ile Val
          1 5 10 15
          Phe Trp Glu His Phe Tyr Arg Ser Ala Glu Lys Phe Leu Leu Pro Lys
                      20 25 30
          Ser Asp Lys His Tyr Phe Val Phe Thr Asp Ser Pro His Ile Leu Gly
                  35 40 45
          Glu Asp His Ser Asn Val Thr Arg Ile Glu Gln Lys Lys Leu Gly Trp
              50 55 60
          Pro Tyr Asp Thr Leu Met Arg Phe Asp Ile Phe Leu Ser Ile Arg Glu
          65 70 75 80
          Thr Leu Glu Asn Phe Asp Tyr Ile Tyr Phe Phe Asn Gly Asn Ser Glu
                          85 90 95
          Ile Leu Val Glu Val Asn Glu Ser Glu Phe Leu Pro Leu Glu Asp Asn
                      100 105 110
          Tyr Asn Leu Val Phe Thr His Gln Pro His Met Phe His Leu Ser Lys
                  115 120 125
          Arg Arg Phe Thr Tyr Asp Arg Asn Pro Glu Ser Cys Ala Tyr Ile Pro
              130 135 140
          Gln Gly Gly Gly Lys Tyr Tyr Phe Thr Gly Ala Leu Asn Gly Gly Lys
          145 150 155 160
          Ala Lys Tyr Tyr Leu Glu Met Cys Glu Lys Leu Ser Gln Asn Thr His
                          165 170 175
          Thr Asp Leu Glu Lys Asn Ile Ile Ala Arg Trp His Asp Glu Ser His
                      180 185 190
          Leu Asn Arg Tyr Ala Ile Gly Arg Met Asp Ile Lys Ile Leu Pro Pro
                  195 200 205
          Tyr Phe Thr Arg Ser Glu Ser Glu Lys Trp Lys Thr Ser Ala Lys Ile
              210 215 220
          Met Phe Ser Asp Lys Thr His Tyr Arg Phe Gly Gly His Ala Tyr Leu
          225 230 235 240
          Arg Gly Glu Ser Glu Asn Lys Ile Thr Pro Thr Glu Trp Glu Glu Lys
                          245 250 255
          Tyr Lys Asn Lys Lys Arg Arg Phe Ser Phe Arg Ile Lys Gln Tyr Ile
                      260 265 270
          Lys Ser Trp Phe Leu
                  275
           <![CDATA[ <210> 5]]>
           <![CDATA[ <211> 286]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Acinetobacter bereziniae]]>
           <![CDATA[ <400> 5]]>
          Met Arg Asp Glu Val Lys Leu Pro Lys Tyr Ser Val Ala Ile Leu Tyr
          1 5 10 15
          Ile Ala Thr Gly Arg Tyr Asn Ile Phe Trp Glu Tyr Phe Tyr Lys Ser
                      20 25 30
          Ala Glu Gln Phe Leu Leu Lys Asp Cys Glu Lys His Phe Phe Ile Phe
                  35 40 45
          Thr Asp Ser Val Glu Pro Met Val Gly Glu Gly Gln Lys Asn Val Thr
              50 55 60
          Arg Ile Glu Gln Lys Lys Leu Gly Trp Pro Phe Asp Thr Leu Leu Arg
          65 70 75 80
          Phe Glu Ile Phe Leu Ser Ile Glu Asp Lys Leu Gln Asp Phe Asp Tyr
                          85 90 95
          Val Phe Phe Phe Asn Gly Asn Thr Glu Ile Leu Ser Glu Ile Lys Ala
                      100 105 110
          Ala Asp Leu Leu Pro Leu Ser Ile His Gln Lys Leu Val Phe Ala His
                  115 120 125
          Gln Pro His Leu Phe His Asn Lys Ile Asn Lys Phe Thr Tyr Asp Arg
              130 135 140
          Asn Pro Glu Ser Ser Ala Tyr Ile Ala Tyr Asn Tyr Gly His Ala Tyr
          145 150 155 160
          Phe Thr Gly Ala Leu Asn Gly Gly Glu Val Phe Ser Tyr Leu Glu Met
                          165 170 175
          Cys Lys Val Leu Ala Lys Asn Ile Gln Arg Asp Leu Ser Lys Asp Ile
                      180 185 190
          Ile Ala Leu Trp His Asp Glu Ser His Leu Asn His Tyr Ala Leu Asn
                  195 200 205
          Arg Asn Asp Ile Lys Ile Leu Pro Pro Tyr Phe Thr Arg Gly Glu Thr
              210 215 220
          Glu Tyr Trp Lys Thr Asp Ser Lys Val Met Phe Ser Asp Lys Thr His
          225 230 235 240
          Phe Arg Phe Gly Gly His Ala Tyr Leu Arg Gly Glu Thr Asp Glu Lys
                          245 250 255
          Ile Ser Gln Asn Glu Trp Glu Asn Lys Tyr Gly Lys Ser Arg Ser Arg
                      260 265 270
          Phe Lys Phe Arg Phe Lys Gln Phe Ile Lys Ser Ile Phe Leu
                  275 280 285
           <![CDATA[ <210> 6]]>
           <![CDATA[ <211> 287]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Acinetobacter boissieri]]>
           <![CDATA[ <400> 6]]>
          Met Cys Thr Asn Lys Pro Lys Tyr Arg Val Ala Ile Leu Tyr Ile Ala
          1 5 10 15
          Thr Gly Arg Tyr Thr Val Phe Trp Asp Gly Phe Phe Lys Ser Ala Glu
                      20 25 30
          Lys Tyr Leu Leu Leu Glu Ser Gln Lys Glu Tyr Phe Ile Phe Thr Asp
                  35 40 45
          Thr Pro His Val Leu Gln Glu Asn Glu Arg Val His Gln His Phe Gln
              50 55 60
          Ser Lys Leu Gly Trp Pro Phe Asp Thr Leu Lys Arg Phe Glu Ile Phe
          65 70 75 80
          Leu Ser Ile Lys Asp Gln Leu Lys Gly Phe Asp Phe Ile Tyr Phe Phe
                          85 90 95
          Asn Gly Asn Thr Glu Phe Val Thr Glu Ile Thr Glu Gln Glu Phe Leu
                      100 105 110
          Pro Leu Asp Lys Gln Gln Asn Leu Thr Leu Leu His Gln Pro His Leu
                  115 120 125
          Phe His Arg Arg Pro Arg His Phe Pro Tyr Asp Arg Asn Lys Glu Ser
              130 135 140
          Leu Ala Cys Ile Pro Tyr Asn Glu Gly Met Tyr Tyr Phe Thr Gly Ala
          145 150 155 160
          Leu Asn Gly Gly Lys Ala Ser Ala Tyr Leu Glu Met Cys Glu Gln Leu
                          165 170 175
          Asn Lys Asn Thr Asn Ile Asp Leu Lys Asn Asn Val Ile Ala Leu Phe
                      180 185 190
          His Asp Glu Ser His Leu Asn Arg Tyr Val Leu Gly Arg Asp Asp Val
                  195 200 205
          Lys Ile Leu Asp Pro Tyr Phe Ala Lys Gly Glu Thr Glu Tyr Trp Lys
              210 215 220
          His Ala Ser Lys Val Met Phe Ser Asp Lys Thr His Tyr Arg Phe Gly
          225 230 235 240
          Gly His Asp Tyr Leu Arg Gly Glu Ser Asp His Lys Ile Thr Gln Asp
                          245 250 255
          Glu Trp Glu Asn Gly Lys Lys Arg Asn Lys Lys Arg Tyr Lys Phe Arg
                      260 265 270
          Leu Arg Gln Ala Ile His Ala Phe Phe Ile Gln Arg Ser Leu Lys
                  275 280 285
           <![CDATA[ <210> 7]]>
           <![CDATA[ <211> 281]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Acinetobacter calcoaceticus]]>
           <![CDATA[ <400> 7]]>
          Met Asn Leu Ser Pro Lys Lys Val Ala Ile Leu Tyr Ile Ala Thr Gly
          1 5 10 15
          Arg Tyr Thr Val Phe Trp Asp Tyr Phe Tyr Gln Ser Ala Glu Ser Asn
                      20 25 30
          Leu Leu Arg Glu Cys Lys Lys His Tyr Phe Val Phe Thr Asp Asn Glu
                  35 40 45
          Glu Leu Leu Lys Lys Lys Thr Asp Gln Asn Val Ser Tyr Ile Ser Gln
              50 55 60
          Asp Lys Leu Gly Trp Pro Tyr Asp Thr Leu Met Arg Phe Asp Ile Phe
          65 70 75 80
          Leu Ser Ile Glu Asp Arg Leu Asn Thr Phe Asp Tyr Ile Tyr Phe Phe
                          85 90 95
          Asn Ala Asn Thr Glu Ile Leu Lys Pro Ile Asp Ala Gln Asp Ile Leu
                      100 105 110
          Pro Ile Asp Gln Gln Asn Leu Ala Phe Ala Ile Gln Pro His Ala Phe
                  115 120 125
          His Arg Asn Lys Lys Lys Tyr Thr Tyr Asp Arg Asn Pro Asn Ser Thr
              130 135 140
          Ala Tyr Ile Ala Met Asp Glu Gly Lys Tyr Tyr Phe Thr Gly Ala Leu
          145 150 155 160
          Asn Gly Gly Arg Ala Gln Ala Tyr Leu Glu Met Cys Arg Gln Leu Ser
                          165 170 175
          Ser Asn Thr His Val Asp Leu Ser Asn Glu Gln Ile Ala Leu Trp His
                      180 185 190
          Asp Glu Ser His Leu Asn Lys Tyr Ala Leu Asn Arg Lys Asp Ile Lys
                  195 200 205
          Val Leu Pro Pro Phe Phe Thr Arg Gly Glu Asn Glu Ile Trp Lys Lys
              210 215 220
          Lys Ala Lys Val Met Phe Ser Asp Lys Ser His Phe Arg Phe Gly Gly
          225 230 235 240
          His Ala Tyr Leu Arg Gly Glu Thr Asp Glu Lys Ile Ser Glu Lys Gln
                          245 250 255
          Trp Glu Val Ser Lys Asn Ala Lys His Lys Gly Trp Gly Phe Arg Ile
                      260 265 270
          Lys Gln Arg Ile Ser Ser Trp Phe Leu
                  275 280
           <![CDATA[ <210> 8]]>
           <![CDATA[ <211> 281]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Acinetobacter johnsonii]]>
           <![CDATA[ <400> 8]]>
          Met Tyr Ser Lys Thr Lys Val Ala Ile Leu Tyr Ile Ala Thr Gly Arg
          1 5 10 15
          Tyr Ile Thr Phe Trp Asp Phe Phe Tyr Lys Ser Ala Glu Gln Asn Leu
                      20 25 30
          Leu Leu Asn Ser Ser Lys His Tyr Phe Val Phe Thr Asp Cys Lys Glu
                  35 40 45
          Leu Leu Glu Ser Asp Ile Glu Lys Asn Ile Thr Tyr Ile Lys Gln Gln
              50 55 60
          Lys Leu Gly Trp Pro Tyr Asp Thr Leu Met Arg Phe Asn Ile Phe Leu
          65 70 75 80
          Thr Gln Lys Asp Gln Leu Lys Lys Phe Asp Tyr Ile Phe Phe Phe Asn
                          85 90 95
          Ala Asn Thr Glu Ile Ile Lys Asn Ile Lys Glu Glu Asp Leu Leu Pro
                      100 105 110
          Leu His Ser Asp Glu Asn Leu Val Leu Thr His Gln Pro His Val Phe
                  115 120 125
          His Lys Asn Lys Lys Gln Phe Thr Tyr Asp Arg Asn Pro Leu Ser Asn
              130 135 140
          Ala Tyr Ile Pro Leu Ser Gln Gly Arg Tyr Tyr Phe Thr Gly Ala Leu
          145 150 155 160
          Asn Gly Gly Lys Ser Val Asn Phe Leu Glu Met Cys Glu His Leu Asn
                          165 170 175
          Arg Asn Thr Lys Glu Asp Leu Asp Gln Asn Ile Ile Ala Leu Trp His
                      180 185 190
          Asp Glu Ser His Leu Asn Lys Tyr Val Leu Asp Arg Thr Asp Val Lys
                  195 200 205
          Ile Leu Pro Pro Tyr Phe Thr Arg Gly Glu Lys Glu Tyr Trp Lys Lys
              210 215 220
          Glu Ala Lys Val Met Phe Ser Asp Lys Ser His Tyr Arg Phe Gly Gly
          225 230 235 240
          His Ala Phe Leu Arg Gly Glu Thr Asp Gln Tyr Ile Asp Gln Ile Glu
                          245 250 255
          Trp Lys Ala Leu Asn Gly Lys Pro Lys Lys Arg Ile Ser Phe Arg Leu
                      260 265 270
          Lys Gln Tyr Ile Lys Ser Phe Phe Ile
                  275 280
           <![CDATA[ <210> 9]]>
           <![CDATA[ <211> 285]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Brachyspira sp. CAG:484]]>
           <![CDATA[ <400> 9]]>
          Met Gly Asn Lys Val Ala Val Leu Tyr Ile Val Thr Gly Arg Tyr Val
          1 5 10 15
          Cys Phe Trp Asp Glu Phe Tyr Pro Ser Cys Glu Lys Tyr Phe Leu Pro
                      20 25 30
          Asp Ala Gln Lys Lys Tyr Phe Val Phe Thr Asp Ala Glu His Leu Asn
                  35 40 45
          Phe Glu Glu Asn Asp Asn Val Leu Lys Ile His Gln Glu Lys Leu Gly
              50 55 60
          Trp Pro Tyr Asp Thr Met Leu Arg Phe Asp Ile Phe Leu Lys Gln Lys
          65 70 75 80
          Glu Ala Leu Lys Glu Tyr Asp Tyr Ile Phe Phe Phe Asn Ala Asn Thr
                          85 90 95
          Lys Phe Leu Asn Tyr Val Arg Glu Glu Ile Leu Pro Asn Glu Glu Asn
                      100 105 110
          Asp Trp Leu Ile Thr Gly Ser His Pro Ala Phe Tyr Asn Lys His Pro
                  115 120 125
          Asp Glu Phe Thr Tyr Asp Arg Asn Pro Glu Ser Gln Ala Tyr Ile Pro
              130 135 140
          Tyr Gly Ala Gly Lys His Tyr Ala Thr Gly Ala Leu Asn Gly Gly Ser
          145 150 155 160
          Gly Ala Ser Phe Leu Glu Met Cys Glu Glu Leu Ser Arg Leu Thr His
                          165 170 175
          Ile Asp Met Asp Asn Gly Val Val Pro Leu Trp His Asp Glu Ser Met
                      180 185 190
          Leu Asn Lys Tyr Met Leu Asn Lys Asn Pro Leu Ile Met Pro Val Asn
                  195 200 205
          Tyr Leu Tyr Pro Glu Glu Arg Trp Met Pro Arg Lys Trp Tyr Arg Asn
              210 215 220
          Asn Pro Phe Lys Lys Asp Ile Lys Ile Leu Ser Thr Asp Lys Thr His
          225 230 235 240
          Pro Arg Tyr Gly Gly Lys Glu Tyr Leu Arg Gly Ile Ser Asp Lys Lys
                          245 250 255
          Ala Lys Met Pro Asn Pro Ile Phe Ser Val Ser Tyr Glu Asp Ala Lys
                      260 265 270
          Lys Val Leu Arg Ile Leu Gly Phe Lys Ile Arg Ile Val
                  275 280 285
           <![CDATA[ <210> 10]]>
           <![CDATA[ <211> 274]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Candidatus Melainabacteria]]>
           <![CDATA[ <400> 10]]>
          Met Leu Lys Phe Thr Tyr Asn Lys Lys Lys Glu Ser Leu Met Lys Ile
          1 5 10 15
          Ala Ile Ile Tyr Ile Gly Ile Gly Arg Tyr Thr Val Phe Trp Asp Glu
                      20 25 30
          Phe Tyr Lys Ser Cys Glu Lys Asn Phe Ile Arg Asn Ala Gln Lys His
                  35 40 45
          Tyr Phe Tyr Phe Thr Asp Ser Lys Glu Tyr Lys Ser Asp Asp Lys Ile
              50 55 60
          Thr Ile Ile Pro Gln Glu Asn Leu Gly Trp Pro Leu Val Thr Cys Leu
          65 70 75 80
          Arg Tyr Lys Phe Ile Asn Thr Ile Lys Asp Ser Leu Lys Asn Tyr Asp
                          85 90 95
          Tyr Ile Phe Phe Phe Asn Gly Asn Tyr Glu Val Tyr Ser Lys Val Thr
                      100 105 110
          Ala Glu Glu Phe Leu Pro Thr Asp Glu Asp Gly Gly Leu Ile Ala Leu
                  115 120 125
          Lys His Asn Tyr Asn Lys Tyr Lys Lys Pro Asp Asp Phe Pro Trp Glu
              130 135 140
          Arg Asn Pro Lys Ser Thr Ser Tyr Ile Pro Tyr Gly Thr Asp Ser Phe
          145 150 155 160
          Tyr Tyr Gln Ala Cys Leu Trp Gly Gly Lys Thr Ser Gln Met Leu Lys
                          165 170 175
          Leu Val Glu Asp Cys Glu Lys Met Met Asp Glu Asp Leu Ala Asn Asp
                      180 185 190
          Ile Val Pro Ile Phe His Asp Glu Ser Leu Phe Asn Lys Tyr Met Leu
                  195 200 205
          Asp Lys Lys His Lys Thr Leu Gly Tyr Glu Tyr Gly Phe Val Pro Glu
              210 215 220
          Gly Lys Pro Phe Trp Lys Tyr Phe Gly Val Lys Met Thr Gln Arg Pro
          225 230 235 240
          Lys Ser Trp Lys Tyr Gly Gly Val Asp Trp Leu Arg Gly Leu Thr Asp
                          245 250 255
          Lys Lys Gln Thr Leu Phe Ser Tyr Ile Leu Glu Lys Leu His Leu Thr
                      260 265 270
          Lys Lys
           <![CDATA[ <210> 11]]>
           <![CDATA[ <211> 253]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Candidatus Paearchaeota]]>
           <![CDATA[ <400> 11]]>
          Met Pro Pro Lys Val Ala Ile Ile Phe Ile Gly Thr Ser Lys Tyr Ala
          1 5 10 15
          Asp Phe Phe Pro Glu Trp Lys Arg Cys Val Asp Lys His Phe Leu Lys
                      20 25 30
          Glu Cys Asp Lys Thr Ile Ile Ala Ile Thr Asp Arg Val Asp Glu Glu
                  35 40 45
          Tyr Phe His Leu Glu Asp Val Tyr Cys Gly Lys Val Ala His Met Glu
              50 55 60
          Trp Pro Phe Ile Thr Val Leu Arg Phe Arg Phe Ile Asn Glu Ile Pro
          65 70 75 80
          Gly Leu Lys Gln Phe Asp Tyr Val Phe Phe Leu Asp Ala Asp Leu Phe
                          85 90 95
          Pro Ser Asn Asp Ile Leu Leu Ser Glu Ile Ile Ser Pro Asp Lys Lys
                      100 105 110
          Leu Val Gly Val Gln His Pro Gly Asn Phe Leu Asp Ser Thr Trp Asn
                  115 120 125
          Thr Leu Asp Arg Thr Pro Gly Ser Thr Ala Cys Val Ser Gly Asp Ile
              130 135 140
          Thr Ser Tyr Gly Thr Thr Phe Tyr His Gln Gly Cys Leu Trp Gly Gly
          145 150 155 160
          Thr Gly Lys Ala Val Ser Glu Met Val Leu Lys Leu Ala Lys Asn Val
                          165 170 175
          Asp Ala Asp Leu Lys Asn Asn Ile Met Ala Ile Trp His Asp Glu Ser
                      180 185 190
          His Met Asn Lys Tyr Phe Leu Glu Asn Ile Ala Asp Val His Thr Leu
                  195 200 205
          His Ser Gly Phe Ala Tyr Pro Glu His Gly Asn Trp Ala Val Ile Glu
              210 215 220
          Asp Asn Leu Glu Ile Lys Met Val His Lys Glu Lys Ser His Glu Asp
          225 230 235 240
          Phe Pro Arg Phe Arg Gly Asn Asn Pro His Asp Lys Gly
                          245 250
           <![CDATA[ <210> 12]]>
           <![CDATA[ <211> 289]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Candidatus Paearchaeota]]>
           <![CDATA[ <400> 12]]>
          Met Ala Ser Lys Ser Leu Arg Glu Lys Leu Met Arg Val Lys Trp Ile
          1 5 10 15
          Lys Lys Leu Leu Arg Ala Leu Pro Thr Leu Leu Arg Leu His Ile Lys
                      20 25 30
          Tyr Phe Glu Asn Arg Lys Tyr Thr Ile Arg Ile Leu Lys Lys Glu Glu
                  35 40 45
          Arg Lys Glu Lys Lys Lys Gln Met Gln Phe Pro Lys Ser Ile Ala Ile Leu
              50 55 60
          Phe Val Gly Thr Gly Ile Tyr Phe Asn Tyr Phe Gly Glu Phe Tyr Glu
          65 70 75 80
          Asn Ile Lys Arg Asn Phe Leu Pro Glu Ile Pro Lys Lys Phe Phe Val
                          85 90 95
          Phe Thr Asp Lys Asp Phe Lys Glu Asn Glu Asp Val Glu Arg Val Lys
                      100 105 110
          Ile Pro Asp Glu Lys Ile Tyr Ala Ile Leu Arg Tyr Leu Gly Asp Ile
                  115 120 125
          Pro Lys Ile Lys Asn Leu Lys Asn Phe Glu Tyr Val Ile Lys Met Asp
              130 135 140
          Ala Asp Leu Val Val Pro Glu Pro Ile Ser Ser Ala Glu Phe Phe Tyr
          145 150 155 160
          His Asn Lys Pro Leu Phe Gly Val Arg His Pro Tyr Phe Leu Cys Arg
                          165 170 175
          Gln Gly Ser Phe Glu Ile Ser Pro Lys Ser Lys Ala Ala Val Ser Pro
                      180 185 190
          Arg Glu Asp Leu Ser Glu Tyr Ile Gln Cys Cys Phe Trp Gly Gly Lys
                  195 200 205
          Thr Asn Tyr Val Val Lys Met Val Lys Glu Met Tyr Lys Asn Ile Lys
              210 215 220
          Ile Asp Leu Asn Asn Gly Ile Ile Ala Arg Ile Phe Asp Glu Ser Tyr
          225 230 235 240
          Leu Asn Lys Tyr Phe Ile Ser Asn Lys Pro Leu Phe Tyr Val Tyr Pro
                          245 250 255
          Pro Asn Tyr Ala Tyr Pro Asp Val Pro Ile Pro Glu Lys Leu Lys Lys
                      260 265 270
          Lys Ile Leu His Val Thr Asn Lys Arg Phe Lys Val Asn Tyr Gln Lys
                  275 280 285
          Lys
           <![CDATA[ <210> 13]]>
           <![CDATA[ <211> 261]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Chitinophaga niabensis]]>
           <![CDATA[ <400> 13]]>
          Met Lys Ile Ala Leu Leu Phe Ile Cys Thr Gly Lys Tyr Ser Ile Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Thr Ser Ala Glu Gln Tyr Phe Val Pro Gly Ala
                      20 25 30
          Glu Lys Ala Tyr Phe Val Phe Thr Asp Asp Ala Asp Leu Pro Phe Lys
                  35 40 45
          Asp Ala Gln Asn Val His Val His His Gln Gln Lys Leu Gly Trp Pro
              50 55 60
          Tyr Asp Thr Leu Met Arg Phe Ser Ile Phe Ser Arg Val Glu Lys Glu
          65 70 75 80
          Leu Ala Ala Phe Asp Tyr Ile Phe Phe Phe Asn Ala Asn Thr Glu Phe
                          85 90 95
          Ile Lys Pro Ile Thr Ala Ala Glu Ile Leu Pro Thr Asp Ala Glu Asp
                      100 105 110
          Gly Leu Thr Val Val Leu His Pro Gly Tyr Tyr Asn Lys Pro Leu Lys
                  115 120 125
          Ala Phe Pro Tyr Glu Lys Thr Gln Lys Lys Ser Thr Ala Tyr Met Pro
              130 135 140
          Ser Asn Glu Arg His Gln Tyr Phe Gln Gly Cys Leu Asn Gly Gly Thr
          145 150 155 160
          Gly Lys Ala Tyr Leu Gln Leu Ile Arg Gln Leu Thr Glu Asn Thr Gln
                          165 170 175
          Lys Asp Leu Asp Asn Gly Ile Ile Ala Ile Trp His Asp Glu Ser Gln
                      180 185 190
          Leu Asn Lys Tyr Val Ala Asn Lys His Pro Lys Val Leu Thr Pro Gly
                  195 200 205
          Tyr Ala Tyr Pro Glu Gly Trp Asp Leu Pro Phe Glu Lys Ala Ile Leu
              210 215 220
          Met Arg Asp Lys Gly Arg Phe Gly Gly Ser Asp Phe Met Arg Gln Thr
          225 230 235 240
          Thr Pro Glu Ala Pro Leu Asn Thr Phe Gln Leu Ile Ile Arg Lys Ile
                          245 250 255
          Lys Arg Leu Phe Ser
                      260
           <![CDATA[ <210> 14]]>
           <![CDATA[ <211> 234]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 14]]>
          Met Val Ile Asn Ile Phe Tyr Ile Cys Thr Gly Glu Tyr Lys Arg Phe
          1 5 10 15
          Phe Asp Lys Phe Tyr Leu Ser Cys Glu Asp Lys Phe Ile Pro Glu Phe
                      20 25 30
          Glu Lys Lys Tyr Tyr Val Phe Thr Asp Ser Asp Arg Ile Tyr Phe Ser
                  35 40 45
          Lys Tyr Leu Asn Val Glu Val Ile Asn Val Glu Lys Asn Cys Trp Pro
              50 55 60
          Leu Asn Thr Leu Leu Arg Phe Ser Tyr Phe Leu Lys Val Ile Asp Lys
          65 70 75 80
          Leu Gln Thr Asn Ser Tyr Thr Phe Phe Phe Asn Ala Asn Ala Val Ile
                          85 90 95
          Val Lys Glu Ile Pro Phe Ser Thr Phe Met Glu Ser Asp Leu Ile Gly
                      100 105 110
          Val Ile His Pro Gly Tyr Lys Asn Arg Ile Ser Ile Leu Tyr Pro Trp
                  115 120 125
          Glu Arg Arg Lys Asn Ala Thr Cys Tyr Leu Gly Tyr Leu Lys Lys Gly
              130 135 140
          Ile Tyr Tyr Gln Gly Cys Phe Asn Gly Gly Lys Thr Ala Ser Phe Lys
          145 150 155 160
          Arg Leu Ile Gln Ile Cys Asn Met Met Thr Met Ala Asp Leu Lys Lys
                          165 170 175
          Asn Leu Ile Ala Lys Val His Asp Glu Ser Tyr Leu Asn Tyr Tyr Tyr
                      180 185 190
          Tyr Tyr Asn Lys Pro Leu Leu Leu Ser Glu Leu Tyr Ser Trp Pro Glu
                  195 200 205
          Lys Tyr Gly Glu Asn Lys Asp Ala Lys Ile Ile Met Arg Asp Lys Glu
              210 215 220
          Arg Glu Ser Trp Tyr Gly Asn Ile Lys Lys
          225 230
           <![CDATA[ <210> 15]]>
           <![CDATA[ <211> 185]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Escherichia coli]]>
           <![CDATA[ <400> 15]]>
          Tyr Leu Asn Val Glu Val Ile Asn Val Glu Lys Asn Cys Trp Pro Leu
          1 5 10 15
          Asn Thr Leu Leu Arg Phe Ser Tyr Phe Leu Lys Val Ile Asp Lys Leu
                      20 25 30
          Gln Thr Asn Ser Tyr Thr Phe Phe Phe Asn Ala Asn Ala Val Ile Val
                  35 40 45
          Lys Glu Ile Pro Phe Ser Thr Phe Met Glu Ser Asp Leu Ile Gly Val
              50 55 60
          Ile His Pro Gly Tyr Lys Asn Arg Ile Ser Ile Leu Tyr Pro Trp Glu
          65 70 75 80
          Arg Arg Lys Asn Ala Thr Cys Tyr Leu Gly Tyr Leu Lys Lys Gly Ile
                          85 90 95
          Tyr Tyr Gln Gly Cys Phe Asn Gly Gly Lys Thr Ala Ser Phe Lys Arg
                      100 105 110
          Leu Ile Gln Ile Cys Asn Met Met Thr Met Ala Asp Leu Lys Lys Asn
                  115 120 125
          Leu Ile Ala Lys Val His Asp Glu Ser Tyr Leu Asn Tyr Tyr Tyr Tyr
              130 135 140
          Tyr Asn Lys Pro Leu Leu Leu Ser Glu Leu Tyr Ser Trp Pro Glu Lys
          145 150 155 160
          Tyr Gly Glu Asn Lys Asp Ala Lys Ile Ile Met Arg Asp Lys Glu Arg
                          165 170 175
          Glu Ser Trp Tyr Gly Asn Ile Lys Lys
                      180 185
           <![CDATA[ <210> 16]]>
           <![CDATA[ <211> 542]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Haemophilus pittmaniae]]>
           <![CDATA[ <400> 16]]>
          Met Ile Lys Glu Thr Ile Ala Val Leu Tyr Ile Val Gln Gly Asn Asp
          1 5 10 15
          Phe Ala Ala Trp Asp Asn Phe Tyr Arg Ser Ser Glu Glu Phe Leu Leu
                      20 25 30
          Pro Arg Gln His Lys Gln Tyr Phe Val Phe Ser Asp Asp Glu Ser Ile
                  35 40 45
          Thr Arg Asn Ser Asn Ile Ser Ile Val Arg Thr Asn Gly Leu Lys Asp
              50 55 60
          Ser Lys Ser Arg Phe Trp Leu Phe Ser Ala Ile Glu Asn Gln Leu Ala
          65 70 75 80
          Glu Phe Thr Tyr Val Tyr Ala Phe Ser Ser His Val Arg Phe Val Ser
                          85 90 95
          Pro Ile Val Ala Glu Asp Ile Thr Pro Thr Pro Asn Ser Pro Phe Val
                      100 105 110
          Val Tyr Arg Gln Tyr Pro Asp Leu Asp His Val Leu Ala Asn Glu Phe
                  115 120 125
          Pro Tyr Glu Arg Val Val Asn Ala Asn Ser Tyr Val Pro Tyr Gly Val
              130 135 140
          Gly Glu Gln Tyr Leu Thr Cys Ala Leu Phe Gly Gly Met Arg Asp Ser
          145 150 155 160
          Phe Ile Ser Ala Cys Arg Cys Ile Asp Ala Ala Ile Glu Asp Asp Arg
                          165 170 175
          Tyr Arg His Ile Ala Ser Leu Asn Ala Glu Asp Lys Gln Leu Asn Gln
                      180 185 190
          Tyr Phe Leu Tyr Lys Asn Asn Met Asn Val Leu Ser Ala Asn Trp Ile
                  195 200 205
          Arg Lys Ala Asn Glu Pro Trp Lys Arg Tyr Ala Lys Met Leu Asp Val
              210 215 220
          Ile Gln Glu Asp Ser Phe Asp Ile Pro Val Asp Val Leu Glu Ser Val
          225 230 235 240
          Lys Asn Ile His Glu Ile Phe Arg Tyr Ala Pro His Ser Phe Phe Leu
                          245 250 255
          Asp Leu Gln Glu Asn Val Ala Lys Ser Trp Arg Ala Leu Leu Lys Ala
                      260 265 270
          Tyr Leu Tyr Gly Gln Leu Thr Thr Phe Asp Phe Pro Ala Lys Lys Pro
                  275 280 285
          Glu Leu Val Gly Lys Asn Ile Ile Trp Gln Tyr Trp Gly Gln Gly Ile
              290 295 300
          Asp Asp Arg Leu Pro Glu Leu Thr Lys Val Cys Phe Ala Ser Val Asp
          305 310 315 320
          Arg Asn Lys Gly Asp Tyr Thr Val Ile Arg Val Asp Asp Ala Ser Leu
                          325 330 335
          Ala Glu Tyr Ile Asp Leu Pro Asp Phe Met Trp Gln Lys Arg Gly Gly
                      340 345 350
          Ala Phe Ser Thr Ala Leu Phe Ser Asp Val Val Arg Leu Ile Leu Leu
                  355 360 365
          Tyr Val Tyr Gly Gly Ile Trp Val Asp Ala Thr Ile Ile Phe Ser Ser
              370 375 380
          Pro Leu Pro Lys Gly Leu Leu Glu Gln Asp Phe Phe Leu Phe His Arg
          385 390 395 400
          Asp Ile Gly Asn Ser Asn Lys Ala Tyr Trp Glu Arg Ile Asn Lys Asp
                          405 410 415
          Tyr Phe Cys Trp Asp Lys Glu His Lys Val Asn Ser Leu Asn Ser Phe
                      420 425 430
          Ile Ile Ala Lys Pro Arg His Val Val Thr Glu Thr Leu Leu Gln Leu
                  435 440 445
          Leu Leu Asn Tyr Trp Lys Thr Gln Asp His Val Pro Cys Tyr Tyr Ile
              450 455 460
          Phe Gln Ile Leu Phe Asp Gln Val Met Lys Tyr Asp Leu Asp Asn Gln
          465 470 475 480
          Arg Leu Leu Val Arg Asp Asp Thr Phe Pro His Glu Leu Ser Met Lys
                          485 490 495
          Leu Trp Ser Asp Tyr Asn Ala Glu Glu Ile Asn Asp Leu Phe Ser Arg
                      500 505 510
          Cys Ser Val His Lys Leu Thr Gly His Ala Asn Leu Ala Asp Cys Gly
                  515 520 525
          Glu Asn Ser Val Trp Gln His Leu Lys Arg Glu Tyr Leu Gly
              530 535 540
           <![CDATA[ <210> 17]]>
           <![CDATA[ <211> 542]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Haemophilus pittmaniae HK 85]]>
           <![CDATA[ <400> 17]]>
          Met Ile Lys Glu Thr Ile Ala Val Leu Tyr Ile Val Gln Gly Asn Asp
          1 5 10 15
          Phe Ala Ala Trp Asp Asn Phe Tyr Arg Ser Ser Glu Glu Phe Leu Leu
                      20 25 30
          Pro Gly Gln His Lys Gln Tyr Phe Val Phe Ser Asp Asp Glu Ser Ile
                  35 40 45
          Thr Arg Asn Ser Asn Val Ser Ile Val Arg Thr Asn Gly Leu Lys Asp
              50 55 60
          Ser Lys Ser Arg Phe Trp Leu Phe Ser Ala Ile Glu Asn Gln Leu Ala
          65 70 75 80
          Glu Phe Thr Tyr Val Tyr Ala Phe Ser Ser His Ile Arg Phe Val Ser
                          85 90 95
          Pro Val Val Ser Glu Asp Ile Thr Pro Thr Pro Asn Ser Pro Phe Val
                      100 105 110
          Val Tyr Arg Gln Tyr Pro Asn Leu Asp His Val Leu Ala Asn Glu Phe
                  115 120 125
          Pro Tyr Glu Arg Ala Val Asn Ala Asn Ser Tyr Val Pro Tyr Gly Ala
              130 135 140
          Gly Glu Gln Tyr Leu Thr Cys Ala Leu Phe Gly Gly Met Arg Asp Ser
          145 150 155 160
          Phe Ile Ser Ala Cys Arg Cys Ile Asp Ala Ala Ile Glu Asp Asp Arg
                          165 170 175
          Tyr Arg His Ile Ala Ser Leu Asn Ala Glu Asp Lys Gln Leu Asn Gln
                      180 185 190
          Tyr Phe Leu Tyr Lys Asn Asn Met Asn Val Leu Ser Ala Asn Trp Ile
                  195 200 205
          Arg Lys Ala Asn Glu Pro Trp Lys Arg Tyr Ala Lys Met Leu Asp Val
              210 215 220
          Ile Gln Glu Gly Ser Phe Asp Ile Pro Val Asp Val Leu Glu Ser Val
          225 230 235 240
          Lys Asn Ile His Glu Ile Phe Arg Tyr Ala Pro His Ser Ser Phe Leu
                          245 250 255
          Asp Leu Gln Glu Asn Val Ala Lys Ser Trp Arg Ala Leu Leu Lys Ala
                      260 265 270
          Tyr Leu Tyr Gly Gln Leu Thr Thr Phe Asp Phe Pro Ala Lys Lys Pro
                  275 280 285
          Asp Leu Val Gly Lys Asn Ile Ile Trp Gln Tyr Trp Gly Gln Gly Ile
              290 295 300
          Asp Asp Gly Leu Pro Glu Leu Thr Lys Val Cys Phe Ala Ser Val Asp
          305 310 315 320
          Arg Asn Lys Gly Asp Tyr Thr Val Ile Arg Val Asp Asp Ala Ser Leu
                          325 330 335
          Ala Glu Tyr Ile Asp Leu Pro Asp Phe Met Trp Gln Lys Arg Gly Gly
                      340 345 350
          Ala Phe Ser Ala Ala Leu Phe Ser Asp Val Val Arg Leu Val Leu Leu
                  355 360 365
          Tyr Val Tyr Gly Gly Ile Trp Val Asp Ala Thr Ile Ile Phe Ser Ser
              370 375 380
          Pro Leu Pro Lys Glu Leu Leu Glu Gln Asp Phe Phe Leu Phe His Arg
          385 390 395 400
          Asp Ile Gly Asn Ser Asn Lys Ala Tyr Trp Glu Arg Ile Asn Lys Asp
                          405 410 415
          Tyr Phe Cys Trp Asn Lys Glu His Lys Val Asn Ser Leu Asn Ser Phe
                      420 425 430
          Ile Ile Ala Lys Pro Trp His Val Val Thr Glu Thr Leu Leu Gln Leu
                  435 440 445
          Leu Leu Asn Tyr Trp Lys Thr Gln Asp His Val Pro Cys Tyr Tyr Ile
              450 455 460
          Phe Gln Ile Leu Phe Asp Gln Val Met Lys Tyr Asp Leu Asp Asn Gln
          465 470 475 480
          Arg Leu Leu Ile Arg Asp Asp Thr Phe Pro His Glu Leu Ser Met Lys
                          485 490 495
          Leu Trp Ser Asp Tyr Asn Ala Glu Glu Ile Asn Asp Leu Phe Ser Arg
                      500 505 510
          Cys Ser Val His Lys Leu Thr Gly His Ala Asn Leu Ala Asp Cys Gly
                  515 520 525
          Glu Asn Ser Val Trp Gln His Leu Lys Arg Glu Tyr Leu Gly
              530 535 540
           <![CDATA[ <210> 18]]>
           <![CDATA[ <211> 641]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Helicobacter sp. 11S02629-2]]>
           <![CDATA[ <400> 18]]>
          Met Lys Leu Asp Leu Asp Lys Ser Tyr Asn Phe Leu Ile Val Arg Leu
          1 5 10 15
          Asp His Ile Gly Asp Val Val Leu Thr Leu Gly Cys Ala Glu Ala Ile
                      20 25 30
          Lys Thr Arg Phe Lys Asn Ala Lys Val Phe Tyr Leu Val Asn Ser Tyr
                  35 40 45
          Thr Ala Pro Leu Phe Glu His His Ala Phe Val Asp Gly Phe Ile Asp
              50 55 60
          Leu Asn Thr Asn Gly Val Phe Asp Gln Lys Ala Leu Ile Ser Arg Ile
          65 70 75 80
          Lys Ala Ala Lys Ile Asp Ile Ser Ile Ser Phe Ala Pro Asp Lys Phe
                          85 90 95
          Ala Leu Pro Ala Ile Phe Lys Ala Arg Val Lys Ile Arg Leu Gly Asn
                      100 105 110
          Phe Ile Lys Leu Tyr Ser Leu Leu Leu Thr Lys Arg Val Ile Gln Asn
                  115 120 125
          Arg Ser Ala Cys Asn Arg Ser Glu Ala Leu Tyr Asp Leu Glu Leu Leu
              130 135 140
          Lys Pro Leu Gly Cys Ser Thr Asn Phe Tyr Pro Lys Leu Phe Val Ser
          145 150 155 160
          Glu Ala Glu Lys Glu Glu Ala Leu Lys Tyr Ile Glu Ser Ser Phe Ala
                          165 170 175
          Asn Lys Arg Pro Leu Val Ile Val His Pro Gly Ser Leu Lys Ser Thr
                      180 185 190
          Val Glu Trp Gly Arg Glu Lys Phe Leu Glu Val Ala Ser Leu Leu Ser
                  195 200 205
          Glu Asn Tyr Asn Val Leu Val Thr Gly Ser Asp Ser Glu Met Lys Glu
              210 215 220
          Leu Leu Thr Phe Lys Arg Gly Asn Leu Lys Glu Ser Asn Phe Leu Lys
          225 230 235 240
          Pro Gly Ser Leu Arg Trp Ile Ile Ser Ile Ile Ser Leu Ala Asp Leu
                          245 250 255
          Ile Val Val Asn Ala Thr Gly Thr Leu His Ile Ala Ala Ala Leu Gly
                      260 265 270
          Val Arg Ile Val Gly Ile Tyr Pro Asp Arg Leu Gln Ile Asn Pro Thr
                  275 280 285
          Arg Trp Ala Ala Phe Thr Lys Glu Asp Asp Asp Val Tyr Ile Thr Pro
              290 295 300
          Ser Gly Ile Phe Tyr Gly Ala Lys Ser Tyr Lys Pro Pro Ser Phe Asp
          305 310 315 320
          Asn Asn Asp Pro Arg Met Val Asn Met Asp Ala Ile Lys Val Asp Glu
                          325 330 335
          Val Tyr Lys Ile Ala Asp Leu Glu Leu Lys Lys Leu Asp Pro Arg Tyr
                      340 345 350
          Lys Lys Ile Ala Ile Leu Tyr Ile Ala Leu Gly Arg Tyr Asp Ile Phe
                  355 360 365
          Phe Asn Asp Phe Tyr Glu Ser Met Glu Lys His Phe Val Thr Ser Ala
              370 375 380
          Lys Lys Thr Tyr Phe Val Phe Thr Asp Ser Ala Asn Ile Ser Thr His
          385 390 395 400
          Asp Asn Val Val Lys Ile Lys Gln Glu Lys Leu Gly Trp Pro Phe Asp
                          405 410 415
          Thr Leu Lys Arg Phe Ala Met Phe Glu Ser Ile Lys Asp Arg Leu Ala
                      420 425 430
          Asn Phe Asp Tyr Ile Phe Phe Phe Asn Ala Asn Ala Leu Val Leu Glu
                  435 440 445
          Asp Ile Gln Ala Lys Glu Val Leu Pro Ser Glu Lys Glu Gly Leu Val
              450 455 460
          Phe Ala Arg His Pro Ser Phe Ser Tyr Ile Lys Glu Asp Leu Thr Trp
          465 470 475 480
          Asp Ser Arg Asp Ser Phe Arg Asp Ser Tyr His Lys Asp Leu Asn Ser
                          485 490 495
          Leu Ala Cys Ile Lys Glu Asp Glu Gly Phe Ala Tyr Val Met Gly Ala
                      500 505 510
          Leu Asn Gly Gly Arg Ala Lys Glu Tyr Leu Glu Leu Ile Ser Thr Leu
                  515 520 525
          His Ala Asn Val Glu Ser Asp Leu Gln Lys Asp Val Ile Ala Val Trp
              530 535 540
          His Asp Glu Ser His Leu Asn Arg Tyr Leu Ile Asp Phe Cys Lys Ala
          545 550 555 560
          Gly His Ala Pro Lys Ile Leu Gly Ala Asn Phe Leu Val Pro Glu Glu
                          565 570 575
          Cys Leu Glu Lys Leu Gly Phe Gly Phe Tyr Lys Asp Thr Pro Phe Leu
                      580 585 590
          Lys Leu Ser Ser Leu Lys Ala Lys Ile Thr Leu Leu Asp Lys Ser His
