Chimeric glycosyltransferases

Abstract

The present invention relates to chimeric GT-B type glycosyltransferases, comprising an N-terminal acceptor binding site, a C-terminal donor binding site, and a linker loop linking the N-terminal acceptor binding site and the C-terminal donor binding site, wherein (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and / or (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 4; and / or (iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and / or (iv) the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a glycosyltransferase distinct from the glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence; to nucleic acid molecules encoding such GT-B type glycosyltransferases; to a method of production of such chimeric GT-B type glycosyltransferases; to a method of production of polysaccharides using such chimeric GT-B type glycosyltransferases and the polysaccharides obtainable by such a method; to polysaccharides defined by formula (I); to compositions comprising such polysaccharides; and further to uses of such polysaccharides as thickening and / or gelling agents and / or as rheology modifiers and / or as 3D-printing inks and / or coatings.

Description

[0001] Universitat Munster

[0002] Our ref.: U08363WO / LH

[0003] CHIMERIC GLYCOSYLTRANSFERASES

[0004] TECHNICAL FIELD

[0005] The present invention relates to polypeptides, comprising an N-terminal acceptor binding site, a C-terminal donor binding site, and a linker loop linking the N-terminal acceptor binding site and the C-terminal donor binding site, wherein (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and / or (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 4; and / or (iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and / or (iv) the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a glycosyltransferase distinct from the glycosyltransferase from which the N- terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence; to nucleic acid molecules encoding such polypeptides; to a method of production of such polypeptides; to a method of production of polysaccharides using such polypeptides and the polysaccharides obtainable by such a method; to polysaccharides defined by formula (I); to compositions comprising such polysaccharides; and further to uses of such polysaccharides as thickening and / or gelling agents and / or as rheology modifiers and / or as 3D-printing inks and / or coatings.

[0006] BACKGROUND OF THE INVENTION

[0007] Xanthan represents one of the most important commercial microbially-generated polysaccharides and is produced in multi thousand-ton scale per year. Particularly, it is produced by bacteria of the genus Xanthomonas, most often by microorganisms of the species X. campestris. Due to its unusual physical properties, i.e., its extremely high specific viscosity and its pseudoplasticity, particularly its thickening properties, it is widely used in various applications, ranging from food, feed, agricultural, and technical to pharmaceutical applications. For instance, it is used as a thickening or rheology modifying agent in foods and pharmaceutical applications, in paper and textile finishing applications, as well as in the oil industry in the context of petroleum extraction.

[0008] Xanthan is an anionic heteropolysaccharide. The repeating unit of the polymer is a pentamer composed of five sugar moieties, specifically two glucose, one glucuronic acid and two mannose units. These sugar residues are arranged such that the D-glucose moieties (beta, 1-4-linked) form the backbone of the polymer chain, with side chains (alpha, 1-3 linked) of beta-D-mannose-(1-4)-beta-D-glucuronic acid-(1-2)-alpha-D- mannose residues generally extending from alternate glucose moieties. Usually, this basic structure is specifically acetylated and pyruvylated, as described, for example, by Janson et al. (Structure of

[0009] LH:KA extracellular polysaccharide from Xanthomonas campestris, Carbohydrate Research, 1975, 45, 275-282). The extent of acetylation and pyruvylation is known to vary. It is thus generally accepted that xanthan consists of D-glucosyl, D-mannosyl, and D-glucuronyl acid residues in a molar ratio of 2:2:1 and variable proportions of O-acetyl and pyruvyl residues.

[0010] The biosynthesis of xanthan is one of the best-explored biosynthesis of microbial polysaccharides. Various glycosyltransferases (GTs) specifically transfer sugars from donor molecules to the growing repeating unit of the polymer, i.e., the acceptor molecule.

[0011] However, despite broad utility of xanthan, its native chemical structure is not perfectly suited for certain specific applications, which are mainly reliant on the rheological behavior of xanthan, particularly in the presence of salts.

[0012] To overcome these limitations, modified xanthan structures are designed and generated based on chemical or genetic engineering approaches. These approaches include the chemical (de)acetylation and / or (de)pyruvylation of native xanthan.

[0013] In addition, first approaches have been described, which use gene deletions of GTs to modify the side chain by deleting specific sugar moieties. However, several drawbacks are connected to said methods: Chemical modification of xanthan is performed under harsh conditions, resulting in non-homogenous final products. (De)acetylation and (de)pyruvylation via genetic engineering mitigate these issues to some degree but are still based and dependent on the chemical structure of the native polymer monomeric sugars and can thus affect the rheological properties of xanthan only to a small extent, which holds also applies to the removal of sugar moieties from the side chains. There is thus a prevailing need in the field for modified versions of native xanthan.