                  595 600 605
          Pro Arg Phe Gly Gly His Glu Tyr Leu Arg Gly Ala Val Val Gln Asp
              610 615 620
          Phe Lys Pro Lys Val Gly Leu Thr Cys Ile Lys Asp Thr Gly Gly Gly
          625 630 635 640
          Gly
           <![CDATA[ <210> 19]]>
           <![CDATA[ <211> 331]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Helicobacter sp. 13S00401-1]]>
           <![CDATA[ <400> 19]]>
          Met Leu Asn Pro Phe Lys Thr Asn Thr Ile Ala Ile Leu Tyr Ile Ala
          1 5 10 15
          Leu Gly Arg Tyr Asp Ile Phe Phe Asn Asp Phe Tyr Glu Asn Met Glu
                      20 25 30
          Lys Asn Phe Val Pro Asn Thr Lys Lys Thr Tyr Phe Val Phe Thr Asp
                  35 40 45
          Ser Lys Asn Ile Thr Ser His Glu Asn Ile Val Arg Ile Glu Gln Ala
              50 55 60
          Lys Leu Gly Trp Pro Tyr Asp Thr Leu Lys Arg Phe Ala Met Phe Glu
          65 70 75 80
          Gly Ile Lys Glu Glu Leu Ala Ser Phe Asp Tyr Ile Phe Phe Phe Asn
                          85 90 95
          Ala Asn Ala Leu Val Leu Glu Lys Ile Thr Ala Pro Met Ile Leu Pro
                      100 105 110
          Ser Lys Asp Glu Gly Leu Val Phe Ala Arg His Pro Ala Phe Ser Cys
                  115 120 125
          Ile Leu Pro Asp Met Asn Trp Glu Ser Arg Glu Ser Phe Arg Gln Ser
              130 135 140
          Tyr Cys Lys Asp Pro Asn Ser Leu Ala Cys Ile Lys Asp Asp Glu Gly
          145 150 155 160
          Phe Cys Tyr Val Met Gly Ala Leu Asn Gly Gly Arg Ala Lys Glu Tyr
                          165 170 175
          Leu Glu Leu Ile Glu Thr Leu Ala Ala Arg Val Glu Ala Asp Leu Gln
                      180 185 190
          Lys Asp Val Val Ala Val Trp His Asp Glu Ser His Leu Asn Arg Tyr
                  195 200 205
          Leu Ile Asp Val Val Lys Asn Gly Lys Lys Pro Lys Ile Ile Gly Ala
              210 215 220
          Asn Phe Leu Val Pro Glu Glu His Leu Glu Ala Leu Gly Phe His Phe
          225 230 235 240
          Tyr Lys Asp Val Pro Phe Leu Lys Leu Ala Lys Leu Arg Ala Asn Ile
                          245 250 255
          Thr Leu Leu Asn Lys Ser His Pro Arg Phe Gly Gly His Glu Tyr Leu
                      260 265 270
          Arg Gly Leu Ser Asp Val Lys Val Glu Leu Gln Lys Gly Asp Glu Val
                  275 280 285
          Asn Leu Tyr Lys Arg Tyr Gly Gly Gly Gly Glu Leu Gly Ala Phe Ser
              290 295 300
          Pro Lys Leu Phe Leu Lys Cys Phe Tyr Leu Asn Leu Lys His Asn Leu
          305 310 315 320
          Ser Ala Lys Lys Gly Leu Lys Asp Lys Asn Ala
                          325 330
           <![CDATA[ <210> 20]]>
           <![CDATA[ <211> 226]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Hyphomonas sp.]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> misc_feature]]>
           <![CDATA[ <222> (54)..(54)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> misc_feature]]>
           <![CDATA[ <222> (74)..(74)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> misc_feature]]>
           <![CDATA[ <222> (114)..(114)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <400> 20]]>
          Met Ile Gly Trp Leu Val Ile Gly Thr Asn Lys Tyr Leu Glu Leu Gly
          1 5 10 15
          Val Glu Cys Leu Glu Ser Ile Lys Glu Lys Tyr Thr Gly Ser Gln Ser
                      20 25 30
          Gln Lys Phe Phe Leu Phe Thr Asp Arg Val Asp Glu Val Lys Gln Asp
                  35 40 45
          Trp Ile Thr Thr Phe Xaa Ile Glu His Glu Val Phe Pro Tyr Ile Ser
              50 55 60
          Met Ser Arg Tyr Arg His Phe Val Asp Xaa Lys Glu Val Leu Ala Glu
          65 70 75 80
          Met Asp Tyr Leu Tyr Tyr Val Asp Ala Asp Ser Leu Phe Leu Asn Val
                          85 90 95
          Gly Asp Glu Ile Leu Gly Glu Arg Val Thr Thr Arg His Pro Gly Trp
                      100 105 110
          Phe Xaa Arg Glu Ser Ile Asp Cys Pro Phe Asp Arg Asn Pro Asn Ser
                  115 120 125
          Asn Ala Phe Val Ser Tyr Asp Tyr Lys Gly Pro Tyr Phe Gln Asn Cys
              130 135 140
          Phe Gln Gly Gly Tyr Ser Lys Glu Phe Leu Lys Met Ser Glu Ile Leu
          145 150 155 160
          Ala Glu Arg Thr Lys Met Asp Leu Gly Asn Asp Val Met Pro Leu Trp
                          165 170 175
          His Asp Glu Ser His Met Asn Lys Tyr Met Ser Glu Asn Pro Pro Thr
                      180 185 190
          Arg Ile Leu Asp Pro Gly Tyr Ala Tyr Pro Glu Asn Trp Arg Ile Pro
                  195 200 205
          Phe Glu Gln Lys Ile Ile Gly Val Ser Lys Asn His Asp Glu Ile Arg
              210 215 220
          Ser Asp
          225
           <![CDATA[ <210> 21]]>
           <![CDATA[ <211> 610]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Neisseria shayeganii 871]]>
           <![CDATA[ <400> 21]]>
          Met His Thr Pro Thr Ile Ala Val Leu Tyr Ile Ala Thr Gly Arg Tyr
          1 5 10 15
          Thr Val Phe Trp Glu Pro Phe Tyr Gln Ser Ala Glu Arg Phe Leu Leu
                      20 25 30
          Thr Asp Cys Arg Lys His Tyr Phe Leu Phe Thr Asp Ser Pro Glu Pro
                  35 40 45
          Leu Ala Gly Glu Ala Glu Gly Lys Val Thr Arg Ile His Gln Asn Lys
              50 55 60
          Leu Gly Trp Pro Tyr Asp Thr Leu Met Arg Phe Glu Met Phe Leu Gly
          65 70 75 80
          Ile Lys Ala Gln Leu Ala Ala Tyr Asp Phe Ile Tyr Phe Phe Asn Gly
                          85 90 95
          Asn Thr Glu Leu Leu Ser Pro Val Ser Arg Glu Asp Leu Leu Pro Leu
                      100 105 110
          Gln Ala His Glu Asn Leu Val Ala Ala Arg Gln Pro His Ile Thr His
                  115 120 125
          Leu Ser Ala Asp Glu Phe Pro Tyr Glu Arg Asn Pro Lys Ser Thr Ala
              130 135 140
          Cys Ile Pro Arg Gly Gln Gly Arg Tyr Tyr Phe Thr Gly Ala Leu Asn
          145 150 155 160
          Gly Gly Arg Ala Ala Ala Tyr Leu Ala Met Cys Glu Thr Leu Asn Arg
                          165 170 175
          His Ile Gln Gln Asp Leu Asp Lys Asn Val Ile Ala Leu Trp His Asp
                      180 185 190
          Glu Ser Gln Leu Asn Arg Tyr Leu Leu Asp Arg Asn Asp Val Lys Ile
                  195 200 205
          Leu Pro Arg Tyr Phe Thr Arg Gly Glu Thr Glu Pro Trp Lys Gln Asn
              210 215 220
          Ala Lys Val Met Phe Ser Asp Lys Thr His Tyr Arg Phe Gly Gly His
          225 230 235 240
          Ala Tyr Leu Arg Gly Glu Ser Glu Gln Lys Ile Ser Arg Glu Glu Trp
                          245 250 255
          Glu Ala Glu Tyr Arg Val Pro Ala Asp Val Ala Ala Thr Ala Arg Gln
                      260 265 270
          Pro His Thr Val Phe Ala Thr Asp Ala Lys Trp Lys Arg Arg Val Asp
                  275 280 285
          Ala Cys Asn Arg Arg Pro Trp Lys Ile Leu Tyr Lys Gly Leu Val Pro
              290 295 300
          Lys Pro Val Arg Asn Arg Leu Asn Lys Lys Ala Gln Leu Ala Gln Gln
          305 310 315 320
          Arg His Val Ala Ala Cys Trp Glu Arg Phe Leu Lys Ala Tyr Phe Tyr
                          325 330 335
          Gly Ile Leu Glu Ser Phe Ser Leu Gln Pro Lys Gln Asp Leu Arg Gly
                      340 345 350
          Arg Lys Ile Ile Trp Gln Tyr Trp Gly Gln Gly Ala Asp Ala Ala Asp
                  355 360 365
          Leu Pro Asp Ile Val Arg Leu Cys Phe His Ser Val Glu Gln His Lys
              370 375 380
          Gly Asp Tyr Asp Ile Ile Arg Leu Asp Asp Gly Asn Val Arg Asp Tyr
          385 390 395 400
          Val Asp Phe Pro Asp Phe Val Trp Glu Lys Arg His Asn Pro Glu Phe
                          405 410 415
          Lys His Ala Phe Phe Ala Asp Leu Leu Arg Leu Ala Leu Leu Asp Leu
                      420 425 430
          Tyr Gly Gly Ala Trp Leu Asp Ala Thr Ile Leu Leu Thr Ala Pro Leu
                  435 440 445
          Pro Glu Gly Tyr Leu Lys Asp Ala Gly Phe Phe Met Phe Gln Arg Asp
              450 455 460
          Pro Ala Ala Ala Asp Gln Ala Ala Trp Glu Lys Leu Asn Ala Asp Tyr
          465 470 475 480
          Phe Gly Trp Gln Pro Asn His Lys Val Ser Val Leu Asn Ser Phe Ile
                          485 490 495
          Met Ala His Pro Gly Asn Thr Val Ile His Thr Cys Leu Asp Leu Leu
                      500 505 510
          Leu Asn Phe Trp Lys Thr Gln Asn Arg Ile Pro His Tyr Phe Phe Phe
                  515 520 525
          Gln Ile Met Phe His Glu Leu Met Arg Leu Tyr Phe Ala Asp Arg Gln
              530 535 540
          Cys Pro Leu Ala Asp Asp Thr Leu Pro His Leu Leu Tyr Arg Gln Ile
          545 550 555 560
          Gln Gln Pro Phe Asp Ala Gly Arg Phe Ala Asp Ile Thr Arg Arg Cys
                          565 570 575
          Gly Val His Lys Leu Ser Tyr Leu Lys His Cys Pro Pro Gly Ser Phe
                      580 585 590
          Tyr His His Leu Arg Thr Glu Ala Gly Leu Pro Pro Ala Asn Ala Asn
                  595 600 605
          Gly His
              610
           <![CDATA[ <210> 22]]>
           <![CDATA[ <211> 277]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Pasteurella aerogenes]]>
           <![CDATA[ <400> 22]]>
          Met Ala Lys Val Ala Ile Leu Tyr Ile Ala Thr Gly Arg Tyr Ile Val
          1 5 10 15
          Phe Trp Glu His Phe Tyr Arg Ser Ala Glu Lys Phe Leu Leu Pro Lys
                      20 25 30
          Ser Asp Lys Asn Tyr Phe Val Phe Thr Asp Ser Pro His Ile Leu Gly
                  35 40 45
          Glu Glu His Ser Asn Val Thr Arg Ile Glu Gln Lys Lys Leu Gly Trp
              50 55 60
          Pro Tyr Asp Thr Leu Met Arg Phe Asp Ile Phe Leu Ser Ile Arg Glu
          65 70 75 80
          Thr Leu Glu Lys Phe Asp Tyr Ile Tyr Phe Phe Asn Gly Asn Ser Glu
                          85 90 95
          Leu Leu Ser Glu Val Asn Glu Thr Glu Phe Leu Pro Cys Glu Asp Asn
                      100 105 110
          Tyr Asn Leu Val Phe Thr His Gln Pro His Met Phe His Leu Pro Lys
                  115 120 125
          Arg Arg Phe Thr Tyr Asp Arg Asn Pro Glu Ser Cys Ala Tyr Ile Pro
              130 135 140
          Gln Gly Asp Gly Lys Tyr Tyr Phe Thr Gly Ala Leu Asn Gly Gly Lys
          145 150 155 160
          Ala Lys Tyr Tyr Leu Glu Met Cys Glu Lys Leu Ser Gln Asn Thr His
                          165 170 175
          Thr Asp Leu Glu Lys Asn Ile Ile Ala Arg Trp His Asp Glu Ser His
                      180 185 190
          Leu Asn Arg Tyr Ala Ile Gly Arg Thr Asp Ile Lys Ile Leu Pro Pro
                  195 200 205
          Tyr Phe Thr Arg Ser Glu Thr Glu Lys Trp Lys Thr Ser Ala Lys Ile
              210 215 220
          Met Phe Ser Asp Lys Thr His Tyr Arg Phe Gly Gly His Ala Tyr Leu
          225 230 235 240
          Arg Gly Glu Ser Glu Asn Lys Ile Thr Pro Thr Glu Trp Glu Glu Lys
                          245 250 255
          Tyr Lys Asn Lys Lys Arg Arg Phe Ser Phe Arg Ile Lys Gln Tyr Ile
                      260 265 270
          Lys Ser Trp Phe Leu
                  275
           <![CDATA[ <210> 23]]>
           <![CDATA[ <211> 301]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Psychrobacter sp. (strain PRwf-1)]]>
           <![CDATA[ <400> 23]]>
          Met Lys Ile Thr Glu Leu Asn Met Ser Leu Ser Thr Ser Ala Leu Ser
          1 5 10 15
          Asn Asn Ser Lys Pro Ser Val Ala Ile Leu Tyr Ile Ala Thr Gly Arg
                      20 25 30
          Tyr Thr Val Phe Trp Asp Tyr Phe Tyr Lys Ser Ala Glu Lys Tyr Leu
                  35 40 45
          Leu Pro Asp Cys Asn Lys His Tyr Ile Leu Phe Thr Asp Ser Asp Ala
              50 55 60
          Leu Ile Asp Ser Phe Arg Thr Lys Ser Asp Gln Val Thr Ala Leu Lys
          65 70 75 80
          Lys Glu Ala Met Glu Trp Pro Phe Cys Thr Leu Met Arg Phe Arg Phe
                          85 90 95
          Phe Leu Asp Ala Glu Asn Ile Ile Lys Gln His Asp Phe Val Phe Phe
                      100 105 110
          Phe Asn Ala Asn Thr Glu Phe Leu Ser Thr Ile Thr Gln Tyr Asp Leu
                  115 120 125
          Leu Pro Leu Gly Ser His Glu Asn Leu Thr Leu Cys Leu Gln Pro His
              130 135 140
          Met Phe His Arg Asn Arg Glu Lys Tyr Thr Tyr Asp Arg Asn Pro Lys
          145 150 155 160
          Ser Thr Ala Tyr Ile Ala Tyr Gly Glu Gly Lys Tyr Tyr Phe Thr Gly
                          165 170 175
          Ala Leu Asn Gly Gly Lys Ser Ala Ala Phe Leu Asp Leu Cys His Thr
                      180 185 190
          Leu Tyr Asn Asn Thr Gln Ser Asp Leu Lys Gln Asp Ile Ile Ala Leu
                  195 200 205
          Trp His Asp Glu Ser His Leu Asn Lys Phe Ala Leu Gly Arg Glu Asp
              210 215 220
          Ile Lys Ile Leu Pro Pro Tyr Phe Thr Arg Gly Glu Arg Glu Tyr Trp
          225 230 235 240
          Lys Lys Thr Ser Lys Leu Met Phe Ser Asp Lys Ser His Tyr Arg Phe
                          245 250 255
          Gly Gly His Ala Tyr Leu Arg Ser Glu Thr Asp Glu Lys Ile Thr Gln
                      260 265 270
          Ala Glu Trp Asn Lys Lys Asn Ala Lys Arg Arg Arg Lys Leu Lys Phe
                  275 280 285
          Arg Ala Lys Gln Tyr Ile Ser Ser Leu Leu Phe Arg Gln
              290 295 300
           <![CDATA[ <210> 24]]>
           <![CDATA[ <211> 284]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Psychrobacter sp. P11F6]]>
           <![CDATA[ <400> 24]]>
          Met Thr Glu Asp Asn Lys Pro Ser Val Ala Ile Leu Tyr Ile Ala Thr
          1 5 10 15
          Gly Arg Tyr Thr Val Phe Trp Asp Tyr Phe Tyr Cys Ser Ala Glu Lys
                      20 25 30
          His Leu Leu Pro Asn Ser Asn Lys His Tyr Val Leu Phe Thr Asp Asp
                  35 40 45
          Leu Ala Leu Ile Ser Arg Gln Thr Asp Tyr Pro Asn Val Thr Met Ile
              50 55 60
          Lys Gln Glu Ala Leu Gly Trp Pro Tyr Ser Thr Leu Met Arg Phe Lys
          65 70 75 80
          Phe Phe Leu Gly Ala Lys Ser Ile Ile Glu Lys Tyr Asp Phe Ile Phe
                          85 90 95
          Tyr Phe Asn Ala Asn Thr Glu Phe Leu Ser Asp Ile Thr Glu Asp Glu
                      100 105 110
          Leu Leu Pro Leu Asp His His Glu Glu Leu Ser Leu Gly Val Gln Pro
                  115 120 125
          His Met Phe His Leu Asn Lys Arg Ala Tyr Thr Tyr Asp Arg Asn Pro
              130 135 140
          Gln Ser Gln Ala Tyr Ile Pro Tyr His Lys Gly Arg Tyr Tyr Phe Thr
          145 150 155 160
          Gly Ala Leu Asn Gly Gly Lys Ser His Ala Tyr Leu Gln Met Cys Glu
                          165 170 175
          Thr Leu Asn Gln Asn Thr Glu Leu Asp Leu Lys Asn Asn Val Ile Ala
                      180 185 190
          Leu Trp His Asp Glu Ser Gln Leu Asn Lys Phe Ala Leu Asp Arg Thr
                  195 200 205
          Asp Ile Lys Val Leu Pro Pro Tyr Phe Thr Arg Gly Glu His Glu Tyr
              210 215 220
          Trp Lys Lys Ser Ser Lys Ile Met Phe Ser Asp Lys Thr His Tyr Arg
          225 230 235 240
          Phe Gly Gly His Ala Tyr Leu Arg Ala Glu Thr Asn Asp Lys Ile Thr
                          245 250 255
          Lys Ser Asp Trp Glu Gln Lys Asn Gly Lys Arg Arg Arg Lys Leu Asn
                      260 265 270
          Thr Arg Phe Lys Gln Tyr Ile Ala Ser Leu Phe Phe
                  275 280
           <![CDATA[ <210> 25]]>
           <![CDATA[ <211> 234]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Salmonella enterica]]>
           <![CDATA[ <400> 25]]>
          Met Thr Ile Asn Ile Leu Tyr Ile Cys Thr Gly Glu Tyr Arg Asn Phe
          1 5 10 15
          Phe Asp Lys Phe Tyr Ser Ser Cys Glu Gly Tyr Phe Ile Pro Glu Tyr
                      20 25 30
          Lys Lys Lys Tyr Tyr Val Phe Thr Asp Ser His Ser Asp Lys Phe Ser
                  35 40 45
          Lys Tyr Ser Asn Val Thr Val Val Pro Val Glu Asn Asn Cys Trp Pro
              50 55 60
          Leu Asn Thr Leu Leu Arg Phe Ser Tyr Phe Ser Lys Ile Val Ser Asp
          65 70 75 80
          Leu Gln Pro Asn Thr Tyr Thr Phe Phe Phe Asn Ala Asn Ala Leu Ile
                          85 90 95
          Val Lys Thr Ile Pro Phe Asp Ile Phe Lys Asn Ala Asn Leu Val Gly
                      100 105 110
          Val Val His Pro Gly Tyr Lys Asn Lys Met Ser Ile Phe Tyr Pro Trp
                  115 120 125
          Glu Arg Lys Lys Ser Ala Val Cys Tyr Leu Ser Tyr Phe Lys Lys Gly
              130 135 140
          Ile Tyr Phe Gln Gly Cys Phe Asn Gly Gly Arg Thr Glu Tyr Phe Cys
          145 150 155 160
          Asp Leu Ile Lys Thr Cys Asn Asp Met Thr Ile Lys Asp Leu Lys Arg
                          165 170 175
          Asn Ile Ile Ala Lys Val His Asp Glu Ser Tyr Leu Asn Tyr Tyr Phe
                      180 185 190
          Tyr Phe Lys Glu Pro Leu Cys Leu Ser Glu Leu Tyr Ser Trp Pro Glu
                  195 200 205
          Lys Tyr Gly Glu Asn Thr Glu Ala Lys Ile Ile Met Arg Asp Lys Glu
              210 215 220
          Arg Glu Asp Trp Tyr Ala Asn Ile Lys Ser
          225 230
           <![CDATA[ <210> 26]]>
           <![CDATA[ <211> 234]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Salmonella enterica]]>
           <![CDATA[ <400> 26]]>
          Met Thr Ile Asn Ile Leu Tyr Ile Cys Thr Gly Glu Tyr Arg Asn Phe
          1 5 10 15
          Phe Asp Lys Phe Tyr Thr Ser Cys Glu Gly Tyr Phe Ile Pro Glu Cys
                      20 25 30
          Lys Lys Lys Tyr Tyr Val Phe Thr Asp Ser His Ser Asp Lys Phe Ser
                  35 40 45
          Lys Tyr Asn Asn Val Thr Val Val Pro Val Glu Asn Asn Cys Trp Pro
              50 55 60
          Leu Asn Thr Leu Leu Arg Phe Ser Tyr Phe Ser Lys Ile Val Pro Asp
          65 70 75 80
          Leu Gln Pro Asn Thr Tyr Thr Phe Phe Phe Asn Ala Asn Ala Leu Ile
                          85 90 95
          Val Lys Thr Ile Pro Phe Asp Thr Phe Lys Asn Ala Asn Leu Val Gly
                      100 105 110
          Val Val His Pro Gly Tyr Lys Asn Lys Met Ser Ile Phe Tyr Pro Trp
                  115 120 125
          Glu Arg Lys Lys Ser Ala Ala Cys Tyr Leu Ser Tyr Phe Lys Asn Gly
              130 135 140
          Ile Tyr Phe Gln Gly Cys Phe Asn Gly Gly Arg Thr Glu Tyr Phe Cys
          145 150 155 160
          Asp Leu Ile Lys Thr Cys Asn Asp Met Thr Ile Lys Asp Leu Lys Arg
                          165 170 175
          Asn Ile Ile Ala Lys Val His Asp Glu Ser Tyr Leu Asn Tyr Tyr Phe
                      180 185 190
          Tyr Phe Lys Glu Pro Leu Cys Leu Ser Glu Leu Tyr Ser Trp Pro Glu
                  195 200 205
          Lys Tyr Gly Glu Asn Thr Glu Ala Arg Ile Ile Met Arg Asp Lys Glu
              210 215 220
          Arg Glu Tyr Trp Tyr Ala Asn Ile Lys Asn
          225 230
           <![CDATA[ <210> 27]]>
           <![CDATA[ <211> 189]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Salmonella enterica I]]>
           <![CDATA[ <400> 27]]>
          Lys Phe Ser Lys Tyr Ser Asn Val Thr Val Val Pro Val Glu Asn Asn
          1 5 10 15
          Cys Trp Pro Leu Asn Thr Leu Leu Arg Phe Ser Tyr Phe Ser Lys Ile
                      20 25 30
          Val Ser Asp Leu Gln Pro Asn Thr Tyr Thr Phe Phe Phe Asn Ala Asn
                  35 40 45
          Ala Leu Ile Val Lys Thr Ile Pro Phe Asp Ile Phe Lys Asn Ala Asn
              50 55 60
          Leu Val Gly Val Val His Pro Gly Tyr Lys Asn Lys Met Ser Ile Phe
          65 70 75 80
          Tyr Pro Trp Glu Arg Lys Lys Ser Ala Val Cys Tyr Leu Ser Tyr Phe
                          85 90 95
          Lys Lys Gly Ile Tyr Phe Gln Gly Cys Phe Asn Gly Gly Arg Thr Glu
                      100 105 110
          Tyr Phe Cys Asp Leu Ile Lys Thr Cys Asn Asp Met Thr Ile Lys Asp
                  115 120 125
          Leu Lys Arg Asn Ile Ile Ala Lys Val His Asp Glu Ser Tyr Leu Asn
              130 135 140
          Tyr Tyr Phe Tyr Phe Lys Glu Pro Leu Cys Leu Ser Glu Leu Tyr Ser
          145 150 155 160
          Trp Pro Glu Lys Tyr Gly Glu Asn Thr Glu Ala Lys Ile Ile Met Arg
                          165 170 175
          Asp Lys Glu Arg Glu Asp Trp Tyr Ala Asn Ile Lys Ser
                      180 185
           <![CDATA[ <210> 28]]>
           <![CDATA[ <211> 234]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Salmonella enterica subsp. enterica serovar Ahuza]]>
           <![CDATA[ <400> 28]]>
          Met Thr Ile Asn Ile Leu Tyr Ile Cys Thr Gly Glu Tyr Arg Asn Phe
          1 5 10 15
          Phe Asp Lys Phe Tyr Pro Ser Cys Glu Gly Tyr Phe Ile Pro Glu Tyr
                      20 25 30
          Lys Lys Lys Tyr Tyr Val Phe Thr Asp Ser His Ser Asp Lys Phe Ser
                  35 40 45
          Lys Tyr Ser Asn Val Thr Val Val Pro Val Glu Asn Asn Cys Trp Pro
              50 55 60
          Leu Asn Thr Leu Leu Arg Phe Ser Tyr Phe Ser Lys Ile Val Ser Asp
          65 70 75 80
          Leu Gln Pro Asn Thr Tyr Thr Phe Phe Phe Asn Ala Asn Ala Leu Ile
                          85 90 95
          Val Lys Thr Ile Pro Phe Asp Ile Phe Lys Asn Ala Asn Leu Val Gly
                      100 105 110
          Val Val His Pro Gly Tyr Lys Asn Lys Met Ser Ile Phe Tyr Pro Trp
                  115 120 125
          Glu Arg Lys Lys Ser Ala Val Cys Tyr Leu Ser Tyr Phe Lys Lys Gly
              130 135 140
          Ile Tyr Phe Gln Gly Cys Phe Asn Gly Gly Arg Thr Glu Tyr Phe Cys
          145 150 155 160
          Asp Leu Ile Lys Thr Cys Asn Asp Met Thr Ile Lys Asp Leu Lys Arg
                          165 170 175
          Asn Ile Ile Ala Lys Val His Asp Glu Ser Tyr Leu Asn Tyr Tyr Phe
                      180 185 190
          Tyr Phe Lys Glu Pro Leu Cys Leu Ser Glu Leu Tyr Ser Trp Pro Glu
                  195 200 205
          Lys Tyr Gly Glu Asn Thr Glu Ala Lys Ile Ile Met Arg Asp Lys Glu
              210 215 220
          Arg Glu Asp Trp Tyr Ala Asn Ile Lys Ser
          225 230
           <![CDATA[ <210> 29]]>
           <![CDATA[ <211> 165]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Salmonella enterica subsp. enterica serovar Kingabwa]]>
           <![CDATA[ <400> 29]]>
          Met Thr Ile Asn Ile Leu Tyr Ile Cys Thr Gly Glu Tyr Arg Asn Phe
          1 5 10 15
          Phe Asp Lys Phe Tyr Ser Ser Cys Glu Gly Tyr Phe Ile Pro Glu Tyr
                      20 25 30
          Lys Lys Lys Tyr Tyr Val Phe Thr Asp Ser His Ser Asp Lys Phe Ser
                  35 40 45
          Lys Tyr Ser Asn Val Thr Val Val Pro Val Glu Asn Asn Cys Trp Pro
              50 55 60
          Leu Asn Thr Leu Leu Arg Phe Ser Tyr Phe Ser Lys Ile Val Ser Asp
          65 70 75 80
          Leu Gln Pro Asn Thr Tyr Thr Phe Phe Phe Asn Ala Asn Ala Leu Ile
                          85 90 95
          Val Lys Thr Ile Pro Phe Asp Ile Phe Lys Asn Ala Asn Leu Val Gly
                      100 105 110
          Val Val His Pro Gly Tyr Lys Asn Lys Met Ser Ile Phe Tyr Pro Trp
                  115 120 125
          Glu Arg Lys Lys Ser Ala Val Cys Tyr Leu Ser Tyr Phe Lys Lys Gly
              130 135 140
          Ile Tyr Phe Gln Gly Cys Phe Asn Gly Gly Arg Thr Glu Tyr Phe Cys
          145 150 155 160
          Asp Leu Ile Lys Thr
                          165
           <![CDATA[ <210> 30]]>
           <![CDATA[ <211> 258]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Selenomonas ruminantium]]>
           <![CDATA[ <400> 30]]>
          Met Lys Ile Ala Ile Leu Tyr Ile Ala Leu Gly Lys Tyr Asp Val Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Leu Ser Ala Glu Met His Phe Ile Lys Ser Ile
                      20 25 30
          Lys Lys Asp Tyr Tyr Ile Phe Thr Asp Ala Gln His Ile Tyr Lys Glu
                  35 40 45
          Asp Ala Asp Asn Val Lys Lys Lys Ile Pro Gln Glu Asn Leu Gly Trp Pro
              50 55 60
          Gly Asn Thr Leu Phe Arg Phe Asn Ile Phe Leu Asn Met Glu Ser Glu
          65 70 75 80
          Leu Glu Lys Tyr Asp Tyr Ile Phe Phe Phe Asn Ala Asn Tyr Ile Phe
                          85 90 95
          Val Lys Asp Ile Asp Val Asp Phe Leu Pro Ile Asn Lys Leu Leu Val
                      100 105 110
          Val Gln His Pro Gly Tyr Tyr Asn Lys Arg Val Asn Lys Tyr Pro Tyr
                  115 120 125
          Glu Lys Asn Pro Asn Ser Leu Ala Tyr Val Ser Asn Lys Glu Lys Lys
              130 135 140
          Thr Tyr Val Gln Gly Cys Leu Glu Gly Gly Ser Lys Lys Glu Phe Ile
          145 150 155 160
          Asn Leu Ile Arg Asp Leu Ala Gly Asn Ile Lys Asn Asp Tyr Ser Asn
                          165 170 175
          Gly Ile Ile Ala Lys Trp His Asp Glu Ser His Leu Asn Lys Tyr Ile
                      180 185 190
          Cys Ser His Glu Tyr Lys Leu Met His Pro Gly Tyr Ala Tyr Pro Glu
                  195 200 205
          Gly Trp Glu Ile Pro Tyr Pro Met Glu Ile Met Thr Arg Asp Lys Arg
              210 215 220
          Lys Ile Ala Ser Tyr Asp Val Leu Arg Gly Thr Asn Ser Lys Gly Gly
          225 230 235 240
          Lys Ala Ile Leu Lys Lys Ile Lys Ile Arg Leu Ile Asp Ile Met Glu
                          245 250 255
          His Phe
           <![CDATA[ <210> 31]]>
           <![CDATA[ <211> 262]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Smithella sp. SDB]]>
           <![CDATA[ <400> 31]]>
          Met Gln Ile Gly Val Leu Tyr Ile Cys Ile Gly Lys Tyr Ser Ile Phe
          1 5 10 15
          Trp Lys Ser Phe Tyr Glu Ser Ser Glu Lys His Phe Leu Val His His
                      20 25 30
          Gln Lys Lys Tyr Phe Val Phe Thr Asp Ala Gln Ile Ile Asp Tyr Gln
                  35 40 45
          Asp Asn Ala Asn Val Val Ile Val Phe Gln Lys Asn Leu Gly Trp Pro
              50 55 60
          Asn Asn Thr Leu Met Arg Phe His Ile Phe Leu Arg His Lys Thr Leu
          65 70 75 80
          Leu Gln Glu Met Asp Phe Leu Phe Phe Cys Asn Ala Asn Leu Leu Phe
                          85 90 95
          Val Asp Asn Val Gly Asp Glu Ile Leu Pro Leu Glu Glu Gly Phe Ala
                      100 105 110
          Ala Leu Gln His Pro Gly Tyr Trp Asn Lys Pro Arg Lys Leu Phe Pro
                  115 120 125
          Tyr Glu Thr Asn Pro Met Ser Leu Ala Asn Val Pro Ala His Gln Gly
              130 135 140
          Lys Tyr Tyr Val Met Gly Ala Phe Asn Gly Gly Gln Ala Lys Ile Phe
          145 150 155 160
          Leu Lys Met Ser Glu Glu Leu Ser Lys Asn Ile Asp Glu Asp Phe Lys
                          165 170 175
          Lys Asn Ile Val Ala Val Trp His Asp Glu Ser His Leu Asn Lys Tyr
                      180 185 190
          Val Val Asp Lys Lys Val Lys Ile Leu Asn Pro Ser Tyr Gly Tyr Pro
                  195 200 205
          Glu Asp Arg Asp Leu Pro Phe Lys Pro Lys Ile Met Ile Arg Asp Lys
              210 215 220
          Ala Lys Tyr Gly Gly His Asn Leu Leu Arg Gly Ile Pro Glu Asn Ser
          225 230 235 240
          Ser Phe Ile Arg Lys Tyr Phe Arg Glu Ile Lys Ser Phe Ile Ala Lys
                          245 250 255
          Tyr Leu Arg Asn Asn Arg
                      260
           <![CDATA[ <210> 32]]>
           <![CDATA[ <211> 243]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Synechococcus phage ACG-2014f]]>
           <![CDATA[ <400> 32]]>
          Met Lys Lys Val Ala Ile Ile Phe Ile Gly Thr Gly Arg Tyr Leu Glu
          1 5 10 15
          Phe Leu Pro Lys Tyr Tyr Glu Gln Ala Glu Ala Asn Leu Phe Pro Asp
                      20 25 30
          Arg Pro Lys His Tyr Tyr Val Phe Thr Asp Gly Asp Leu Gly Asn Glu
                  35 40 45
          Leu Pro Asp Asn Val Thr Val Tyr Glu Gln Glu His Leu Gln Trp Pro
              50 55 60
          Tyr Ile Thr Leu Tyr Arg Phe Gly Ile Ile Gln Lys His Leu Glu Glu
          65 70 75 80
          Ile Glu Lys Glu Cys Gly Phe Leu Leu Phe Met Asp Ala Asp Thr Gln
                          85 90 95
          Val Val Ser Pro Val Ser Phe Asp Glu Val Phe Lys Lys Gly Lys Pro
                      100 105 110
          Tyr Thr Gly Val His His Pro Cys His Ala Leu Asn Met Pro Pro His
                  115 120 125
          Asn Glu Phe Pro Gly Ser Leu Glu Thr Asn Thr Ala Ser Lys Ala Ala
              130 135 140
          Cys Lys Pro Gly Asp Asp Phe Ser Val Tyr Trp Gln Gly Cys Val Trp
          145 150 155 160
          Gly Gly Asn Ile Lys Gly Ala Arg Lys Ile Ile Asp Thr Leu His His
                          165 170 175
          Arg Thr Lys Gln Asp Glu Glu Asn Gly Ile Val Ala Leu Trp His Asp
                      180 185 190
          Glu Ser His Ile Asn Arg Tyr Phe Leu Asp Asn Lys Asp Lys Val Asn
                  195 200 205
          Thr Leu Ser Pro Ser Phe Ala Tyr Pro Glu Ser Phe Thr Glu Tyr Met
              210 215 220
          Glu Asp Tyr Glu Pro Lys Ile Val His Leu Ala Lys Glu Asn Ser Lys
          225 230 235 240
          Tyr Gln Val
           <![CDATA[ <210> 33]]>
           <![CDATA[ <211> 243]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Synechococcus phage ACG-2014f]]>
           <![CDATA[ <400> 33]]>
          Met Lys Lys Val Ala Ile Ile Phe Ile Gly Thr Gly Arg Tyr Leu Glu
          1 5 10 15
          Phe Leu Pro Lys Tyr Tyr Glu Gln Ala Glu Ala Asn Leu Phe Pro Asp
                      20 25 30
          Arg Pro Lys His Tyr Tyr Val Phe Thr Asp Gly Asp Leu Gly Asn Glu
                  35 40 45
          Leu Pro Asp Asn Val Thr Val Tyr Glu Gln Glu His Leu Gln Trp Pro
              50 55 60
          Tyr Ile Thr Leu Tyr Arg Phe Gly Ile Ile Gln Lys His Leu Glu Glu
          65 70 75 80
          Ile Glu Lys Glu Cys Gly Phe Leu Leu Phe Met Asp Ala Asp Thr Gln
                          85 90 95
          Val Val Ser Pro Val Ser Phe Asp Glu Val Phe Lys Lys Gly Lys Pro
                      100 105 110
          Tyr Thr Gly Val His His Pro Cys His Ala Leu Asn Met Pro Pro His
                  115 120 125
          Asn Glu Phe Pro Gly Ser Leu Glu Thr Asn Thr Ala Ser Lys Ala Ala
              130 135 140
          Cys Lys Pro Gly Asp Asp Phe Ser Val Tyr Trp Gln Gly Cys Val Trp
          145 150 155 160
          Gly Gly Asn Ile Lys Gly Ala Arg Lys Ile Ile Asp Thr Leu His His
                          165 170 175
          Arg Thr Lys Gln Asp Glu Glu Asn Gly Ile Ile Ala Lys Trp His Asp
                      180 185 190
          Glu Ser His Ile Asn Arg Tyr Phe Leu Asp Asn Lys Asp Lys Val Asn
                  195 200 205
          Thr Leu Ser Pro Ser Phe Ala Tyr Pro Glu Ser Phe Thr Glu Tyr Met
              210 215 220
          Glu Asp Tyr Glu Pro Lys Ile Val His Leu Ala Lys Glu Asn Ser Lys
          225 230 235 240
          Tyr Gln Val
           <![CDATA[ <210> 34]]>
           <![CDATA[ <211> 243]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Synechococcus phage ACG-2014f]]>
           <![CDATA[ <400> 34]]>
          Met Lys Lys Val Ala Ile Ile Phe Ile Gly Thr Asp Arg Tyr Leu Glu
          1 5 10 15
          Phe Leu Pro Lys Tyr Tyr Glu Gln Ala Glu Ala Asn Leu Phe Pro Asp
                      20 25 30
          Arg Pro Lys His Tyr Tyr Val Phe Thr Asp Gly Asp Leu Gly Asn Glu
                  35 40 45
          Leu Pro Asp Asn Val Thr Val Tyr Glu Gln Glu His Leu Gln Trp Pro
              50 55 60
          Tyr Ile Thr Leu Tyr Arg Phe Gly Ile Ile Gln Lys His Leu Glu Glu
          65 70 75 80
          Ile Glu Lys Glu Cys Gly Phe Leu Leu Phe Met Asp Ala Asp Thr Gln
                          85 90 95
          Val Val Ser Pro Val Ser Phe Asp Glu Val Phe Lys Lys Gly Lys Pro
                      100 105 110
          Tyr Thr Gly Val His His Pro Cys His Ala Leu Asn Met Pro Pro His
                  115 120 125
          Asn Glu Phe Pro Gly Ser Leu Glu Thr Asn Thr Ala Ser Lys Ala Ala
              130 135 140
          Cys Lys Pro Gly Asp Asp Phe Ser Val Tyr Trp Gln Gly Cys Val Trp
          145 150 155 160
          Gly Gly Asn Ile Lys Gly Ala Arg Lys Ile Ile Asp Thr Leu His His
                          165 170 175
          Arg Thr Lys Gln Asp Glu Glu Asn Gly Ile Ile Ala Lys Trp His Asp
                      180 185 190
          Glu Ser His Ile Asn Arg Tyr Phe Leu Asp Asn Lys Asp Lys Val Asn
                  195 200 205
          Thr Leu Ser Pro Ser Phe Ala Tyr Pro Glu Ser Phe Thr Glu Tyr Met
              210 215 220
          Glu Asp Tyr Glu Pro Lys Ile Val His Leu Ala Lys Glu Asn Ser Lys
          225 230 235 240
          Tyr Gln Val
           <![CDATA[ <210> 35]]>
           <![CDATA[ <211> 238]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Synechococcus phage Bellamy]]>
           <![CDATA[ <400> 35]]>
          Met Lys Leu Ala Val Val Phe Ile Gly Thr Gly Asp Tyr Ile Asn Phe
          1 5 10 15
          Leu Pro Ser Tyr Tyr Glu Ala Cys Glu Glu Phe Leu Val Pro Asn Thr
                      20 25 30
          Glu Lys Thr Tyr Phe Val Phe Thr Asp Gly Asp Ile Gly Asp Pro Pro
                  35 40 45
          Glu Asn Val Lys Leu Tyr Glu Gln Glu His Leu Pro Trp Pro Tyr Ile
              50 55 60
          Thr Leu Glu Arg Phe Lys Tyr Ile Leu Lys Ala Glu Ser Asp Leu Ala
          65 70 75 80
          Glu Phe Asp Tyr Val Leu Phe Leu Asp Ala Asp Thr Arg Val Val Glu
                          85 90 95
          Thr Val Thr Glu Glu Glu Leu Phe Thr Asp Lys Lys Tyr Ile Gly Val
                      100 105 110
          His His Pro Cys His Phe Leu Gly Met Pro Pro His Asp Asn Pro Pro
                  115 120 125
          Gly Ala Phe Glu Thr Arg Phe Glu Ser Ala Ala Gly Ile Ser Gly Asp
              130 135 140
          Asp Asp Thr Ser Ile Tyr Phe Gln Gly Cys Leu Trp Gly Gly Lys Met
          145 150 155 160
          Pro Tyr Val Leu Asp Met Ile Arg Glu Leu Ala Gln Arg Thr Gln Phe
                          165 170 175
          Asp Leu Asn Arg Asp Val Ile Ala Gln Trp His Asp Glu Ser Gln Met
                      180 185 190
          Asn Lys Phe Phe Cys Glu Arg Arg Glu Asp Val His Val Met Gly Pro
                  195 200 205
          Glu Tyr Ala Tyr Pro Glu Cys Phe Gly Ala Tyr Cys Thr Phe Glu Pro
              210 215 220
          Lys Ile Val His Leu Ala Lys Asp Asn Ser Lys Tyr Gln Gln
          225 230 235
           <![CDATA[ <210> 36]]>
           <![CDATA[ <211> 238]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Synechococcus phage S-CAM9]]>
           <![CDATA[ <400> 36]]>
          Met Lys Val Ala Val Val Phe Ile Gly Thr Glu Lys Tyr Leu Asp Phe
          1 5 10 15
          Leu Pro Ser Trp Tyr Glu Arg Cys Glu Glu Asn Phe Leu Pro Gly Val
                      20 25 30
          Glu Lys Lys Tyr Leu Val Phe Thr Asp Gly Asp Val Pro Glu Ser Pro
                  35 40 45
          Asp Asn Ala Val Val Tyr Lys Gln Glu His Leu Asp Trp Pro Tyr Ile
              50 55 60
          Thr Leu Tyr Arg Phe Lys Ile Ile Gln Lys Ala Leu Asp Glu Ile Val
          65 70 75 80
          Gly Cys Asp Trp Leu Val Phe Leu Asp Ala Asp Met Ala Val Val Asp
                          85 90 95
          Thr Val Thr Ala Pro Glu Ile Phe Thr Asp Lys Pro Tyr Ile Gly Val
                      100 105 110
          His His Pro Cys His Phe Leu Lys Phe Pro Pro His Asn Gln Pro Pro
                  115 120 125
          Gly Ser Phe Glu Thr Asn Pro Leu Ser Thr Ala Lys Val Pro Asp Asp
              130 135 140
          Tyr Asp Phe Ser Ile Tyr Trp Gln Gly Cys Leu Trp Gly Gly Lys Thr
          145 150 155 160
          Ser Glu Val Ile Ser Met Met Glu Glu Leu Asn Ala Arg Ile Ser Leu
                          165 170 175
          Asp Glu Glu Asn Asn Val Ile Ala Gln Trp His Asp Glu Ser His Leu
                      180 185 190
          Asn Ala Phe Tyr Ala Gln Asn Lys Asn Leu Val His Thr Leu Gly Pro
                  195 200 205
          Glu Phe Ala Phe Pro Glu Val Phe Ala Glu Ala Cys Glu Phe Gln Ala
              210 215 220
          Lys Ile Val His Leu Ala Lys Asp Asn Ser Lys Tyr His Val
          225 230 235
           <![CDATA[ <210> 37]]>
           <![CDATA[ <211> 236]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Yersinia kristensenii]]>
           <![CDATA[ <400> 37]]>
          Met Thr Val Asn Ile Leu Tyr Val Cys Thr Gly Asn Tyr Phe Ser Phe
          1 5 10 15
          Phe Lys Asp Phe Tyr Val Ser Cys Glu Lys Leu Phe Leu Pro Asp Cys
                      20 25 30
          Lys Lys Lys Tyr Phe Val Phe Thr Asp Ile Asp Thr Ser Ser Phe Asp
                  35 40 45
          Ile Asn Asp Asp Ile Glu Tyr Ile Lys Ile Glu Lys Asn Cys Trp Pro
              50 55 60
          Leu Asn Thr Leu Leu Arg Phe Ser Tyr Phe Asn Ile Val Arg Asn Lys
          65 70 75 80
          Ile Leu Lys Ser Asp Tyr Val Phe Phe Phe Asn Ala Asn Ala Leu Ile
                          85 90 95
          Val Lys Glu Phe Ser Ser Asp Leu Leu Pro Thr Glu Asp Glu Asn Tyr
                      100 105 110
          Leu Val Gly Val Val His Pro Gly Tyr Glu Asn Lys Pro Ser Phe Leu
                  115 120 125
          Tyr Pro Trp Glu Arg Arg Ile Lys Ser Gln Cys Arg Ile Gly Tyr Leu
              130 135 140
          Cys Lys Gly Thr Tyr Tyr Gln Gly Cys Phe Ser Gly Gly Arg Thr Asn
          145 150 155 160
          Glu Tyr Val Asp Leu Ile Asp Thr Cys Arg Leu Asn Thr Glu Lys Asp
                          165 170 175
          Leu Lys Lys Asn Ile Ile Ala Lys Val His Asp Glu Ser Tyr Leu Asn
                      180 185 190
          His Tyr Phe Lys Asn Lys Lys Lys Pro Lys Ser Leu Ser Ser Leu Tyr Ser
                  195 200 205
          Trp Pro Glu Lys Tyr Gly Asp Asn Glu Asn Ala Ile Ile Ile Met Arg
              210 215 220
          Asp Lys Glu Lys Tyr Glu Trp Tyr Ser Leu Ile Lys
          225 230 235
           <![CDATA[ <210> 38]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> motif 1]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (2)..(2)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> VARIANT]]>
           <![CDATA[ <222> (3)..(3)]]>
           <![CDATA[ <223> Xaa can be Ala, Cys, Ile or Leu]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (4)..(4)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (6)..(7)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> VARIANT]]>
           <![CDATA[ <222> (8)..(9)]]>
           <![CDATA[ <223> Xaa can be Ala, Cys or Gly]]>
           <![CDATA[ <400> 38]]>
          Tyr Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa
          1 5
           <![CDATA[ <210> 39]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> motif 2]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (2)..(2)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> VARIANT]]>
           <![CDATA[ <222> (3)..(3)]]>
           <![CDATA[ <223> Xaa can be Ala or Gly]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (4)..(4)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> UNSURE]]>
           <![CDATA[ <222> (6)..(7)]]>
           <![CDATA[ <223> Xaa can be any naturally occurring amino acid]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> VARIANT]]>
           <![CDATA[ <222> (8)..(9)]]>
           <![CDATA[ <223> Xaa can be Ala, Cys or Gly]]>
           <![CDATA[ <400> 39]]>
          Tyr Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa
          1 5
           <![CDATA[ <210> 40]]>
           <![CDATA[ <211> 306]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Helicobacter mustelae]]>
           <![CDATA[ <400> 40]]>
          Met Gln Ser Thr Ala Gln Asn Thr Gln Gln Asn Thr His Phe Ala Gly
          1 5 10 15
          Ser Ser Gln Thr Thr Pro Gln Ala Ala Gln Ser Val Gln Gln Ala Ser
                      20 25 30
          Leu Ala Leu Pro Lys Ser Ser Pro Thr Cys Tyr Lys Ile Ala Ile Leu
                  35 40 45
          Tyr Ile Cys Thr Gly Ala Tyr Ser Ile Phe Trp Gln Asp Phe Tyr Asp
              50 55 60
          Ser Ala Lys Val His Leu Leu Pro Ala His Arg Leu Thr Tyr Phe Val
          65 70 75 80
          Phe Thr Asp Ala Asp Ser Leu Tyr Ala Glu Glu Ala Ser Asp Val Arg
                          85 90 95
          Lys Ile Tyr Gln Glu Asn Leu Gly Trp Pro Phe Asn Thr Leu Lys Arg
                      100 105 110
          Phe Glu Met Phe Leu Gly Gln Glu Glu Ala Leu Arg Glu Phe Asp Phe
                  115 120 125
          Val Phe Phe Phe Asn Ala Asn Cys Leu Phe Phe Gln His Ile Gly Asp
              130 135 140
          Glu Phe Leu Pro Ile Glu Glu Asp Ile Leu Val Thr Gln His Tyr Gly
          145 150 155 160
          Phe Arg Asp Ala Ser Pro Glu Cys Phe Thr Tyr Glu Arg Asn Pro Lys
                          165 170 175
          Ser Leu Ala Tyr Val Pro Phe Gly Lys Gly Lys Ala Tyr Val Tyr Gly
                      180 185 190
          Ser Thr Asn Gly Gly Lys Ala Gly Ala Phe Leu Ala Leu Ala Arg Thr
                  195 200 205
          Leu Gln Glu Arg Ile Gln Glu Asp Leu Ser Arg Gly Ile Ile Ala Ile
              210 215 220
          Trp His Asp Glu Ser His Leu Asn Ala Tyr Ile Ile Asp His Pro Asn
          225 230 235 240
          Tyr Lys Met Leu Asp Tyr Gly Tyr Gly Phe Pro Glu Gly Tyr Gly Arg
                          245 250 255
          Val Pro Gly Gly Gly Val Tyr Ile Phe Leu Arg Asp Lys Ser Arg Val
                      260 265 270
          Ile Asp Val Asn Ala Ile Lys Gly Met Gly Ser Pro Ala Asn Arg Arg
                  275 280 285
          Leu Lys Asn Ala Leu Arg Lys Leu Lys His Phe Ser Lys Arg Leu Leu
              290 295 300
          Gly Arg
          305
           <![CDATA[ <210> 41]]>
           <![CDATA[ <211> 268]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Clostridium bolteae 90A9]]>
           <![CDATA[ <400> 41]]>
          Met Thr Lys Ile Ala Ile Leu Tyr Ile Cys Ile Gly Lys Tyr Asp Thr
          1 5 10 15
          Phe Trp Lys Asp Phe Tyr Ile Ser Phe Glu Glu Arg Phe Met Thr Glu
                      20 25 30
          Cys Glu Lys Glu Tyr Phe Val Phe Thr Asp Ser Lys Phe Ile Tyr Gly
                  35 40 45
          Glu Asn Val Thr Glu Arg Ile His Arg Ile His Gln Glu Asn Leu Gly
              50 55 60
          Trp Pro Gly Asn Thr Leu Phe Arg Phe Lys Met Phe Leu Gln Ile Ile
          65 70 75 80
          Pro Glu Leu Lys Lys Phe Asp Tyr Thr Phe Phe Met Asn Ala Asn Val
                          85 90 95
          Ile Cys Lys Glu Arg Val Thr Glu Glu Met Met Leu Pro Lys Asp Glu
                      100 105 110
          Lys Leu Val Val Val Gln His Pro Gly Tyr Tyr Lys Gln Lys Pro Tyr
                  115 120 125
          Glu Phe Glu Tyr Asp Arg Asn Arg Lys Ser Lys Ala Tyr Ile Pro Tyr
              130 135 140
          Tyr Lys Gly Glu Val Tyr Ile Cys Gly Gly Ile Asn Gly Gly Arg Thr
          145 150 155 160
          Glu Ala Tyr Ile Glu Leu Ile Lys Thr Leu Asn Lys Asn Ile Asn Ser
                          165 170 175
          Asp Ile Glu Asn Gly Ile Ile Ala Arg Trp His Asp Glu Ser His Ile
                      180 185 190
          Asn Arg Tyr Ile Leu Asp Asn Thr Cys Tyr Lys Leu Leu Ser Pro Ala
                  195 200 205
          Tyr Cys Tyr Pro Glu Asn Trp Asp Ile Pro Phe Thr Pro Ile Leu Val
              210 215 220
          Val Leu Asp Lys Lys Asp Arg Ile Cys Leu Asp Ser Ala Lys Thr Ala
          225 230 235 240
          Glu Gln Cys Ala Asp Ile Phe Phe Leu Glu Lys Ile Lys Lys Gln Phe
                          245 250 255
          Ile Gln Phe Phe Trp Lys Leu Ile Tyr Ile Leu Lys
                      260 265
           <![CDATA[ <210> 42]]>
           <![CDATA[ <211> 786]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Akkermansia muciniphila]]>
           <![CDATA[ <400> 42]]>
          Met Lys Cys Val Leu Ile Val Ser Pro Gly Glu Lys Ser Glu Gly Ala
          1 5 10 15
          Ser Glu Leu His Arg Met Gly Tyr Glu Leu Glu Leu Tyr Pro Ser Thr
                      20 25 30
          Ala Asp Leu Ser Pro Leu Arg Asp Ala Arg Glu Glu Glu Ser Ala Ser
                  35 40 45
          Tyr Leu Gly Arg Ser Pro Ala Ser Ala Glu Arg Ser His Val Arg Ser
              50 55 60
          Leu Arg Ala Ser Phe Ile Arg Leu Leu Glu Asp Arg Asn Tyr Ala Gly
          65 70 75 80
          Ser Asp Leu Ile Ile Phe Gly Glu Ser Asp Ala Val Pro Met Val Ala
                          85 90 95
          Ser Ser Arg Leu Glu Thr Ala Leu Arg Lys Glu Met Lys Glu His Pro
                      100 105 110
          Glu Thr Asp Ile Phe Arg Leu Phe His His Ala Val Trp Ser Pro Gln
                  115 120 125
          Gly Ala Pro Gly Glu Ser Asp Glu Ile Leu Phe Glu Asp Phe Lys Thr
              130 135 140
          Gly Lys Thr Asp Ala Asn Thr Ser Tyr Val Trp Gly Thr His Ala Leu
          145 150 155 160
          Val Ile Pro Ala Ala Arg Arg Pro Arg Val Ala Arg Val Phe Ala Asp
                          165 170 175
          Tyr Arg Leu Pro Thr Asp Ile Ala Leu Glu Ala Ala Asn Ser His Gly
                      180 185 190
          Asp Leu Lys Ile Arg Val Ala Arg His Asn Leu Phe Tyr Gln His Glu
                  195 200 205
          Arg Thr Lys Gln Arg Pro Asp Cys Lys Ile Ala Val Cys Leu Ser Ser
              210 215 220
          Tyr Lys Arg Leu Thr Asp Leu Gln Arg Gln Ile Trp Cys Met Met Asp
          225 230 235 240
          Gln Ser Tyr Pro Asn Leu His Val Phe Ala Ala Val Lys Gly Ile Pro
                          245 250 255
          Glu Gly Thr Tyr Arg Arg Thr Val Leu Pro Leu Phe Glu His Phe Ile
                      260 265 270
          His Glu Gly Arg Leu Thr Met Arg Leu Phe Pro Asn Lys Asn Gln Leu
                  275 280 285
          Ser Asn Phe Leu Asp Thr Ile Arg Asp Leu Asn Val Ser Asp Tyr Asp
              290 295 300
          Leu Phe Ala Lys Ile Asp Asp Asp Asp Leu Tyr Gly Arg Asp Tyr Phe
          305 310 315 320
          Lys Ser Val Asn Lys Phe His Leu His Leu Pro Pro Glu Phe Ser Ser
                          325 330 335
          Phe Tyr Cys Gly Pro Gly Glu Tyr Leu Ser Val Arg Gly Gly Tyr Pro
                      340 345 350
          Phe Ser Gly Asn Gly Phe Phe Gly Cys Phe Gly Pro Thr Leu Val Leu
                  355 360 365
          Ser Arg Asp Val Leu Glu Lys Leu Ile Ile Cys Glu Thr Asn Pro His
              370 375 380
          Met Ile Ser Gln Ile Ser Pro Arg Leu Arg His Ala Gly Tyr Gly Phe
          385 390 395 400
          Thr Glu Asp Asn Phe Met His Met Met Met Leu Asp Thr Gly Ser Ser
                          405 410 415
          Asn Arg Thr Arg Tyr Val Gln Glu Met Ala Leu Pro Met His Leu Ala
                      420 425 430
          Ile Gln Thr Gly Asn Ala Ser Val Met Arg Gly Gly Leu Val Pro Gly
                  435 440 445
          Asp Phe Arg Gly Arg Asn Trp Asn Ile Ser Thr Asn Gln Val Asn Glu
              450 455 460
          Glu Arg Leu Met Glu Val Tyr His Pro Gln Trp His Asp Ile Val Arg
          465 470 475 480
          Val Phe Gly Asn Arg Ala Arg Arg Phe Glu Arg Asp Asp Glu Ala Asp
                          485 490 495
          Val Leu Ser Val Thr Asp Glu Lys Ile Thr Leu Lys Trp Asp Cys Trp
                      500 505 510
          Gly Val Glu Ala Phe Lys Lys Met Glu Asp Gly Thr Phe Tyr Leu Ser
                  515 520 525
          Ser Gly Gly Arg Gln Glu Glu Pro Phe Ser Pro Arg Lys Lys Val Ala
              530 535 540
          Val Leu Phe Ile Ala Thr Gly Arg Tyr Met Thr Phe Trp Glu Glu Phe
          545 550 555 560
          Tyr Ala Ala Ser Lys Gln Tyr Phe Leu Thr Gly His Asp Val His Tyr
                          565 570 575
          Phe Leu Phe Thr Asp His Pro Glu Val Glu Thr Gly Asp Asp Val Thr
                      580 585 590
          Leu Val Arg Lys Pro Phe Tyr Pro Trp Pro Met Glu Thr Leu Arg Arg
                  595 600 605
          Phe Glu Thr Phe Leu Thr Val Arg Glu Glu Leu Gln Gln Tyr Asp Tyr
              610 615 620
          Ile Tyr Phe Met Asn Gly Thr Leu Leu Pro Val Gly Pro Val Gly Gln
          625 630 635 640
          Glu Ile Phe Pro Met Asn Arg Gln Gly Leu Met Val Thr Leu His Pro
                          645 650 655
          Gly Tyr Tyr Gln Arg Pro Arg Ser Thr Tyr Pro Tyr Glu Lys Asn Gly
                      660 665 670
          Met Ser Arg Ala Arg Val Leu His Ser Glu Gly Glu Tyr Tyr Val Ala
                  675 680 685
          Gly Gly Phe Asn Gly Gly Arg Ala Glu Asp Tyr Leu Arg Met Cys Arg
              690 695 700
          Glu Leu Ala Asp Ala Val Arg Arg Asp Leu Glu Asp Gly Val Ile Ala
          705 710 715 720
          Val Trp His Asp Glu Ser His Leu Asn Lys Tyr Val Ile Gly Arg His
                          725 730 735
          Pro Leu Val Leu Ser Pro Glu Tyr Leu Phe Pro Glu Thr Leu Asp Phe
                      740 745 750
          Asn Gln Lys Asn Leu Met Ala Ile Lys Pro Lys Val Lys Met Ile Val
                  755 760 765
          Lys Asp Lys Ser Leu Gln Lys His Gly Gly His Ala Trp Leu Arg Gln
              770 775 780
          Gln Ile
          785
           <![CDATA[ <210> 43]]>
           <![CDATA[ <211> 786]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Akkermansia muciniphila]]>
           <![CDATA[ <400> 43]]>
          Met Lys Cys Val Leu Ile Val Ser Pro Gly Glu Lys Ser Glu Gly Ala
          1 5 10 15
          Ser Glu Leu His Arg Met Gly Tyr Glu Leu Glu Leu Tyr Pro Ser Thr
                      20 25 30
          Ala Asp Leu Ser Pro Leu Arg Asp Ala Arg Glu Glu Glu Ser Ala Ser
                  35 40 45
          Tyr Leu Gly Arg Ser Pro Ala Ser Ala Glu Arg Ser His Val Arg Ser
              50 55 60
          Leu Arg Ala Ser Phe Ile Arg Leu Leu Glu Asp Arg Asn Tyr Ala Gly
          65 70 75 80
          Ser Asp Leu Ile Ile Phe Gly Glu Ser Asp Ala Val Pro Met Val Ala
                          85 90 95
          Ser Ser Arg Leu Glu Thr Ala Leu Arg Lys Glu Met Lys Glu His Pro
                      100 105 110
          Glu Thr Asp Ile Phe Arg Leu Phe His His Ala Val Trp Ser Pro Gln
                  115 120 125
          Gly Ala Pro Gly Glu Ser Asp Glu Ile Leu Phe Glu Asp Phe Lys Thr
              130 135 140
          Gly Lys Thr Asp Ala Asn Thr Ser Tyr Val Trp Gly Thr His Ala Leu
          145 150 155 160
          Val Ile Pro Ala Ala Arg Arg Pro Arg Val Ala Arg Val Phe Ala Asp
                          165 170 175
          Tyr Arg Leu Pro Thr Asp Ile Ala Leu Glu Ala Ala Asn Ser His Gly
                      180 185 190
          Asp Leu Lys Ile Arg Val Ala Arg His Asn Leu Phe Tyr Gln His Glu
                  195 200 205
          Arg Thr Lys Gln Arg Pro Asp Cys Lys Ile Ala Val Cys Leu Ser Ser
              210 215 220
          Tyr Lys Arg Leu Thr Asp Leu Gln Arg Gln Ile Trp Cys Met Met Asp
          225 230 235 240
          Gln Ser Tyr Pro Asn Leu His Val Phe Ala Ala Val Lys Gly Ile Pro
                          245 250 255
          Glu Gly Thr Tyr Arg Arg Thr Val Leu Pro Leu Phe Glu His Phe Ile
                      260 265 270
          His Glu Gly Arg Leu Thr Met Arg Leu Phe Pro Asn Lys Asn Gln Leu
                  275 280 285
          Ser Asn Phe Leu Asp Thr Ile Arg Asp Leu Asn Val Ser Asp Tyr Asp
              290 295 300
          Leu Phe Ala Lys Ile Asp Asp Asp Asp Leu Tyr Gly Arg Asp Tyr Phe
          305 310 315 320
          Lys Ser Val Asn Lys Phe His Leu His Leu Pro Pro Glu Phe Ser Ser
                          325 330 335
          Phe Tyr Cys Gly Pro Gly Glu Tyr Leu Ser Val Arg Gly Gly Tyr Pro
                      340 345 350
          Phe Ser Gly Asn Gly Phe Phe Gly Cys Phe Gly Pro Thr Leu Val Leu
                  355 360 365
          Ser Trp Asp Val Leu Glu Lys Leu Ile Ile Cys Glu Thr Asn Pro His
              370 375 380
          Met Ile Ser Gln Ile Ser Pro Arg Leu Arg His Ala Gly Tyr Gly Phe
          385 390 395 400
          Thr Glu Asp Asn Phe Met His Met Met Met Leu Asp Thr Gly Ser Ser
                          405 410 415
          Asn Arg Thr Arg Tyr Val Gln Glu Met Ala Leu Pro Met His Leu Ala
                      420 425 430
          Ile Gln Thr Gly Asn Ala Ser Val Met Arg Gly Gly Leu Val Pro Gly
                  435 440 445
          Asp Phe Arg Gly Arg Asn Trp Asn Ile Ser Thr Asn Gln Val Asn Glu
              450 455 460
          Glu Arg Leu Met Glu Val His His Pro Gln Trp His Asp Ile Val Arg
          465 470 475 480
          Val Phe Gly Asn Arg Ala Arg Arg Phe Glu Arg Asp Asp Glu Ala Asp
                          485 490 495
          Val Leu Ser Val Thr Asp Glu Lys Ile Thr Leu Lys Trp Asp Cys Trp
                      500 505 510
          Gly Val Glu Ala Phe Lys Lys Met Glu Asp Gly Thr Phe Tyr Leu Ser
                  515 520 525
          Ser Gly Gly Arg Gln Glu Glu Pro Phe Ser Pro Arg Lys Lys Val Ala
              530 535 540
          Val Leu Phe Ile Ala Thr Gly Arg Tyr Met Thr Phe Trp Glu Glu Phe
          545 550 555 560
          Tyr Ala Ala Ser Lys Gln Tyr Phe Leu Thr Gly His Asp Val His Tyr
                          565 570 575
          Phe Leu Phe Thr Asp His Pro Glu Val Glu Thr Gly Asp Asp Val Thr
                      580 585 590
          Leu Val Arg Lys Pro Phe Tyr Pro Trp Pro Met Glu Thr Leu Arg Arg
                  595 600 605
          Phe Glu Thr Phe Leu Thr Val Arg Glu Glu Leu Gln Gln Tyr Asp Tyr
              610 615 620
          Ile Tyr Phe Met Asn Gly Thr Leu Leu Pro Val Gly Pro Val Gly Gln
          625 630 635 640
          Glu Ile Phe Pro Met Asp Arg Gln Gly Leu Met Val Thr Leu His Pro
                          645 650 655
          Gly Tyr Tyr Gln Arg Pro Arg Ser Thr Tyr Pro Tyr Glu Lys Asn Gly
                      660 665 670
          Met Ser Arg Ala Arg Val Leu His Ser Glu Gly Glu Tyr Tyr Val Ala
                  675 680 685
          Gly Gly Phe Asn Gly Gly Arg Ala Glu Asp Tyr Leu Arg Met Cys Arg
              690 695 700
          Glu Leu Ala Asp Ala Val Arg Arg Asp Leu Glu Asp Gly Val Ile Ala
          705 710 715 720
          Val Trp His Asp Glu Ser His Leu Asn Lys Tyr Val Ile Gly Arg His
                          725 730 735
          Pro Leu Val Leu Ser Pro Glu Tyr Leu Phe Pro Glu Thr Leu Asp Phe
                      740 745 750
          Asn Gln Lys Asn Leu Met Ala Ile Lys Pro Lys Val Lys Met Ile Val
                  755 760 765
          Lys Asp Lys Ser Leu Gln Lys His Gly Gly His Ala Trp Leu Arg Gln
              770 775 780
          Gln Ile
          785
           <![CDATA[ <210> 44]]>
           <![CDATA[ <211> 787]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Akkermansia sp. BIOML-A61]]>
           <![CDATA[ <400> 44]]>
          Met Lys Cys Val Leu Ile Ile Ser Ser Gly Glu Met Ala Glu Gly Ala
          1 5 10 15
          Ser Glu Leu His Arg Met Gly Tyr Glu Leu Glu Leu Tyr Pro Ser Thr
                      20 25 30
          Arg Asp Leu Ser Ser Leu Lys Asp Thr Arg Glu Lys Glu Ser Ala Ala
                  35 40 45
          Phe Ile Gly Arg Asp Pro Cys Ser Ala Glu Arg Ser His Val Arg Ser
              50 55 60
          Leu Arg Ala Ser Phe Ile Arg Met Leu Glu Asp Arg Arg Tyr Ala Gly
          65 70 75 80
          Asn Asp Leu Ile Ile Phe Gly Glu Ser Asp Ala Val Pro Met Val Ala
                          85 90 95
          Ser Ser Arg Leu Glu Ala Ala Leu Arg Lys Glu Met Lys Glu His Pro
                      100 105 110
          Glu Thr Asp Ile Phe Arg Leu Phe His His Ala Val Trp Ser Pro Gln
                  115 120 125
          Gly Asn Pro Phe Glu Ser Asp Glu Leu Leu Phe Glu Asp Phe Lys Thr
              130 135 140
          Gly Lys Thr Asp Phe Asn Thr Pro Tyr Val Trp Gly Thr His Ala Met
          145 150 155 160
          Val Ile Pro Ser Cys Lys Arg Glu Lys Val Ile Gln Val Phe Ala Asp
                          165 170 175
          Tyr Arg Leu Pro Thr Asp Ile Ala Leu Glu Ala Ala Asn Ser Asn Gly
                      180 185 190
          Glu Leu His Ile Arg Val Ala Arg His Asn Leu Phe Tyr Gln His Glu
                  195 200 205
          Arg Thr Lys Lys Arg Pro Ala Cys Arg Ile Ala Ala Cys Leu Ser Ser
              210 215 220
          Tyr Arg Arg Leu Thr Asp Leu Gln Arg Gln Ile Trp Cys Met Met Asp
          225 230 235 240
          Gln Ser Tyr Glu Asn Phe His Val Phe Ala Ala Val Lys Gly Ile Pro
                          245 250 255
          Glu Ala Thr Tyr Arg Lys Thr Val Leu Pro Leu Phe Glu His Phe Ile
                      260 265 270
          Gln Glu Gly Arg Leu Thr Met Arg Leu Phe Pro Asn Lys Asn Gln Leu
                  275 280 285
          Ser Asn Phe Leu Asp Ala Ile Arg Asp Leu Asp Ile Ser Asp Tyr Asp
              290 295 300
          Leu Phe Ala Lys Ile Asp Asp Asp Asp Leu Tyr Gly Arg Asp Tyr Phe
          305 310 315 320
          Lys Ser Ile Asn Asp Phe His Gln His Leu Pro Arg Glu Phe Ser Ser
                          325 330 335
          Tyr Tyr Cys Gly Phe Gly Gln Tyr Leu Asn Ala Arg Gly Gly Tyr Pro
                      340 345 350
          Leu Cys Gly Asn Gly Phe Phe Ser Cys Phe Gly Pro Thr Met Val Phe
                  355 360 365
          Ser Arg Asp Val Leu Glu Lys Leu Ile Thr Cys Glu Gln Asp Pro Gly
              370 375 380
          Arg Ile Ser Glu Ile Ser Pro Arg Leu Gly His Ser Gly Tyr Gly Phe
          385 390 395 400
          Thr Glu Asp Asn Leu Met His Lys Leu Met Ile Asp Thr Gly Ser Cys
                          405 410 415
          Asn Arg Ile Arg Tyr Val Gln Glu Met Ser Leu Pro Met His Leu Val
                      420 425 430
          Ile Gln Thr Asn Asn Ala Ser Val Met Arg Gly Gly Leu Val Pro Gly
                  435 440 445
          Asp Phe Arg Gly Arg Asn Trp Gln Ile Ser His Ser Arg Phe Asn Ala
              450 455 460
          Glu Ser Phe Met Glu Ile Gly His Pro Gln Trp Tyr Asp Ile Val Arg
          465 470 475 480
          Ile Phe Gly Gly Arg Ala Cys Arg Phe Gln Arg Asn Asp Trp Ala Asp
                          485 490 495
          Val Leu Ser Leu Thr Asp Glu Glu Val Thr Leu Lys Trp Asp Gln Trp
                      500 505 510
          Gly Thr Glu Thr Phe Arg Arg Arg Asp Asp Gly Ser Phe Phe Leu Ser
                  515 520 525
          Gly Asn Gly Glu Gln Gln Asn Ser Pro Ser Ser Gln Arg Lys Lys Val
              530 535 540
          Ala Val Leu Tyr Ile Ala Thr Gly Arg Tyr Met Ala Phe Trp Lys Asp
          545 550 555 560
          Phe Tyr Ala Ala Ala Lys Gln Tyr Phe Leu Pro Gly His Asp Val Arg
                          565 570 575
          Tyr Phe Leu Phe Thr Asp His Asn Glu Val Lys Thr Pro Asp Asp Val
                      580 585 590
          Thr Leu Val Ile Lys Pro Phe Tyr Pro Trp Pro Met Glu Thr Leu Arg
                  595 600 605
          Arg Phe Glu Thr Phe Leu Ser Val Gln Lys Glu Leu Gln Glu Tyr Asp
              610 615 620
          Tyr Ile Tyr Phe Met Asn Gly Thr Leu Leu Pro Val Ser Pro Ile Gly
          625 630 635 640
          Glu Glu Ile Phe Pro Asn Asp Arg Gln Gly Ile Ala Val Thr Leu His
                          645 650 655
          Pro Gly Tyr Tyr Gly Asn Thr Arg Ser Cys Tyr Pro Tyr Glu Lys Asn
                      660 665 670
          Gly Met Ser Glu Ala Arg Ile Leu Pro Glu Gln Gly Glu Tyr Tyr Val
                  675 680 685
          Ala Gly Gly Phe Asn Gly Gly Arg Thr Lys Asp Phe Leu Ser Met Cys
              690 695 700
          Arg Glu Leu Ala Gly Ala Val Lys Arg Asp Leu Asp Asn Gly Ile Ile
          705 710 715 720
          Ala Val Trp His Asp Glu Ser His Leu Asn Lys Tyr Val Val Gly Arg
                          725 730 735
          His Pro Leu Val Leu Gly Pro Glu Tyr Leu Phe Pro Glu Thr Leu Val
                      740 745 750
          Phe Asn Arg Tyr Tyr Leu Met Gly Leu Lys His Arg Val Lys Ile Leu
                  755 760 765
          Val Lys Asp Lys Ser Leu Ser Lys Tyr Gly Gly His Ala Trp Leu Arg
              770 775 780
          Lys Leu Val
          785
           <![CDATA[ <210> 45]]>
           <![CDATA[ <211> 786]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Akkermansia sp. CAG:344]]>
           <![CDATA[ <400> 45]]>
          Met Lys Cys Val Leu Ile Val Ser Ser Gly Glu Lys Ser Glu Gly Ala
          1 5 10 15
          Ser Glu Leu His Arg Met Gly Tyr Glu Leu Glu Leu Tyr Pro Ser Thr
                      20 25 30
          Ala Asp Leu Ser Pro Leu Arg Asp Ala Arg Glu Glu Glu Ser Ala Ser
                  35 40 45
          Tyr Leu Gly Arg Ser Pro Ala Ser Ala Glu Arg Ser His Val Arg Ser
              50 55 60
          Leu Arg Ala Ser Phe Ile Arg Leu Leu Glu Asp Arg Asn Tyr Ala Gly
          65 70 75 80
          Ser Asp Leu Ile Ile Phe Gly Glu Ser Asp Ala Val Pro Met Val Ala
                          85 90 95
          Ser Ser Arg Leu Glu Thr Ala Leu Arg Lys Glu Met Lys Glu His Pro
                      100 105 110
          Glu Thr Asp Ile Phe Arg Leu Phe His His Ala Val Trp Ser Pro Gln
                  115 120 125
          Gly Ala Pro Gly Glu Ser Asp Glu Ile Leu Phe Glu Asp Phe Lys Thr
              130 135 140
          Gly Lys Thr Asp Ala Asn Thr Ser Tyr Val Trp Gly Thr His Ala Leu
          145 150 155 160
          Val Ile Pro Ala Ala Arg Arg Pro Arg Val Ala Arg Val Phe Ala Asp
                          165 170 175
          Tyr Arg Leu Pro Thr Asp Ile Ala Leu Glu Ala Ala Asn Ser His Gly
                      180 185 190
          Asp Leu Lys Ile Arg Val Ala Arg His Asn Leu Phe Tyr Gln His Glu
                  195 200 205
          Arg Thr Lys Gln Arg Pro Asp Cys Lys Ile Ala Val Cys Leu Ser Ser
              210 215 220
          Tyr Lys Arg Leu Thr Asp Leu Gln Arg Gln Ile Trp Cys Met Met Asp
          225 230 235 240
          Gln Ser Tyr Pro Asn Leu His Val Phe Ala Ala Val Lys Gly Ile Pro
                          245 250 255
          Glu Gly Thr Tyr Arg Arg Thr Val Leu Pro Leu Phe Glu His Phe Ile
                      260 265 270
          His Glu Gly Arg Leu Thr Met Arg Leu Phe Pro Asn Lys Asn Gln Leu
                  275 280 285
          Ser Asn Phe Leu Asp Thr Ile Arg Asp Leu Asn Val Ser Asp Tyr Asp
              290 295 300
          Leu Phe Ala Lys Ile Asp Asp Asp Asp Leu Tyr Gly Arg Asp Tyr Phe
          305 310 315 320
          Lys Ser Val Asn Lys Phe His Leu His Leu Pro Pro Glu Phe Ser Ser
                          325 330 335
          Phe Tyr Cys Gly Pro Gly Glu Tyr Leu Ser Val Arg Gly Gly Tyr Pro
                      340 345 350
          Phe Ser Gly Asn Gly Phe Phe Gly Cys Phe Gly Pro Thr Leu Val Leu
                  355 360 365
          Ser Arg Asp Val Leu Glu Lys Leu Ile Ile Cys Glu Thr Asn Pro His
              370 375 380
          Met Ile Ser Gln Ile Ser Pro Arg Leu Arg His Ala Gly Tyr Gly Phe
          385 390 395 400
          Thr Glu Asp Asn Phe Met His Met Met Met Leu Asp Thr Gly Ser Ser
                          405 410 415
          Asn Arg Thr Arg Tyr Val Gln Glu Met Ala Leu Pro Met His Leu Ala
                      420 425 430
          Ile Gln Thr Gly Asn Ala Ser Val Met Arg Gly Gly Leu Val Pro Gly
                  435 440 445
          Asp Phe Arg Gly Arg Asn Trp Asn Ile Ser Thr Asn Gln Val Asn Glu
              450 455 460
          Glu Arg Leu Met Glu Val His His Pro Gln Trp His Asp Ile Val Arg
          465 470 475 480
          Val Phe Gly Asn Arg Ala Arg Arg Phe Glu Arg Asp Asp Glu Ala Asp
                          485 490 495
          Val Leu Ser Val Thr Asp Glu Lys Ile Thr Leu Lys Trp Asp Cys Trp
                      500 505 510
          Gly Val Glu Ala Phe Lys Lys Met Glu Asp Gly Thr Phe Tyr Leu Ser
                  515 520 525
          Ser Gly Gly Arg Gln Glu Glu Pro Phe Ser Pro Arg Lys Lys Val Ala
              530 535 540
          Val Leu Phe Ile Ala Thr Gly Arg Tyr Met Thr Phe Trp Glu Glu Phe
          545 550 555 560
          Tyr Ala Ala Ser Lys Gln Tyr Phe Leu Thr Gly His Asp Val His Tyr
                          565 570 575
          Phe Leu Phe Thr Asp His Pro Glu Val Glu Thr Gly Asp Asp Val Thr
                      580 585 590
          Leu Val Arg Lys Pro Phe Tyr Pro Trp Pro Met Glu Thr Leu Arg Arg
                  595 600 605
          Phe Glu Thr Phe Leu Thr Val Arg Glu Glu Leu Gln Gln Tyr Asp Tyr
              610 615 620
          Ile Tyr Phe Met Asn Gly Thr Leu Leu Pro Val Gly Pro Val Gly Gln
          625 630 635 640
          Glu Ile Phe Pro Met Asn Arg Gln Gly Leu Met Val Thr Leu His Pro
                          645 650 655
          Gly Tyr Tyr Gln Arg Pro Arg Ser Thr Tyr Pro Tyr Glu Lys Asn Gly
                      660 665 670
          Met Ser Arg Ala Arg Val Leu His Ser Glu Gly Glu Tyr Tyr Val Ala
                  675 680 685
          Gly Gly Phe Asn Gly Gly Arg Ala Glu Asp Tyr Leu Arg Met Cys Arg
              690 695 700
          Glu Leu Ala Asp Ala Val Arg Arg Asp Leu Glu Asp Gly Val Ile Ala
          705 710 715 720
          Val Trp His Asp Glu Ser His Leu Asn Lys Tyr Val Ile Gly Arg His
                          725 730 735
          Pro Leu Val Leu Ser Pro Glu Tyr Leu Phe Pro Glu Thr Leu Asp Phe
                      740 745 750
          Asn Gln Lys Asn Leu Met Ala Ile Lys Pro Lys Val Lys Met Ile Val
                  755 760 765
          Lys Asp Lys Ser Leu Gln Lys His Gly Gly His Ala Trp Leu Arg Gln
              770 775 780
          Gln Ile
          785
           <![CDATA[ <210> 46]]>
           <![CDATA[ <211> 787]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Akkermansia sp. KLE1798]]>
           <![CDATA[ <400> 46]]>
          Met Lys Cys Val Leu Ile Val Ser Ser Gly Glu Met Ala Glu Gly Ala
          1 5 10 15
          Ser Glu Leu His Arg Met Gly Tyr Glu Leu Glu Leu Tyr Pro Ser Thr
                      20 25 30
          Arg Asp Leu Ser Ser Leu Lys Asp Thr Arg Glu Lys Glu Ser Ala Ala
                  35 40 45
          Phe Ile Gly Arg Asp Pro Cys Ser Ala Glu Arg Ser His Val Arg Ser
              50 55 60
          Leu Arg Ala Ser Phe Ile Gln Val Leu Glu Asp Arg Arg Tyr Ala Gly
          65 70 75 80
          Asn Asp Leu Ile Ile Phe Gly Glu Ser Asp Ala Val Pro Met Val Ala
                          85 90 95
          Ser Ser Arg Leu Glu Thr Ala Leu Arg Lys Glu Met Glu Glu His Pro
                      100 105 110
          Glu Thr Asp Ile Phe Arg Leu Phe His His Ala Val Trp Ser Pro Gln
                  115 120 125
          Gly Asn Pro Phe Glu Ser Asp Glu Leu Leu Phe Glu Asp Phe Lys Thr
              130 135 140
          Gly Gly Thr Asp Ser Asn Thr Ala Tyr Val Trp Gly Thr His Ala Met
          145 150 155 160
          Val Ile Pro Ser Cys Lys Arg Lys Lys Val Ile Gln Val Phe Ala Asp
                          165 170 175
          Tyr Arg Leu Pro Thr Asp Val Ala Leu Glu Ala Ala Asn Ser Ser Gly
                      180 185 190
          Glu Leu Asn Ile Arg Val Ala Arg His Asn Leu Phe Tyr Gln His Glu
                  195 200 205
          Arg Thr Lys Lys Arg Pro Ala Cys Arg Ile Ala Ala Cys Leu Ala Ser
              210 215 220
          Tyr Arg Arg Leu Ala Asp Leu Gln Arg Gln Ile Trp Cys Met Met Asp
          225 230 235 240
          Gln Ser Tyr Glu Asn Phe His Val Phe Ala Ala Val Lys Gly Ile Pro
                          245 250 255
          Glu Thr Thr Tyr Arg Lys Thr Val Leu Pro Leu Phe Glu His Phe Ile
                      260 265 270
          Gln Glu Gly Arg Leu Thr Met Arg Leu Phe Pro Asn Lys Asn Gln Leu
                  275 280 285
          Ser Asn Phe Leu Asp Thr Val Arg Gly Leu Asp Ile Ser Asn Tyr Asp
              290 295 300
          Leu Phe Ala Lys Ile Asp Asp Asp Asp Leu Tyr Gly Arg Asp Tyr Phe
          305 310 315 320
          Lys Ser Val Asn Asp Phe His Gln His Leu Pro Pro Glu Phe Ser Ser
                          325 330 335
          Tyr Tyr Cys Gly Phe Gly Gln Tyr Leu Asn Asn Arg Gly Gly Tyr Pro
                      340 345 350
          Leu Cys Gly Asn Gly Phe Phe Ser Cys Phe Gly Pro Thr Met Val Phe
                  355 360 365
          Ser Lys Asp Val Leu Glu Lys Leu Ile Thr Cys Glu Gln Glu Pro Gly
              370 375 380
          Arg Ile Ser Glu Ile Phe Pro Arg Leu Gly His Ser Gly Tyr Gly Phe
          385 390 395 400
          Thr Glu Asp Asn Leu Met His Lys Leu Met Ile Asp Thr Gly Ser Cys
                          405 410 415
          Asn Arg Ile Arg Tyr Val Gln Glu Met Ser Leu Pro Met His Leu Val
                      420 425 430
          Ile Gln Thr Asn Asn Ala Ser Val Ile Arg Gly Gly Leu Val Pro Gly
                  435 440 445
          Asp Phe Arg Gly Arg Asn Trp His Ile Ser Thr Ser Arg Ala Asn Ala
              450 455 460
          Glu Ser Leu Ile Glu Ile Ser His Pro Gln Trp Tyr Asp Ile Val Arg
          465 470 475 480
          Ile Phe Gly Gly Arg Ala Cys Arg Phe Gln Arg Asn Asp Trp Ala Asp
                          485 490 495
          Val Leu Ser Leu Thr Asp Glu Glu Val Thr Leu Lys Trp Asp Gln Trp
                      500 505 510
          Gly Thr Glu Thr Phe Arg Arg Lys Glu Asp Gly Ser Phe Phe Leu Ser
                  515 520 525
          Glu Asn Gly Asn Gln Gln His Ser Pro Ser Ser Arg Lys Arg Lys Val
              530 535 540
          Ala Val Leu Tyr Ile Ser Thr Gly Arg Tyr Ile Thr Phe Trp Lys Asp
          545 550 555 560
          Phe Tyr Ala Ala Ser Lys Gln Tyr Phe Leu Pro Gly His Asp Val Arg
                          565 570 575
          Tyr Phe Leu Phe Thr Asp His Asp Glu Val Lys Thr Ala Asp Asp Val
                      580 585 590
          Thr Leu Val Ser Lys Pro Phe Tyr Pro Trp Pro Met Glu Thr Leu Arg
                  595 600 605
          Arg Phe Glu Thr Phe Leu Ser Ile Glu Lys Glu Leu Gln Glu Tyr Asp
              610 615 620
          Tyr Ile Tyr Phe Met Asn Gly Thr Leu Leu Pro Val Ser Pro Ile Gly
          625 630 635 640
          Glu Glu Ile Phe Pro Asn Asp Arg Gln Gly Leu Ala Val Thr Leu His
                          645 650 655