[0014] SUMMARY OF THE INVENTION

[0015] The present invention addresses this need by providing for chimeric GT-B type glycosyltransferases (GTBs), nucleic acids encoding the same, methods for the production of such chimeric GT-B type GTs, methods for the production polysaccharides using said chimeric GT-B type GTs, polysaccharides obtainable by such methods and as described herein.

[0016] The polypeptides, e.g., the chimeric GT-B type glycosyltransferases, of the present invention are capable of generating polysaccharides that are derivatives of native xanthan in featuring, instead of the terminal side chain D-mannosyl moiety, a different monosaccharide, the precise structure of which depends on the respective type of GT that is used for C-terminal donor binding site domain swapping, as will be explained in more detail herein below.

[0017] The polypeptides of the present invention are thus able to generate modified xanthan molecules that exhibit different rheology properties and thickening behavior, and that can even be tailored to retain their advantageous rheological properties in the presence of salts, which broadens the area of possible applications. In a first aspect, the present invention therefore relates to a polypeptide, said polypeptide comprising an N- terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C-terminal donor binding site domain, wherein

[0018] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and / or

[0019] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 4; and / or

[0020] (iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and / or

[0021] (iv) the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a GT-B type glycosyltransferase distinct from the GT- B type glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same GT-B type glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence, preferably through a peptide bond.

[0022] In various embodiments, the polypeptide is a chimeric GT-B type glycosyltransferase. Particularly, the polypeptide, i.e., the chimeric GT-B type glycosyltransferase, of the present invention is characterized in that it is capable of catalyzing the transfer of a monosaccharide moiety from a donor (glycosyl donor molecule), such as nucleotide-sugar or lipid-phospho-sugar donors, to an acceptor substrate (glycosyl acceptor molecule) selected from monosaccharides, disaccharides, polysaccharides, lipids and proteins, preferably polysaccharides.

[0023] The polypeptide of the present invention can, in some embodiments, be isolated, cytosolic or membranebound. In various embodiments, the present invention thus relates to an isolated polypeptide, i.e., an isolated chimeric GT-B type glycosyltransferase, that may have been recombinantly produced and thus may be a recombinant isolated polypeptide, e.g., recombinant isolated chimeric GT-B type glycosyltransferase.

[0024] In various embodiments, the polypeptide of the present invention is capable of catalyzing the transfer of a monosaccharide moiety from a donor, preferably a sugar nucleotide or lipid-phospho-sugar, to p-D-GIcA- (1 ^2)-a-D-Man-(1 ^3)-p-D-Glc-(1 ^4)-D-Glc, which preferably may be linked to a carrier molecule, such as an isoprenoid lipid carrier molecule, optionally through bound through a pyrophosphate linking group, such as in the form of p-D-GlcA-(1 -^2)-a-D-Man-(1 -^3)-p-D-Glc-(1 — >4)-D-Glc-1 -diphospho-ditrans.octacis- undecaprenol. In some embodiments, the N-terminal acceptor binding site domain and the C-terminal donor binding site domain originate from different GT-B type glycosyltransferases. In some such embodiments, the C-terminal donor binding site domain is not a chimeric C-terminal donor binding site domain as defined in item (iv) herein above).

[0025] In some embodiments, the N-terminal acceptor binding site domain and the linker loop originate from different GT-B type glycosyltransferases.

[0026] In some embodiments, the C-terminal donor binding site domain and the linker loop originate from different GT-B type glycosyltransferases.

[0027] In various embodiments, the N-terminal acceptor binding site domain and the linker loop originate from different GT-B type glycosyltransferases or the C-terminal donor binding site domain and the linker loop originate from different GT-B type glycosyltransferases.

[0028] In some embodiments, the N-terminal acceptor binding site domain and the linker loop originate from identical GT-B type glycosyltransferases and the C-terminal donor binding site domain originates from a different GT-B than the N-terminal acceptor binding site domain and the linker loop. In some such embodiments, the C-terminal donor binding site domain is not a chimeric C-terminal donor binding site domain as defined in item (iv) herein above.

[0029] In various embodiments, variants comprising an N-terminal acceptor binding site domain that has less than 100 % sequence identity to the amino acid set forth in SEQ ID NO: 1 retain their functionality in that the N- terminal acceptor binding site domain functions as an acceptor binding site to the same extent as the domain having the exact sequence of SEQ ID NO: 1 or retains at least 80 %, preferably at least 90 % of this activity. In various embodiments, variants comprising a C-terminal donor binding site domain that has less than 100 % sequence identity to the amino acid set forth in SEQ ID NO: 2 retain their functionality in that the C-terminal donor binding site domain functions as a donor binding site to the same extent as the domain having the exact sequence of SEQ ID NO: 2 or retains at least 80 %, preferably at least 90 % of this activity. Likewise, in various embodiments, variants comprising a C-terminal donor binding site domain that has less than 100 % sequence identity to the amino acid set forth in SEQ ID NO: 3 retain their functionality in that the C-terminal donor binding site domain functions as a donor binding site to the same extent as the domain having the exact sequence of SEQ ID NO: 3 or retains at least 80%, preferably at least 90 % of this activity.