          Pro Gly Phe Tyr Glu Leu Pro Leu Ser Cys Tyr Pro Tyr Glu Lys Asn
                      660 665 670
          Gly Met Ser Glu Ala Arg Ile Ser Pro Gly Gln Gly Glu Tyr Tyr Val
                  675 680 685
          Ala Gly Gly Phe Asn Gly Gly Lys Ala Lys Asp Phe Leu Ser Met Cys
              690 695 700
          Gln Glu Leu Ala Gly Ala Val Lys Arg Asp Leu Asp Asn Gly Ile Ile
          705 710 715 720
          Ala Val Trp His Asp Glu Ser His Ile Asn Lys Tyr Val Ile Gly Arg
                          725 730 735
          His Pro Leu Val Leu Gly Pro Glu Tyr Leu Phe Pro Glu Thr Leu Val
                      740 745 750
          Phe Asn Arg Tyr His Leu Met Gly Leu Lys His Arg Val Lys Ile Leu
                  755 760 765
          Val Lys Asp Lys Ser Leu Ser Lys Tyr Gly Gly His Ala Trp Leu Arg
              770 775 780
          Lys Gln Ser
          785
           <![CDATA[ <210> 47]]>
           <![CDATA[ <211> 232]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Algoriphagus sp.]]>
           <![CDATA[ <400> 47]]>
          Met Lys Ile Cys Ile Leu Thr Ile Ala Thr Asn Gln Tyr Leu Gln Phe
          1 5 10 15
          Ile Glu Lys Leu Tyr Ser Asp Ile Ala Glu Lys Phe Ile Pro Glu Ser
                      20 25 30
          Glu Ile Asn Cys Leu Leu Phe Thr Asp His Glu Ile Glu Glu Thr Ser
                  35 40 45
          Asp Asn Val Lys Val His Tyr Ile Asp His Glu Pro Trp Pro Met Pro
              50 55 60
          Thr Leu Lys Arg Tyr Asn Tyr Phe Val Lys Glu Lys Asp Phe Ile Leu
          65 70 75 80
          Gln His Asp Tyr Cys Phe Tyr Met Asp Ala Asp Met Arg Ile Asp Ala
                          85 90 95
          Pro Val Gly Gln Glu Ile Leu Gly Asp Leu Val Ala Thr Arg His Gly
                      100 105 110
          Tyr Gln Ser Tyr His Asp Pro Lys Asn Gln Ser Phe Asp Arg Asn Pro
                  115 120 125
          Lys Ser Leu Ala Tyr Val Asp Pro Ser Glu Lys Thr Val Thr Tyr Tyr
              130 135 140
          Ala Gly Gly Phe Asn Gly Gly Lys Thr Gln Asn Phe Met Lys Met Ser
          145 150 155 160
          Glu Val Ile Ala Asp Arg Val Asn Lys Asp Leu Glu Asn Asn Val Val
                          165 170 175
          Ala Leu Trp His Asp Glu Ser His Met Asn Arg Tyr Leu Ile Asp Asn
                      180 185 190
          Pro Pro Thr Leu Asp Leu Ser Pro Glu Tyr Cys Tyr Ala Glu Glu Phe
                  195 200 205
          Ile Gly Ser Asn Tyr Pro Leu Gln Asn Pro Lys Ile Ile Ala Leu Lys
              210 215 220
          Lys Asn His Ala Glu Leu Arg Ser
          225 230
           <![CDATA[ <210> 48]]>
           <![CDATA[ <211> 263]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides ovale]]>
           <![CDATA[ <400> 48]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Leu Ser Ala Glu Arg Tyr Phe Met Gln Asp Gln
                      20 25 30
          Ser Phe Ile Ile Glu Tyr Tyr Val Phe Thr Asp Ser Pro Lys Leu Tyr
                  35 40 45
          Asp Glu Glu Asn Asn Lys His Ile His Arg Ile Lys Gln Lys Asn Leu
              50 55 60
          Gly Trp Pro Asp Asn Thr Leu Lys Arg Phe His Ile Phe Leu Arg Ile
          65 70 75 80
          Lys Glu Gln Leu Glu Arg Glu Thr Asp Tyr Leu Phe Phe Phe Asn Ala
                          85 90 95
          Asn Leu Leu Phe Thr Ser Pro Ile Gly Lys Glu Ile Leu Pro Pro Ser
                      100 105 110
          Asp Ser Asn Gly Leu Leu Gly Thr Met His Pro Gly Phe Tyr Asn Lys
                  115 120 125
          Pro Asn Ser Glu Phe Thr Tyr Glu Arg Arg Asp Ala Ser Thr Ala Tyr
              130 135 140
          Ile Pro Glu Gly Glu Gly Arg Tyr Tyr Tyr Ala Gly Gly Leu Ser Gly
          145 150 155 160
          Gly Cys Thr Lys Ala Tyr Leu Lys Leu Cys Thr Thr Ile Cys Ser Trp
                          165 170 175
          Val Asp Arg Asp Ala Thr Asn His Ile Ile Pro Ile Trp His Asp Glu
                      180 185 190
          Ser Leu Ile Asn Lys Tyr Phe Leu Asp Asn Pro Pro Ala Ile Thr Leu
                  195 200 205
          Ser Pro Ala Tyr Leu Tyr Pro Glu Gly Trp Leu Leu Pro Phe Glu Pro
              210 215 220
          Ile Ile Leu Ile Arg Asp Lys Asn Asn Pro Gln Tyr Gly Gly His Glu
          225 230 235 240
          Leu Leu Arg Arg Lys Asn Ser Leu Trp Glu Arg Ile Lys Leu Ile Cys
                          245 250 255
          Gln Lys Phe Lys Ser Ala Asp
                      260
           <![CDATA[ <210> 49]]>
           <![CDATA[ <211> 263]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides ovale]]>
           <![CDATA[ <400> 49]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Leu Ser Ala Glu Arg Tyr Phe Met Gln Asp Gln
                      20 25 30
          Ser Phe Ile Ile Glu Tyr Tyr Val Phe Thr Asp Ser Pro Lys Leu Tyr
                  35 40 45
          Asp Glu Glu Asn Asn Lys His Ile His Arg Ile Lys Gln Lys Asn Leu
              50 55 60
          Gly Trp Pro Asp Asn Thr Leu Lys Arg Phe His Ile Phe Leu Arg Ile
          65 70 75 80
          Lys Glu Gln Leu Glu Arg Glu Thr Asp Tyr Leu Phe Phe Phe Asn Ala
                          85 90 95
          Asn Leu Leu Phe Thr Ser Pro Ile Gly Lys Glu Ile Leu Pro Pro Ser
                      100 105 110
          Asp Ser Asn Gly Leu Leu Gly Thr Met His Pro Gly Phe Tyr Asn Lys
                  115 120 125
          Pro Asn Ser Glu Phe Thr Tyr Glu Arg Arg Asp Ala Ser Thr Ala Tyr
              130 135 140
          Ile Pro Glu Gly Glu Gly Arg Tyr Tyr Tyr Ala Gly Gly Leu Ser Gly
          145 150 155 160
          Gly Cys Thr Lys Ala Tyr Leu Lys Leu Cys Thr Thr Ile Cys Ser Trp
                          165 170 175
          Val Asp Arg Asp Ala Thr Asn His Ile Ile Pro Ile Trp His Asp Glu
                      180 185 190
          Ser Leu Ile Asn Lys Tyr Phe Leu Asp Asn Pro Pro Ala Ile Thr Leu
                  195 200 205
          Ser Pro Ala Tyr Leu Tyr Pro Glu Gly Trp Leu Leu Pro Phe Glu Pro
              210 215 220
          Ile Ile Leu Ile Arg Asp Lys Asn Lys Pro Gln Tyr Gly Gly His Glu
          225 230 235 240
          Leu Leu Arg Arg Lys Asn Ser Leu Trp Glu Arg Ile Lys Leu Ile Cys
                          245 250 255
          Gln Lys Phe Lys Ser Ala Asp
                      260
           <![CDATA[ <210> 50]]>
           <![CDATA[ <211> 263]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides ovale]]>
           <![CDATA[ <400> 50]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe
          1 5 10 15
          Trp Lys Asn Phe Tyr Leu Ser Ala Glu Arg Tyr Phe Leu Gln Asp Gln
                      20 25 30
          Ser Phe Ile Leu Glu Tyr Tyr Val Phe Thr Asp Ser Pro Lys Leu Tyr
                  35 40 45
          Asp Glu Asp Asn Asn Lys His Ile His Arg Ile Lys Gln Lys Asn Leu
              50 55 60
          Gly Trp Pro Asp Asn Thr Leu Lys Arg Phe His Ile Phe Leu Arg Ile
          65 70 75 80
          Lys Gln Gln Leu Leu Gln Glu Thr Asp Phe Leu Phe Phe Cys Asn Ala
                          85 90 95
          Asn Leu Leu Phe Lys Gln Asn Ile Gly Pro Glu Ile Ile Pro Leu Lys
                      100 105 110
          Thr Glu Asn Gln Leu Val Gly Thr Ile His Pro Gly Phe Tyr Asn Ser
                  115 120 125
          Pro Asn Ser Glu Phe Thr Tyr Glu Arg Arg Tyr Asn Ser Lys Ala Tyr
              130 135 140
          Ile Pro Glu Gly Glu Gly Ile His Tyr Tyr Ala Gly Gly Phe Ser Gly
          145 150 155 160
          Gly Tyr Thr Glu Arg Tyr Leu Gln Leu Cys Glu Thr Ile Lys Ser Trp
                          165 170 175
          Val Asp Ile Asp Asn Ser Lys Lys Ile Val Ala Ile Trp His Asp Glu
                      180 185 190
          Ser His Ile Asn Lys Tyr Phe Leu Glu Asn Pro Pro Phe Thr Leu Ser
                  195 200 205
          Pro Ala Tyr Leu Tyr Pro Glu Gly Trp Ser Ile Pro Phe Lys Glu Ile
              210 215 220
          Ile Met Ile Arg Asp Lys Ser Lys Glu Glu Tyr Gly Gly His Thr Leu
          225 230 235 240
          Leu Arg Lys Lys Glu Pro Trp Ser Ser Lys Leu Leu Tyr Ala Leu Lys
                          245 250 255
          Arg Phe Phe Arg Leu Ser Glu
                      260
           <![CDATA[ <210> 51]]>
           <![CDATA[ <211> 263]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides ovale]]>
           <![CDATA[ <400> 51]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Leu Ser Ala Glu Arg Tyr Phe Met Gln Asp Gln
                      20 25 30
          Ser Phe Ile Ile Glu Tyr Tyr Val Phe Thr Asp Ser Pro Gln Leu Tyr
                  35 40 45
          Asp Glu Glu Asn Asn Glu His Ile His Arg Ile Lys Gln Lys Asn Leu
              50 55 60
          Gly Trp Pro Asp Asn Thr Leu Lys Arg Phe His Thr Phe Leu Arg Ile
          65 70 75 80
          Lys Glu Gln Leu Glu Arg Glu Thr Asp Tyr Leu Phe Phe Phe Asn Ala
                          85 90 95
          Asn Leu Leu Phe Thr Cys Pro Ile Gly Lys Glu Met Leu Pro Ser Ser
                      100 105 110
          Asn Ser Asn Gly Leu Leu Gly Thr Ile His Pro Gly Phe Tyr Asn Lys
                  115 120 125
          Pro Asn Ser Glu Phe Thr Tyr Glu Arg Arg Val Ala Ser Thr Ala Tyr
              130 135 140
          Ile Pro Glu Gly Lys Gly Leu Tyr Tyr Tyr Ala Gly Gly Leu Ser Gly
          145 150 155 160
          Gly Cys Thr Glu Ser Tyr Leu Gln Leu Cys Thr Thr Ile Cys Ser Trp
                          165 170 175
          Val Asp Lys Asp Ala Ala Asn His Ile Ile Pro Ile Trp His Asp Glu
                      180 185 190
          Ser Leu Ile Asn Lys Tyr Phe Leu Asp Asn Pro Pro Ala Ile Thr Leu
                  195 200 205
          Pro Pro Ala Tyr Leu Tyr Pro Glu Gly Trp Ser Leu Pro Phe Lys Pro
              210 215 220
          Ile Ile Leu Ile Arg Asp Lys Asn Lys Pro Glu Tyr Gly Gly His Glu
          225 230 235 240
          Phe Leu Arg Arg Lys Asn Ser Leu Trp Val Lys Ile Lys Leu Ile Cys
                          245 250 255
          Gln Lys Ile Lys Leu Ala Asp
                      260
           <![CDATA[ <210> 52]]>
           <![CDATA[ <211> 257]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides ovale SD CMC 3f]]>
           <![CDATA[ <400> 52]]>
          Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe Trp Lys Asp Phe Tyr Leu
          1 5 10 15
          Ser Ala Glu Arg Tyr Phe Met Gln Asp Gln Ser Phe Ile Ile Glu Tyr
                      20 25 30
          Tyr Val Phe Thr Asp Ser Pro Lys Leu Tyr Asp Glu Glu Asn Asn Lys
                  35 40 45
          His Ile His Arg Ile Lys Gln Lys Asn Leu Gly Trp Pro Asp Asn Thr
              50 55 60
          Leu Lys Arg Phe His Ile Phe Leu Arg Ile Lys Glu Gln Leu Glu Arg
          65 70 75 80
          Glu Thr Asp Tyr Leu Phe Phe Phe Asn Ala Asn Leu Leu Phe Thr Ser
                          85 90 95
          Pro Ile Gly Lys Glu Ile Leu Pro Pro Ser Asp Ser Asn Gly Leu Leu
                      100 105 110
          Gly Thr Met His Pro Gly Phe Tyr Asn Lys Pro Asn Ser Glu Phe Thr
                  115 120 125
          Tyr Glu Arg Arg Asp Ala Ser Thr Ala Tyr Ile Pro Glu Gly Glu Gly
              130 135 140
          Arg Tyr Tyr Tyr Ala Gly Gly Leu Ser Gly Gly Cys Thr Lys Ala Tyr
          145 150 155 160
          Leu Lys Leu Cys Thr Thr Ile Cys Ser Trp Val Asp Arg Asp Ala Thr
                          165 170 175
          Asn His Ile Ile Pro Ile Trp His Asp Glu Ser Leu Ile Asn Lys Tyr
                      180 185 190
          Phe Leu Asp Asn Pro Pro Ala Ile Thr Leu Ser Pro Ala Tyr Leu Tyr
                  195 200 205
          Pro Glu Gly Trp Leu Leu Pro Phe Glu Pro Ile Ile Leu Ile Arg Asp
              210 215 220
          Lys Asn Asn Pro Gln Tyr Gly Gly His Glu Leu Leu Arg Arg Lys Asn
          225 230 235 240
          Ser Leu Trp Glu Arg Ile Lys Leu Ile Cys Gln Lys Phe Lys Ser Ala
                          245 250 255
          Asp
           <![CDATA[ <210> 53]]>
           <![CDATA[ <211> 263]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides reticulotermitis JCM 10512]]>
           <![CDATA[ <400> 53]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Ser Ile Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Leu Ser Ala Glu Arg Tyr Leu Met Gln Ser Pro
                      20 25 30
          Ala Tyr Thr Arg Glu Tyr Tyr Val Phe Thr Asp Ser Leu Lys Leu Tyr
                  35 40 45
          Asp Glu Glu Asn Asn Lys His Ile His Arg Ile Lys Gln Lys Asn Leu
              50 55 60
          Gly Trp Pro Asp Asn Thr Leu Lys Arg Phe His Met Phe Leu Gln Ile
          65 70 75 80
          Lys Gln Gln Leu Leu Gln Glu Thr Asp Phe Leu Ile Phe Cys Asn Ala
                          85 90 95
          Asn Leu Leu Phe Lys Gln Asn Val Gly His Glu Ile Ile Pro Gln Lys
                      100 105 110
          Gly Lys Asn Gln Phe Val Gly Thr Ile His Pro Gly Phe Tyr Asn Ser
                  115 120 125
          His Asn Tyr Asp Phe Thr Tyr Glu Arg Arg His Asn Ser Lys Ala Tyr
              130 135 140
          Ile Pro Glu Gly Glu Gly Val His Tyr Tyr Ala Gly Gly Phe Ser Gly
          145 150 155 160
          Gly Tyr Thr Lys Ala Tyr Leu Gln Leu Cys Glu Thr Ile Lys Ser Trp
                          165 170 175
          Val Asp Ile Asp Lys Ser Asn Lys Ile Val Ala Ile Trp His Asp Glu
                      180 185 190
          Ser His Ile Asn Arg Tyr Phe Leu Lys Asn Pro Pro Leu Thr Leu Ser
                  195 200 205
          Pro Gly Tyr Leu Tyr Pro Glu Gly Trp Ser Ile Pro Phe Glu Glu Ile
              210 215 220
          Ile Thr Ile Arg Asp Lys Asn Lys Glu Glu Tyr Gly Gly His Ile Leu
          225 230 235 240
          Leu Arg Lys Lys Glu Ser Trp Arg Asn Lys Ile Leu Lys Ile Ile Lys
                          245 250 255
          Lys Thr Leu Phe Pro Leu Pro
                      260
           <![CDATA[ <210> 54]]>
           <![CDATA[ <211> 256]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides sp. OM08-11]]>
           <![CDATA[ <400> 54]]>
          Met Lys Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Leu Ser Ser Glu Ser His Phe Phe Ser Asp Asp
                      20 25 30
          Pro Asn Cys Ile Arg Glu Tyr Tyr Val Phe Thr Asp Ser Lys Leu Leu
                  35 40 45
          Phe Gly Glu Lys Glu Asn Gln His Ile His Arg Ile Tyr Gln Lys Asn
              50 55 60
          Leu Gly Trp Pro Asn Asn Thr Leu Lys Arg Phe His Ile Phe Leu Glu
          65 70 75 80
          Ile Lys Glu Arg Leu Leu Lys Glu Thr Asp Tyr Leu Phe Phe Cys Asn
                          85 90 95
          Ala Asn Leu Leu Phe Lys Gln Thr Val Gly Leu Glu Ile Leu Pro Pro
                      100 105 110
          Ala Ile Gly Asn Gly Leu Val Gly Thr Leu His Pro Gly Phe Phe Asn
                  115 120 125
          Lys Asn Asn Asn Glu Phe Thr Tyr Glu Arg Ser Pro His Ser Thr Ala
              130 135 140
          Tyr Ile Ala Glu Gly Glu Gly Ile Tyr Tyr Tyr Ala Gly Gly Phe Ser
          145 150 155 160
          Gly Gly Lys Thr Lys Glu Tyr Ile Lys Leu Cys Glu Thr Ile Lys Arg
                          165 170 175
          Arg Ile Asp Gln Asp Leu Gln Gln Arg Phe Ile Ala Val Trp His Asp
                      180 185 190
          Glu Ser His Ile Asn Arg Tyr Phe Leu Glu Asn Pro Pro Thr Thr Leu
                  195 200 205
          Ser Pro Ser Tyr Leu Tyr Pro Glu Gly Ser Ile Leu Pro Phe Glu Glu
              210 215 220
          Lys Ile Met Ile Arg Asp Lys Ser Lys Lys Glu Tyr Gly Gly His Lys
          225 230 235 240
          Phe Leu Arg Lys Lys Asp Ser Trp Leu His Arg Leu Ile Lys Lys Leu
                          245 250 255
           <![CDATA[ <210> 55]]>
           <![CDATA[ <211> 263]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides xylanisolvens]]>
           <![CDATA[ <400> 55]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Leu Ser Ala Glu Arg Tyr Phe Met Gln Asp Gln
                      20 25 30
          Ser Phe Thr Ile Glu Tyr Tyr Val Phe Thr Asp Thr Ser Lys Leu Tyr
                  35 40 45
          Asp Glu Glu Asn Asn Lys His Ile His Arg Ile Lys Gln Lys Asn Leu
              50 55 60
          Gly Trp Pro Asp Asn Thr Leu Lys Arg Phe His Ile Phe Leu Arg Ile
          65 70 75 80
          Lys Glu Gln Leu Glu Arg Glu Thr Asp Tyr Leu Phe Phe Phe Asn Ala
                          85 90 95
          Asn Leu Leu Phe Thr Ser Ser Ile Gly Lys Glu Ile Leu Pro Pro Ser
                      100 105 110
          Asp Ser Asn Gly Leu Leu Gly Thr Met His Pro Gly Phe Tyr Asn Lys
                  115 120 125
          Pro Asn Ser Glu Phe Thr Tyr Glu Arg Arg Asp Ala Ser Thr Ala Tyr
              130 135 140
          Ile Pro Glu Gly Glu Gly Leu Tyr Tyr Tyr Ala Gly Gly Leu Ser Gly
          145 150 155 160
          Gly Cys Thr Lys Ala Tyr Leu Lys Leu Cys Thr Thr Ile Cys Ser Trp
                          165 170 175
          Val Asp Arg Asp Ala Thr Asn His Ile Ile Pro Ile Trp His Asp Glu
                      180 185 190
          Ser Leu Ile Asn Lys Tyr Phe Leu Asp Asn Pro Pro Ala Ile Thr Leu
                  195 200 205
          Pro Pro Ala Tyr Leu Tyr Pro Glu Gly Trp Leu Leu Pro Phe Glu Pro
              210 215 220
          Ile Ile Leu Ile Arg Asp Lys Asn Lys Pro Lys Tyr Gly Gly His Glu
          225 230 235 240
          Leu Leu Arg Arg Lys Asn Ser Leu Trp Glu Arg Ile Lys Leu Ile Cys
                          245 250 255
          Gln Lys Phe Lys Ser Ala Asp
                      260
           <![CDATA[ <210> 56]]>
           <![CDATA[ <211> 309]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bisgaard taxon 44 str. 111]]>
           <![CDATA[ <400> 56]]>
          Met Gln Gln Pro Lys Val Ala Phe Leu Ser Ile Asn Thr Gly Ser Tyr
          1 5 10 15
          Asp Thr Phe Phe Lys Ala Val Phe Ala His Asn Gln Gln Asn Phe Leu
                      20 25 30
          Pro Asp Cys Gln Val Gln Tyr Phe Val Phe Thr Asp Ser Glu Asp Leu
                  35 40 45
          Ala Thr Thr Tyr Ala Asn Thr Glu Asn Val Thr Leu Ile Pro Gln Glu
              50 55 60
          His Leu Ala Trp Pro Gly Ala Thr Leu His Arg Phe Lys Met Phe Asn
          65 70 75 80
          Arg Pro Glu Val Arg Glu Leu Leu Ser Glu Tyr Asp Tyr Val Phe Phe
                          85 90 95
          Ala Asn Ala Asn Trp Tyr Ala Lys Asn Pro Ile Leu Gly Lys Asn Phe
                      100 105 110
          Leu Gln Pro Ala Thr Gly Asp Ala Ser Lys Asp Leu Tyr Leu Val Tyr
                  115 120 125
          His Tyr Gly Gln Asn Ala Val Pro Glu Ala Ala Lys Ser Tyr Glu Arg
              130 135 140
          Asn Pro Gln Ser Leu Ala Tyr Ile Pro Glu Asn Ala Thr Thr Thr Tyr
          145 150 155 160
          Val Ala Gly Gly Phe Phe Gly Gly Thr Ser Ala Ala Phe Met His Met
                          165 170 175
          Ile Ala Thr Leu Glu Arg Asn Ile Asp Phe Asp Leu Ala Lys Gly Ile
                      180 185 190
          Ile Ala Leu Trp His Asp Glu Ser His Leu Asn His Tyr Leu Tyr Thr
                  195 200 205
          Thr Gly Tyr Gln Ala His Ile Met Pro Pro Ile Phe Met Val Pro Gln
              210 215 220
          Glu Tyr His Pro Ile Ser Ser Tyr Ile Gly Glu Arg Pro Glu Trp Leu
          225 230 235 240
          Gly Val Cys Leu Asn Lys Asn Leu Leu Val Gln Asp Leu Asn Ala Leu
                          245 250 255
          Arg Asn Lys Gln Val Gly Phe Ser Leu Glu Gln Ile Gln Gln Leu Leu
                      260 265 270
          Glu His Glu Lys Asp Leu Asp Ala Ile Trp Arg Glu Gln Arg Ala Glu
                  275 280 285
          Phe Glu Pro Tyr Trp Gln Gln Asn Leu Gly Phe Val Gly Leu Ile Tyr
              290 295 300
          Asn Gln Val Glu Gln
          305
           <![CDATA[ <210> 57]]>
           <![CDATA[ <211> 301]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bisgaard Taxon 44 str. B96_3]]>
           <![CDATA[ <400> 57]]>
          Met Gln Lys Pro Lys Val Ala Leu Val Ser Ile Asn Thr Gly Ala Tyr
          1 5 10 15
          Asp Thr Tyr Phe Lys Val Leu Phe Pro Tyr Phe Tyr Thr Asn Phe Leu
                      20 25 30
          Pro Asp Cys Glu Leu Thr Phe Val Val Phe Thr Asp Ser Ser Glu Leu
                  35 40 45
          Glu Glu Leu Tyr Arg Tyr Asn Pro Ile Val Lys Ile Ile Lys Thr Pro
              50 55 60
          Tyr Glu Ala Trp Pro Gly Ala Thr Leu Lys Arg Phe His Tyr Phe Ser
          65 70 75 80
          Gln Ala Ser Ser His Leu Glu Gln Phe Asp Tyr Ile Phe Phe Ala Asn
                          85 90 95
          Ala Asn Tyr Tyr Cys Lys Asn Lys Ile Leu Ala Ser Glu Leu Leu Leu
                      100 105 110
          Pro Glu Gly Glu Lys Gly Leu Ile Phe Val Glu His Phe Gly Gln Asn
                  115 120 125
          His Leu Pro Glu Arg Leu Arg Ser Tyr Glu Arg Asn Pro Ala Ser Leu
              130 135 140
          Ala Tyr Ile Pro Glu Glu Gln Ala Thr Thr Tyr Val Ala Gly Ala Phe
          145 150 155 160
          Tyr Gly Gly Thr Ala Gln Glu Phe Leu Thr Met Ala Lys Thr Leu Ala
                          165 170 175
          Gln Arg Val Asp Gln Asp Leu Ala Asn Gly Ile Ile Ala Ile Trp His
                      180 185 190
          Asp Glu Ser His Leu Asn Cys Tyr Ala Leu Gln Ile Gly Tyr Gln Ala
                  195 200 205
          Lys Val Leu Pro Pro Arg Tyr Leu Val Pro Gln Glu Tyr Tyr Phe Ala
              210 215 220
          Ser Ser Tyr Ile Gly Glu Arg Gln Asp Trp Pro Cys Val Leu Leu Asn
          225 230 235 240
          Lys Asn Ala Leu Pro Ile Ala Ala Gln Asp Val Arg Asp Ser Lys Ala
                          245 250 255
          Lys Leu Asp Ala Arg Leu Val Glu Arg Leu Leu Ile Lys Glu Arg Glu
                      260 265 270
          Leu Glu Gln Leu Trp Leu Asp Lys Arg Glu Val Tyr Leu Glu Gln Ala
                  275 280 285
          Lys Ser Asn Pro Gly Phe Ile Val Phe Asn Trp Gln Val
              290 295 300
           <![CDATA[ <210> 58]]>
           <![CDATA[ <211> 303]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bisgaard taxon 44 str. B96_4]]>
           <![CDATA[ <400> 58]]>
          Met Lys Val Ala Phe Leu Ser Val Asn Thr Gly Ala Tyr Asp Thr Phe
          1 5 10 15
          Phe Lys Val Leu Phe Pro Tyr Asn Tyr Gln Asn Phe Leu Pro Asp Cys
                      20 25 30
          Gln Val Thr Phe Phe Val Phe Thr Asp Ser Lys Asp Leu Glu Gln Ser
                  35 40 45
          Phe Ala Leu Asn Pro Arg Val Lys Val Ile Tyr Gln Glu Tyr Glu Pro
              50 55 60
          Trp Pro Ala Pro Thr Leu Asp Arg Phe Ala Tyr Phe Leu Ser Gln Ala
          65 70 75 80
          Glu Gln Leu Gln Glu Phe Asp Tyr Ile Phe Phe Ala Asn Ala Asn Tyr
                          85 90 95
          Tyr Cys Lys Asn Pro Ile Lys Ala Glu Gln Ile Leu Phe Ala Pro Thr
                      100 105 110
          Gly Asp Leu Gly Lys Asp Leu Ile Met Val Glu His Phe Gly Gln Asn
                  115 120 125
          Phe Ile Ala Glu His Leu Arg Ser Tyr Glu Arg Asn Pro Ser Ser Gln
              130 135 140
          Ala Tyr Ile Ala Pro Gln Pro Glu Arg Pro Thr Thr Tyr Val Ala Gly
          145 150 155 160
          Gly Phe Tyr Gly Gly Thr Ala Gln Ala Phe Leu Ala Leu Ala Arg Thr
                          165 170 175
          Leu Ala Gln Arg Ile Gln Ala Asp Lys Glu Gln Gly Ile Val Ala His
                      180 185 190
          Trp His Asp Glu Ser His Leu Asn Arg Tyr Leu Tyr Asp Leu Asn Tyr
                  195 200 205
          Ala Cys His Phe Leu Pro Pro Cys Tyr Cys Val Pro Gln Glu Tyr Asp
              210 215 220
          Phe Glu Ser Arg Tyr Ile Gly Glu Arg Gln Asp Trp Pro Cys Val Leu
          225 230 235 240
          Leu Asn Lys Asn Ala Leu Pro Ser Pro Ala Gln Asp Ile Arg Ser Asn
                          245 250 255
          Gln Ala Ser Tyr Asp Pro Arg Trp Ile Glu Ile Leu Ile Met Gln Glu
                      260 265 270
          Arg Glu Leu Glu Ser Trp Trp Leu Arg Asp Arg His Ile Phe Tyr Pro
                  275 280 285
          Asn Ala Ile Lys Asn Gln Cys Phe Asn Thr Leu Leu Trp Glu Ile
              290 295 300
           <![CDATA[ <210> 59]]>
           <![CDATA[ <211> 308]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bisgaard taxon 44 str. EEAB3T1]]>
           <![CDATA[ <400> 59]]>
          Met Ser Arg Thr Lys Val Ala Val Leu Ser Val Asn Thr Gly Ala Tyr
          1 5 10 15
          Ala Ser Phe Phe Lys Val Leu Phe Pro Tyr Asn Tyr Gln Asn Phe Leu
                      20 25 30
          Pro Asp Cys Glu Val Thr Phe Phe Val Phe Thr Asp Ser Lys Glu Leu
                  35 40 45
          Ala Gln Leu Tyr Ala Tyr Asn Pro Gln Val Lys Ile Ile Pro Leu Asp
              50 55 60
          Tyr Gln Pro Trp Pro Leu Pro Thr Leu Phe Arg Phe Lys Tyr Phe Leu
          65 70 75 80
          Glu Leu Glu Ser Thr Leu Ala Glu Phe Ala Tyr Val Phe Phe Met Asn
                          85 90 95
          Ala Asn Phe Tyr Cys Lys Arg Pro Leu Tyr Ala Gln Asp Leu Leu Phe
                      100 105 110
          Ala Pro Thr Gly Asn Trp Ala Gln Asp Leu Ile Val Val Glu His Phe
                  115 120 125
          Gly Gln Asn Cys Leu Pro Glu Glu Leu Arg Ser Tyr Glu Arg Asn Pro
              130 135 140
          Gln Ser Gln Ala Tyr Ile Ser Pro Thr Pro Glu Lys Ala Thr Thr Tyr
          145 150 155 160
          Ile Ala Gly Ala Phe Asn Gly Gly Thr Ser Gln Ala Phe Leu Thr Met
                          165 170 175
          Ser Arg Glu Leu Ala Gln Arg Thr Leu Thr Asp Tyr Gln Asn Asn Leu
                      180 185 190
          Ile Ala Val Trp His Asp Glu Ser His Leu Asn Arg Leu Leu Tyr Asp
                  195 200 205
          Leu Asp Tyr Gln Ala His Ile Leu Pro Pro His Tyr Val Met Pro Gln
              210 215 220
          Glu Tyr Asp Phe Glu Ser Arg Tyr Val Gly Glu Arg Gln Asp Trp Phe
          225 230 235 240
          Ala Val Leu Leu Asn Lys Asn Ala Leu Pro Phe Asp Pro Gln Leu Ala
                          245 250 255
          Arg Asp Asn Gln Gln Glu Phe Asp Pro Arg His Leu Glu Leu Leu Val
                      260 265 270
          Leu Gln Glu Arg Gln Leu Glu Asn Ile Trp Leu Thr Tyr Arg Asp Thr
                  275 280 285
          Phe Tyr Pro Asn Ala Ile Lys Asn Asn Ser Phe Asn Cys Phe Ile Trp
              290 295 300
          Lys Ile Glu Pro
          305
           <![CDATA[ <210> 60]]>
           <![CDATA[ <211> 527]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Candidatus Magasanikbacteria]]>
           <![CDATA[ <400> 60]]>
          Met Lys Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Lys Ile Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Leu Ser Cys Glu Lys His Phe Ile Ser Glu Val
                      20 25 30
          Glu Lys His Tyr Phe Val Phe Thr Asp Ser Glu Ser Ile Glu Phe Glu
                  35 40 45
          Asn Glu Asn Ser Arg Ile His Arg Val Tyr Gln Gln Asn Leu Gly Trp
              50 55 60
          Pro Gly Asn Thr Leu Arg Arg Tyr Glu Met Phe Leu Lys Lys Lys Glu
          65 70 75 80
          Glu Leu Lys Lys Phe Asp Phe Leu Phe Phe Phe Asn Ala Asn Leu Gln
                          85 90 95
          Phe Leu Glu Lys Ile Thr Ser Asp Glu Phe Val Pro Val Gly Gln Glu
                      100 105 110
          Lys Leu Val Ala Cys Leu His Pro Gly Tyr Tyr Asp Lys Lys Lys Glu
                  115 120 125
          Ser Phe Thr Tyr Glu Arg Asn Ser Lys Ser Thr Ala Phe Ile Pro Lys
              130 135 140
          Gly Gln Gly Val Tyr Tyr Phe Ala Gly Gly Ile Asn Gly Gly Leu Ser
          145 150 155 160
          Lys Asp Phe Ile Glu Ala Met Glu Val Met Asp Glu Asn Ile Lys Lys
                          165 170 175
          Asp Phe His Asn Asn Ile Ile Ala Val Trp His Asp Glu Ser His Trp
                      180 185 190
          Asn Tyr Phe Leu Asn Asn Asn Ile Glu Asp Ile Lys Ile Leu Asp Pro
                  195 200 205
          Ser Tyr Leu Tyr Pro Glu Gly Gly Leu Leu Pro Phe Val Pro Lys Ile
              210 215 220
          Leu Val Arg Asp Lys Lys Ile Leu Gly Gly His Thr Lys Leu Arg Asp
          225 230 235 240
          Asn Phe Asn Phe Ile Leu Tyr Ile Asn Glu Ile Lys Ser Tyr Met Lys
                          245 250 255
          Lys Leu Ile Cys Lys Leu Lys Phe Glu Tyr Ile Ile Lys Leu Lys Gly
                      260 265 270
          Gly Leu Gly Asn Gln Met Phe Gln Tyr Ala His Gly Arg Ser Leu Glu
                  275 280 285
          Phe Ser Gly Lys Lys Val Ile Phe Asp Ile Ser Phe Phe Glu Asn Asn
              290 295 300
          Lys Ala Lys Arg Asp Ile Ala Arg Asp Phe Lys Leu Asp Asn Phe Asn
          305 310 315 320
          Ile Asp Thr Arg Val Lys Phe Val Asn Lys Lys Asn Ile Tyr Leu Asp
                          325 330 335
          Phe Val Asn Lys Ile Lys Arg Lys Ile Gly Phe Ser Leu Glu Glu Ser
                      340 345 350
          Phe Gln Gly Glu Lys Tyr Phe Glu Asn Ile Glu Asp Ile Ile Arg Lys
                  355 360 365
          Glu Leu Thr Leu Lys Lys Glu Leu Tyr Glu Lys Val Asp Lys Asn Leu
              370 375 380
          Leu Asn Lys Ile Leu Leu Ser Asn Ser Val Ser Ile His Ile Arg Arg
          385 390 395 400
          Thr Asp Tyr Val Thr Ser Lys Ile Ala Asn Lys Val Leu Gly Val Cys
                          405 410 415
          Ser Leu Asp Tyr Tyr Lys Ile Ser Ile Ser Lys Ile Ala Ser Leu Leu
                      420 425 430
          Asp Asn Pro His Phe Tyr Ile Phe Ser Asp Asp Ile Glu Trp Val Arg
                  435 440 445
          Ser Asn Leu Phe Met Glu Tyr Pro Phe Thr Tyr Val Ser Asn Gly Val
              450 455 460
          Tyr Lys Asp Tyr Glu Glu Leu Val Leu Met Ser Ser Cys Lys His Asn
          465 470 475 480
          Ile Ile Ala Asn Ser Thr Phe Ser Trp Trp Ala Ala Trp Leu Asn Lys
                          485 490 495
          Asn Gln Asn Lys Ile Val Val Ala Pro Ser Lys Trp Phe Asn Asp Lys
                      500 505 510
          Thr Tyr Ser Glu Asn Asn Leu Val Pro Lys Lys Trp Ile Arg Ile
                  515 520 525
           <![CDATA[ <210> 61]]>
           <![CDATA[ <211> 526]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Candidatus Nomurabacteria]]>
           <![CDATA[ <400> 61]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Ser Ile Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Leu Ser Met Glu Glu Arg Phe Ile Thr Asp Ser
                      20 25 30
          Glu Lys Tyr Tyr Phe Val Phe Thr Asp Ser Ala Glu Leu Asp Phe Glu
                  35 40 45
          Lys Glu Asn Pro Arg Val His Arg Ile Tyr Gln Glu Asn Leu Gly Trp
              50 55 60
          Pro Glu Asn Thr Leu Met Arg Phe His Val Phe Leu Asn Lys Glu Lys
          65 70 75 80
          Glu Leu Glu Asp Met Asn Tyr Leu Phe Phe Phe Asn Ala Asn Leu Ile
                          85 90 95
          Val Leu Glu Lys Ile Thr Ala Asp Asn Phe Leu Pro Asn Glu Asn Glu
                      100 105 110
          Asn Leu Val Ala Thr Leu His Pro Gly Phe Tyr Asn Lys Asn Arg Lys
                  115 120 125
          Lys Phe Thr Tyr Glu Asn Asn Lys Lys Ser Thr Ala Phe Ile Ser Lys
              130 135 140
          Asp Gln Gly Gln Tyr Tyr Phe Ala Gly Gly Leu Asn Gly Gly Lys Thr
          145 150 155 160
          Thr Asn Phe Ile Glu Ala Met Lys Val Met Arg Asp Asn Val Asp Ile
                          165 170 175
          Asp Lys Lys Asn Asn Ile Ile Ala Lys Trp His Asp Glu Ser His Trp
                      180 185 190
          Asn Arg Tyr Val Leu Asn Arg Thr Asp Val Lys Ile Leu Pro Pro Ser
                  195 200 205
          Tyr Leu Tyr Pro Glu Gly Trp Pro Leu Pro Phe Asn Pro Ile Ile Leu
              210 215 220
          Ile Arg Asp Lys Asn Lys Tyr Gly Gly His Ala Ile Leu Arg Ser Ile
          225 230 235 240
          Lys Val Asn Lys Phe Lys Val His Phe Leu Lys Met Lys Lys Ile Phe
                          245 250 255
          His Lys Phe Tyr Asn Lys Tyr Leu Glu Phe Lys Met Val Leu Phe Glu
                      260 265 270
          Phe Lys Lys Pro Thr Tyr Ser Asn Leu Asn Lys Phe Asn Leu Lys Asn
                  275 280 285
          Thr Lys Phe Ile Leu Ile Thr Ile Ala Phe Asn Asn Val Glu Ile Ile
              290 295 300
          Lys Phe Gln Asn Glu Lys Val Met Glu Asn Leu Lys Asp Asp Phe Ser
          305 310 315 320
          His Ile Ile Val Asp Asn Ser Ser Thr Lys Asn Val Ser Gly Glu Ile
                          325 330 335
          Phe Lys Tyr Cys Lys Ile Asn Asn Ile Pro Tyr Val Lys Leu Pro Asn
                      340 345 350
          Asn Thr Phe Glu Lys Ser Pro Ser Lys Ser His Gly Lys Ala Leu Asn
                  355 360 365
          Trp Ala Tyr Arg Asn Ile Ile Asn Lys Tyr Glu Pro Ala Tyr Phe Gly
              370 375 380
          Phe Ile Asp His Asp Ile Ile Pro Phe Lys Glu Thr Ser Ile Thr Asn
          385 390 395 400
          Tyr Ile Lys Asn Gly Ala Trp Gly Leu Ile Gln Glu Arg Glu Glu Lys
                          405 410 415
          Trp Tyr Leu Trp Pro Gly Phe Cys Phe Phe Lys Phe Ala Glu Val Arg
                      420 425 430
          Lys Tyr Lys Met Asn Phe Met Pro Tyr Arg Gly Leu Asp Thr Gly Gly
                  435 440 445
          Ser Asn Tyr His Ser Leu Tyr Lys Asn Ile Asn Lys Asn Asn Ile Leu
              450 455 460
          Lys Ile Arg Gln Thr Tyr Phe Asp Leu Asp Lys Asn Glu Lys Val Thr
          465 470 475 480
          Lys Phe Asp Thr Ser Glu Asn Ile Val Glu Val Leu Asp Asp Trp Val
                          485 490 495
          His Ile Met Arg Thr Ser Asn Trp Asn Asn Gln Val Ser Ser Lys Asn
                      500 505 510
          Ser Lys Phe Asn Glu Ile Ile Tyr Ile Ile Lys Glu Lys Phe
                  515 520 525
           <![CDATA[ <210> 62]]>
           <![CDATA[ <211> 231]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Chlamydiae bacterium]]>
           <![CDATA[ <400> 62]]>
          Met Trp Cys Phe Ala His Glu Pro Thr Ile Gly Phe Cys Ile Val Ala
          1 5 10 15
          Thr Gly Lys Tyr Ile Asp Phe Thr Pro Pro Leu Ile Glu Ser Ala Glu
                      20 25 30
          Lys Tyr Phe Cys Arg Gly Thr Pro Lys Arg Tyr Phe Val Phe Ser Asp
                  35 40 45
          Arg Thr Ser Glu Leu Pro Lys Asn Ala Glu Ile Ile Glu Val Arg His
              50 55 60
          Phe Ser Trp Pro Phe Ser Thr Ala Met Arg Asn Thr Phe Tyr Val Leu
          65 70 75 80
          His Lys Glu Arg Leu Lys Glu Cys Asp Tyr Leu Phe Ala Ile Asp Ala
                          85 90 95
          Asp Met Arg Phe Val Ser Pro Ile Ala Lys Glu Glu Val Leu Gly Thr
                      100 105 110
          Leu Val Ala Thr Gln His Pro Gly Phe Tyr Arg Met Arg Gly Ser Tyr
                  115 120 125
          Glu Ser Asn Ser Ile Ser Lys Ala Phe Val Ala Pro Asn Glu Gly Glu
              130 135 140
          Tyr Tyr Phe Cys Gly Gly Phe Phe Gly Gly Lys Arg Glu Glu Phe Ile
          145 150 155 160
          Lys Leu Cys Gln Lys Thr Ser Asp Asn Phe Phe Glu Asp Leu Lys Lys
                          165 170 175
          Gly Phe Ile Ala Glu Trp His Asp Glu Ser His His Asn Arg Tyr Leu
                      180 185 190
          Ile Asp Tyr Pro Pro Thr Lys Ile Leu Ser Pro Ala Tyr Cys Tyr Pro
                  195 200 205
          Glu Ser Trp Lys Leu Pro Phe Glu Lys Lys Leu Leu Ala Leu Asp Lys
              210 215 220
          Asn His Ala Glu Phe Gln Lys
          225 230
           <![CDATA[ <210> 63]]>
           <![CDATA[ <211> 234]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Chlamydiae bacterium]]>
           <![CDATA[ <400> 63]]>
          Met Gln Glu Gly Phe Ala Arg Asp Ser Thr Pro Lys Gln Ile Gly Leu
          1 5 10 15
          Phe Ile Val Ala Thr Gly Lys Tyr Ile Gln Phe Val Asn Pro Leu Ile
                      20 25 30
          Thr Ser Ala Arg His Trp Phe Cys Thr Asp His His Val Val Phe Phe
                  35 40 45
          Val Phe Thr Asp Gln Glu Met Gln Glu Glu Phe Asp Val Ile Arg Ile
              50 55 60
          Pro Val Arg His Leu Gly Trp Pro Tyr Ala Thr Leu Met Arg Phe His
          65 70 75 80
          Met Tyr Ala Glu Tyr Gln Glu Gln Phe Asp Cys Leu Asp Tyr Ile Phe
                          85 90 95
          Ala Ile Asp Ala Asp Ala Leu Phe Val Ala Pro Val Gly Glu Glu Ile
                      100 105 110
          Phe Ser Asp Arg Val Phe Thr Leu His Pro Gly Phe Val Asn Arg Ala
                  115 120 125
          Gly Thr Tyr Glu Arg Asn Pro Leu Ser Ala Ala Cys Val Ala Ser His
              130 135 140
          Glu Gly Thr Phe Tyr Phe Ala Gly Gly Phe Tyr Gly Gly Ser Pro Lys
          145 150 155 160
          Glu Phe Phe Arg Phe Val Asn Thr Ala Lys Glu Lys Val Asp Gln Asp
                          165 170 175
          Leu Ala Lys Gly Cys Ile Ala Leu Trp His Asp Glu Ser His Leu Asn
                      180 185 190
          Arg Tyr Ala Ile Asp Tyr Pro Pro Thr Leu Ile Leu Thr Pro Ser Tyr
                  195 200 205
          Cys Tyr Pro Glu Ser Trp Arg Leu Pro Tyr Val Lys Lys Ile Leu Val
              210 215 220
          Leu Asp Lys Asp His Cys Ala Met Arg Asn
          225 230
           <![CDATA[ <210> 64]]>
           <![CDATA[ <211> 282]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Clostridium hathewayi CAG:224]]>
           <![CDATA[ <400> 64]]>
          Met Val Tyr Cys Asp Met Ile Arg Asn Lys Ile Gly Ile Leu Tyr Val
          1 5 10 15
          Cys Thr Gly Glu Tyr Asn Ile Phe Trp Glu Asp Phe Tyr Lys Ser Phe
                      20 25 30
          Glu Glu Lys Phe Cys Thr Asn Ser Asp Lys Ile Tyr Met Val Phe Thr
                  35 40 45
          Asp Ala Asn Ser Ile Ala Tyr Glu Glu Met Thr Asn Val Ile Lys Ile
              50 55 60
          Tyr Gln Asp Cys Leu Gly Trp Pro Tyr Asp Thr Leu Met Arg Tyr Ser
          65 70 75 80
          Met Phe Glu Lys Ile Lys Asp Ile Ile Gly Lys Cys Glu Tyr Val Phe
                          85 90 95
          Phe Phe Asn Ala Asn Met Ile Cys Asn Leu Ala Val Tyr Glu Glu Asp
                      100 105 110
          Ile Leu Pro Arg Arg Ser Lys Gly Glu Ser Leu Ser Val Val Leu His
                  115 120 125
          Pro Gly Tyr Gly Gly Lys Lys Ala Arg Phe Cys Pro Leu Glu Arg Asn
              130 135 140
          Lys Lys Ser Leu Ala Tyr Ile Pro Tyr Asn Cys Asn Ala Lys Tyr Val
          145 150 155 160
          Cys Gly Gly Val Asn Gly Gly Glu Ser Gln Ala Tyr Ile Glu Leu Ile
                          165 170 175
          Glu Glu Leu Asn Arg Arg Ile Asn Ile Asp Leu Asp Asn Ala Ile Val
                      180 185 190
          Ala Arg Val His Asp Glu Ser His Leu Asn Lys Tyr Ile Tyr Gly Arg
                  195 200 205
          Gln Gly Val Arg Tyr Leu Gly Pro Glu Phe Cys Asn Pro Asp Asp Leu
              210 215 220
          Thr Leu Met Val Glu Lys Lys Ile Arg Leu Leu Asp Lys Asn Lys Tyr
          225 230 235 240
          Leu Asn Ile Asn Lys Leu Lys Asn Ile Lys Asn Glu Asn Phe Phe Gln
                          245 250 255
          Lys Trp Arg Arg Arg Phe Ala Lys Tyr Ser Val Cys Glu Ile Gly Tyr
                      260 265 270
          Leu Lys Asp Val Phe Met Arg Lys Arg Leu
                  275 280
           <![CDATA[ <210> 65]]>
           <![CDATA[ <211> 177]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Coxiella sp.]]>
           <![CDATA[ <400> 65]]>
          Met Asp Lys Asn Ile Gly Lys Tyr Lys Ile Val Met Val Ser Ile Cys
          1 5 10 15
          Leu Asn Gln Pro Tyr Trp Gln Tyr Ile Ser Pro Met Ile Glu Ser Ala
                      20 25 30
          Arg Lys Phe Leu Leu Lys Gly His Asp Val Asp Phe Phe Val Trp Thr
                  35 40 45
          Asp Met Pro Glu Glu Thr Asn Leu Gly Gln Gly Val Lys Ile Phe Pro
              50 55 60
          Thr Ala Pro Cys Asp Trp Pro Leu Pro Thr Leu Phe Arg Tyr His Leu
          65 70 75 80
          Phe Leu Gln Gln Glu Glu Leu Leu Lys Gln Tyr Asp Tyr Ile Phe Tyr
                          85 90 95
          Cys Asp Ala Asp Met Leu Phe Val Ser Arg Val Gly Asn Glu Ile Leu
                      100 105 110
          Gly Glu Gly Leu Thr Ala Ala Ala His Pro Met Tyr Ala Leu Arg Pro
                  115 120 125
          Glu Tyr Ile His Pro Tyr Glu Pro Asn Ser Gln Ser Thr Ala Tyr Ile
              130 135 140
          Pro Ser Leu Gly Arg Val Leu Glu Asn Pro Lys Arg Phe Glu Pro Phe
          145 150 155 160
          Tyr Ala Ala Gly Gly Phe Gln Gly Gly Arg Thr Glu Asn Phe Ile Gln
                          165 170 175
          Ala
           <![CDATA[ <210> 66]]>
           <![CDATA[ <211> 557]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Desulfocurvibacter africanus PCS]]>
           <![CDATA[ <400> 66]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Thr Val Phe
          1 5 10 15
          Trp Asn His Phe Phe Thr Ser Cys Glu Gln His Phe Leu Arg Glu His
                      20 25 30
          Glu Lys His Tyr Tyr Ile Phe Thr Asp Gly Glu Ile Ala His Leu Asn
                  35 40 45
          Cys Asn Arg Val His Arg Ile Glu Gln Gln His Leu Gly Trp Pro Asp
              50 55 60
          Ser Thr Leu Lys Arg Phe His Met Phe Glu Arg Ile Ala Asp Thr Leu
          65 70 75 80
          Arg Gln Asn Ser Asp Phe Ile Val Phe Phe Asn Ala Asn Met Val Phe
                          85 90 95
          Leu Arg Asp Val Gly Lys Glu Phe Leu Pro Thr Arg Glu Gln Ala Leu
                      100 105 110
          Val Phe His Arg His Pro Gly Leu Phe Arg Arg Pro Ala Trp Leu Leu
                  115 120 125
          Pro Tyr Glu Arg Arg Pro Glu Ser Thr Ala Tyr Ile Pro Tyr Gly Ser
              130 135 140
          Gly Ser Ile Tyr Val Cys Gly Gly Val Asn Gly Gly Tyr Thr Gln Pro
          145 150 155 160
          Tyr Leu Asp Phe Val Ala Met Leu Arg Arg Asn Ile Asp Ile Asp Val
                          165 170 175
          Glu Arg Gly Ile Ile Ala Arg Trp His Asp Glu Ser His Ile Asn Arg
                      180 185 190
          Phe Val Ile Gly Arg His Tyr Lys Ile Gly His Pro Gly Tyr Val Tyr
                  195 200 205
          Pro Asp Arg Arg Asn Leu Pro Phe Pro Arg Ile Ile Arg Val Ile Asp
              210 215 220
          Lys Ala Ser Val Gly Gly His Thr Phe Leu Arg Gly Gln Thr Pro Glu
          225 230 235 240
          Pro Ala Pro Glu Glu Gln Ser Lys Thr Val Ala Lys Lys Leu Arg Ser
                          245 250 255
          Gln Leu Lys Arg Pro Cys Met Pro Arg Ala Ala Gln Asp Glu Pro Ile
                      260 265 270
          Ile Leu Ala Arg Met Met Gly Gly Leu Gly Asn Gln Met Phe Ile Tyr
                  275 280 285
          Ala Ala Ala Arg Val Leu Ala Glu Arg Gln Gly Ala Gln Leu His Leu
              290 295 300
          Asp Thr Gly Lys Leu Ser Gly Asp Ser Ile Arg Gln Tyr Asp Leu Pro
          305 310 315 320
          Ala Phe Ser Ile Asp Ala Pro Leu Trp His Ile Pro Cys Gly Cys Asp
                          325 330 335
          Arg Ile Val Gln Ala Trp Phe Ala Leu Arg His Val Ala Ala Gly Cys
                      340 345 350
          Gly Met Pro Lys Pro Thr Met Gln Val Leu Arg Ser Gly Phe His Leu
                  355 360 365
          Asp Gln Arg Phe Phe Ser Ile Arg His Ser Ala Tyr Leu Ile Gly Tyr
              370 375 380
          Trp Gln Ser Pro His Tyr Trp Arg Gly His Glu Asp Arg Val Arg Ser
          385 390 395 400
          Ser Phe Asp Leu Thr Arg Phe Glu Arg Pro His Leu Arg Glu Ala Leu
                          405 410 415
          Ala Ala Val Ser Gln Pro Asn Thr Ile Ser Val His Leu Arg Arg Gly
                      420 425 430
          Asp Phe Arg Ala Pro Lys Asn Ser Asp Lys His Leu Leu Ile Asp Gly
                  435 440 445
          Ser Tyr Tyr Glu Arg Ala Arg Lys Leu Leu Leu Glu Met Thr Pro Gln
              450 455 460
          Ser His Phe Tyr Ile Phe Ser Asp Glu Pro Glu Glu Ala Gln Arg Leu
          465 470 475 480
          Phe Ala His Trp Glu Asn Thr Ser Phe Gln Pro Arg Arg Ser Gln Glu
                          485 490 495
          Glu Asp Leu Leu Leu Met Ser Arg Cys Ser Ala Ser Ile Ile Ala Asn
                      500 505 510
          Ser Ser Phe Ser Trp Trp Gly Ala Trp Leu Gly Arg Pro Lys Gln His
                  515 520 525
          Val Ile Ala Pro Arg Met Trp Phe Thr Arg Asp Val Leu Met His Thr
              530 535 540
          Tyr Thr Leu Asp Leu Phe Pro Glu Lys Trp Ile Leu Leu
          545 550 555
           <![CDATA[ <210> 67]]>
           <![CDATA[ <211> 239]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Desulfocurvibacter africanus PCS]]>
           <![CDATA[ <400> 67]]>
          Met Arg Val Ala Val Leu Tyr Ile Cys Thr Gly Lys Tyr Thr Val Phe
          1 5 10 15
          Trp Asp Gly Phe Phe Arg Ser Ser Glu Leu Phe Phe Met Arg Ser His
                      20 25 30
          Glu Lys His Tyr Phe Val Phe Thr Asp Gly His Ile Asp His Thr Asn
                  35 40 45
          Asp Ser Arg Val His Arg Ile Gln Gln Lys Lys Leu Gly Trp Pro Tyr
              50 55 60
          Asp Thr Leu His Arg Phe His Met Phe Ser Cys Ile Glu Ser Glu Leu
          65 70 75 80
          Gln Ser Phe Asp Phe Ile Leu Tyr Ile Asn Ala Asn Ser Tyr Phe Val
                          85 90 95
          Thr Glu Cys Gly Asp Asp Val Leu Pro Lys His Lys Asp His Leu Leu
                      100 105 110
          Leu Thr Leu His Pro Gly Tyr Trp His Ser Lys Tyr Arg Leu Leu Arg
                  115 120 125
          Trp Lys Leu Pro Tyr Glu Arg Asp Val Arg Ser Thr Ala Tyr Ile Pro
              130 135 140
          Tyr Trp Lys Gly Gly Arg Tyr Val Cys Gly Gly Leu Asn Gly Gly Trp
          145 150 155 160
          Arg Asp Ser Tyr Leu Arg Leu Ile Arg Glu Leu Lys Glu Ala Ile Asp
                          165 170 175
          Val Asp Gly Ile Asn Gly Ile Val Ala Arg Trp His Asp Glu Ser His
                      180 185 190
          Leu Asn Arg Tyr Ala Leu Glu His Pro Ala Lys Leu Leu His Pro Gly
                  195 200 205
          Tyr Met His Pro Ala Gly Glu Lys Leu Pro Phe Pro Lys Ile Val His
              210 215 220
          Leu Phe His Lys Lys Asp Phe Gly Gly His Asp Phe Leu Arg Ser
          225 230 235
           <![CDATA[ <210> 68]]>
           <![CDATA[ <211> 282]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Gemmiger formicilis]]>
           <![CDATA[ <400> 68]]>
          Met Lys Thr Leu Ala Ile Leu Tyr Ile Cys Thr Gly Pro Tyr Ala Val
          1 5 10 15
          Phe Trp His Asp Phe Tyr Pro Asn Phe Lys Ala Asn Phe Leu Pro Asp
                      20 25 30
          Cys Asp Arg Ile Phe Tyr Val Phe Thr Asp Ala Ala His Ile Asp Tyr
                  35 40 45
          Glu Asp Ala Pro Asp Val Arg Arg Ile Tyr Gln Lys Ala Leu Pro Trp
              50 55 60
          Pro Gln Ser Thr Met Leu Arg Phe Asp Ala Phe Leu Gly Gln Ala Asp
          65 70 75 80
          Ala Leu Gln Gly Tyr Asp Tyr Leu Phe Phe Ala Asn Ala Asn Leu His
                          85 90 95
          Cys Thr Arg Val Ile Arg Ala Asp Glu Leu Leu Pro Asp Pro Ala Ala
                      100 105 110
          Gly Gln Ser Leu Thr Ala Val Cys His Leu Pro Tyr Tyr Gly Lys Asn
                  115 120 125
          Pro Ile Phe His Pro Tyr Asp Arg Ser Gly Lys Ser Arg Ala Ser Ile
              130 135 140
          Pro Tyr Ser Cys Gly Gln Tyr Tyr Val Ala Gly Gly Leu Asn Gly Gly
          145 150 155 160
          Thr Ala Ala Ala Tyr Leu Ala Leu Cys Arg Glu Leu Lys Lys Arg Thr
                          165 170 175
          Asp Glu Asp Leu Gln Asn Asn Val Ile Ala Arg Phe His Asp Glu Ser
                      180 185 190
          Gln Leu Asn Arg Leu Val Ala Glu Thr Pro Gly Lys Phe Arg Ile Leu
                  195 200 205
          Pro Pro Asp Tyr Cys Thr Pro Glu Glu Thr Pro Thr Gly His Glu Ala
              210 215 220
          Ile Leu Val Leu Gln Lys Ser Arg Cys Ile Asn Val Glu Ser Val Lys
          225 230 235 240
          Gly Ala Ala Lys Pro Gln Asn Phe Val Gln Arg Lys Trp Glu Ala Phe
                          245 250 255
          Arg Leu Asn Trp Leu Pro Tyr Leu Trp Leu Ala Arg Asp Thr Leu Leu
                      260 265 270
          Arg Arg Arg Ile Asp Phe Lys Asn Asp Leu
                  275 280
           <![CDATA[ <210> 69]]>
           <![CDATA[ <211> 276]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Gemmiger formicilis]]>
           <![CDATA[ <400> 69]]>
          Met Thr Lys Val Ala Ala Leu Tyr Ile Ala Thr Gly Arg Tyr Thr Val
          1 5 10 15
          Phe Trp Pro Glu Phe Tyr Glu Ser Ala Glu Lys Tyr Leu Leu Lys Asp
                      20 25 30
          Cys Glu Val His Tyr Phe Val Phe Thr Asp Thr Ala Thr Leu Pro Gly
                  35 40 45
          Asp Asp Asn Pro Arg Val His Ile Cys Ala Gln Glu Ala Tyr Ser Trp
              50 55 60
          Pro Phe Ala Thr Leu Arg Arg Phe Glu Ile Phe Leu Lys Gln Glu Gln
          65 70 75 80
          Ala Leu Lys Ala Phe Asp Tyr Ile Phe Phe Phe Asn Ala Asn Ala Glu
                          85 90 95
          Phe Met Gln Pro Val Thr Arg Glu Met Leu Leu Pro Arg Ala Glu Lys
                      100 105 110
          Gly Glu His Leu Leu Val Val Gln His Pro Ser Phe Tyr Ala Lys Pro
                  115 120 125
          Asn Tyr Glu Phe Thr Tyr Asp Arg Asn Pro Arg Ser Thr Ala Cys Ile
              130 135 140
          Pro Tyr Gly Leu Gly Lys Tyr Tyr Val Cys Gly Gly Val Asn Gly Gly
          145 150 155 160
          Glu Ala Ala Ala Phe Leu Gln Leu Cys His Thr Leu Asp Ala Arg Ile
                          165 170 175
          Arg Arg Asp Leu Gln Arg Asn Val Ile Ala Leu Trp His Asp Glu Ser
                      180 185 190
          Gln Ile Asn Arg Tyr Ile Leu Phe Arg Lys Asp Phe Arg Val Leu Thr
                  195 200 205
          Pro Ala Phe Cys Tyr Pro Glu Gly Trp Asp His Leu Pro Phe Pro Cys
              210 215 220
          Ile Ile Arg Ile Arg Ser Lys Ala Arg Tyr Ile Asp Ile Pro Ala Leu
          225 230 235 240
          Arg Lys Asp Ala Pro Glu Thr Lys Leu Ser Pro Ala Val Ala Arg Trp
                          245 250 255
          Asn His Phe Ala Met Arg Ala Ala Arg Trp Thr Gln Asn His Ile Phe
                      260 265 270
          Lys Lys Gly Ser
                  275
           <![CDATA[ <210> 70]]>
           <![CDATA[ <211> 437]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Guillardia theta]]>
           <![CDATA[ <400> 70]]>
          Met Arg Arg Leu Ile Phe Phe Leu Leu Leu Leu Leu Gln Ala Arg Gly
          1 5 10 15
          Ala Glu Asp Arg Thr Asp Ser Gln Asp Val Ala Val Thr Ile Thr Arg
                      20 25 30
          Pro Glu Asp Gly Glu Arg Val Arg Gly Asp Val Val Pro Leu Glu Val
                  35 40 45
          Ser Ala Met Ser Ser Arg Arg Gly Ser Arg Val Ile Leu Tyr Met Asp
              50 55 60
          Gly Arg Glu Val Tyr Arg Thr Glu Glu Arg Ala Val Ser Leu Gln Met
          65 70 75 80
          Ser Gln Leu Gln Val Gly Tyr His Val Met Glu Val Gln Leu Thr Glu
                          85 90 95
          Glu Asp Glu Ala Val Ala Tyr Asp Ser Val Gly Arg Thr Ala Gln Glu
                      100 105 110
          Phe Phe Tyr Val Gly Asp Gly Met Leu Ala Pro Asp Leu Asp Asp Thr
                  115 120 125
          Glu His Val Asn Ile Leu Ser Asn Val Thr Thr Ile Asp Asp Met Glu
              130 135 140
          Arg Val Ala Ile Gln His Tyr Glu Arg Gly Gln Leu Asp Leu Ser Gly
          145 150 155 160
          Arg Glu Met Ser Glu Gln Ser Tyr Val Arg Leu Ile Ser Ala Met Ser
                          165 170 175
          Asn Ala Pro Gly Ala Thr Met Pro Arg Leu Leu Ala Ser Leu Gly Arg
                      180 185 190
          Val Leu Met Ala Lys Lys Asp Tyr Ile Gly Ala Leu Gln Ala Tyr Arg
                  195 200 205
          Gly Asp Glu Val Arg Leu Phe Glu Met His Asp Lys Thr Ser Lys Arg
              210 215 220
          Leu Glu Ala Arg Ala Ala Cys Pro Val Lys Ala Ile Gly Thr Pro His
          225 230 235 240
          Ala Pro Gln Leu Leu Lys Val Gly Ile Leu Thr Val Ala Ser Gly Arg
                          245 250 255
          Tyr Ala Ser Phe Val Arg Ser Thr Val Ser Ser Ala Glu Ser Tyr Leu
                      260 265 270
          Leu Arg Ile Tyr Gly Leu Leu Ser Asp Leu Cys Met Gly Arg Glu Gly
                  275 280 285
          Lys Gly Leu Met Leu Lys Gly Pro Trp Glu Asp Gly Arg Val His Ala
              290 295 300
          Val Tyr Arg Lys His Asp Gly Trp Pro Ser Ala Ser Met Lys Arg Ala
          305 310 315 320
          His Ser Tyr Leu Glu His Ala Glu Leu Trp Gly Ala Met Asp Tyr Val
                          325 330 335
          Phe Ala Val Asp Val Gly Glu Thr Val Gly Thr Leu His Ala Asp Asn
                      340 345 350
          Ala Phe Tyr Asp Gly Ser Glu Val Val Gly Arg Phe Gln Lys Ala Trp
                  355 360 365
          Ser Val Asn Ala Glu Pro Gly Thr Met Trp Arg Gln His His Gly Asn
              370 375 380
          Ala Ala Leu Val Thr Arg Ala Val Tyr Glu Lys Arg Glu Glu Ser Thr
          385 390 395 400
          Ala Gly Met Arg Ala Gly Glu Gly Arg His Tyr Phe Ala Gly Gly Phe
                          405 410 415
          Tyr Gly Gly Arg Ser Gln Lys Val Leu Glu Met Leu Lys Glu Leu Val
                      420 425 430
          Lys Arg Thr Asn Gln
                  435
           <![CDATA[ <210> 71]]>
           <![CDATA[ <211> 927]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Guillardia theta]]>
           <![CDATA[ <400> 71]]>
          Met Ala Met Trp Arg Leu Ala Met Ala Met Leu Val Val Val Ser Thr
          1 5 10 15
          Met Gly Gly Gly Gly Asp Ala Arg Gly Arg Arg Glu Asp Asp Glu Ile
                      20 25 30
          Pro Ile Arg Val His Ala Thr Ser Pro Met Arg Gly Trp Val Leu Val
                  35 40 45
          Arg Gly Lys Leu Pro Val Ser Phe Phe Leu Ser Tyr Gly Asn Ser Ser
              50 55 60
          Arg Ser Gly Ala Cys Arg Ala Gly Ser Gln Arg Leu Ser Met Arg Ile
          65 70 75 80
          Ile Val Asp Glu Glu Thr Tyr Glu Glu Val Asp Ile Ser Asn Gly Ala
                          85 90 95
          Ala Tyr Ser Lys Val Phe Pro Ala Asp Leu Ser Pro Gly Val His Thr
                      100 105 110
          Ile Arg Ile Glu Gly Ala Glu Ser Thr Arg Pro Phe Leu Gln Gln Glu
                  115 120 125
          Glu Ile Ser Phe Ser Val Val Asp Ser Glu Asp Asp Phe His Asp Val
              130 135 140
          Arg His Tyr Asp Glu Ile Met Gly Ser Ala Pro Thr Ile Asp Asp Ser
          145 150 155 160
          Arg Gly Phe Thr Ser Gly Lys Pro Leu Lys Gly Val Ala Ile Leu Tyr
                          165 170 175
          His Lys Gln Ala Arg Leu Lys Tyr Glu Asp Arg Trp Ile Glu Lys Cys
                      180 185 190
          Ile Glu Ser Ile Leu Asn Gln Asn Tyr Pro Phe Phe Asp Ile Val Glu
                  195 200 205
          Leu Asn Tyr Gly Gly Glu Tyr His Ser Phe Met Gln Gln Tyr Leu His
              210 215 220
          Arg Leu Glu Gly Lys Arg Tyr Thr Phe Phe Ser Arg Glu Phe Gln Ser
          225 230 235 240
          His Ala Val Ala Met Asn Phe Leu Leu Asp Trp Val Phe Ser Glu Asp
                          245 250 255
          Tyr Asp Val Ala Phe Asn Val Asn Leu Asp Asp Tyr Tyr Ser Pro Asp
                      260 265 270
          Arg Phe Lys Leu Gln Ala Glu Ala Val Met Glu Gly Ala Asp Leu Val
                  275 280 285
          Ser Ser Tyr Phe Val Arg Val Val Val Glu Ala Gly Glu Gly Val Asp Val
              290 295 300
          Ile Asn Thr Lys Met Asn Pro Val Tyr Leu Thr Ala Gln Glu Leu Leu
          305 310 315 320
          Gly Arg Asp His Val Ser Glu Glu Asp Ile Phe Ser Gln Leu Arg Glu
                          325 330 335
          Asp His Asn Val Ile Cys His Pro Gly Val Ala Tyr Ser Arg Ser Phe
                      340 345 350
          Trp Lys Thr Leu Ser Ser Leu Asn Cys Lys Gln Gly Leu Ala Glu Ser
                  355 360 365
          Ser Gln Asp Ala Gln Trp His Ala Arg Ser Asn Thr Leu His Gln His
              370 375 380
          Ala Arg Val Cys Ser Asp Ser Ala Asp Leu Glu Asn Leu Phe Arg Pro
          385 390 395 400
          Leu Arg Tyr Arg Pro Glu Leu Pro Ala Glu Asp Leu Arg Leu Trp Gln
                          405 410 415
          Arg Ala Val Leu Met Ser Ser Val Lys Thr Val Ile Val Pro Arg Val
                      420 425 430
          Leu Val Phe Tyr Arg Ile His Glu Ser Gln Leu Ser Pro Ser Asp Glu
                  435 440 445
          Ala Val Asn Val Lys Tyr Arg Ala Asp Leu Thr Val Ser Gly Ala Met
              450 455 460
          Leu Asn Lys Met Arg Val Gly Ile Leu Thr Ile Cys Thr Gly Arg Gln
          465 470 475 480
          Glu Thr Tyr Ser Gly Arg Leu Arg Tyr Cys Ala Tyr Leu Pro Glu His
                          485 490 495
          Ile Arg Thr Val Arg Glu Arg Phe Val Ser Glu His His Leu Cys Phe
                      500 505 510
          Phe Thr Phe Thr Asp Asp Pro Arg Gly Ala Glu Glu Ile Phe Ser Arg
                  515 520 525
          Met Pro Ser Pro Ser Glu Val Ile Pro Ile Arg Gly Arg Gly Phe Pro
              530 535 540
          Ala Asp Thr Leu Tyr Arg Tyr His Tyr Phe Leu Ser Gln Ser Ser Lys
          545 550 555 560
          Leu Lys Thr Glu Thr Asp Val Val Phe Tyr Leu Asp Val Asp Val Ile
                          565 570 575
          Val Glu Lys Gly Ile Ala Ala Ala Pro Cys Cys His Pro Thr Tyr Asp
                      580 585 590
          Ser Arg Arg His Gly Asn Thr Arg Asp Lys Asn Ser Phe Glu Ser Ile
                  595 600 605
          Tyr Arg Ser Trp Ser Ser Ala Val Leu Leu Val Leu Val Leu Val Leu
              610 615 620
          Val Leu Val Leu Val Leu Val Leu Val Leu Val Leu Val Leu Leu Pro
          625 630 635 640
          Leu Cys Ile Val Ser Phe Leu Ser His Arg Asp Cys Ser Phe Ala Asn
                          645 650 655
          Ser Tyr His Arg His Ile Leu Asp His Pro Ile Ser Glu His Leu Arg
                      660 665 670
          Pro Cys Tyr Phe Ala Gly Gly Phe Ile Gly Gly Arg Thr Asp Glu Phe
                  675 680 685
          Leu Gln Met Ser Ala Ala Ile Ser Glu Ala Ile Asp Arg Asp Asp Glu
              690 695 700
          Asn Asp Val Ile Asn Glu Ser Leu Gln Pro Arg Leu Lys Leu Ala Met
          705 710 715 720
          Gln Val Val Ala Leu Trp His Asp Glu Ser His Leu Asn Arg Tyr Leu
                          725 730 735
          Ser His His Pro Gln Leu Val Arg Ile Leu Ser Pro Ser Tyr Leu Tyr
                      740 745 750
          Pro Asp Gly Trp Asp Ile Pro Phe Pro Arg Arg Ile Ala Val Gln Lys
                  755 760 765
          Ile Pro His Glu Ala Thr Arg Phe Ser Arg Glu Arg Phe Val Ser Val
              770 775 780
          Cys Ile Gln His Ala Asn Gly Val Cys Asn Met Gly Glu Ser Leu Gly
          785 790 795 800
          Arg Met Met Thr Ala Val Ala Ser Gly Val Ala Leu Ala Ala Ala Leu
                          805 810 815
          Asn Asp Asp Gln Asp Glu Asp Ala Gly Arg Leu Gln Val Leu Leu Pro
                      820 825 830
          Leu Asn Trp Cys Tyr Gly Asp Ser Lys Val Ile Cys Gly Gly Asn Arg
                  835 840 845
          Ser Lys Glu Arg Leu Ser Tyr Arg His Ser Trp Leu Ser Asn Phe Arg
              850 855 860
          Arg Ser Asp Ser Ile His Glu Ile Pro Phe His Leu Leu His Thr Ser
          865 870 875 880
          Ser Ile Gly Ala Val Gly Gln Glu Gly Ser Phe Ala Ile Pro Glu Pro
                          885 890 895
          Ser Met Arg Ala Val Ser Ser Gly Ile Pro Gly Gly Val Phe Leu Met
                      900 905 910
          Glu Val Pro Leu Gln Leu Ser His Leu Arg Ser Ser His Arg Val
                  915 920 925
           <![CDATA[ <210> 72]]>
           <![CDATA[ <211> 382]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Helicobacter sp. 11S02629-2]]>
           <![CDATA[ <400> 72]]>
          Met Gln Asp Ile Glu Leu Glu Ser Lys Lys Asp Thr Phe Glu Ser Pro
          1 5 10 15
          Pro Pro Pro Ile Asn Asn Leu Ser Gln Lys Asp Ile Asp Lys Met Lys
                      20 25 30
          Ile Ala Ile Leu Tyr Ile Ala Thr Gly Arg Tyr Asp Val Phe Phe Glu
                  35 40 45
          Asp Phe Tyr Lys Ser Met Glu Lys Phe Phe Ile Lys Asp Ala Ser Lys
              50 55 60
          His Tyr Phe Val Trp Thr Asp Ser Lys Lys Ile Glu Thr Asn Gly Asn
          65 70 75 80
          Ile Thr Lys Ile Tyr Gln Glu Lys Leu Gly Trp Pro Tyr Asp Thr Leu
                          85 90 95
          Leu Arg Tyr Asp Met Phe Trp Lys Ile Lys Asp Glu Leu Ser Asn Phe
                      100 105 110
          Asp Tyr Ile Phe Phe Phe Asn Ala Asn Met Val Val Lys Gln Glu Ile
                  115 120 125
          Phe Lys Asp Glu Phe Leu Pro Asp Thr Lys Ser Gly Leu Val Gly Cys
              130 135 140
          Leu His Pro Gly Phe Ile Lys Ile Gly Leu Asp Leu Lys Ile Tyr Pro
          145 150 155 160
          Ser Arg Asn Ala Lys Lys Phe Thr Tyr Asp Lys Asn Pro Lys Ser Leu
                          165 170 175
          Ala Phe Ile Glu Glu Gly Arg Gly Ser Ala Tyr Tyr Ala Gly Gly Leu
                      180 185 190
          Asn Gly Gly Ser Lys Asp Ala Tyr Leu Lys Leu Ile Lys Thr Leu Lys
                  195 200 205
          Asp Asn Ile Gln Thr Asp Met Asp Asn Gly Val Thr Ala Leu Trp His
              210 215 220
          Asp Glu Ser His Ile Asn Lys Tyr Phe Leu Asp Lys Glu Ile Lys Ala
          225 230 235 240
          Leu Ser Ser Met Phe Leu Lys Pro Glu Gly Trp Tyr Phe Asn Ile Asp
                          245 250 255
          Lys Ala Phe Val Met Asp Glu Glu Cys Met Gln Asp Gly Leu Lys Arg
                      260 265 270
          Lys Glu Phe Thr Glu Asn Leu Cys Ser Lys Ala Thr Leu Ile Pro Arg
                  275 280 285
          Asp Leu Leu Ile Lys Leu Leu Glu Lys Gln Tyr Gly Phe Lys Asn Tyr
              290 295 300
          Glu Ser Arg Tyr His Phe Met Lys Asp Ala Ile Asn Asp Tyr Phe Asp
          305 310 315 320
          Leu Glu Lys His Thr Lys Ile Leu Leu Leu Asp Lys Ala Asn Pro Lys
                          325 330 335
          Tyr Gly Gly His Ala Tyr Leu Arg Gly Glu Lys Arg Phe Lys Ser Ile
                      340 345 350
          Ser Ile Glu Tyr Arg Phe Lys Ser Ile Lys Thr Leu Lys Lys Ile Ala
                  355 360 365
          Ser Ser Ile Lys Ala Lys Leu Lys Arg Phe Lys Ser Leu Arg
              370 375 380
           <![CDATA[ <210> 73]]>
           <![CDATA[ <211> 389]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Helicobacter sp. 13S00401-1]]>
           <![CDATA[ <400> 73]]>
          Met Ile His Ala Ser Lys Lys Tyr Lys Ser Tyr Thr Lys Leu His Ser
          1 5 10 15
          Tyr Pro Pro Pro Pro Ser Asn Ile Gln Thr Leu Pro Ser Glu Asp Lys
                      20 25 30
          Ser Lys Met Lys Ile Ala Ile Leu Tyr Ile Ser Thr Gly Arg Tyr Asp
                  35 40 45
          Ile Phe Phe Lys Lys Phe His Lys Thr Met Gln Lys Phe Phe Ile Lys
              50 55 60
          Gly Ala Gln Lys His Tyr Phe Val Trp Thr Asp Ser Lys Lys Ile Lys
          65 70 75 80
          Asn Thr Lys Asp Ile Thr Lys Ile Tyr Gln Glu Lys Leu Gly Trp Pro
                          85 90 95
          Tyr Asp Thr Leu Met Arg Tyr His Met Phe Tyr Glu Ile Arg Asp Arg
                      100 105 110
          Leu Lys Glu Phe Asp Tyr Ile Tyr Phe Phe Asn Ala Asn Ile Val Ile
                  115 120 125
          Lys Gln Glu Ile Thr Lys Asp Glu Phe Leu Pro Asn Thr Lys Ser Gly
              130 135 140
          Leu Val Gly Cys Leu His Pro Gly Phe Ile Asp Leu Asp Leu Glu Phe
          145 150 155 160
          Asn Ile Val Pro Lys Lys Asp Ala Ala Lys Phe Thr Tyr Asp Arg Asn
                          165 170 175
          Glu Lys Ser Leu Ala Tyr Ile Lys Glu Gly Asp Gly Leu Ala Tyr Tyr
                      180 185 190
          Ala Gly Gly Leu Asn Gly Gly Ala Lys Asp Ala Tyr Leu Lys Leu Ile
                  195 200 205
          Lys Asp Leu Arg Asp Asn Ile Gln Gln Asp Leu Asp Lys Gly Ile Val
              210 215 220
          Ala Leu Trp His Asp Glu Ser His Ile Asn Lys Tyr Phe Leu Asp Lys
          225 230 235 240
          Glu Ile Lys Ala Leu Pro Ser Thr Phe Leu Val Pro Glu Gly Trp Glu
                          245 250 255
          Phe Ser Ile Ser Asp Lys Phe Ile Met Asp Glu Glu Cys Met Lys Asp
                      260 265 270
          Glu Leu Lys Lys Lys Glu Phe Thr Lys Asn Leu Leu Ser Arg Leu Asn
                  275 280 285
          Leu Ile Pro Ser Leu Glu Leu Glu Lys Leu Leu Ala Lys Gln Ser Glu
              290 295 300
          Leu Lys Ser Tyr Glu Asp Arg Arg Cys Phe Met Gln Thr Ala Ile Asn
          305 310 315 320
          Gly Tyr Phe Asp Leu Lys Lys His Thr Lys Ile Leu Leu Leu Leu Glu Lys
                          325 330 335
          Ser Asn Pro Lys Tyr Gly Gly His Asp Tyr Leu Arg Gly Glu Lys Gln
                      340 345 350
          His Lys Asn Val Ser Leu Arg Tyr Tyr Ile Ser Arg Val Asn Val Ala
                  355 360 365
          Arg Lys Leu Ala Ser Leu Ile Lys Arg Arg Leu Lys Lys Tyr Ile Lys
              370 375 380
          Arg Asn Asp Lys Ile
          385
           <![CDATA[ <210> 74]]>
           <![CDATA[ <211> 275]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Lachnospiraceae bacterium]]>
           <![CDATA[ <400> 74]]>
          Met Val Ser Arg Met Asp Ser Asn Leu Lys Ile Ala Ile Leu Tyr Ile
          1 5 10 15
          Cys Thr Gly Glu Tyr Asn Val Phe Trp Lys Asp Phe Tyr Ile Ser Phe
                      20 25 30
          Glu Lys Phe Phe Leu Thr Ser Tyr Glu Lys His Tyr Phe Val Phe Thr
                  35 40 45
          Asp Ala Lys Lys Ile Tyr Asn Glu Asp Cys Tyr Lys Arg Ile His Lys
              50 55 60
          Ile Tyr Gln Lys Asn Leu Gly Trp Pro Glu Asn Thr Leu Phe Arg Tyr
          65 70 75 80
          Glu Met Phe Phe Ser Ile Arg Glu Tyr Leu Lys Glu Phe Asp Tyr Thr
                          85 90 95
          Phe Phe Phe Asn Ala Asn Val Ile Cys Lys Asp Val Ile Val Gly Glu
                      100 105 110
          Glu Phe Leu Pro Leu Lys Glu Gly Leu Leu Val Val Gln His Pro Gly
                  115 120 125
          Phe Phe Asp Val Pro Asn Tyr Arg Phe Pro Tyr Asp Arg Asn Lys Lys
              130 135 140
          Ser Ser Ala Tyr Ile Pro Tyr Gly Lys Gly Gln Val Tyr Val Cys Gly
          145 150 155 160
          Gly Ile Asn Gly Gly Lys Thr Asn Val Phe Leu Asp Leu Ile Lys Glu
                          165 170 175
          Leu Lys Asn Arg Ile Glu Leu Asp Tyr Lys Lys Gly Ile Ile Ala Leu
                      180 185 190
          Trp His Asp Glu Ser Gln Ile Asn Lys Tyr Ile Leu Glu His Ser Ala
                  195 200 205
          Tyr Lys Leu Leu Ser Pro Ser Tyr Cys Tyr Pro Glu Gly Trp Asn Ile
              210 215 220
          Pro Phe Ile Pro Lys Leu Val Val Leu Asp Lys Asn Lys Phe Ile Asp
          225 230 235 240
          Val Ser Asn Ile Lys Lys Thr Asn Glu Arg Asn Glu Phe Ile Ile Lys
                          245 250 255
          Ile Lys Arg Tyr Leu Ile Cys Lys Phe Tyr Asp Leu Tyr Tyr Trp Phe
                      260 265 270
          Arg Arg Gly
                  275
           <![CDATA[ <210> 75]]>
           <![CDATA[ <211> 258]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Marinomonas polaris]]>
           <![CDATA[ <400> 75]]>
          Met Ser Lys Ile Gly Val Leu Tyr Ile Cys Thr Gly Lys Tyr Ala Ala
          1 5 10 15
          Phe Trp Asp Gly Phe Tyr Ala Ser Ala Lys Glu Asn Leu Cys Ile Asp
                      20 25 30
          Ser Gln Leu Ile Phe Tyr Val Phe Thr Asp Cys Glu Ala Leu Leu Asn
                  35 40 45
          Leu Gln Leu Asp Asp Val Arg Phe Ile Tyr Lys Lys Ser Glu Ser Trp
              50 55 60
          Pro Met Pro Thr Leu Met Arg Phe Ser Thr Phe Leu Ser Gln Glu Lys
          65 70 75 80
          Lys Tyr Leu Glu Val Asp Tyr Leu Leu Phe Cys Asn Ala Asn Leu Ile
                          85 90 95
          Ile Glu Gln Pro Ile Ala Thr Ala Glu Ile Phe Phe Asp Lys Pro Tyr
                      100 105 110
          Phe Ala Thr Ile His Pro Gly His Val Gly Lys Asp Pro Gln Lys Phe
                  115 120 125
          Pro Tyr Glu Lys Asn Ser Asn Ser Leu Ala Tyr Ile Asn Asn Ala Ala
              130 135 140
          Pro Tyr Tyr Val Cys Gly Gly Phe Asn Gly Gly Arg Arg Glu Asp Phe
          145 150 155 160
          Val Lys Met Cys Glu Leu Leu Ser Arg Asn Ile Asp Lys Asp Leu Glu
                          165 170 175
          Asn Asn Ile Ile Ala Val Trp His Asp Glu Thr His Phe Asn Lys Phe
                      180 185 190
          Tyr Ser Glu Arg Leu Asn Leu Phe Asn Val Leu Pro Ala Lys Tyr Cys
                  195 200 205
          Gln Pro Gln Gly Trp Pro Ala Lys Asp Asp Pro Ile Ile Thr Val Leu
              210 215 220
          Asn Lys Glu Phe Val Ile Gly Val Ser Asn Lys Gly Ala Phe Tyr Ser
          225 230 235 240
          Ile Arg Tyr Tyr Leu Ser Lys Leu Tyr Arg Arg Ile Arg Ser Ile Leu
                          245 250 255
          Ile Ser
           <![CDATA[ <210> 76]]>
           <![CDATA[ <211> 266]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Marmoricola scoriae]]>
           <![CDATA[ <400> 76]]>
          Met Ser Ala Ala Thr Thr Ala Gly Pro Arg Val Ser Leu Ile Val Ile
          1 5 10 15
          Ala Thr Gly Arg Tyr Leu Ser Phe Leu Glu Pro Leu Leu Val Ser Ala
                      20 25 30
          Arg Arg His Val Val Gly Leu Asp Arg Val Phe Val Leu Ser Asp Leu
                  35 40 45
          Arg Pro Pro Asp Asp Pro Thr Val Gln Trp Leu Pro Trp Gly His Leu