[0030] In various embodiments, the linker loop consists of 28 to 36 amino acid residues, preferably of 29 to 36 amino acid residues, for instance, 30 to 35 amino acid residues.

[0031] In some embodiments, the polypeptide according to the present invention comprises or consists of an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

[0032] In some embodiments, the polypeptide is characterized in that the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain as defined in item (iv) above and further in that the second amino acid sequence of the chimeric C-terminal donor binding site domain constitutes about 5 to 70 %, such as about 8 to 20 %, or 10 to 15 % or 10 to 30 %, or 20 to 40 %, or 30 to 50 %, or 40 to 60 %, or 50 to 70 %, of the entire length of the chimeric C-terminal donor binding site domain; and / or the first amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 13; and / or the second amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 14; and / or the polypeptide comprises or consists of amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0033] In some embodiments, the polypeptide comprising the chimeric C-terminal donor binding site domain is further characterized in that

[0034] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and / or

[0035] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4.

[0036] In some embodiments, the polypeptide comprising the chimeric C-terminal donor binding site domain is further characterized in that

[0037] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0038] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID In a further aspect, the present invention relates to a nucleic acid encoding the polypeptide of the present invention.

[0039] In another aspect, the present invention also relates to a vector comprising a nucleic acid molecule according to the present invention, wherein the vector may be a plasmid.

[0040] In yet another aspect, the present invention relates to a host cell comprising a nucleic acid molecule according to the present invention or a vector according to the present invention.

[0041] The host cell may be a prokaryotic host cell.

[0042] In another aspect, the present invention relates to a method for the production of a polypeptide of the present invention, comprising

[0043] (1) cultivating the host cell of the invention under conditions that allow the expression of the polypeptide;

[0044] (2) optionally isolating the expressed polypeptide from the host cell.

[0045] In various embodiments, said method is a biotechnological method.

[0046] In yet another aspect, the present invention further relates to a method for the production of a polysaccharide using the polypeptide according to the present invention.

[0047] In some embodiments, the method comprises

[0048] (a) optionally expressing a polypeptide of the invention in a suitable host cell; and

[0049] (b) contacting said polypeptide with at least one suitable donor substrate (glycosyl donor molecule) and at least one suitable acceptor substrate (glycosyl acceptor molecule) under conditions that allow the enzymatic production of the polysaccharide, wherein the polysaccharide is preferably xanthan or a xanthan derivative.

[0050] In various embodiments, the donor substrate is a nucleotide-sugar or a lipid-phospho-sugar.

[0051] In various embodiments, the acceptor substrate is a polysaccharide, preferably a polyprenyl phospho oligosaccharide, such as an oligosaccharide bound to an undecaprenylphosphate lipid carrier, more preferably p-D-GlcA-(1 -^2)-a-D-Man-(1 -^3)-p-D-Glc-(1 — >4)-D-Glc-1 -diphospho-ditrans.octacis- undecaprenol.

[0052] In various embodiments, the method further comprises a step of isolating the produced polysaccharide.

[0053] The host cell may be Xanthomonas campestris. In some embodiments, the host cell is Xanthomonas campestris with or without deletion of the terminal mannose-transferring GTB Guml.

[0054] In still another aspect, the present invention relates to a polysaccharide obtainable by a method of the present invention. In a still further aspect, the present invention also relates to a polysaccharide of the following formula (I): -glucose — D-glucose - - | J n

[0055] D-mannose

[0056] D-glucuronic acid wherein the D-glucose moieties are linked in a p-[1 ,4] configuration; the D-mannose moiety, optionally acetylated at the 6-0 position, is linked in an a-[1 ,3] configuration to the D-glucose moiety; the D-glucuronic acid moiety is linked in a p-[1 ,2] configuration to the D-mannose moiety;

[0057] X is a monosaccharide moiety or a monosaccharide derivative moiety, wherein may be optionally substituted and wherein further optionally one or more, preferably one or two, monosaccharide hydroxy groups may be replaced by halogen, alkyl, preferably Ci-Ce alkyl, more preferably methyl or ethyl, alkoxy, preferably methoxy, monocarboxylic acids having 2 to 8 carbon atoms, preferably acetyl, and dicarboxylic acids having 2 to 9 carbon atoms, preferably pyruvyl, wherein the dicarboxylic acid may optionally be ketal- linked to the monosaccharide, NH2, -NHR1, and -N(R1)2, wherein each R1is independently selected from alkyl, preferably C1-C6 alkyl, monocarboxylic acids having 2 to 8 carbon atoms, preferably acetyl, and dicarboxylic acids having 2 to 9 carbon atoms, preferably pyruvyl, wherein the dicarboxylic acid may optionally be ketal-linked to the monosaccharide; wherein each alkyl, alkoxy, monocarboxylic acid, and dicarboxylic acid group may be independently substituted with one or more substituents selected from halogen, OH, CH3, OCH3, SH, SCH3, NH2, NHCH3, N(CH3)2; n is an integer from 2 to 2000; with the proviso that X is not a p-[1 ,4]-li nked D-mannose moiety or a p-[1 ,4]-lin ked D-mannose moiety containing a ketal-linked pyruvic acid at the 4,6 position; or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