              50 55 60
          Pro Trp Pro Tyr Pro Thr Leu Leu Arg Tyr Arg Ala Ile Ser Ala Tyr
          65 70 75 80
          Arg Arg Val Leu Glu Gln Thr Asp Val Leu Leu Tyr Val Asp Val Asp
                          85 90 95
          Met Leu Phe Val Gly Thr Phe Asp Val Ser Ala Thr Ala Gly Leu Val
                      100 105 110
          Ala Val Arg His Pro Gly Phe Ala Glu Ser Ser Arg Ala Gln Leu Pro
                  115 120 125
          Tyr Glu Thr Asp Val Arg Ser Arg Ala Phe Val Pro Pro Glu Leu Gly
              130 135 140
          Thr Val Tyr Val Ala Gly Gly Val Gln Gly Gly Arg Ala Gly Asp Tyr
          145 150 155 160
          Leu Asp Ala Cys Glu Leu Met Ala Glu Glu Val Gln Leu Asp Leu Asp
                          165 170 175
          Gly Gly Ile Val Pro Thr Trp His Asp Glu Ser Val Trp Asn Ala Phe
                      180 185 190
          Cys Ala Arg Arg Pro Pro Asp Thr Leu Leu Ser Val His His Cys Thr
                  195 200 205
          Pro Glu Lys Glu Val Gly Pro Glu Thr Leu Leu Val Ala Leu Asp Lys
              210 215 220
          Asp His Asp His Phe Arg Glu Val Pro His Leu Glu Arg Ala Arg Arg
          225 230 235 240
          Arg Leu Leu Gln Gln Leu Gln Arg Val Arg Ala Ala Val Val Arg Ala
                          245 250 255
          Val Arg Pro Ala Val Arg Val Val Arg Arg
                      260 265
           <![CDATA[ <210> 77]]>
           <![CDATA[ <211> 250]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Muribaculaceae bacterium]]>
           <![CDATA[ <400> 77]]>
          Met Lys Ile Gly Met Leu Tyr Ile Gly Ile Gly Arg Tyr Ala Ala Phe
          1 5 10 15
          Trp Pro Glu Phe Tyr Arg Ser Ala Arg Glu Tyr Phe Leu Pro Asp Ala
                      20 25 30
          Thr Lys His Phe Phe Val Phe Ala Asp Ala Pro Leu Glu Asp Ala Gly
                  35 40 45
          Asp Asp Val Ser Val Phe His Asn Asp Asp Met Gly Trp Pro Leu Asn
              50 55 60
          Ser Leu Trp Arg Tyr His Met Phe Leu Arg Ile Ala Asp Arg Leu Lys
          65 70 75 80
          Glu Tyr Asp Tyr Leu Phe Phe Phe Asn Ala Asn Cys Lys Phe Val Arg
                          85 90 95
          Arg Val Glu Pro Ser Asp Ile Leu Pro Gln Gly Asp Val Glu Tyr Cys
                      100 105 110
          Ala Met Cys Thr Gln Thr Asp Pro Ala Lys Met Ser Leu Glu Ser Arg
                  115 120 125
          Pro Glu Cys Ala Ser Tyr Val Ala Pro Gly Ser Val Ser Arg Tyr Trp
              130 135 140
          Ala Gly Gly Ile Asn Gly Gly Arg Ala Glu Ala Phe Leu Arg Leu Ala
          145 150 155 160
          Arg Glu Cys Ala Ala Ile Ala Glu Arg Asp Leu Ala Asn Gly Phe Met
                          165 170 175
          Pro Val Trp His Asp Glu Ser Val Val Asn His Phe Phe Ala Asp Lys
                      180 185 190
          Lys Val Arg Ala Leu Asp Arg Arg Met Gly Cys Pro Ser Gln Trp Lys
                  195 200 205
          Ser Pro Ala Asp Pro Phe Val Ile Leu Arg Arg Lys Asp Asp Val Leu
              210 215 220
          Gly Arg Ser Trp Leu Arg Thr Tyr Lys Gly Arg Lys His Ser Ser Phe
          225 230 235 240
          Trp Lys Lys Leu Phe Arg Lys Leu Arg Lys
                          245 250
           <![CDATA[ <210> 78]]>
           <![CDATA[ <211> 258]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Neisseriaceae bacterium]]>
           <![CDATA[ <400> 78]]>
          Met Lys Ile Ala Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe
          1 5 10 15
          Trp Ser Asp Phe Tyr Ser Thr Ser Gln Lys Tyr Phe Cys Thr Thr Glu
                      20 25 30
          Asp Lys His Tyr Phe Val Phe Thr Asp Ser Glu Gln Ile Lys Ala Asp
                  35 40 45
          His Asn Val Ser Val Ile Tyr Gln Asp Ser Leu Gly Trp Pro Phe Asn
              50 55 60
          Thr Leu Tyr Arg Tyr Arg Met Phe Leu Arg Val Gln His Lys Leu Ser
          65 70 75 80
          Lys Phe Asp Lys Val Ile Phe Phe Asn Gly Asn Cys Thr Phe Val Asp
                          85 90 95
          Gln Ile Asp Tyr Glu Asn Phe Phe Gly Arg Ser Ser Thr Leu Val Ala
                      100 105 110
          Cys Leu His Pro Gly Phe Leu Asn Lys Asn Cys Glu Glu Phe Thr Tyr
                  115 120 125
          Glu Lys Arg Lys Asn Ser Leu Ala Phe Val Gly Ser Pro Trp Lys Tyr
              130 135 140
          Phe Ala Gly Gly Ile Asn Gly Gly Asn Ala Asn Glu Ile Leu Lys Ile
          145 150 155 160
          Phe Gln Ile Leu Ser His Asn Ile Glu Asp Asp Leu Lys Asn Gly Ile
                          165 170 175
          Val Ala Ile Trp His Asp Glu Ser His Trp Asn Ala Tyr Leu Asn Asn
                      180 185 190
          Asn Tyr Glu Val Leu Lys Asp Lys Leu His Ile Leu Ser Pro Glu Tyr
                  195 200 205
          Leu Tyr Pro Glu Gly Trp Asp Leu Pro Phe Glu Lys Lys Lys Ile Ile Leu
              210 215 220
          Arg Asp Lys Asn Gln Tyr Gly Gly His Asn Leu Leu Arg Gly Ala Ala
          225 230 235 240
          Gln His Asn Phe Pro Asn Thr Ile Lys Lys Ile Leu Lys Lys Ile Ile
                          245 250 255
          Cys Arg
           <![CDATA[ <210> 79]]>
           <![CDATA[ <211> 265]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Nocardioides sp. PD653]]>
           <![CDATA[ <400> 79]]>
          Met Ser Ser Glu Thr Thr Arg Val Gly Leu Ile Val Ile Ala Thr Gly
          1 5 10 15
          Arg Tyr Val Glu Phe Val Asp Gln Leu Leu Ala Ser Ala His Glu His
                      20 25 30
          Val Ala Gly Leu His Arg Leu Tyr Val Leu Ser Asp Arg Arg Pro Pro
                  35 40 45
          Asp Asp Pro Arg Ile Val Trp Leu Pro Trp Gly His Ile Gly Trp Pro
              50 55 60
          Tyr Pro Thr Leu Leu Arg Tyr Arg Ala Ile Ala Ala His Gln Asp Ile
          65 70 75 80
          Leu Arg Glu Cys Asp Ile Leu Val Tyr Ser Asp Val Asp Met Arg Phe
                          85 90 95
          Val Ala Ser Phe Asp Met Thr Gln Ile Arg Gly Ile Phe Ala Val Ser
                      100 105 110
          His Pro Gly Tyr Val Gly Ala Thr Pro Asp Ser Leu Pro Tyr Glu Arg
                  115 120 125
          Asn Pro Ala Ser Gln Ala Tyr Val Pro Val Gly Ser Gly Leu Glu Tyr
              130 135 140
          Phe Ala Gly Gly Val Gln Gly Gly Arg Ala Glu Ile Tyr Leu Asp Ala
          145 150 155 160
          Cys Glu Gln Met Ala Ala Arg Val Gln Glu Asp Leu Asn Ala Gly Ile
                          165 170 175
          Val Pro Val Trp His Asp Glu Ser Ile Trp Asn Gly Trp Leu Ile Asp
                      180 185 190
          His Pro Pro Asp Leu Val Leu Gly Ser Glu Tyr Cys Thr Pro Glu Thr
                  195 200 205
          Ala Ala Gly Pro Gln Ser Val Leu Leu Ala Leu Asp Lys Asp His Ala
              210 215 220
          Arg Leu Arg Gly Thr Pro Trp Gln Val Arg Ser Val Glu Arg Leu Val
          225 230 235 240
          Arg Ala Arg Arg Ala Leu Arg Arg Arg Ser Arg Ala Ala Ala Arg Val
                          245 250 255
          Ala Ala Arg Ala Trp Gly Arg Arg Arg
                      260 265
           <![CDATA[ <210> 80]]>
           <![CDATA[ <211> 274]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Parabacteroides goldsteinii]]>
           <![CDATA[ <400> 80]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Arg Tyr Ser Ile Phe
          1 5 10 15
          Trp Lys Lys Phe Tyr Gln Ser Thr Glu Lys Ser Phe Met Gln Gly Leu
                      20 25 30
          Pro Cys Ile Arg Glu Tyr Tyr Val Phe Thr Asp Asn Pro Cys Leu Tyr
                  35 40 45
          Gly Glu Lys Lys Asn Lys Arg Ile His Arg Ile Tyr Gln Glu Asn Leu
              50 55 60
          Gly Trp Pro Asp Asn Thr Leu Met Arg Phe Ser Met Phe Leu Lys Ile
          65 70 75 80
          Lys Glu Arg Leu Glu Lys Glu Thr Asp Tyr Leu Tyr Phe Phe Asn Ala
                          85 90 95
          Asn Met Val Ile Arg Glu Lys Ile Gly Lys Glu Phe Leu Pro Glu Glu
                      100 105 110
          Ser Ser Asn Gly Leu Val Gly Leu Ile His Pro Gly Gly Tyr Asp Arg
                  115 120 125
          Glu Val Asn Glu Phe Thr Tyr Asp Arg Asn Glu Lys Ser Thr Ala Tyr
              130 135 140
          Ile Pro Tyr Gly Glu Gly Arg Tyr Tyr Tyr Ala Gly Gly Leu Asn Gly
          145 150 155 160
          Gly Arg Thr Pro Ala Phe Leu Lys Met Ser Glu Thr Leu Arg Asp Asn
                          165 170 175
          Thr Glu Glu Asp Lys Arg Asn Gly Val Met Ala Leu Trp His Asp Glu
                      180 185 190
          Ser His Ile Asn Arg Tyr Phe Leu Asp His Pro Pro Tyr Ser Leu Thr
                  195 200 205
          Pro Ala Tyr Cys Tyr Pro Glu Gly Trp Asn Met Pro Phe Pro Gln Ile
              210 215 220
          Ile Leu Leu Leu Asp Lys Ser Phe Ile Cys Gly Gly His Lys Tyr Leu
          225 230 235 240
          Arg Gly Gly Lys Arg Asn Phe His Asp Tyr Thr Ser Tyr Leu Lys Arg
                          245 250 255
          Ser Leu Val Arg Phe Ala Arg Lys Val Ile Gly Val Leu Arg Gly Phe
                      260 265 270
          Gly Leu
           <![CDATA[ <210> 81]]>
           <![CDATA[ <211> 274]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Parabacteroides goldsteinii]]>
           <![CDATA[ <400> 81]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Arg Tyr Ser Ile Phe
          1 5 10 15
          Trp Lys Lys Phe Tyr Gln Ser Thr Glu Lys Ser Phe Met Gln Gly Ser
                      20 25 30
          Pro Cys Ile Arg Glu Tyr Tyr Val Phe Thr Asp Asn Pro Cys Leu Tyr
                  35 40 45
          Gly Glu Lys Lys Asn Lys Arg Ile His Arg Ile Tyr Gln Glu Asn Leu
              50 55 60
          Gly Trp Pro Asp Asn Thr Leu Met Arg Phe Ser Met Phe Leu Lys Ile
          65 70 75 80
          Lys Glu Arg Leu Glu Lys Glu Thr Asp Tyr Leu Tyr Phe Phe Asn Ala
                          85 90 95
          Asn Met Val Ile Arg Glu Lys Ile Gly Lys Glu Phe Leu Pro Glu Glu
                      100 105 110
          Ser Ser Asn Gly Leu Val Gly Leu Ile His Ser Gly Gly Tyr Asp Arg
                  115 120 125
          Glu Val Asn Glu Phe Thr Tyr Asp Arg Asn Glu Lys Ser Thr Ala Tyr
              130 135 140
          Ile Pro Tyr Gly Glu Gly Arg Tyr Tyr Tyr Ala Gly Gly Leu Asn Gly
          145 150 155 160
          Gly Arg Thr Pro Ala Phe Leu Lys Met Ala Glu Thr Leu Arg Asp Asn
                          165 170 175
          Thr Glu Glu Asp Lys Arg Asn Gly Val Met Ala Leu Trp His Asp Glu
                      180 185 190
          Ser His Ile Asn Arg Tyr Phe Leu Asp His Pro Pro Tyr Ser Leu Thr
                  195 200 205
          Pro Ala Tyr Cys Tyr Pro Glu Gly Trp Asn Met Pro Phe Pro Gln Ile
              210 215 220
          Ile Leu Leu Leu Asp Lys Ser Phe Ile Cys Gly Gly His Lys Tyr Leu
          225 230 235 240
          Arg Gly Gly Lys Arg Asn Phe His Asp Tyr Thr Ser Tyr Leu Lys Arg
                          245 250 255
          Ser Leu Val Arg Phe Ala Arg Lys Val Ile Gly Val Leu Arg Gly Phe
                      260 265 270
          Gly Leu
           <![CDATA[ <210> 82]]>
           <![CDATA[ <211> 274]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Parabacteroides goldsteinii]]>
           <![CDATA[ <400> 82]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Arg Tyr Ser Ile Phe
          1 5 10 15
          Trp Lys Lys Phe Tyr Gln Ser Thr Glu Lys Ser Phe Met Gln Gly Leu
                      20 25 30
          Pro Cys Ile Arg Glu Tyr Tyr Val Phe Thr Asp Asn Pro Cys Leu Tyr
                  35 40 45
          Gly Glu Lys Lys Asn Lys Arg Ile His Arg Ile Tyr Gln Glu Asn Leu
              50 55 60
          Gly Trp Pro Asp Asn Thr Leu Met Arg Phe Ser Met Phe Leu Lys Ile
          65 70 75 80
          Lys Glu Arg Leu Glu Lys Glu Thr Asp Tyr Leu Tyr Phe Phe Asn Ala
                          85 90 95
          Asn Met Val Ile Arg Glu Lys Ile Gly Lys Glu Phe Leu Pro Glu Glu
                      100 105 110
          Ser Ser Asn Gly Leu Val Gly Leu Ile His Ser Gly Gly Tyr Asp Arg
                  115 120 125
          Glu Val Asn Glu Phe Thr Tyr Asp Arg Asn Glu Lys Ser Thr Ala Tyr
              130 135 140
          Ile Pro Tyr Gly Glu Gly Arg Tyr Tyr Tyr Ala Gly Gly Leu Asn Gly
          145 150 155 160
          Gly Arg Thr Pro Ala Phe Leu Lys Met Ala Glu Thr Leu Arg Asp Asn
                          165 170 175
          Thr Glu Glu Asp Lys Arg Asn Gly Val Met Ala Leu Trp His Asp Glu
                      180 185 190
          Ser His Ile Asn Arg Tyr Phe Leu Asp His Pro Pro Tyr Ser Leu Thr
                  195 200 205
          Pro Ala Tyr Cys Tyr Pro Glu Gly Trp Asn Met Pro Phe Pro Gln Ile
              210 215 220
          Ile Leu Leu Leu Asp Lys Ser Phe Ile Cys Gly Gly His Lys Tyr Leu
          225 230 235 240
          Arg Gly Gly Lys Arg Asn Phe His Asp Tyr Thr Ser Tyr Leu Lys Arg
                          245 250 255
          Ser Leu Val Arg Phe Ala Arg Lys Val Ile Gly Val Leu Arg Gly Phe
                      260 265 270
          Gly Leu
           <![CDATA[ <210> 83]]>
           <![CDATA[ <211> 272]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Parabacteroides gordonii MS-1]]>
           <![CDATA[ <400> 83]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Ser Ile Phe
          1 5 10 15
          Trp Lys Lys Phe Tyr Lys Ser Ala Glu Arg Tyr Leu Met Gln Gly Tyr
                      20 25 30
          Pro Cys Ile Arg Glu Tyr Tyr Val Phe Thr Asp Ala Pro Ser Val Tyr
                  35 40 45
          Gly Glu Lys Glu Asn Gly His Ile His Arg Ile Tyr Gln Glu Asn Leu
              50 55 60
          Gly Trp Pro Arg Asn Thr Leu Met Arg Phe His Met Phe Leu Arg Ile
          65 70 75 80
          Lys Lys Gln Leu Glu Arg Glu Thr Asp Tyr Leu Tyr Phe Phe Asn Ala
                          85 90 95
          Asn Met Gln Phe Arg Val Pro Val Gly Lys Glu Phe Leu Pro Asp Asp
                      100 105 110
          Phe Ser Asn Gly Leu Val Gly Cys Met Phe Pro Trp Ser Tyr Asn Glu
                  115 120 125
          Thr Asn Leu Glu Phe Gly Tyr Asp Arg Asn Pro Met Ser Thr Ala Tyr
              130 135 140
          Ile Pro Glu Gly Glu Gly Asp Phe Tyr Tyr Ala Gly Ala Leu Ile Gly
          145 150 155 160
          Gly Lys Thr Glu Ala Phe Leu Lys Met Ser Glu Thr Ile Leu Asn Asn
                          165 170 175
          Ile Gln Glu Asp Glu Lys Lys Gly Val Ile Ala Leu Trp His Asp Glu
                      180 185 190
          Ser His Leu Asn Arg Tyr Phe Met Asp Asn Pro Pro Lys Cys Leu Thr
                  195 200 205
          Pro Ala Tyr Cys Tyr Pro Glu Arg Trp Lys Ser Pro Phe Pro Glu Ile
              210 215 220
          Ile Arg Leu Phe Asp Lys Asn Gly Ser Trp Gly Gly Tyr Ala Tyr Leu
          225 230 235 240
          Arg Gly Glu Lys Ala Gly Val Lys Asp Tyr Leu Arg Ser Tyr Lys Val
                          245 250 255
          Lys Ile Lys Tyr Met Ile Met Pro Phe Tyr Arg Phe Val Cys Arg Lys
                      260 265 270
           <![CDATA[ <210> 84]]>
           <![CDATA[ <211> 256]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Parachlamydia acanthamoebae]]>
           <![CDATA[ <400> 84]]>
          Met Val Thr Arg Gly Phe Cys Met Leu Thr Arg Ser Leu Lys Ile Leu
          1 5 10 15
          Ile Gly Leu Cys Leu Leu Phe Ser His Ala Leu Tyr Ala Ala Asn Val
                      20 25 30
          Gly Leu Leu Val Met Ala Thr Gly Lys Tyr Val Ser Phe Val Pro Pro
                  35 40 45
          Leu Val Lys Ser Ala Asp His Phe Phe Cys Lys Asn His Lys Val Thr
              50 55 60
          Tyr Phe Val Phe Thr Asp Gly Tyr Leu Glu Pro Met Pro Asn Val Val
          65 70 75 80
          Pro Ile Phe His Ala Lys Met Gly Trp Pro Tyr Asp Thr Met Met Arg
                          85 90 95
          Tyr His Val Tyr Asp Met His Arg Asp Ala Phe Ala Gly Gln Asp Tyr
                      100 105 110
          Leu Tyr Ala Cys Asp Ala Asp Met Leu Phe Val Gly Glu Val Gly Asp
                  115 120 125
          Glu Ile Leu Gly Asn Arg Val Ala Thr Arg His Pro Gly Phe Ile Asn
              130 135 140
          Arg Pro Lys Ser Ser Tyr Thr Tyr Glu Arg Asn Pro Leu Ser Thr Ala
          145 150 155 160
          Tyr Ile Pro Gln Gly Glu Gly Asn Asp Tyr Phe Ala Gly Gly Phe Tyr
                          165 170 175
          Gly Gly Thr Lys Asp Glu Phe Leu Asn Ile Val His Thr Asn Ala Val
                      180 185 190
          Asn Ile Asp Gln Asp Met Gln Asn Gly Ile Ile Ala Val Trp His Asp
                  195 200 205
          Glu Ser His Trp Asn Arg Phe Cys Ile Asn Asn Pro Pro Thr Val Ile
              210 215 220
          Leu Ser Pro Ser Tyr Cys Tyr Pro Gln Gly Leu Arg Ile Pro Phe Leu
          225 230 235 240
          Pro Lys Leu Ile Ala Leu Asp Lys Asn His Glu Glu Met Arg Lys Gly
                          245 250 255
           <![CDATA[ <210> 85]]>
           <![CDATA[ <211> 249]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Parachlamydia acanthamoebae]]>
           <![CDATA[ <400> 85]]>
          Met Leu Thr Arg Ser Leu Lys Ile Leu Ile Gly Leu Cys Leu Leu Phe
          1 5 10 15
          Ser His Ala Leu Tyr Ala Ala Asn Val Gly Leu Leu Val Met Ala Thr
                      20 25 30
          Gly Lys Tyr Val Ser Phe Val Pro Pro Leu Val Lys Ser Ala Asp His
                  35 40 45
          Phe Phe Cys Lys Asn His Lys Val Thr Tyr Phe Val Phe Thr Asp Gly
              50 55 60
          Tyr Leu Glu Pro Met Pro Asn Val Val Pro Ile Phe His Ala Lys Met
          65 70 75 80
          Gly Trp Pro Tyr Asp Thr Met Met Arg Tyr His Val Tyr Asp Met His
                          85 90 95
          Arg Asp Ala Phe Ala Gly Gln Asp Tyr Leu Tyr Ala Cys Asp Ala Asp
                      100 105 110
          Met Leu Phe Val Gly Glu Val Gly Asp Glu Ile Leu Gly Asn Arg Val
                  115 120 125
          Ala Thr Arg His Pro Gly Phe Ile Asn Arg Pro Lys Ser Ser Tyr Thr
              130 135 140
          Tyr Glu Arg Asn Pro Leu Ser Thr Ala Tyr Ile Pro Gln Gly Glu Gly
          145 150 155 160
          Asn Asp Tyr Phe Ala Gly Gly Phe Tyr Gly Gly Thr Lys Asp Glu Phe
                          165 170 175
          Leu Asn Ile Val His Thr Asn Ala Val Asn Ile Asp Gln Asp Met Gln
                      180 185 190
          Asn Gly Ile Ile Ala Val Trp His Asp Glu Ser His Trp Asn Arg Phe
                  195 200 205
          Cys Ile Asn Asn Pro Pro Thr Val Ile Leu Ser Pro Ser Tyr Cys Tyr
              210 215 220
          Pro Gln Gly Leu Arg Ile Pro Phe Leu Pro Lys Leu Ile Ala Leu Asp
          225 230 235 240
          Lys Asn His Glu Glu Met Arg Lys Gly
                          245
           <![CDATA[ <210> 86]]>
           <![CDATA[ <211> 251]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Parachlamydia sp.]]>
           <![CDATA[ <400> 86]]>
          Met Asn Ser Lys Cys Val Arg Ile Leu Ile Thr Leu Leu Leu Leu Ser
          1 5 10 15
          Ser Pro Ser Leu Tyr Ala Ala Lys Val Gly Leu Leu Val Met Ala Thr
                      20 25 30
          Gly Lys Tyr Ile Thr Phe Val Pro Pro Leu Val Ala Ser Ala Asp Lys
                  35 40 45
          Tyr Phe Cys Lys Asn His Asp Val Thr Tyr Phe Val Phe Thr Asp Gly
              50 55 60
          Gln Phe Asp Val Val Pro Asn Lys Val Val Pro Ile Phe His Pro Arg
          65 70 75 80
          Met Gly Trp Pro Phe Asp Thr Met Met Arg Asn His Val Tyr Glu Met
                          85 90 95
          Asn Ser Asp Ala Phe Ala Asp Gln Asp Tyr Leu Tyr Ala Cys Asp Ala
                      100 105 110
          Asp Met Leu Phe Val Gly Asn Val Gly Asp Glu Ile Leu Gly Lys Arg
                  115 120 125
          Met Ala Thr Glu His Pro Gly Phe Tyr Gly Lys Asn Arg Lys Val Phe
              130 135 140
          Ser Phe Glu Thr Asn Pro Leu Ser Lys Ala Tyr Ile Ala Pro Asn Glu
          145 150 155 160
          Gly Thr Lys Tyr Phe Cys Gly Gly Phe Phe Gly Gly Glu Arg Glu Ala
                          165 170 175
          Phe Leu Asp Ile Val Arg Thr Thr Ser Glu Arg Val Asp Glu Asp Leu
                      180 185 190
          Ala Asn Asp Ile Val Ala Val Trp His Asp Glu Ser His Trp Asn Arg
                  195 200 205
          Tyr Cys Ile Asp Tyr Pro Pro Thr Val Ile Leu Thr Pro Ser Tyr Cys
              210 215 220
          Phe Pro Gln Gly Ser Lys Leu Pro Phe Val Pro Lys Leu Ile Ala Leu
          225 230 235 240
          Asn Lys Asn His Gln Asp Met Arg Phe Asn Asp
                          245 250
           <![CDATA[ <210> 87]]>
           <![CDATA[ <211> 272]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Piromyces sp.]]>
           <![CDATA[ <400> 87]]>
          Met Lys Lys Asp Tyr Phe Val Phe Thr Asp Ser Glu Thr Ile Tyr Gly
          1 5 10 15
          Asp Glu Asn Pro Asn Val His Ile Ile Pro Gln Glu Asn Leu Gly Trp
                      20 25 30
          Pro Gly Asn Thr Leu Tyr Arg Phe His Met Phe Leu Ser Gln Lys Glu
                  35 40 45
          Glu Leu Glu Lys Phe Lys Tyr Ile Phe Phe Leu Asn Ala Asn Val Glu
              50 55 60
          Cys Tyr Glu Glu Ile Lys Glu Asn Asp Phe Leu Pro Lys Lys Glu Gly
          65 70 75 80
          Leu Leu Phe Val Lys His Phe Asn Phe His Asp Lys Gln Asn Thr Leu
                          85 90 95
          Phe Ser Tyr Glu Arg Asn Ser Asn Ser Thr Ala Tyr Ile Pro Met Gly
                      100 105 110
          Glu Gly Lys Tyr Tyr Val Cys Gly Gly Ala Asn Gly Gly Lys Ala Lys
                  115 120 125
          Asn Tyr Leu Asp Met Cys Glu Glu Leu Arg Arg Arg Ile Asp Ile Asp
              130 135 140
          Asp Glu Asn Gly Val Thr Ala Ile Trp His Asp Glu Ser Gln Ile Asn
          145 150 155 160
          Arg Tyr Leu Tyr Asp Leu Asp Lys Glu Asn Lys Pro Tyr Lys Ile Leu
                          165 170 175
          Asp Pro Gly Tyr Cys Phe Pro Glu Met Phe Leu Glu Asn Lys Leu Lys
                      180 185 190
          Asn Pro Asp Ser Phe Pro Tyr Asp Pro Ile Leu Leu Tyr Arg Arg Lys
                  195 200 205
          Gln Asp Tyr Ile Asn Val Asn Lys Ile Lys Gly Asp Tyr Asn Glu Met
              210 215 220
          Gln Gly Asn Asn Lys Asn Asn Asn Lys Lys Ile His Tyr Tyr Asn Ser
          225 230 235 240
          Lys Thr Asn Lys Ile Asn Lys Gly Asn Ser Thr Lys Glu Glu Ile Ser
                          245 250 255
          Lys Glu Glu Asn Lys Glu Ser Gln Lys Lys Val Ile Lys Asn Asn Tyr
                      260 265 270
           <![CDATA[ <210> 88]]>
           <![CDATA[ <211> 284]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Piromyces sp.]]>
           <![CDATA[ <400> 88]]>
          Lys Ser Val Asp Ser Ala Asp Ser Ile Glu Ile Ser Glu Leu Asn Lys
          1 5 10 15
          Asn Thr His Glu Lys Asp Ile Ala Ile Leu Tyr Ile Cys Thr Gly Lys
                      20 25 30
          Tyr Asp Val Phe Trp Lys Glu Phe Tyr Glu Ser Val Glu Glu Lys Phe
                  35 40 45
          Ile Pro His Met Lys Lys Asp Tyr Phe Val Phe Thr Asp Ser Lys Asp
              50 55 60
          Ile Tyr Lys Lys Glu Asn Asn Asn Val His Ile Ile Lys Gln Lys Asn
          65 70 75 80
          Leu Gly Trp Pro Gly Asn Thr Leu Tyr Arg Phe His Met Phe Leu Ser
                          85 90 95
          Gln Lys Glu Lys Leu Gln Asn Tyr Lys Tyr Ile Phe Phe Met Asn Ala
                      100 105 110
          Asn Ile Ile Cys Asn Phe Gly Val Gly Glu Glu Phe Leu Pro Lys Asp
                  115 120 125
          Glu Gly Leu Leu Phe Val Gln His His Ala Tyr Tyr Lys Ala Pro Asn
              130 135 140
          Thr Lys Phe Ser Tyr Glu Arg Asn Ser Asn Ser Thr Ala Tyr Ile Pro
          145 150 155 160
          Met Gly Gln Gly Lys Tyr Tyr Val Cys Gly Gly Val Asn Gly Gly Arg
                          165 170 175
          Ala Lys Glu Tyr Leu His Met Cys Glu Val Leu Lys Ser Arg Ile Asp
                      180 185 190
          Glu Asp Asp Lys Asn Asp Val Val Ala Val Trp His Asp Glu Ser His
                  195 200 205
          Ile Asn Lys Tyr Leu Leu Glu Leu Glu Lys Ser Gln Tyr Lys Leu Leu
              210 215 220
          Asn Val Ser Tyr Cys Phe Pro Glu Tyr Lys Met Asn Arg Lys Ser Phe
          225 230 235 240
          Pro Phe Asp Pro Ile Leu Phe Phe Arg Asn Lys Lys Lys Tyr Ile Asn
                          245 250 255
          Leu Lys Glu Ile Lys Gly Asp Ser His Glu Met Met Gly Asn Asn Lys
                      260 265 270
          Asn Lys Asn Lys Ala Lys Asn Lys Ala Ile Ile Asn
                  275 280
           <![CDATA[ <210> 89]]>
           <![CDATA[ <211> 250]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Porphyromonadaceae bacterium]]>
           <![CDATA[ <400> 89]]>
          Met Lys Ile Gly Met Leu Tyr Ile Gly Ile Gly Arg Tyr Ala Ala Phe
          1 5 10 15
          Trp Pro Glu Phe Tyr Arg Ser Ala Arg Glu Tyr Phe Leu Pro Asp Ala
                      20 25 30
          Thr Lys His Phe Phe Val Phe Ala Asp Ala Pro Leu Glu Asp Ala Gly
                  35 40 45
          Asp Asp Val Ser Val Phe His Asn Asp Asp Met Gly Trp Pro Leu Asn
              50 55 60
          Ser Leu Trp Arg Tyr His Met Phe Leu Arg Ile Ala Asp Leu Leu Lys
          65 70 75 80
          Glu Tyr Asp Tyr Leu Phe Phe Phe Asn Ala Asn Cys Lys Phe Val Arg
                          85 90 95
          Arg Val Glu Pro Ser Asp Ile Leu Pro Gln Gly Asp Val Glu Tyr Cys
                      100 105 110
          Ala Met Cys Thr Gln Thr Asp Pro Ala Lys Met Ser Leu Glu Ser Arg
                  115 120 125
          Pro Glu Cys Ala Ser Tyr Val Ala Pro Gly Ser Val Ser Arg Tyr Trp
              130 135 140
          Ala Gly Gly Ile Asn Gly Gly Arg Ala Glu Ala Phe Leu Arg Leu Ala
          145 150 155 160
          Arg Glu Cys Ala Ala Ile Ala Glu Arg Asp Leu Ala Asn Gly Phe Met
                          165 170 175
          Pro Val Trp His Asp Glu Ser Val Val Asn His Phe Phe Ala Asp Lys
                      180 185 190
          Lys Val Arg Ala Leu Asp Arg Arg Met Gly Cys Pro Ser Gln Trp Lys
                  195 200 205
          Ser Pro Ala Asp Pro Phe Val Ile Leu Arg Arg Lys Asp Asp Val Leu
              210 215 220
          Gly Arg Ser Trp Leu Arg Thr Tyr Lys Gly Arg Lys His Ser Ser Phe
          225 230 235 240
          Trp Lys Lys Leu Phe Arg Lys Leu Arg Lys
                          245 250
           <![CDATA[ <210> 90]]>
           <![CDATA[ <211> 231]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Prochlorococcus phage P-SSM2]]>
           <![CDATA[ <400> 90]]>
          Met Lys Ile Cys Ile Leu Thr Ile Ala Thr Asn Lys Tyr Ile Gln Phe
          1 5 10 15
          Val Glu Lys Leu Tyr Asp Asn Ile Asp Asp His Phe Leu Asn Gly His
                      20 25 30
          Glu Ile Glu Gly Ile Ile Phe Thr Asp Gln Glu Val Glu Ser Ser Asp
                  35 40 45
          Asn Ile Lys Ile Ser Gln Ile Glu His Glu Pro Trp Pro Val Pro Thr
              50 55 60
          Leu Lys Arg Tyr Asn Tyr Phe Met Lys Glu Ala Glu His Ile Ser Lys
          65 70 75 80
          Tyr Asp Tyr Cys Phe Tyr Phe Asp Val Asp Met Gly Ile Val Asp Lys
                          85 90 95
          Val Gly Asp Glu Val Leu Gly Asp Leu Val Ala Thr Met His Pro Tyr
                      100 105 110
          Gln Ser Phe Ala Pro Lys Ile Gln Arg Ser Tyr Asp Arg Asn Pro Lys
                  115 120 125
          Ser Leu Ala Tyr Val Pro Leu Tyr Asp Glu Gly Glu His Tyr Tyr Ala
              130 135 140
          Gly Gly Phe Asn Gly Gly Ser Thr Lys Arg Phe Leu Glu Met Ala Glu
          145 150 155 160
          Val Ile Ala Asp Arg Val Asn Lys Asp Leu Glu Asn Asp Val Ile Ala
                          165 170 175
          Leu Trp His Asp Glu Ser His Leu Asn Arg Tyr Leu Ile Asp Asn Pro
                      180 185 190
          Pro Thr Ile Ser Leu Thr Pro Ser Tyr Cys Phe Ala Glu Glu Gln Met
                  195 200 205
          Ser Asn Leu Glu Tyr Pro Tyr Lys Pro Lys Ile Ile Ala Leu Lys Lys
              210 215 220
          Asp His Asn Glu Leu Arg Ser
          225 230
           <![CDATA[ <210> 91]]>
           <![CDATA[ <211> 269]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> R. glucophaga]]>
           <![CDATA[ <400> 91]]>
          Met Asp Lys Asn Pro Arg Asn Phe Met Lys Glu Ser Asp Met Asn Lys
          1 5 10 15
          Val Ala Val Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Val Phe Trp Lys
                      20 25 30
          Asp Phe Tyr Ile Ser Tyr Glu Lys Tyr Phe Leu Pro Asp Cys Glu Lys
                  35 40 45
          His Tyr Tyr Val Phe Thr Asp Ala Ala Glu Ile Tyr Met Glu Lys Glu
              50 55 60
          Asn Leu Arg Ile His Lys Phe Tyr Gln Glu Ser Leu Gly Trp Pro Asp
          65 70 75 80
          Asn Thr Leu Met Arg Phe His Met Phe Leu Arg Gln Lys Ala Glu Leu
                          85 90 95
          Glu Lys Tyr Asp Tyr Ile Phe Phe Met Asn Ala Asn Cys Gln Ala Leu
                      100 105 110
          Asp Thr Ile Thr Glu Glu Glu Phe Leu Pro Lys Lys Lys Asp Ile Ile
                  115 120 125
          Val Val Gln His Pro Gly Tyr Tyr Asn Lys Thr Asn Lys Gln Phe Ala
              130 135 140
          Tyr Asp Arg Asn Pro Lys Ser Thr Ala Tyr Ile Pro Lys Gly Gln Gly
          145 150 155 160
          Lys Tyr Tyr Val Cys Gly Gly Val Asn Gly Gly Arg Ala Gln Ala Phe
                          165 170 175
          Ile Gln Leu Met Glu Glu Leu Lys His Asn Ile Asp Val Asp Lys Lys
                      180 185 190
          Asn Gly Glu Leu Ala Leu Trp His Asp Glu Ser His Ile Asn His Tyr
                  195 200 205
          Val Trp Thr His Asp Asn Tyr Glu Val Leu Pro Pro Ser Tyr Cys Trp
              210 215 220
          Pro Glu Asp Trp Asn Leu Pro Met Pro Gly Lys Ile Leu Ile Arg Glu
          225 230 235 240
          Lys Ser Lys Trp Ile Phe Val Asp Met Val Lys Ser Gln Ser Leu Ser
                          245 250 255
          Gly Lys Ile Lys Ala Val Ile Lys Lys Ile Ile Arg Arg
                      260 265
           <![CDATA[ <210> 92]]>
           <![CDATA[ <211> 1759]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Salpingoeca rosetta]]>
           <![CDATA[ <400> 92]]> Met Val Val Gly Leu Arg Cys Ser Cys Arg Arg Met Val Pro Arg Leu 1 5 10 15 Arg Arg Leu Gln Leu Leu Trp Gln Arg Arg Trp Arg Thr Ile Val Val 20 25 30 Ile Val Leu Cys Ile Thr Leu Leu Ala Ala Leu Arg Arg Asn Arg Ser 35 40 45 Ala Arg Tyr Asn Ala Ser Asp Val Pro Asp Leu Thr Lys Cys Arg Ala 50 55 60 Pro Ser Leu Glu Val Ala Pro Leu Ile Thr Ala Ser Leu Ile His Asp 65 70 75 80 Thr Ala Asp Ala Ala Ala Phe Leu Asp Leu His Arg Val Ala Glu Ala 85 90 95 Pro Phe Asn Asp Leu Ile Leu Ser Ala Ser Ser Ala Arg Ser Gly Asp 100 105 110 Ala Asn Arg Asn His Ala Arg Ala Asp Val Asp Arg Asn Ala Leu Arg 115 120 125 Pro Val Thr His Arg Gln Thr Leu Ala His Cys Ile Ala Gln His Tyr 130 135 140 Leu Gly Ala Ile Gln Gln Pro Thr Trp Ser His Pro Ser Val Trp Phe 145 150 155 160 Ala Ala Ile Gln Ala Ala Gln His Glu Gly Ala Ala Ala Ala Gln Gln Gln 165 170 175 Arg Ala Ala Arg Ser Ala His Ala Lys Asp Ala Asn Asp Asn Ala Asp 180 185 190 Ser Ile Ser Ser Ser Ser Ser Ser Ser Glu Gly Gly Thr Ala Ala Thr 195 200 205 Met Asp Gln Val Arg Arg Lys Trp Trp Ala His Met Val Gln Leu Ala 210 215 220 Arg Leu His Ile His Gln Gln Ala Asp Gly Asp Pro Ala Ser Glu Gln 225 230 235 240 Gly Lys Val Leu Glu Val Pro Ser Gly Leu Pro Ile His Asp Glu Tyr 245 250 255 Ile Ser Ser Leu Val Ser Ala Leu Leu Met Pro Met Asp Ser Asp Ala 260 265 270 Leu Ala Tyr Ser Ala His Val Pro Lys His Ile Asn Val His Thr Thr 275 280 285 Ile Cys Gly Ala Ser Pro Ala Thr Ala Arg Arg Cys Arg Ser Gly Arg 290 295 300 Pro Met Leu Ser Pro Ser Ala Ser Leu Gln Ala Ala Thr Ala Val Phe 305 310 315 320 Ala Ser Arg Ser Leu Ala Leu Thr Asn Val Ala Asp Thr Trp Leu Leu 325 330 335 Gln Leu Ala Gln His Val Arg Ala Gly Gly Val Val Ala Phe His Asp 340 345 350 Val Tyr Phe Ala Gly Asp Asp Pro Ser Pro Cys Val Leu Ala Asn Ala 355 360 365 Leu His Ser Pro Ala Gly Asp Thr Ser Val Val Tyr Ala Thr Glu Ala 370 375 380 Thr Ile Ala Phe Phe Leu Ala His Phe Asp Val Glu Trp Tyr Arg Asp 385 390 395 400 Val Arg Val Ser Phe His Pro Ser Leu Ala Cys Glu Lys Arg Glu Val 405 410 415 Phe Ala Leu Leu Arg Arg Arg Thr Thr Leu Arg Pro Gly Ile Ala Thr 420 425 430 Pro Arg Arg Val Asp Leu Thr Ile Thr Ser Leu Arg Ala Ile Lys Thr 435 440 445 Ala Leu Ala Ala Ala Pro Ser Ser Arg His Pro Ser Gln Trp Leu Gln 450 455 460 Arg Leu Leu Asp Ala Ala Leu His His Ser Pro Asn Ala Trp Pro Asn 465 470 475 480 Leu Ala His Arg Pro His Thr Thr Asn Thr Asn Thr Asn Thr Asn Thr 485 490 495 Asn Thr Asn Thr Asn Thr Asn Ala Asn Asn Asp Gly Gly Ala Val Ser 500 505 510 Leu Phe His Glu Ala Ser Arg Pro Arg Arg Trp Ala Thr Asn Gly Leu 515 520 525 Leu His Asp Thr Met Ser Gln Leu Glu Ala Phe Asp Ala Val Glu Arg 530 535 540 Leu Leu Glu Gly Gly Val Pro Asp Gly Cys Asn Pro Thr Ser Asp Ile 545 550 555 560 Cys Asn Ile Phe Phe Val Trp Thr Ser His Arg Asp Thr Trp Ser Phe 565 570 575 Leu Asn Arg Leu Ala Val Glu Ser Ala Leu Arg Ile Phe Pro Arg Ala 580 585 590 Arg Val Ile Ile Val Ser Asn Thr Leu Pro Val Thr Phe Phe Asn Ser 595 600 605 Leu Gln Ala Ser His Arg Val Tyr Val Trp Arg Ile Val Pro Thr Arg 610 615 620 Leu Val Arg Ala Gly Val Ala Gly Gly Arg Trp Leu Arg Ala Ala Leu 625 630 635 640 Arg Glu Gln Gly Pro His Leu Pro Thr His Gln Ser Asp Phe Leu Arg 645 650 655 Tyr Val Val Leu Tyr Lys Tyr Gly Gly Leu Phe Ser Asp Thr Asp Leu 660 665 670 Val Trp Leu Asp Ala Ser Pro Leu Ala His Ala Ile Gly Arg Asn Phe 675 680 685 Leu Gly Lys Ile Asp Ser Arg Pro Ile Leu Ala Arg Cys Pro Trp Cys 690 695 700 Val Asp Ser Thr Trp Tyr Leu Ala Asn Gly Val Leu Arg Phe Gln Ala 705 710 715 720 Arg His Lys Met Leu Ala Ser Ile Leu Gly His Ile Asp Thr Leu Arg 725 730 735 Tyr Asp Pro Ser Asp Arg Leu Ala Ile Gly Pro His Leu Val Thr Lys 74 0 745 750 Thr Phe Asn Ala Leu Gln Asp Pro Ser Val Ile Leu Val Asp Glu His 755 760 765 Val Leu Phe Pro Met Ser Gly Pro Asp Val Leu Gln Tyr Met Asp Pro 770 775 780 Arg Pro Pro His Thr Arg Leu Ala Asp Leu Leu Thr Ser Ala Ala Val 785 790 795 800 His Val Phe Glu Ala Thr Tyr Lys Ala Ala Pro Tyr Ala Pro Thr Ser 805 810 815 Ala Met Gln Gln Leu Leu Ala Leu Thr Pro Trp Val Gln Ile Asp Pro 820 825 830 Val Cys Glu Cys Val Trp Gln Gln Asp Asp Thr Asp Thr Asp Thr Asn 835 840 845 Glu Asp Lys Gly Asp Asp Asp Ser Asn Ser Asn Ser Asn Ser Asn Ser 850 855 860 Asn Ser Gly Gly Ala Ala Val Gly Gly Gly Asp Ser Thr Lys Arg Lys Lys 865 870 875 880 Thr Asn Leu Cys Leu Pro Tyr Ser Ala Thr Ala Arg Tyr Ar g Gln Gly 885 890 895 Ser Arg Asp His Leu Val Arg Met Cys Val Lys Ile Arg Gly Ile His 900 905 910 Ser Asp Ala Asp Gln Ser Ser Thr Pro Asn Ser Asp Arg Asn Glu Arg 915 920 925 Ala Ala Gly Asp Gly Asp Asn Asp Glu Gly Ala Gly Gly Glu Glu Lys 930 935 940 Ser Arg Asp Gly Thr Ser Val Val Gly Leu Pro Glu Asp Gly Val Leu 945 950 955 960 Val Leu Glu Ala Arg Leu Gly Arg Val Gln Thr Ala Tyr Gln Ala Ala 965 970 975 Asp Lys Arg Ile Val Val Pro Leu His Arg Asp Met Thr His Thr Glu 980 985 990 Leu Leu Glu Leu Ala Gln Leu Trp Tyr Val His Gly Glu Glu Tyr Cys 995 1000 1005 Asn Asp His Val Thr Val Gln Val Val Leu Ala Ser Gly Val Val 1010 1015 1020 Tyr Ala Glu Gly Gly Val Asp Val Met Thr Pro Cys Phe Gly Val 1025 1030 1035 Ala Glu Ala Gln Gln Arg Tyr Ile Gly Pro Pro Leu Al a Lys Gly 1040 1045 1050 Thr Tyr Thr Trp Thr Asn Ala Glu His Met Leu Gln Cys Thr Arg 1055 1060 1065 Phe Ser Glu Glu Tyr Ala Gly Pro Asp Pro Leu Met Phe Trp Pro 1070 1075 1080 Arg Lys Tyr Arg Lys Leu Val Gly Asp Met Val Ser Pro Ala Thr 1085 1090 1095 Ala Lys Tyr Ala Phe Gly Tyr Thr Pro Ala Leu Thr Ala Ala Asn 1100 1105 1110 Gly Ala Gly Glu Gln Gln Gln Arg Gln Gln Pro Arg Tyr Lys Val 1115 1120 1125 Gly Leu Val Val Val Ala Thr Gly Trp Tyr Tyr Ala Phe Leu Asp 1130 1135 1140 Asp Phe Val Ala Ser Ala Glu Glu Phe Phe Met Pro Gly His Glu 1145 1150 1155 Val His Tyr Phe Val Phe Thr Asp Asn Arg Pro Phe Ala Ala Gly 1160 1165 1170 Pro Ala Asp Arg Met His Ile Leu Arg Gln Pro Val Tyr Gly Trp 1175 1180 1185 Pro Phe Asp Ser Met Phe Arg Tyr Glu Ser Ile Leu Arg Gln Arg 1190 1195 1200 His His Phe Arg Asn Met Asp Tyr Ile Phe Met Leu Asp Ser Asp 1205 1210 1215 Ile Val Phe Ser Asn Phe Val Arg Glu Glu Ile Leu Gly Glu Met 1220 1225 1230 Val Gly Val Thr Gln Ala Phe Ala Phe Gly Leu Ala Arg Ser Glu 1235 1240 1245 Tyr Pro Leu Glu Ser Asn Pro Ala Ser Val Ala Tyr Val Pro Gln 1250 1255 1260 Arg Ser Thr Pro Cys Tyr Tyr Ala Gly Gly Ile Phe Gly Gly Thr 1265 1270 1275 Val Glu Gly Ala Val Arg Phe Leu Gln His Thr Ala Trp Leu Met 1280 1285 1290 Glu Trp Asp Ile Met Gln Gln Val Ser Ala Gly His Asp Asp Glu 1295 1300 1305 Ser Tyr Leu Asn Arg Ile Phe Ala Trp Asn Pro Pro Asp Val Val 1310 1315 1320 Leu Pro Ala Ser Tyr Ile Tyr Pro Glu Pro Pro Cys Asp Arg Ala 1325 1330 1335 Trp Gln Ala Gly Gly Arg Arg Tyr Asp Gly Thr Tyr Pro Pro Arg 1340 1345 1350 Ile Leu Asn Val Gly Cys Arg Lys Val Leu Gly Leu Gln Pro Gly 1355 1360 1365 Met Gly Arg Lys Thr Arg Thr Glu Asp Ala Gly Thr Pro Lys Asp 1370 1375 1380 Phe Met Leu Arg Glu Ala Arg Ala His Ala Ala Asp Met Thr Pro 1385 1390 1395 Gly Thr Ala Asn Glu Gln Gln Gln Gln Glu Gln Gln Gln Lys Gln 1400 1405 1410 Gln His Gly Gly Glu Gly Ala Glu Asp Ala Leu Leu Ser Thr Pro 1415 1420 1425 Leu Trp Met Val Ser Cys Ile Asp Ser Leu Pro Asp Asp Leu Asp 1430 1435 1440 Asp Gly Ala His Asp Asp M et Trp Ala Asp Ser Ile Ala Ala Ala 1445 1450 1455 Pro Arg Arg Leu Ala Asn His Thr Arg His Ala Gly Val Val Tyr 1460 1465 1470 Gly Met Ser Asn Ala Leu Leu Gln Arg Phe Trp Ala Ala Arg Ala 1475 1480 1485 Ser Gln Leu His Pro Pro Thr Leu His Val Val Ala Val Ser Arg 1490 1495 1500 Cys Asp Ala Gln Ala Val Ala Ala Leu Leu Gln Leu Val Gln Ala 1505 1510 1515 Asp Val Val Leu Val Thr Asp Pro Gly Leu Thr Pro Gly Trp Gln 1520 1525 1530 Val Asn Phe Gly Val Trp Arg Arg Met Leu Gln Asp Asn Ser Arg 1535 1540 1545 Leu Trp Lys Ala Ala Gly Gln Ser Ala Gly Gly Asp Gly Ala Gly 1550 1555 1560 Asp Asp Val Thr Ala Pro Leu Arg Ile Ala Ala Ala Cys Pro Phe 1565 1570 1575 Pro Ser Ser Lys Tyr Arg Gln Met Asn Leu Gly Thr Arg Ala Val 1580 1585 1590 Gly Asn Leu Leu Cys Gly Trp Asp Thr Gln Ala Ala Ser His Pro 1595 1600 1605 Ser Asp Gly Gln Arg Glu Arg Val Leu Ala Thr Asp Phe Arg Thr 1610 1615 1620 Tyr Val Cys Arg Ala Lys Pro Leu His Asp Val Glu Asp His Gln 1625 1630 1635 Ala Phe Met Ala Leu Lys Ala Pro Leu Leu Ser Ala Me t Asn Lys 1640 1645 1650 Ile Gly Ala Pro Val Val Met Glu Ala Asp Ala Arg Ser Asn Leu 1655 1660 1665 Gly Leu Pro Thr Arg Ser Gly Ile Asp Thr Gln Pro Arg Ser Arg 1670 1675 1680 Gly Gly Gly Ala Ala Gly Arg Gly Gly Asp Val Thr Gly Ser Phe 1685 1690 1695 Ala Arg Arg Thr Met Ala Ala Leu Arg Ser Leu Met Gln Ser Glu 1700 1705 1710 Gly Gly Ala Ala Val Glu Val Ala Ser Cys Phe Thr Asp Met Gln 1715 1720 1725 Pro Phe Ile Ala Ala Pro Val Val Ala Arg Thr Ser Ala Lys Met 1730 1735 1740 Cys Cys Arg Thr Arg Phe Leu Gly Met Cys Leu Ser Tyr Arg Glu 1745 1750 1755 Cys <![CDATA[ <210> 93]]>