[0058] In some embodiments, the polysaccharide of the present invention is characterized in that X is selected from a pentose, a hexose, a heptose, or an octose, as well as from their respective uronic acids, ulosonic acids, aldonic acids and aldaric acids, as well as from their respective amino sugar derivatives, preferably selected from a pentose and a hexose or a uronic acid or aminosugar derived therefrom, more preferably selected from ribose, arabinose, cladinose, xylose, lyxose, deoxyribose, ribulose, xylulose, allose, altrose, glucose, fructose, mannose, mannuronic acid, guluronic acid, glucuronic acid, sorbose, gulose, idose, iduronic acid, psicose, galactose, talose, tagatose, fuculose, fucose, rhamnose, qinovose, ketodeoxyoctulosonic acid, neuraminic acid, glucosamine, N-acetyl-glucosamine, muramic acid, N- acetylmuramic acid, galactosamine, quinovosamine, rhamnosamine, 2-deoxy-glucose, fluorodeoxyglucose, 6-deoxyfructose, 1 ,6-dichlorofructose, 3,6-anhydrogalactose, 1-O-methyl galactose, 6-O-methyl galactose, 1-O-methyl glucose, 1-O-methyl fructose, 3-O-methyl fructose, sedoheptulose, mannoheptulose, 2-keto-3-deoxy-mannooctanoic acid, and sialinic acids, such as N-acetylneuraminic acid and N-glycolylneuraminic acid.

[0059] In various embodiments, X is selected from glucose, galactose, xylose, / V-acetyl-glucosamine, / V-acetyl- galactosamine, glucuronic acid, galactofuranose, L-mannose, fucose, rhamnose, ketodeoxyoctulosonic acid, neuraminic acid, sialic acids such as / V-acetylneuraminic acid and / V-glycolylneuraminic acid, and 2- keto-3-deoxy-mannooctanoic acid. In some embodiments, X is not D-mannose. In some embodiments, X is not L-mannose. In some embodiments, X is not mannose. In some embodiments, X is galactose or glucuronic acid.

[0060] In yet another aspect, the present invention relates to the use of a polysaccharide according to the present invention as a thickening and / or gelling agent and / or as a rheology modifier, preferably in foods and / or feeds, cosmetics, medicinal and pharmaceutical formulations, inks and printing formulations, sizings for papers and / or textiles, drilling muds, or concrete or further technical applications, such as film compositions, and coating compositions.

[0061] In a final aspect, the present invention relates to a composition comprising the polysaccharide of the present invention, wherein preferably the composition is selected from a food composition, a feed composition, a cosmetic composition, a medicinal composition, a pharmaceutical composition, an ink composition, a printing composition, a paper sizing composition, a textile sizing composition, a drilling mud composition, or a concrete composition as well as films and coatings made from such formulations (e.g., film compositions and coating compositions).

[0062] BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 depicts the repeating enzymatic steps for the biosynthesis of xanthan, i.e., the assembly of the pentasaccharide repeat unit of xanthan linked to an isoprenoid lipid carrier molecule. The assembly of this pentasaccharide is shown to proceed by sequential addition, in a specific and defined order, of the five individual sugar moieties that are comprised in the pentasaccharide repeat unit. Each unique sugar addition step is catalyzed by a specific enzyme specific to that particular step. The sugars are donated by sugar nucleotides. The sequential enzymatic addition steps are numbered from l-V, wherein in step V the transfer of a p-mannosyl unit from GDP-a-D-mannose to the p-D-GlcA-(1 ^2)-a-D-Man-(1 ^3)-p-D-Glc-(1 ^4)-D- Glc unit linked to the carrier molecule is catalyzed. Typically, the carrier molecule, an undecaprenylphosphate lipid carrier, is ditrans, octacis-undecaprenol, linked to the oligosaccharide via a diphospho group.

[0064] Figure 2 depicts concentrations of sugar monomers glucose, mannose and galactose in different xanthan variants produced by campestris Aguml (producing “xanthan Aguml”), X. campestris Aguml pSRK_GT1 (producing “xanthan Aguml GT 1 ”), X. campestris Agum / pSRK_GumlNT_GT1 CT+loop (producing “xanthan Aguml GumlNT_GT1 CT+loop”), and X. campestris Aguml pSRK_GumlNT+loop_GT1 CT (producing “xanthan Aguml GumlNT+loop_GT1 CT”); determined according to PMP-methode. “Aguml" refers to gene alteration within the genome of the respective organism by deletion of the gene guml, coding for a GT, which catalyzes attachment of the terminal mannose moiety within the xanthan side chain, the respectively produced xanthan derivative being referred to as “xanthan Agum , “Aguml pSRK_GT 1 ” refers to the mutant which, in addition to gene deletion of guml, performs plasmid-based expression of GT1 from Kozakia baliensis, the respective GT catalyzing attachment of glucuronic acid within the side chain of xanthan in K. baliensis, the respectively produced xanthan derivative being referred to as “xanthan Aguml GT1”; “Agum\ pSRK_GumlNT_GT1CT+loop” refers to the mutant which, in addition to gene deletion of guml, performs plasmid-based expression of a chimeric GT consisting of the N-terminal domain of Guml GT from X. campestris, the C-terminal domain of GT1 from K. baliensis, and the linker loop from GT1 linking the N- terminal domain with the C-terminal domain, the respectively produced xanthan derivative being referred to as “xanthan Aguml GumlNT_GT1CT+loop”; “Aguml pSRK_GumlNT+loop_GT1CT” refers to the mutant which, in addition to gene deletion o guml, performs plasmid-based expression of a chimeric GT consisting of the N-terminal domain of Guml GT from X. campestris, the C-terminal domain of GT1 from K. baliensis, and the linker loop from Guml linking the N-terminal domain with the C-terminal domain, the respectively produced xanthan derivative being referred to as “xanthan Aguml GumlNT+loop_GT1CT”.

[0065] Figure 3 depicts concentrations of sugar monomers glucose, mannose, and galactose in different xanthan variants produced by campestris Aguml (producing “xanthan Aguml’"), X. campestris Aguml pSRK_GT1 (producing “xanthan Aguml GT1”), and X. campestris Aguml pSRK_GumlNT+loop 2_GT1CT (producing “xanthan Aguml GumlNT+loop 2_GT1CT”), and X. campestris Aguml pSRK_GumlNT+loop 3_GT1CT (producing “xanthan Aguml GumlNT+loop 3_GT1 CT”); wherein “loop 2” indicates the linker loop shortened by 5 amino acids at the C-terminal end and “loop 3” indicates the linker loop shortened by 10 amino acids at the C-terminal end.

[0066] Figure 4 depicts concentrations of glucuronic acid in different xanthan variants produced byX campestris Aguml (producing “xanthan Aguml") and X campestris Aguml pSRK_GumlNT+loop_GumKCT (producing “xanthan Aguml GumlNT+loop_GumKCT”); “Aguml pSRK_GumlNT+loop_GumKCT” refers to the mutant which, in addition to gene deletion of guml, performs plasmid-based expression of a chimeric GT consisting of the N-terminal domain of Guml GT from X campestris, the C-terminal domain of GumK from Xanthomonas campestris, and the linker loop from Guml linking the N-terminal domain with the C-terminal domain, the respectively produced xanthan derivative being referred to as “xanthan Aguml GumlNT+loop_GumKCT”.

[0067] Figure 5 depicts viscosity curves of 1 % xanthan solution from different xanthan derivatives at shear rates between 0.05 and 16 s-1(logarithmic scale). 1% (w / v) xanthan samples in ultra pure water were analysed with a Modular Compact Rheometer 302 from Anton Paar equipped with a CP 50-1 cone-and-plate measuring system, 50 mm diameter, 1 ° cone angle and 50 pm cone truncation (Anton Paar GmbH, Austria) and a Peltier controlled TEK 150 P temperature unit (Anton Paar GmbH, Austria). All measurements were performed at 20 °C and all samples were incubated in the measuring system at 20 °C for 5 min prior to the measurement “dguml” refers to xanthan produced by the strain X. campestris Aguml, which is missing the terminal mannose. GumIGTI chimera refers to a xanthan variant produced by the strain X. campestris Aguml which also expresses the chimeric GT GumlNT+loop_GT1CT. GumIGumK chimera refers to a xanthan variant produced by the strain X. campestris Aguml which also expresses the chimeric GT GumlNT+loop_GumKCT. Figure 6 depicts Frequency sweeps of 1 % xanthan solutions from xanthan variants chimera at circular frequencies between 0.6 and 63 rad / s (logarithmic scale). 1 % (w / v) xanthan samples in ultra pure water were analysed with a Modular Compact Rheometer 302 from Anton Paar equipped with a CP 50-1 cone- and-plate measuring system, 50 mm diameter, 1 ° cone angle and 50 pm cone truncation (Anton Paar GmbH, Austria) and a Peltier controlled TEK 150 P temperature unit (Anton Paar GmbH, Austria). All measurements were performed at 20 °C and all samples were incubated in the measuring system at 20 °C for 5 min prior to the measurement “dguml” refers to xanthan produced by the strain X. campestris Aguml, which is missing the terminal mannose. GumIGTI chimera refers to a xanthan variant produced by the strain X. campestris Aguml which also expresses the chimeric GT GumlNT+loop_GT1 CT. GumIGumK chimera refers to a xanthan variant produced by the strain X. campestris Aguml which also expresses the chimeric GT GumlNT+loop_GumKCT.