           <![CDATA[ <211> 282]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Subdoligranulum sp.]]>
           <![CDATA[ <400> 93]]>
          Met Lys Thr Leu Ala Ile Leu Tyr Ile Cys Thr Gly Pro Tyr Ala Val
          1 5 10 15
          Phe Trp His Asp Phe Tyr Pro Asn Phe Lys Ala Asp Phe Leu Pro Asp
                      20 25 30
          Cys Asp Arg Thr Phe Tyr Val Phe Thr Asp Ala Ala His Ile Asp Tyr
                  35 40 45
          Glu Asp Ala Pro Asp Val Arg Arg Ile Tyr Gln Lys Ala Leu Pro Trp
              50 55 60
          Pro Gln Ser Thr Met Leu Arg Phe Asp Ala Phe Leu Gly Gln Ala Asp
          65 70 75 80
          Ala Leu Gln Gly Tyr Asp Tyr Leu Phe Phe Ala Asn Ala Asn Leu His
                          85 90 95
          Cys Thr Arg Val Ile Arg Ala Asp Glu Leu Leu Pro Asp Pro Ala Ala
                      100 105 110
          Gly Gln Ser Leu Thr Ala Val Cys His Leu Pro Tyr Tyr Gly Lys Asn
                  115 120 125
          Pro Ile Phe His Pro Tyr Asp Arg Ser Gly Lys Ser Arg Ala Ser Ile
              130 135 140
          Pro Tyr Ser Cys Gly Gln Tyr Tyr Val Ala Gly Gly Leu Asn Gly Gly
          145 150 155 160
          Thr Ala Ala Ala Tyr Leu Ala Leu Cys Arg Glu Leu Lys Lys Arg Thr
                          165 170 175
          Asp Glu Asp Leu Gln Asn Asn Val Ile Ala Arg Phe His Asp Glu Ser
                      180 185 190
          Gln Leu Asn Arg Leu Val Ala Glu Thr Pro Gly Lys Phe Arg Ile Leu
                  195 200 205
          Pro Pro Asp Tyr Cys Thr Pro Glu Glu Thr Pro Thr Gly His Glu Ala
              210 215 220
          Ile Leu Val Leu Gln Lys Ser Arg Cys Ile Asn Val Glu Ser Val Lys
          225 230 235 240
          Gly Thr Ala Lys Pro Gln Asn Phe Phe Gln Arg Lys Trp Glu Ala Phe
                          245 250 255
          Arg Leu Asn Trp Leu Pro Tyr Leu Trp Leu Ala Arg Asp Thr Leu Leu
                      260 265 270
          Arg Arg Arg Ile Asp Phe Lys Asn Asp Leu
                  275 280
           <![CDATA[ <210> 94]]>
           <![CDATA[ <211> 276]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Subdoligranulum sp.]]>
           <![CDATA[ <400> 94]]>
          Met Thr Lys Val Ala Ala Leu Tyr Ile Ala Thr Gly Arg Tyr Thr Val
          1 5 10 15
          Phe Trp Pro Glu Phe Tyr Glu Ser Ala Glu Lys Tyr Leu Leu Lys Asp
                      20 25 30
          Cys Glu Val His Tyr Phe Val Phe Thr Asp Ala Ala Thr Leu Pro Gly
                  35 40 45
          Gly Asp Asn Pro Arg Val His Ile Cys Ala Gln Asp Ala Tyr Ser Trp
              50 55 60
          Pro Phe Ala Thr Leu Arg Arg Phe Glu Ile Phe Leu Lys Gln Glu Gln
          65 70 75 80
          Ala Leu Lys Ala Phe Asp Tyr Ile Phe Phe Phe Asn Ala Asn Ala Glu
                          85 90 95
          Phe Met Gln Pro Val Thr Arg Glu Met Leu Leu Pro Arg Ala Glu Lys
                      100 105 110
          Gly Glu His Leu Leu Val Val Gln His Pro Ser Phe Tyr Ala Lys Pro
                  115 120 125
          Asn Tyr Glu Phe Thr Tyr Asp Arg Asn Pro Arg Ser Thr Ala Cys Ile
              130 135 140
          Pro Tyr Gly Leu Gly Lys Tyr Tyr Val Cys Gly Gly Val Asn Gly Gly
          145 150 155 160
          Glu Ala Ala Ala Phe Leu Gln Leu Cys His Thr Leu Asp Ala Arg Ile
                          165 170 175
          Arg Arg Asp Leu Gln Arg Asn Val Ile Ala Leu Trp His Asp Glu Ser
                      180 185 190
          Gln Ile Asn Arg Tyr Ile Leu Phe Arg Lys Asp Phe Arg Val Leu Thr
                  195 200 205
          Pro Ala Phe Cys Tyr Pro Glu Gly Trp Asp His Leu Pro Phe Pro Cys
              210 215 220
          Ile Ile Arg Ile Arg Ser Lys Ala Arg Tyr Ile Asp Ile Pro Ala Leu
          225 230 235 240
          Arg Lys Asp Ala Pro Glu Thr Lys Leu Ser Pro Ala Val Ala Arg Trp
                          245 250 255
          Asn His Phe Ala Met Arg Ala Ala Arg Trp Thr Gln Asn His Ile Phe
                      260 265 270
          Lys Lys Gly Ser
                  275
           <![CDATA[ <210> 95]]>
           <![CDATA[ <211> 280]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Subdoligranulum sp.]]>
           <![CDATA[ <400> 95]]>
          Met Lys Lys Val Ala Val Leu Tyr Ile Ala Thr Gly Lys Tyr Val Arg
          1 5 10 15
          Leu Trp Pro Gly Phe Leu Glu Ser Ala Glu Lys Tyr Leu Leu Lys Ser
                      20 25 30
          Cys Glu Val Glu Tyr Phe Val Phe Thr Asp Val Asp His Leu Ala Glu
                  35 40 45
          Glu Glu Asp Asn Pro Arg Ile His Arg Ile Phe Gln Glu Pro Met Pro
              50 55 60
          Trp Pro Tyr Thr Thr Leu Leu Arg Phe Glu Ile Phe Leu Lys Ala Glu
          65 70 75 80
          Glu Gln Leu Lys Ala Phe Asp Tyr Ile Tyr Phe Phe Asn Ala Asn Cys
                          85 90 95
          Glu Phe Lys Gln Pro Ile Thr Glu Glu Met Leu Leu Pro Arg Pro Lys
                      100 105 110
          Lys His Glu His Met Val Phe Val Leu His Pro Ala Phe Tyr Trp Arg
                  115 120 125
          Tyr Asn Tyr Glu Phe Thr Tyr Asp His Asn Pro Arg Cys Lys Ala Tyr
              130 135 140
          Ile Pro Met Gly Leu Gly Arg Asp Tyr Val Cys Gly Gly Ile Asn Gly
          145 150 155 160
          Gly Asp Arg Asp Ala Tyr Leu Lys Phe Cys His Thr Leu Gln Lys Arg
                          165 170 175
          Ile Arg Gln Asp Lys Asp Arg Gly Ile Ile Ala Leu Trp His Asp Glu
                      180 185 190
          Ser His Ile Asn Trp Tyr Ala Phe Thr His Pro His Tyr Arg Leu Leu
                  195 200 205
          Asp Ala Ser Phe Cys Phe Phe Pro Gly Trp Asp Thr Val Lys Pro Cys
              210 215 220
          Tyr Ile Tyr Ile Arg Pro Lys Glu Glu Tyr Phe Asp Val Asp Ala Phe
          225 230 235 240
          Lys Arg Asp Pro Pro Lys Thr Gln Leu Ser Pro Lys Val Glu Lys Tyr
                          245 250 255
          Asn Glu Phe Met Leu Lys Ala Ala Arg Lys Ile Gln Arg His Met Pro
                      260 265 270
          Trp Leu Pro Arg Arg Lys Arg Glu
                  275 280
           <![CDATA[ <210> 96]]>
           <![CDATA[ <211> 282]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Subdoligranulum sp.]]>
           <![CDATA[ <400> 96]]>
          Met Lys Thr Leu Ala Ile Leu Tyr Ile Cys Thr Gly Pro Tyr Ala Val
          1 5 10 15
          Phe Trp His Asp Phe Tyr Pro Asn Phe Lys Ala Asn Phe Leu Pro Asp
                      20 25 30
          Cys Asp Arg Thr Phe Tyr Val Phe Thr Asp Ala Ala His Ile Asp Tyr
                  35 40 45
          Glu Asp Ala Pro Asp Val Arg Arg Ile Tyr Gln Lys Ala Leu Pro Trp
              50 55 60
          Pro Gln Ser Thr Met Leu Arg Phe Asp Ala Phe Leu Gly Gln Ala Asp
          65 70 75 80
          Ala Leu Gln Gly Tyr Asp Tyr Leu Phe Phe Ala Asn Ala Asn Leu His
                          85 90 95
          Cys Thr Arg Ile Ile Arg Ala Asp Glu Leu Leu Pro Asp Pro Ala Ala
                      100 105 110
          Gly Gln Ser Leu Thr Ala Val Cys His Leu Pro Tyr Tyr Gly Lys Asn
                  115 120 125
          Pro Ile Phe His Pro Tyr Asp Arg Ser Gly Lys Ser Arg Ala Ser Ile
              130 135 140
          Pro Tyr Asn Cys Gly Gln Tyr Tyr Val Ala Gly Gly Leu Asn Gly Gly
          145 150 155 160
          Thr Ala Ala Ala Tyr Leu Ala Leu Cys Arg Glu Leu Lys Lys Arg Thr
                          165 170 175
          Asp Glu Asp Leu Gln Asn Asn Val Ile Ala Arg Phe His Asp Glu Ser
                      180 185 190
          Gln Leu Asn Arg Leu Val Ala Glu Thr Pro Gly Lys Phe Arg Ile Leu
                  195 200 205
          Pro Pro Asp Tyr Cys Thr Pro Glu Glu Thr Pro Thr Gly His Glu Ala
              210 215 220
          Ile Leu Val Leu Gln Lys Ser Arg Cys Ile Asn Val Glu Ser Val Lys
          225 230 235 240
          Gly Ala Ala Lys Pro Gln Asn Phe Phe Gln Arg Lys Trp Glu Ala Phe
                          245 250 255
          Arg Leu Asn Trp Leu Pro Tyr Leu Trp Leu Val Arg Asp Thr Leu Leu
                      260 265 270
          His Arg Arg Ile Asp Phe Lys Asn Asp Leu
                  275 280
           <![CDATA[ <210> 97]]>
           <![CDATA[ <211> 280]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Subdoligranulum variabile]]>
           <![CDATA[ <400> 97]]>
          Met Lys Arg Val Ala Ala Leu Tyr Ile Cys Thr Gly Glu Tyr Leu Arg
          1 5 10 15
          Leu Trp Pro Glu Phe Ile Ala Ser Ala Glu Lys Tyr Leu Leu Lys Gln
                      20 25 30
          Cys Glu Ile His Tyr Phe Val Phe Thr Asp Ala Asp His Ile Glu Gly
                  35 40 45
          Glu Glu Asn Asn Pro Arg Ile His Arg Ile Tyr Gln Lys Pro Gln Pro
              50 55 60
          Trp Pro Tyr Thr Thr Leu Lys Arg Phe Glu Ile Phe Leu Arg Cys Glu
          65 70 75 80
          Glu Gln Leu Lys Ala Phe Asp Tyr Ile Tyr Phe Phe Asn Ala Asn Cys
                          85 90 95
          Glu Phe Thr Gln Pro Ile Thr Glu Glu Glu Met Phe Leu Pro Arg Pro Glu
                      100 105 110
          Lys His Glu His Met Val Phe Val Leu His Pro Ala Phe Tyr Trp Arg
                  115 120 125
          Pro Asn Tyr Glu Phe Thr Tyr Asp Arg Asn Pro Arg Ser Lys Ala Phe
              130 135 140
          Ile Pro Met Gly Leu Gly Lys Asp Tyr Val Cys Gly Gly Ile Asn Gly
          145 150 155 160
          Gly Glu Ala Arg Ala Tyr Leu Lys Phe Cys His Leu Leu Asp Lys Arg
                          165 170 175
          Ile Asn Gln Asp Leu Asp Arg Gly Ile Ile Ala Trp Trp His Asp Glu
                      180 185 190
          Ser His Ile Asn Trp Tyr Ala Phe Thr His Arg Lys Tyr Arg Leu Leu
                  195 200 205
          Asp Ala Ser Phe Cys Phe Phe Glu Gly Trp His Thr Lys Lys Pro Cys
              210 215 220
          Tyr Ile Leu Ile Arg Ala Lys Glu Arg Tyr Phe Asp Val Asp Thr Phe
          225 230 235 240
          Lys Lys Asn Ser Pro Ala Thr Gln Leu Ser Pro Arg Val Glu Lys Tyr
                          245 250 255
          Asn His Phe Met Met Arg Val Ser Arg Tyr Leu Gln Arg Arg Met Pro
                      260 265 270
          Trp Leu Pro Arg Arg Pro Arg Glu
                  275 280
           <![CDATA[ <210> 98]]>
           <![CDATA[ <211> 287]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Subdoligranulum variabile]]>
           <![CDATA[ <400> 98]]>
          Met Ser Ala Asn Arg Pro Arg Val Ala Val Leu Tyr Leu Cys Thr Gly
          1 5 10 15
          Ala Tyr Gln Val Phe Trp Lys Asp Phe Tyr Pro Asn Phe Arg Ala His
                      20 25 30
          Phe Leu Pro Asp Cys Glu Arg Thr Phe Phe Val Phe Thr Asp Ala Pro
                  35 40 45
          Ala Ile Asp Tyr Glu Asp Ala Pro Asp Val Arg Arg Ile Pro Gln Glu
              50 55 60
          Ala Leu Pro Trp Pro Tyr Ser Thr Met Gln Arg Phe Asp Ala Phe Leu
          65 70 75 80
          Gly Gln Ala Thr Ala Leu Ala Gly Tyr Asp Tyr Leu Phe Phe Ala Asn
                          85 90 95
          Ala Asn Leu Arg Cys Leu Arg Asp Val Thr Ala Ala Glu Leu Leu Pro
                      100 105 110
          Asp Ala Ala Ala Gly Gln Ala Leu Thr Val Val Cys His Leu Pro Tyr
                  115 120 125
          Tyr Gly Lys Asp Pro Leu Phe His Pro Tyr Glu Arg Arg Arg Lys Ser
              130 135 140
          Arg Ala Cys Ile Pro Tyr Asn Cys Gly Thr Trp Tyr Val Ala Gly Gly
          145 150 155 160
          Leu Asn Gly Gly Gln Ser Ala Ala Tyr Leu Glu Leu Cys Arg Glu Leu
                          165 170 175
          Lys Ala Arg Thr Asp Glu Asp Leu Arg Arg Gly Val Ile Ala Arg Phe
                      180 185 190
          His Asp Glu Ser Gln Leu Asn Arg Leu Val Ala Glu Gln Pro Gly Arg
                  195 200 205
          Phe Arg Val Leu Gly Pro Asp Tyr Cys Thr Pro Glu Glu Thr Pro Thr
              210 215 220
          Gly His Glu Ala Ile Arg Val Leu Gln Lys Ala His Tyr Ile Asp Val
          225 230 235 240
          Gln Ala Val Arg Gly Ala Ala Lys Pro Gln Asn Trp Val Gln Cys Lys
                          245 250 255
          Trp Glu Ala Phe Cys Leu Asn Trp Leu Pro Tyr Leu Trp Arg Ala Arg
                      260 265 270
          Asp Ala Leu Leu Arg Arg Arg Val Glu Pro Pro Gln Lys Met Pro
                  275 280 285
           <![CDATA[ <210> 99]]>
           <![CDATA[ <211> 281]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Subdoligranulum variabile]]>
           <![CDATA[ <400> 99]]>
          Met Thr Lys Val Ala Ala Leu Tyr Ile Cys Thr Gly Lys Tyr Ile Ala
          1 5 10 15
          Phe Trp Pro Glu Phe Tyr Asp Ser Ala Glu Gln Asn Leu Leu Pro Gly
                      20 25 30
          Cys Glu Val His Tyr Phe Val Phe Thr Asp Ala Pro Val Leu Tyr Gly
                  35 40 45
          Glu Glu Ala Asn Pro Arg Ile His Arg Cys Pro Gln Glu Ala Tyr Ser
              50 55 60
          Trp Pro Phe Ala Thr Leu Arg Arg Phe Glu Ile Phe Leu Ser Arg Glu
          65 70 75 80
          Glu Glu Leu Lys Ala Phe Asp Tyr Ile Phe Phe Phe Asn Ala Asn Ala
                          85 90 95
          Gln Ile Met Thr Thr Ile Thr Pro Glu Met Phe Leu Pro Arg Ala Asp
                      100 105 110
          Arg Gly Glu His Leu Leu Val Val Gln His Pro Ser Phe Tyr Thr Lys
                  115 120 125
          Pro Asn Tyr Glu Phe Thr Tyr Asp Arg Asn Pro Arg Cys Arg Ala Phe
              130 135 140
          Ile Pro Met Gly Leu Gly Arg Tyr Tyr Val Cys Gly Gly Ile Asn Gly
          145 150 155 160
          Gly Glu Ala Ala Ala Phe Leu Lys Leu Cys His Thr Leu Asp Lys Arg
                          165 170 175
          Ile Arg Lys Asp Leu Ala His Asn Val Ile Ala Gln Trp His Asp Glu
                      180 185 190
          Ser His Ile Asn Arg Tyr Ile Leu Trp Arg Arg Asp Val Arg Val Leu
                  195 200 205
          Ser Pro Ser Tyr Cys Trp Pro Glu Gly Trp Asn Leu Pro Leu Pro Cys
              210 215 220
          Arg Ile Leu Ile Arg Ser Lys Ala Arg Tyr Phe Asp Val Gln Gln Leu
          225 230 235 240
          Arg Lys Asp Ala Pro Ala Thr Glu Leu Pro Arg Tyr Val Val Arg Cys
                          245 250 255
          Asn Asp Phe Met Lys Arg Ala Ala Arg Trp Leu Gln Arg Arg Leu Pro
                      260 265 270
          Pro Lys Lys Glu Asp Ile Asn Asp Glu
                  275 280
           <![CDATA[ <210> 100]]>
           <![CDATA[ <211> 286]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Subdoligranulum variabile]]>
           <![CDATA[ <400> 100]]>
          Met Ser Glu Ser Arg Ile Arg Val Ala Val Leu Tyr Leu Cys Thr Gly
          1 5 10 15
          Ala Tyr Gln Val Phe Trp His Asp Phe Tyr Pro Asn Phe Arg Gln His
                      20 25 30
          Phe Leu Pro Asp Cys Asp Arg Thr Phe Phe Val Phe Thr Asp Ala Ala
                  35 40 45
          Ser Ile Asp Tyr Glu Asp Gln Pro Asp Val Arg Arg Phe Gln Gln Glu
              50 55 60
          Ala Leu Pro Trp Pro Tyr Ser Thr Met Gln Arg Phe Asp Ala Phe Leu
          65 70 75 80
          Ser Gln Ala Glu Ala Leu Ala Asp Tyr Asp Tyr Leu Phe Phe Ala Asn
                          85 90 95
          Ala Asn Leu His Cys Leu Arg Asp Val Thr Ala Gly Glu Leu Leu Pro
                      100 105 110
          Asp Ala Ala Lys Gly Gln Glu Leu Thr Val Val Cys His Leu Pro Tyr
                  115 120 125
          Tyr Gly Arg Asn Pro Ile Phe His Pro Tyr Glu Arg Arg Arg Lys Cys
              130 135 140
          Arg Ala Gly Ile Pro Tyr Asn Cys Gly Thr Tyr Tyr Val Ala Gly Gly
          145 150 155 160
          Ile Asn Gly Gly Ala Ser Gly Ala Phe Leu Glu Met Cys Arg Glu Leu
                          165 170 175
          Lys Ala Arg Thr Asp Glu Asp Leu Gln Arg Gly Ile Ile Ala Arg Cys
                      180 185 190
          His Asp Glu Ser Gln Leu Asn Arg Leu Val Ala Glu Cys Pro Glu Arg
                  195 200 205
          Phe Arg Ile Leu Pro Pro Glu Tyr Cys Thr Pro Glu Glu Thr Pro Thr
              210 215 220
          Gly Lys Glu Ala Ile Arg Val Leu Gln Lys Ser His Tyr Ile Asp Met
          225 230 235 240
          Ser Ala Val Arg Gln Gln Gly Arg Arg Gln Asn Tyr Leu Gln Arg Lys
                          245 250 255
          Trp Glu Ala Phe Cys Leu Asn Trp Leu Pro Tyr Leu Trp Trp Ala Arg
                      260 265 270
          Asp Thr Leu Leu Arg Arg Arg Val Asp Pro Pro Arg Thr Arg
                  275 280 285
           <![CDATA[ <210> 101]]>
           <![CDATA[ <211> 258]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Sulfurospirillum deleyianum]]>
           <![CDATA[ <400> 101]]>
          Met Asn Lys Ile Gly Ile Leu Tyr Ile Cys Thr Gly Asp Tyr Trp Lys
          1 5 10 15
          Phe Trp Glu Asn Phe Tyr Lys Ser Ser Glu Glu Leu Phe Leu Thr Asn
                      20 25 30
          Glu Glu Lys His Tyr Phe Leu Phe Thr Asp Asn Arg Glu Leu Leu Asn
                  35 40 45
          Ile Asn Asn Glu Arg Ile His Ser Phe Phe Gln Glu Lys Met Asp Trp
              50 55 60
          Pro Tyr Pro Thr Leu Tyr Arg Tyr Lys Thr Phe Ile Lys Tyr Lys Thr
          65 70 75 80
          Val Phe Gln Asp Met Asp Tyr Leu Ile Phe Cys Asn Ala Asn Leu Leu
                          85 90 95
          Phe Asn Glu Lys Ile Ser Arg Asn Asp Leu Phe Ala Asn Lys Glu Leu
                      100 105 110
          Phe Ala Thr Leu His Pro Gly Phe Phe Asp Lys Lys Pro Gln Lys Phe
                  115 120 125
          Thr Tyr Glu Thr Asn Ile Lys Ser Leu Ala Tyr Thr Glu Lys Lys Val
              130 135 140
          Asp Ser Ile Tyr Val Cys Gly Gly Phe Asn Gly Gly Ile Lys Asn Asp
          145 150 155 160
          Phe Leu Lys Met Ala Glu Ile Leu Asp Asp Asn Ile Asp Lys Asp Phe
                          165 170 175
          Ser Glu Ser Ile Ile Ala Ile Trp His Asp Glu Ser His Ile Asn Asn
                      180 185 190
          Tyr Val Gln Asn Asn Lys Glu Lys Phe Asn Ile Leu Ser Pro Ser Phe
                  195 200 205
          Cys Tyr Pro Gln His Tyr Ser Ile Asp Ile Asn Lys Lys Ile Ile Val
              210 215 220
          Gln Asp Lys Glu Lys Ile Ile Ser Ile Lys His Lys Gly Val Phe Tyr
          225 230 235 240
          Asn Ile Arg Phe Leu Ile Ile Lys Met Leu Lys Lys Met Phe Arg His
                          245 250 255
          Arg Arg
           <![CDATA[ <210> 102]]>
           <![CDATA[ <211> 246]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Bacteroides ovale]]>
           <![CDATA[ <400> 102]]>
          Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe
          1 5 10 15
          Trp Lys Asp Phe Tyr Leu Ser Ala Glu Arg Tyr Phe Met Gln Asp Gln
                      20 25 30
          Ser Phe Ile Ile Glu Tyr Tyr Val Phe Thr Asp Ser Pro Lys Leu Tyr
                  35 40 45
          Asp Glu Glu Asn Asn Lys His Ile His Arg Ile Lys Gln Lys Asn Leu
              50 55 60
          Gly Trp Pro Asp Asn Thr Leu Lys Arg Phe His Ile Phe Leu Arg Ile
          65 70 75 80
          Lys Glu Gln Leu Glu Arg Glu Thr Asp Tyr Leu Phe Phe Phe Asn Ala
                          85 90 95
          Asn Leu Leu Phe Thr Ser Pro Ile Gly Lys Glu Ile Leu Pro Pro Ser
                      100 105 110
          Asp Ser Asn Gly Leu Leu Gly Thr Met His Pro Gly Phe Tyr Asn Lys
                  115 120 125
          Pro Asn Ser Glu Phe Thr Tyr Glu Arg Arg Asp Ala Ser Thr Ala Tyr
              130 135 140
          Ile Pro Glu Gly Glu Gly Arg Tyr Tyr Tyr Ala Gly Gly Leu Ser Gly
          145 150 155 160
          Gly Cys Thr Lys Ala Tyr Leu Lys Leu Cys Thr Thr Ile Cys Ser Trp
                          165 170 175
          Val Asp Arg Asp Ala Thr Asn His Ile Ile Pro Ile Trp His Asp Glu
                      180 185 190
          Ser Leu Ile Asn Lys Tyr Phe Leu Asp Asn Pro Pro Ala Ile Thr Leu
                  195 200 205
          Ser Pro Ala Tyr Leu Tyr Pro Glu Gly Trp Leu Leu Pro Phe Glu Pro
              210 215 220
          Ile Ile Leu Ile Arg Asp Lys Asn Lys Pro Gln Tyr Gly Gly His Glu
          225 230 235 240
          Leu Leu Arg Arg Lys Asn
                          245
          
      

Claims (94)

A method for producing an α-1,3 glycosylated form of fucose-α-1,2-galactose-R (fucose-alpha-1,2-galactose-R, Fucose) by a cell, preferably a single cell -a1,2-Gal-R), wherein the alpha-1,3 glycation occurs at the end of fucose-alpha-1,2-galactose-R (Fuc-a1,2-Gal-R)" Fucose-a1,2-galactose"-group, wherein the method comprises the steps of: i. Provide the ability to synthesize Fuc-a1,2-Gal-R, express an alpha-1,3-glycosyltransferase (alpha-1,3-glycosyltransferase), and have the ability to synthesize the alpha-1,3-sugar A nucleotide-sugar capable cell that is a donor of syltransferase, and ii After allowing synthesis of the Fuc-a1,2-Gal-R, expression of the α-1,3-glycosyltransferase, synthesis of the nucleotide-sugar and synthesis of the α-1,3 glycosylated form of Fuc-a1, The cells were cultured under the condition of 2-Gal-R, iii. Preferably the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is isolated from the culture. The method of claim 1, wherein the galactose (Gal) residue in the Fuc-a1,2-Gal-R is via a beta-1,3 or a beta-1,4 glycosidic linkage combined with R. The method of claim 1 or 2, wherein the R comprises a monosaccharide, a disaccharide, an oligosaccharide, a peptide, a protein, a glycopeptide, a sugar A glycoprotein, a lipid or a glycolipid. The method of any one of claims 1 to 3, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-R, preferably the Fuc-a1,2-Gal -R is Fuc-a1,2-Gal-b1,3-GlcNAc-R. The method of claim 4, wherein the N-acetylglucosamine (GlcNAc) residue in the Fuc-a1,2-Gal-b1,3-GlcNAc-R is via a β-1,3 or A β-1,4 glycosidic bond binds to R. The method of claim 4 or 5, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R, preferably, wherein the Fuc-a1 , 2-Gal-R is Fuc-a1, 2-Gal-b1, 3-GlcNAc-b1, 3-Gal-R, more preferably, wherein Fuc-a1, 2-Gal-R is milk-N-fucoid Pentasaccharide I (lacto-N-fucopentaose I, LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc). The method of any one of claims 1 to 3, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,4-R, preferably, wherein the Fuc-a1,2 -Gal-R is Fuc-a1,2-Gal-b1,4-Glc, as required, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,4-(Fuc -a1,3)-Glc. The method of any one of claims 1 to 7, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is a structure of the histo blood group antigen (HBGA) system. The method of any one of claims 1 to 8, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase (alpha-1,3-galactosyltransferase), which is Has the transfer of galactose (Gal) residues from UDP-galactose (UDP-galactose, UDP-Gal) to fucose-a1,2-galactose-R (Fuc-a1,2-Gal- R) is a glycosyltransferase capable of the terminal "fucose-al,2-galactose"-group. The method of any one of claims 1 to 6, 8 or 9, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase which is a A glycosyltransferase capable of transferring (Gal) residues from UDP-Gal to the terminal "fucose-al,2-galactose"-group of LNFP-I. The method of any one of claims 1 to 3, 7, 9, or 10, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase that has half the Transglycosylation of the ability of a lactose (Gal) residue to transfer from UDP-Gal to the terminal "fucose-al,2-galactose"-group of Fuc-a1,2-Gal-b1,4-Glc The enzyme, optionally, the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated, preferably α-1,3-fucosylated. The method of any one of claims 1 to 6 or 8 to 10, wherein the α-1,3 glycated form of Fuc-a1,2-Gal-R is an α-1,3 glycated form of milk-N- Fucopentaose I (LNFP-I), which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-a1 ,3-LNFP-I), the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase, and the nucleotide-sugar is UDP-galactose (UDP-galactose, UDP-Gal). The method of any one of claims 1 to 3, 7 or 9 to 11, wherein the α-1,3 glycated form of Fuc-a1,2-Gal-R is an α-1,3 glycated form of Fuc- a1,2-Gal-b1,4-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, as required, an α-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3 )-Glc, the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase, and the nucleotide-sugar is UDP-galactose (UDP-Gal). The method of any one of claims 9 to 13, wherein the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain, and a. Include the motif with SEQ ID NO: 01 YX[FHMQT]XAXX[ACG][ACG], where X can be any amino acid residue, or b. Include the motif YXQXCXX[ACG][ACG] with SEQ ID NO: 02, where X can be any amino acid residue, or c. Include serial identification numbers such as: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, a polypeptide sequence of any of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, or d. is the serial identification number: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 any one of functional homologs, variants or derivatives Derivative with and with serial identification numbers: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, Any of the alpha-1,3-galactosyltransferase polypeptides of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 At least 80% of the overall sequence similarity of the full length, and the terminal "rock" of fucose-a1,2-galactose-R (fucose-a1,2-galactose-R, Fuc-a1,2-Gal-R) The algalose-a1,2-galactose"-group has a-1,3-galactosyltransferase activity, or e. is a functional fragment, including from SEQ ID NO: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37. An oligopeptide sequence of 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues, and to fucose-a1,2-galactose-R (Fuc-a1,2 The terminal "fucose-al,2-galactose"-group of -Gal-R) has a-1,3-galactosyltransferase activity. The method of any one of claims 1 to 8, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase (alpha-1,3- N-acetylgalactosaminyltransferase), which is capable of transferring an N-acetylgalactosamine (N-acetylgalactosamine, GalNAc) residue from UDP-N-acetylgalactosamine (UDP-N-acetylgalactosamine, UDP-GalNAc) to rock A glycosyltransferase capable of the terminal "fucose-al,2-galactose"-group of Fucose-al,2-galactose-R (Fuc-al,2-Gal-R). The method of any one of claims 1 to 6, 8 or 15, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase, which is Has a terminal "fucose-a1,2-galactose" that transfers an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to LNFP-I- A glycosyltransferase with the ability of a group. The method of any one of claims 1 to 3, 7 or 15, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase, which is Has a terminal "fucose-a1,2-galactose"-group that transfers an N-acetylgalactosamine (GalNAc) residue from UDP-GalNAc to Fuc-a1,2-Gal-b1,4-Glc A glycosyltransferase with the ability to group, as needed, the glucose residues in the Fuc-a1,2-Gal-b1,4-Glc are fucosylated, preferably α-1,3 - Fucosification. The method of any one of claims 1 to 6, 8, 15 or 16, wherein the α-1,3 glycated form of Fuc-a1,2-Gal-R is an α-1,3 glycated form of milk- N-fucopentaose I (LNFP-I), which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc -a1,3-LNFP-I), the α-1,3-glycosyltransferase is an α-1,3-N-acetylgalactosamine transferase, and the nucleotide-sugar is UDP-N - Acetylgalactosamine (UDP-GalNAc). The method of any one of claims 1 to 3, 7, 15 or 17, wherein the α-1,3 glycated form of Fuc-a1,2-Gal-R is an α-1,3 glycated form of Fuc- a1,2-Gal-b1,4-Glc, which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc (alpha-tetrasaccharide or A-tetrasaccharide (A-tetrasaccharide)), as required - Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc in α-1,3 glycosylated form, which is GalNAc-a1,3- (Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3)-Glc, the α-1,3-glycosyltransferase is an α-1,3-N-acetylgalactosamine transferase, and the nucleotide-sugar is UDP-N-acetylgalactosamine (UDP-GalNAc). The method of any one of claims 15 to 19, wherein the α-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain, and a. Include the motif YX[ACIL]XGXX[ACG][ACG] with SEQ ID NO: 38, where X can be any amino acid residue, or b. Include the motif YX[AG]XAXX[ACG][ACG] with SEQ ID NO: 39, where X can be any amino acid residue, or c. Include such as serial identification numbers: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, a polypeptide sequence of any one of 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, or d. is the serial identification number: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, a functional homologue, variant or derivative of any of 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, With and with serial identification numbers: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 of the α-1,3-N-acetylgalactosyltransferase ( a-1,3-N-acetylgalactosyltransferase) polypeptides have at least 80% overall sequence similarity over the full length of any of the polypeptides and are The terminal "fucose-a1,2-galactose" group of R) has a-1,3-N-acetylgalactosyltransferase activity, or e. is a functional fragment, including from SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102. An oligopeptide sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues, and to fucose-a1,2-galactose-R The terminal terminal "fucose-al,2-galactose" group of (Fuc-a1,2-Gal-R) has a-1,3-N-acetylgalactosyltransferase activity. The method of any one of claims 6, 10, 12, 14, 16, 18 or 20, wherein fucose is transferred from GDP-fucose to milk-N by the action of a glycosyltransferase -The terminal galactose residue of lacto-N-tetraose (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), the LNFP-I is synthesized in the cell, the The glycosyltransferases are: a. an alpha-1,2-fucosyltransferase (alpha-1,2-fucosyltransferase) selected from the group consisting of Brachyspira pilosicoli with UniProt ID A0A2N5RQ26, with List of Polypeptides of Dysgonomonas mossii with UniProt ID F8X274, Dechlorosoma suillum with UniProt ID G8QLF4, Desulfovibrio alaskensis with UniProt ID Q316B5 and Polaribacter vadi with UniProt ID A0A1B8TNT0, or b. terminal galactose residues for LNT Alpha-1,2-fucosyltransferase activity from Brachyspira trichomes (UniProt ID A0A2N5RQ26), from D. mossii (UniProt ID F8X274), from D. suillum (UniProt ID G8QLF4), a functional fragment of any