[0068] Figure 7 depicts temperature sweeps of 1 % xanthan solutions. 1 % (w / v) xanthan samples in ultra pure water were analysed with a Modular Compact Rheometer 302 from Anton Paar equipped with a CP 50-1 cone-and-plate measuring system, 50 mm diameter, 1 ° cone angle and 50 pm cone truncation (Anton Paar GmbH, Austria) and a Peltier controlled TEK 150 P temperature unit (Anton Paar GmbH, Austria). All samples were incubated in the measuring system at 20 °C for 5 min prior to the measurement “dguml” refers to xanthan produced by the strain X. campestris Aguml, which is missing the terminal mannose. GumIGTI chimera refers to a xanthan variant produced by the strain X. campestris Aguml which also expresses the chimeric GT GumlNT+loop_GT1 CT. GumIGumK chimera refers to a xanthan variant produced by the strain X. campestris Aguml which also expresses the chimeric GT GumlNT+loop_GumKCT.

[0069] Figure 8 depicts viscosity sweeps of 1 % xanthan solutions, GumIGumK chimera variant (‘Aguml pSRK_GumlNT+loop_GumKCT”) with NaCI or CaCh. 1 % (w / v) xanthan samples in ultra pure water were analysed with a Modular Compact Rheometer 302 from Anton Paar equipped with a CP 50-1 cone-and- plate measuring system, 50 mm diameter, 1 ° cone angle and 50 pm cone truncation (Anton Paar GmbH, Austria) and a Peltier controlled TEK 150 P temperature unit (Anton Paar GmbH, Austria). All measurements were performed at 20 °C and all samples were incubated in the measuring system at 20 °C for 5 min prior to the measurement.

[0070] Figure 9 depicts amplitude sweeps of 1 % xanthan solutions, GumIGumK chimera variant (‘Aguml pSRK_GumlNT+loop_GumKCT”) with NaCI or CaCh. 1 % (w / v) xanthan samples in ultra pure water were analysed with a Modular Compact Rheometer 302 from Anton Paar equipped with a CP 50-1 cone-and- plate measuring system, 50 mm diameter, 1 ° cone angle and 50 pm cone truncation (Anton Paar GmbH, Austria) and a Peltier controlled TEK 150 P temperature unit (Anton Paar GmbH, Austria). All measurements were performed at 20 °C and all samples were incubated in the measuring system at 20 °C for 5 min prior to the measurement.

[0071] Figure 10 depicts Temperature sweeps of 1 % xanthan solutions, GumIGumK chimera variant ‘Aguml pSRK_GumlNT+loop_GumKCT”) with NaCI or CaCh 1 % (w / v) xanthan samples in ultra pure water were analysed with a Modular Compact Rheometer 302 from Anton Paar equipped with a CP 50-1 cone-and- plate measuring system, 50 mm diameter, 1 ° cone angle and 50 pm cone truncation (Anton Paar GmbH, Austria) and a Peltier controlled TEK 150 P temperature unit (Anton Paar GmbH, Austria). All samples were incubated in the measuring system at 20 °C for 5 min prior to the measurement.

[0072] DETAILED DESCRIPTION OF THE INVENTION

[0073] Before describing in detail exemplary embodiments of the present invention, definitions that are important for understanding the present invention are given.

[0074] The present invention will be described with respect to particular embodiments and with reference to certain figures, but the invention is not limited thereto but only by the claims.

[0075] As used in this specification and in the appended claims, the singular forms of “a” and “an” also include the respective plurals unless the context clearly dictates otherwise.

[0076] “At least one”, as used herein, relates to one or more, in particular 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. “At least” if used in relation to a numerical value and in particular a list of numerical values relates to each of said separate numerical values and defines said numerical value as the minimum value. “At least 80, 81 , 82, etc.” thus means at least 80, at least 81 , at least 82 and so on.

[0077] In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10 %, preferably ±5 %, more preferably ±2 %.

[0078] The term “monosaccharide” is used herein to designate the monomeric building blocks of carbohydrates in general, more specifically the monomeric building blocks of disaccharides, such as sucrose, lactose, and maltose, oligosaccharides, and polysaccharides, such as cellulose, starch, and xanthan. Preferably, the term “monosaccharide” as used herein refers to triose, tetrose, pentose, hexose, heptose, and octose molecules. The term monosaccharide may be understood to refer to compounds having the chemical formula (CH2O)X, with x being selected from 3, 4, 5, 6, 7, and 8. Monosaccharide derivatives, which may also be referred to as modified monosaccharides, include, without limitation, amino sugars, such as galactosamine, glucosamine, sialic acid, and / V-acetylglucosamine; sulfosugars such as sulfoquinovose; sugar acids, for instance uronic acids, such as glucuronic acid, galacturonic acid, and iduronic acid; ulosonic acids, such as neuramic acid and ketodeoxyoctulosonic acid; aldonic acids, such as gluconic acid and xylonic acid; aldaric acids, such as glucaric acid and xylaric acid. The term “oligosaccharide” is used herein to designate saccharide molecules or moieties containing three to ten monosaccharide moieties and / or monosaccharide derivative moieties. Non-limiting examples of oligosaccharides include raffinose, stachyose, and verbascose. The term “polysaccharide” is used herein to designate saccharide polymers containing more than ten monosaccharide moieties and / or monosaccharide derivative moieties. Nonlimiting examples of polysaccharides include cellulose, chitin, xanthan, amylose, laminarin, xylan, mannan, and galactomannan. The term “peptide” is used throughout the specification to designate a polymer of amino acid residues connected to each other by peptide bonds. The terms “protein” and “polypeptide” are used interchangeably throughout the specification to designate a polymer of amino acid residues connected to each other by peptide bonds. A protein or polypeptide according to the present invention has preferably more than 100 amino acid residues.

[0079] “Nucleic acid” as used herein includes all natural forms of nucleic acids, such as DNA and RNA. Preferably, the nucleic acid molecules of the invention are DNA.

[0080] Generally, the skilled person understands that for putting the present invention into practice any nucleotide sequence described herein may comprise an additional start and / or stop codon or that a start and / or stop codon included in any of the sequences described herein may be deleted, depending on the nucleic acid construct used. The skilled person will base this decision, e.g., on whether a nucleic acid sequence comprised in the nucleic acid molecule of the present invention is to be translated and / or is to be translated as a fusion protein. In various embodiments, the isolated polypeptides of the invention additionally comprise the amino acid M on the N-terminus.

[0081] “Isolated” as used herein in relation to a molecule means that said molecule has been at least partially separated from other molecules it naturally associates with or other cellular components. “Isolated” may mean that the molecule has been purified to separate it from other molecules and components, such as other proteins and nucleic acids and cellular debris, in particular those that accompany it due to its recombinant production in host cells.

[0082] “Membrane-bound” as used herein in relation to a molecule means that said molecule is in a state of association with a membrane, particularly a cell membrane or cell membrane fragments, i.e., a lipid bilayer.

[0083] “Cytosolic” as used herein in relation to a molecule means that said molecule is not associated with a membrane and is freely available in the cytosol of the prokaryotic host cell.

[0084] Determination of the sequence identity of nucleic acid or amino acid sequences can be done by a sequence alignment based on well-established and commonly used BLAST algorithms (See, e.g. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403- 410, and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”; Nucleic Acids Res., 25, S.3389-3402). Such an alignment is based on aligning similar nucleotide or amino acid sequences stretches with each other. Another algorithm known in the art for said purpose is the FASTA algorithm. Alignments, in particular multiple sequence comparisons, are typically done by using computer programs. Commonly used are the Clustal series (See, e.g., Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31 , 3497-3500), T-Coffee (See, e.g., Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217) or programs based on these known programs or algorithms. Also possible are sequence alignments using computer programs such as Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA, USA) with the set standard parameters, with the AlignX module for sequence comparisons being based on the ClustalW. If not indicated otherwise, the sequence identity is determined using the BLAST algorithm.

[0085] Such a comparison also allows the determination of the similarity of the compared sequences. Said similarity is typically expressed in percent identity, i.e., the portion of identical nucleotides / amino acids at the same or corresponding (in an alignment) sequence positions relative to the total number of the aligned nucleotides / amino acids. For example, if in an alignment 90 amino acids of a 100 aa long query sequence are identical to the amino acids in corresponding positions of a template sequence, the sequence identity is 90 %. The broader term “homology” additionally considers conserved amino acid substitutions, i.e. amino acids that are similar regarding their chemical properties, since those typically have similar chemical properties in a protein. Accordingly, such homology can be expressed in percent homology. If not indicated otherwise, sequence identity and sequence homology relate to the entire length of the aligned reference sequence, i.e., in the present case, e.g., SEQ ID NO: 1. In some embodiments, a polypeptide described herein has, over its entire length, the sequence identity as herein defined. For instance, but without limitation, the polypeptide may be a shortened variant of SEQ ID NO: 1 that comprises at least 107 continuous amino acids of the amino acid sequence of SEQ ID NO: 1 (equaling 80 % sequence identity).

[0086] It is to be understood that the term “comprising” is not limiting. For the purposes of the present invention the term “consisting of’ is considered to be a narrower embodiment of the term “comprising”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group that preferably consists of these embodiments only.