of a polypeptide from D. alaskensis (UniProt ID Q316B5) and a polypeptide from P. vadi (UniProt ID A0A1B8TNTO), or c. a polypeptide with UniProt Any of the polypeptides of Brachyspira trichomes with ID A0A2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5, and P. vadi with UniProt ID A0A1B8TNT0 A functional homolog, variant or derivative of the same with Brachyspira trichomes from UniProt ID A0A2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. suillum with UniProt ID Q316B5, respectively D. alaskensi s has at least 80% overall sequence similarity to the full length of any of the polypeptides of P. vadi with UniProt ID A0A1B8TNT0 and has an alpha to the terminal galactose residue of lacto-N-saccharide (LNT) -1,2-fucosyltransferase activity, or d. a polypeptide comprising, or consisting of, an amino acid sequence having the same Polypeptide from (UniProt ID A0A2N5RQ26), Polypeptide from D. mossii (UniProt ID F8X274), Polypeptide from D. suillum (UniProt ID G8QLF4), Polypeptide from D. alaskensis (UniProt ID Q316B5) The peptide has at least 80% sequence similarity to the full-length amino acid sequence of any of the polypeptides from P. vadi (UniProt ID A0A1B8TNTO) and has alpha-1,2-rock to the terminal galactose residue of LNT Fluorosyltransferase activity. The method of any one of claims 1 to 21, wherein the cell is modified in the expression or activity of a glycosyltransferase. The method of any of the preceding claims, wherein the cell expresses a membrane transporter protein or a polypeptide having transport activity to transport the compound across the outer membrane of the cell wall. The method of claim 23, wherein the membrane transporter protein or the polypeptide having transport activity is selected from a list including a transporter, a P-P-bond-hydrolysis-driven transporter (P-P -bond-hydrolysis-driven transporter), b-barrel porins, auxiliary transport proteins, putative transport proteins and phosphate transfer-driven group translocators ( phosphotransfer-driven group translocator), Preferably, the transporter includes MFS transporter, sugar efflux transporter and siderophore exporters, or Preferably, the P-P-bond-hydrolysis-driven transporter includes ABC transporter and chelatin exporter. The method of any one of claims 23 or 24, wherein the membrane transporter or polypeptide having transport activity controls the α-1,3 glycosylated form of Fuc-a1,2-Gal-R and/or is used for The alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R produces one or more precursors and/or the flow of acceptors on the outer membrane of the cell wall. The method of any one of claims 23 to 25, wherein the membrane transporter or polypeptide having transport activity provides improved production of the α-1,3 glycosylated form of Fuc-a1,2-Gal-R and/or activated and/or enhanced outflow. The method of any of the preceding claims, wherein the cell is a metabolically engineered cell. The method of claim 27, wherein the cell line is modified with a gene expression module characterized in that the expression from any of the expression modules is constitutive, or by a natural inducer )Creative. The method of any one of claims 27 or 28, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. The medium of any one of claims 27 to 29, wherein the cell comprises a modification for reducing the production of acetic acid. The method of any one of claims 27 to 29, wherein the cell comprises lower or reduced expression and/or eliminated, impaired, reduced or delayed activity of any one or more proteins, the Any one or more proteins including beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase (N-acetylglucosamine-6-phosphate deacetylase), glucosamine-6-phosphate deaminase (glucosamine-6-phosphate deaminase), N-acetylglucosamine repressor, ribonucleotide monophosphate Enzyme (ribonucleotide monophosphatase), EIICBA-Nag, UDP-glucose:undecene-phosphate glucose-1-phosphate transferase (UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase), L-fucokinase (L-fuculokinase), L-fucose isomerase (L-fucose isomerase), N-acetylneuraminate lyase (N-acetylneuraminate lyase), N-acetylmannosamine kinase (N-acetylmannosamine kinase) ), N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1 -Galactose-1-phosphate uridylyltransferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP - ATP-dependent 6-phosphofructokinase isozyme 1 (ATP-dependent 6-phosphofructokinase isozyme 1), ATP-dependent 6-phosphofructokinase isozyme 2 (ATP-dependent 6-phosphofructokinase isozyme 2), glucose -6-phosphate isomerase (glucose-6-phosphate is omerase), aerobic respiration control protein, transcriptional repressor IclR (transcriptional repressor IclR), lon protease, glucose-specific translocating phosphotransferase IIBC component ptsG (glucose-specific translocating phosphotransferase enzyme) IIBC component ptsG), glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX (glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX), enzyme IIA ^Glc , β-glucoside specific PTS enzyme II (beta -glucoside specific PTS enzyme II), fructose-specific PTS multiphosphoryl transfer protein FruA and FruB (fructose-specific PTS multiphosphoryl transfer protein FruA and FruB), ethanol dehydrogenase (ethanol dehydrogenase), aldehyde dehydrogenase (aldehyde dehydrogenase), Pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvate decarboxylase. The method of any of the preceding claims, wherein the cell has the ability to produce phosphoenolpyruvate (PEP). The method of any of the preceding claims, wherein the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP). The method of any of the preceding claims, wherein the cell comprises an at least partially inactivated catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide that participates in and/or is affected by the alpha - Required for this production of the 1,3 glycated form of Fuc-a1,2-Gal-R. The method of any of the preceding claims, wherein the cell is resistant to lactose killing when grown in an environment in which lactose is combined with one or more other carbon sources. The method of any of the preceding claims, wherein the cells produce 90 g/L or more of the alpha-1,3 glycated form of Fuc-a1 in whole broth and/or supernatant ,2-Gal-R, and/or wherein in the whole broth and/or supernatant, the α-1,3 glycated form of Fuc-a1,2-Gal-R is based on the α-1,3 glycated form The total amount of Fuc-a1,2-Gal-R and its precursors in the whole broth and/or supernatant, respectively, was measured to be at least 80% pure. The method of any of the preceding claims, wherein the cells are stably cultured in a medium. A method as in any preceding claim, wherein the condition includes: (i) using a medium comprising at least one precursor and/or acceptor for the production of the α-1,3 glycosylated form of Fuc-a1,2-Gal-R, and/or (ii) adding to the medium at least one precursor and/or acceptor feed for the production of the α-1,3 glycated form of Fuc-a1,2-Gal-R. The method of any of the preceding claims, comprising at least one of the following steps: (i) using a medium comprising at least one precursor and/or acceptor; (ii) for the medium in a reactor Add at least one precursor and/or acceptor feed with a total reactor volume in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters), preferably in a continuous manner, and preferably such that the The final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before the addition of the precursor and/or recipient feed; (iii) for a reactor The medium in which is supplemented with at least one precursor and/or acceptor feed, wherein the total reactor volume is in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters), preferably in a continuous manner, and relatively Preferably, the final volume of the medium is no greater than three times, preferably no greater than two times, more preferably less than two times the volume of the medium before the addition of the precursor and/or recipient feed, and wherein more Preferably the pH of the precursor and/or acceptor feed is set between 3 and 7, and wherein preferably the temperature of the precursor and/or acceptor feed is maintained between 20°C and 80°C; (iv) adding at least one precursor and/or receiving in a continuous manner during the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution (v) adding at least one precursor and/or receiving in a continuous manner during the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution is fed to the medium, and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained between 20°C and 80°C between °C; the method results in 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 in the final culture Fuc-a1,2 in an alpha-1,3 saccharified form at a concentration of 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 -Gal-R. The method of any one of claims 1 to 38, the method comprising at least one of the following steps: (i) using a culture medium comprising at least 50, more preferably at least 75, more preferably at least 75 per liter of initial reactor volume preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose, wherein the reactor volume is in the range of 250 mL (milliliters) to 10.000 ^m3 (cubic meters); (ii) the medium Add a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor the volume is in the range of 250 mL (milliliter) to 10.000 m ³ (cubic meter), preferably in a continuous form, and preferably such that the final volume of the medium is no more than three times, preferably no more than twice, More preferably less than twice the volume of the medium prior to adding the lactose feed; (iii) adding a lactose feed to the medium comprising at least 50, more preferably at least 75 per liter of initial reactor volume , more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose, wherein the reactor volume is in the range of 250 mL (milliliter) to 10.000 m ³ (cubic meter), preferably with Continuous format, and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium before the addition of the lactose feed, and preferably the pH of the lactose feed is set between 3 and 7, and wherein preferably the temperature of the lactose feed is maintained between 20°C and 80°C; (iv) by a By means of a feeding solution, during the course of 1 day, 2 days, 3 days, 4 days, 5 days, a lactose feed was added to the medium in a continuous manner; (v) by means of a feeding solution, in During the course of 1 day, 2 days, 3 days, 4 days, 5 days, a lactose feed was added to the medium in a continuous manner, and wherein the concentration of the lactose feed solution was 50 g/L, preferably 75 g /L, better is 100 g/L, better is 125 g/L, better is 150 g/L, better is 175 g/L, better is 200 g/L, better is 225 g/L , better is 250 g/L, better is 275 g/L, better is 300 g/L, better is 325 g/L, better is 350 g/L, better is 375 g/L, more preferably 400 g/L, more preferably 450 g/L, more preferably 500 g/L, still more preferably 550 g/L, and most preferably 600 g/L; and preferably the feed solution The pH is set between 3 and 7, and wherein preferably the temperature of the feed solution is maintained between 20°C and 80°C; the method results in a final volume of the culture in have 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 it is an alpha-1,3 glycated form of Fuc-a1,2-Gal-R at a concentration of at least 200 g/L. The method of claim 39, wherein the lactose feed is obtained by starting the culture at a concentration of at least 5 mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, More preferably this is done by adding lactose at a concentration of >300 mM. The method of any one of claims 39 or 40, wherein the lactose feeding is achieved by adding lactose to the culture at a concentration such that at least 5 mM is obtained throughout the production phase of the culture, preferably Lactose concentrations of 10 mM or 30 mM. The method of any of the preceding claims, wherein the cells are cultured for at least about 60, 80, 100, or about 120 hours or in a continuous fashion. The method of any one of the preceding claims, wherein the cells are in a medium comprising a carbon source comprising monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol, including molasses, corn steep liquor ), peptone (peptone), tryptone (tryptone) or yeast extract (yeast extract) complex medium; preferably, wherein the carbon source is selected from the group consisting of glucose, glycerol, fructose, sucrose, maltose, lactose, arabinoside Sugar (arabinose), malto-oligosaccharides (malto-oligosaccharides), maltotriose (maltotriose), sorbitol (sorbitol), xylose (xylose), rhamnose (rhamnose), galactose, mannose, methanol , ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvic acid List of salts. The method of any one of the preceding claims, wherein the medium comprises at least one precursor selected from the group consisting of lactose, galactose, fucose, sialic acid, GlcNAc, GalNAc, lacto-N-disaccharide (lacto-N-disaccharide) N-biose, LNB), N-acetyllactosamine (N-acetyllactosamine, LacNAc) group. The method of any of the preceding claims, wherein exponential cell growth is provided by adding a carbon-based substrate, preferably glucose or sucrose, to the medium comprising a precursor, preferably lactose The first stage is followed by the second stage, in which only a one-carbon based substrate, preferably glucose or sucrose, is added to the medium. The method of any one of claims 1 to 45, wherein the first stage of exponential cell growth is provided by adding a carbon substrate, preferably glucose or sucrose, to the medium comprising a precursor, preferably lactose , followed by a second stage in which a carbon substrate, preferably glucose or sucrose, and a precursor, preferably lactose, are added to the medium. The method of any one of the preceding claims, wherein the cell produces a charged, preferably sialylated and/or a mixture of neutral disaccharides and oligosaccharides comprising an alpha-1,3 glycated form of Fuc-a1,2-Gal-R. The method of any one of the preceding claims, wherein the cell produces a mixture of charged, preferably sialylated and/or neutral oligosaccharides comprising an alpha-1,3 glycosylated form of Fuc-a1,2- Gal-R. A metabolically engineered cell to produce the alpha-1,3 glycated form of fucose-alpha-1,2-galactose-R (fucose-alpha-1,2-galactose-R, Fuc-a1,2 -Gal-R), wherein the α-1,3 glycation occurs at the terminal "fucose-a1, 2-galactose-R" (Fuc-a1,2-Gal-R) 2-galactose"-group, and in which the cell - Synthesis of Fuc-a1,2-Gal-R, with - expresses an alpha-1,3-glycosyltransferase, and -has the ability to generate a nucleotide-sugar, wherein the nucleotide-sugar is the donor of the alpha-1,3-glycosyltransferase. The cell of claim 50, wherein the galactose (Gal) residue in the Fuc-a1,2-Gal-R is via a beta-1,3 or a beta-1,4 glycosidic linkage combined with R. The cell of any one of claims 50 or 51, wherein the R comprises a monosaccharide, a disaccharide, an oligosaccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a lipid or a glycolipid (glycolipid). The cell according to any one of claims 50 to 52, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-R, preferably the Fuc-a1,2-Gal -R is Fuc-a1,2-Gal-b1,3-GlcNAc-R. The cell of claim 53, wherein the N-acetylglucosamine (GlcNAc) residue in the Fuc-a1,2-Gal-b1,3-GlcNAc-R is via a β-1,3 or a β-1 , 4 glycosidic bonds are combined with R. The cell of any one of claims 53 or 54, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-R, preferably, wherein The Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-R, more preferably, wherein Fuc-a1,2-Gal-R is milk- N-fucopentaose I (LNFP-I, Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc). The cell of any one of claims 50 to 52, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,4-R, preferably, wherein the Fuc-a1,2 -Gal-R is Fuc-a1,2-Gal-b1,4-Glc, as required, wherein the Fuc-a1,2-Gal-R is Fuc-a1,2-Gal-b1,4-(Fuc -a1,3)-Glc. The cell of any one of claims 50 to 56, wherein the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is a structure of the tissue blood group antigen (HBGA) system. The cell of any one of claims 50 to 57, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase having a galactose (Gal) residue The base is transferred from UDP-galactose (UDP-Gal) to the terminal "fucose-a1,2-galactose of Fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R)" "-group capacity of a glycosyltransferase. The cell of any one of claims 50 to 55, 57 or 58, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase that is a A glycosyltransferase capable of transferring (Gal) residues from UDP-Gal to the terminal "fucose-al,2-galactose"-group of LNFP-I. The cell of any one of claims 50 to 52, 56, 58, or 59, wherein the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase that has half the Transglycosylation of the ability of a lactose (Gal) residue to transfer from UDP-Gal to the terminal "fucose-al,2-galactose"-group of Fuc-a1,2-Gal-b1,4-Glc The enzyme, optionally, the glucose residue in the Fuc-a1,2-Gal-b1,4-Glc is fucosylated, preferably α-1,3-fucosylated. The cell of any one of claims 50 to 55 or 57 to 59, wherein the α-1,3 glycated form of Fuc-a1,2-Gal-R is an α-1,3 glycated form of milk-N- Fucopentaose I (LNFP-I), which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-a1 ,3-LNFP-I), the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase, and the nucleotide-sugar is UDP-galactose (UDP-Gal) . The cell of any one of claims 50 to 52, 56 or 58 to 60, wherein the α-1,3 glycated form of Fuc-a1,2-Gal-R is an α-1,3 glycated form of Fuc- a1,2-Gal-b1,4-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc, as required, an α-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc, which is Gal-a1,3-(Fuc-a1,2)-Gal-b1,4-(Fuc-a1,3 )-Glc, the α-1,3-glycosyltransferase is an α-1,3-galactosyltransferase, and the nucleotide-sugar is UDP-galactose (UDP-Gal). The cell of any one of claims 58 to 62, wherein the alpha-1,3-galactosyltransferase has a PFAM PF03414 domain, and a. Include the motif with SEQ ID NO: 01 YX[FHMQT]XAXX[ACG][ACG], where X can be any amino acid residue, or b. Include the motif YXQXCXX[ACG][ACG] with SEQ ID NO: 02, where X can be any amino acid residue, or c. Include serial identification numbers such as: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, a polypeptide sequence of any of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, or d. is the serial identification number: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, A functional homologue, variant or derivative of any of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37, having the same number as having a sequence identification number : 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 have at least 80% overall sequence similarity over the full length of any of the alpha-1,3-galactosyltransferase polypeptides, And to the terminal "fucose-al,2-galactose"-group of Fucose-al,2-galactose-R (Fuc-al,2-Gal-R) has a-1,3-galactose Lactosyltransferase activity, or e. is a functional fragment, including from SEQ ID NO: 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37. An oligopeptide sequence of 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues, and to fucose-a1,2-galactose-R (Fuc-a1,2 The terminal "fucose-al,2-galactose"-group of -Gal-R) has a-1,3-galactosyltransferase activity. The cell of any one of claims 50 to 57, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase, which is a - Transfer of acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to fucose-a1,2-galactose-R (Fuc-a1,2-Gal-R ) of the terminal "fucose-a1,2-galactose"-group capable of a glycosyltransferase. The cell of any one of claims 50 to 55, 57 or 64, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase, which is Has a terminal "fucose-a1,2-galactose" that transfers an N-acetylgalactosamine (GalNAc) residue from UDP-N-acetylgalactosamine (UDP-GalNAc) to LNFP-I- A glycosyltransferase with the ability of a group. The cell of any one of claims 50 to 52, 56 or 64, wherein the α-1,3-galactosyltransferase is an α-1,3-N-acetylgalactosamine transferase, which is Has a terminal "fucose-a1,2-galactose"-group that transfers an N-acetylgalactosamine (GalNAc) residue from UDP-GalNAc to Fuc-a1,2-Gal-b1,4-Glc A glycosyltransferase with the ability to group, as needed, the glucose residues in the Fuc-a1,2-Gal-b1,4-Glc are fucosylated, preferably α-1,3 - Fucosification. The cell of any one of claims 50 to 55, 57, 64 or 65, wherein the α-1,3 glycated form of Fuc-a1,2-Gal-R is an α-1,3 glycated form of milk- N-fucopentaose I (LNFP-I), which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc -a1,3-LNFP-I), the α-1,3-glycosyltransferase is an α-1,3-N-acetylgalactosamine transferase, and the nucleotide-sugar is UDP-N - Acetylgalactosamine (UDP-GalNAc). The cell of any one of claims 50 to 52, 56, 64 or 66, wherein the α-1,3 glycated form of Fuc-a1,2-Gal-R is an α-1,3 glycated form of Fuc- a1,2-Gal-b1,4-Glc, which is GalNAc-a1,3-(Fuc-a1,2)-Gal-b1,4-Glc (α-Galose or A-Galose), as required Define an α-1,3 glycosylated form of Fuc-a1,2-Gal-b1,4-(Fuc-a1,3)-Glc, which is GalNAc-a1,3-(Fuc-a1,2)-Gal- b1,4-(Fuc-a1,3)-Glc, the α-1,3-glycosyltransferase is an α-1,3-N-acetylgalactosamine transferase, and the nucleotide-sugar It is UDP-N-acetylgalactosamine (UDP-GalNAc). The cell of any one of claims 64 to 68, wherein the α-1,3-N-acetylgalactosamine transferase has a PFAM PF03414 domain, and a. Include the motif YX[ACIL]XGXX[ACG][ACG] with SEQ ID NO: 38, where X can be any amino acid residue, or b. Include the motif YX[AG]XAXX[ACG][ACG] with SEQ ID NO: 39, where X can be any amino acid residue, or c. Include such as serial identification numbers: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, a polypeptide sequence of any one of 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, or d. is the serial identification number: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, a functional homologue, variant or derivative of any of 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102, With and with serial identification numbers: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102 of the α-1,3-N-acetylgalactosyltransferase ( a-1,3-N-acetylgalactosyltransferase) polypeptides have at least 80% overall sequence similarity over the full length of any of the polypeptides and are The terminal "fucose-a1,2-galactose" group of R) has a-1,3-N-acetylgalactosyltransferase activity, or e. is a functional fragment, including from SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 or 102. An oligopeptide sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 consecutive amino acid residues, and to fucose-a1,2-galactose-R The terminal terminal "fucose-al,2-galactose" group of (Fuc-a1,2-Gal-R) has a-1,3-N-acetylgalactosyltransferase activity. The cell of any one of claims 55, 59, 61, 63, 65, 67 or 69, wherein fucose is transferred from GDP-fucose to milk-N by the action of a glycosyltransferase -The terminal galactose residue of saccharide (LNT, Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc), the LNFP-I is synthesized in the cell, and the glycosyltransferase is a . an alpha-1,2-fucosyltransferase (alpha-1,2-fucosyltransferase), selected from including from Brachyspira trichomes with UniProt ID A0A2N5RQ26, Dysgonomonas mossii with UniProt ID F8X274, with UniProt ID G8QLF4 List of polypeptides of Dechlorosoma suillum , Desulfovibrio alaskensis with UniProt ID Q316B5 and Polaribacter vadi with UniProt ID A0A1B8TNT0, or b. α-1,2-fucosylation to terminal galactose residues of LNT Enzyme Activity Polypeptide from Brachyspira trichomes (UniProt ID A0A2N5RQ26), Polypeptide from D. mossii (UniProt ID F8X274), Polypeptide from D. suillum (UniProt ID G8QLF4), Polypeptide from D. alaskensis A functional fragment of either the polypeptide of (UniProt ID Q316B5) and the polypeptide from P. vadi (UniProt ID A0A1B8TNTO), or c. Brachyspira trichomoniasis with UniProt ID A0A2N5RQ26, with UniProt ID A functional homolog, variant or of any of the polypeptides of D. mossii with F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5, and P. vadi with UniProt ID A0A1B8TNT0 Derivatives with Brachyspira trichomes from UniProt ID A0A2N5RQ26, D. mossii with UniProt ID F8X274, D. suillum with UniProt ID G8QLF4, D. alaskensis with UniProt ID Q316B5 and P. alaskensis with UniProt ID A0A1B8TNT0, respectively The full length of any of the polypeptides of vadi at least 80% overall sequence similarity, and has α-1,2-fucosyltransferase activity on the terminal galactose residue of lacto-N-tetraose (LNT), or d. a multi-peptide comprising An amino acid sequence, or consisting of an amino acid sequence having as many as the polypeptide from Brachyspira trichomes (UniProt ID A0A2N5RQ26), from D. mossii (UniProt ID F8X274) The full length of any of the peptide, the polypeptide from D. suillum (UniProt ID G8QLF4), the polypeptide from D. alaskensis (UniProt ID Q316B5), and the polypeptide from P. vadi (UniProt ID A0A1B8TNT0) The amino acid sequence has at least 80% sequence similarity and has α-1,2-fucosyltransferase activity on the terminal galactose residue of LNT. The cell of any one of claims 50 to 70, wherein the cell is modified in the expression or activity of a glycosyltransferase. The cell of any one of claims 50 to 71, wherein the cell expresses a membrane transporter or a polypeptide having transport activity to transport the compound across the outer membrane of the cell wall. The cell of claim 72, wherein the membrane transporter or the polypeptide having transport activity is selected from a list including transporter, P-P-bond-hydrolysis-driven transporter, b-barrel porin, cotransporter , putative transporters and phosphotransfer driven group translocators, Preferably, the transporter includes MFS transporter, sugar efflux transporter and siderophore exporters, or Preferably, the P-P-bond-hydrolysis-driven transporter includes ABC transporter and chelatin exporter. The cell of any one of claims 72 or 73, wherein the membrane transporter or polypeptide having transport activity controls the α-1,3 glycosylated form of Fuc-a1,2-Gal-R and/or is used for The alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R produces one or more precursors and/or the flow of acceptors on the outer membrane of the cell wall. The cell of any one of claims 72 to 74, wherein the membrane transporter or polypeptide having transport activity provides improved production of the α-1,3 glycosylated form of Fuc-a1,2-Gal-R and/or activated and/or enhanced outflow. The cell of any one of claims 50 to 75, wherein the cell line is modified with a gene expression module, characterized in that expression from any of the expression modules is constitutive or created by a natural inducer. The cell of any one of claims 50 to 76, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein. The cell of any one of claims 50 to 77, wherein the cell comprises a modification for reducing the production of acetic acid. The cell of any one of claims 50 to 78, wherein the cell comprises lower or reduced expression and/or eliminated, impaired, reduced or delayed activity of any or more proteins, the Any or more of the proteins include beta-galactosidase, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine arrestin, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecene-phosphoglucose-1-phosphotransferase, L-fucokinase, L-rock Fucose isomerase, N-acetylneuraminic acid dissociation enzyme, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man , EIID-Man, ushA, galactose-1-phosphate uridylate transferase, glucose-1-phosphate adenylyl transferase, glucose-1-phosphatase, ATP-dependent fructose-6-phosphate activase isoenzyme 1. ATP-dependent fructose-6-phosphate activating enzyme isoenzyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional inhibitory protein IclR, lon protease, glucose-specific translocation phosphotransferase IIBC components ptsG, glucose-specific translocation phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIA ^Glc , β-glucoside-specific PTS enzyme II, fructose-specific PTS polyphosphate transfer proteins FruA and FruB, alcohol dehydrogenase aldehyde Dehydrogenase, pyruvate formate lyase, acetate kinase, phosphoacyltransferase, phosphoacetyltransferase, pyruvate decarboxylase. The cell of any one of claims 50 to 79, wherein the cell has the ability to produce phosphoenolpyruvate (PEP). The cell of any one of claims 50 to 80, wherein the cell is modified to enhance the production and/or supply of phosphoenolpyruvate (PEP). The cell of any one of claims 50 to 81, wherein the cell comprises an at least partially inactivated catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide that participates in and/or is affected by This production of the alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R is required. The cell of any one of claims 50 to 82, wherein the cell is resistant to the phenomenon of lactose killing when grown in an environment in which lactose is combined with one or more other carbon sources. The cell of any one of claims 50 to 83, wherein the cell produces 90 g/L or more of the alpha-1,3 glycated form of Fuc in whole broth and/or supernatant -a1,2-Gal-R, and/or wherein in the whole broth and/or supernatant, the α-1,3 glycated form of Fuc-a1,2-Gal-R is based on the α-1,3 The saccharified form of Fuc-al,2-Gal- and its precursors, measured in total in the whole broth and/or supernatant, respectively, were at least 80% pure. The cell of any one of claims 50 to 84, wherein the cell produces a charged, preferably sialylated and/or neutral mixture of disaccharides and oligosaccharides comprising an alpha-1,3 glycosylated form of Fuc -a1,2-Gal-R. A cell according to any one of claims 50 to 85, wherein the cell produces a mixture of charged, preferably sialylated and/or neutral oligosaccharides comprising an α-1,3 glycosylated form of Fuc-a1, 2-Gal-R. The method of any one of claims 1 to 49 or the cell of any one of claims 50 to 86, wherein the cell is a bacterium, fungus, yeast, a plant cell, an animal cell or a protozoan cell (protozoan cell), preferably, the bacteria is an Escherichia coli ( Escherichia coli ) strain (strain), more preferably an Escherichia coli strain, which is a K-12 strain, still more preferably, the Escherichia coli K- 12 strains were Escherichia coli MG1655. Preferably, the fungus belongs to a genus selected from the genus Rhizopus , Dictyostelium , Penicillium , Mucor or Aspergillus ), preferably, the yeast belongs to a genus selected from the group consisting of Saccharomyces , Zygosaccharomyces , Pichia , Kemagtler ( Komagataella ), Hansenula , Yarrowia , Starmerella , Kluyveromyces or Debaromyces The group, preferably, the plant cell is an algal cell or is derived from tobacco (tobacco), alfalfa (alfalfa), rice (rice), tomato, cotton, rapeseed (rapeseed), soybean, Maize or corn plants, preferably, the animal cell line is derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians Amphibians or insects, or a genetically modified cell line derived from human cells excluding embryonic stem cells, more preferably the human and non-human mammalian cells are an epithelial cell, One embryonic kidney cell (embryonic kidney cell), one fibroblast cell (fibroblast cell), one COS cell, one Chinese hamster ovary (Chinese hamster ovary, CHO) cell, one murine myeloma cell (murine myeloma cell), one NIH- 3T3 cells, a non-mammary adult stem cell (non-mammary adult stem cell) or its derivatives, more preferably the insect cell line is derived from fall armyworm ( Spodoptera frugiperda ), silkworm ( Bombyx mori ), cabbage armyworm ( Mamestra ) brassicae ), Trichoplusia ni or Drosophila melanogaster , preferably, the protozoan cell is a tarantula Leishmania tarentola e ) cells. The method of any one of claims 1 to 49 and 87, or the cell of any one of claims 50 to 87, wherein the cell is a bacterium, preferably an E. coli strain, more preferably a K One cell of one Escherichia coli strain of 12 strains, still more preferably, the Escherichia coli K-12 strain is Escherichia coli MG1655. The method of claim 88, or the cell of claim 88, wherein the cell is a live Gram-negative bacterium comprising a reduced or eliminated poly-N-acetyl-glucosamine ( poly-N-acetyl-glucosamine, PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, osmoregulatory periplasmic glucan ( Synthesis of Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose. The method of any one of claims 1 to 4 and 87, or the cell of any one of claims 50 to 87, wherein the cell is a yeast cell. The method of any one of claims 1 to 49 and 87 to 90, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partition ( two-phase partitioning), reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion ( enzymatic digestion), tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction Chromatography (hydrophobic interaction chromatography) and/or gel filtration (gel filtration), ligand exchange chromatography (ligand exchange chromatography). The method of any one of claims 1 to 49 and 87 to 91, further comprising Fuc-a1,2-Gal-R in any of the α-1,3 glycosylated forms from the cell, preferably from the cell Purification of the α-1,3 glycosylated form of LNFP-I. The method of any one of claims 1 to 49 and 87 to 92, wherein the purification comprises at least one of the following steps: use of activated charcoal or carbon, charcoal, nanofiltration, ultrafiltration, electrophoresis , use of enzymes or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, lyophilization, spray freeze drying freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drum drying, roller drying, vacuum drum drying or vacuum roller drying. A cell according to any one of claims 50 to 90, or use of the method according to any one of claims 1 to 49 or 87 to 93, for the α-1,3 glycosylated form of Fuc-a1,2 -Gal-R, preferably the production of an alpha-1,3 glycosylated form of LNFP-I.