[0087] When referring to compositions and the weight percent of the therein comprised ingredients it is to be understood that according to the present invention, the overall amount of ingredients does not exceed 100 % (± 1 % due to rounding).

[0088] It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0089] For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. a polysaccharide is defined to be obtainable from a specific source or by means of a specific method, this is also to be understood to disclose a polysaccharide which is obtained from this source or by this method. The definitions set forth in this section are intended to clarify terms used throughout this application. In this section, the definition applies to compounds of formula (I) unless otherwise stated. The term “herein” means the entire application.

[0090] Unless specified otherwise within this specification, the nomenclature used in this specification generally follows the examples and rules stated in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979, which is incorporated by references herein for its exemplary chemical structure names and rules on naming chemical structures.

[0091] The term “Cm-n” or “Cm-ngroup” used alone or as a prefix, refers to any group having m to n carbon atoms. For instance, a Ci-Ce group refers to a group having 1 to 6 carbon atoms.

[0092] As used in this application, the term “optionally substituted” means that substitution is optional and therefore it is possible for the designated atom to be unsubstituted. In the event a substitution is desired then such substitution means that any number of hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the normal valency of the designated atom is not exceeded, and that the substitution results in a stable compound. For example, when a substituent is oxo ( / .e., =O), then 2 hydrogens on the atom are replaced. When a group is indicated to be “optionally substituted” or “substituted” unless otherwise expressly stated examples of suitable substituents include the following: halogen, SH, amino, alkyl, preferably Ci-Ce alkyl, more preferably methyl, alkoxy, preferably Ci-Ce alkoxy, more preferably OCH3, -S-alkyl, preferably -S(Ci-Ce alkyl), more preferably SCH3, alkenyl, preferably C2- Ce alkenyl, cycloalkyl, preferably cyclopropyl, -OCO-alkyl, -CO-amino, -CO-alkyl, CO2-alkyl, monocarboxylic acids having 2 to 8 carbon atoms, preferably acetyl, and dicarboxylic acids having 2 to 9 carbon atoms, preferably pyruvyl, wherein the dicarboxylic acid may optionally be ketal-linked, -SO(alkyl), -SO2-alkyl, -S02-amino, -NHCHO, -N-(alkyl)-CHO, -NH-CO-amino, -N-(alkyl)-CO-amino, -NH-COalkyl, -N- (alkyl)-COalkyl, wherein each alkyl, alkenyl, cycloalkyl, alkoxy, monocarboxylic acid, and dicarboxylic acid group may be independently substituted with one or more substituents selected from halogen, OH, CH3, OCH3, SH, SCH3, NH2, NHCH3, N(CH3)2. If the group to be substituted is a ring, the optional substituents could also be selected from: vicinal -O(alkyl)O-, vicinal -OC(alkyl)O-, vicinal -CH2O(alkyl)O-, vicinal - S(alkyl)S- and - O(alkyl)S-, wherein each alkyl group may be independently substituted with one or more substituents selected from halogen, OH, CH3, OCH3, SH, SCH3, NH2, NHCH3, and N(CH3)2.

[0093] The term “alkyl” used alone or as a suffix or prefix, refers to monovalent straight or branched chain hydrocarbon radicals comprising 1 to about 12 carbon atoms that are saturated.

[0094] The term “hydrocarbon” used alone or as a suffix or prefix, refers to any structure comprising only carbon and hydrogen atoms up to 12 carbon atoms.

[0095] The term “hydrocarbon radical” or “hydrocarbyl” used alone or as a suffix or prefix, refers to any structure as a result of removing one or more hydrogens from a hydrocarbon. The term “alkylene” used alone or as suffix or prefix, refers to divalent straight or branched chain hydrocarbon radicals comprising 1 to about 12 carbon atoms, which serves to links two structures together.

[0096] The term “alkenyl” used alone or as suffix or prefix, refers to a monovalent straight or branched chain hydrocarbon radical having at least one carbon-carbon double bond and comprising at least 2 carbon atoms.

[0097] The term “cycloalkyl” used alone or as suffix or prefix, refers to a monovalent ring- containing hydrocarbon radical comprising at least 3 carbon atoms. When cycloalkyl contains more than one ring, the rings can be fused or unfused and include bicyclo radicals. Fused rings generally refer to at least two rings sharing two atoms therebetween.

[0098] The term “alkoxy” used alone or as a suffix or prefix, refers to radicals of the general formula -O-R, wherein -R is selected from a hydrocarbon radical, preferably comprising 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, for instance 1 to 6 carbon atoms Exemplary alkoxy includes methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, isobutoxy, cyclopropylmethoxy, allyloxy, and propargyloxy.

[0099] The term “substituted” used as a suffix of a first structure, molecule or group, followed by one or more names of chemical groups refers to a second structure, molecule or group, which is a result of replacing one or more hydrogens of the first structure, molecule or group with the one or more named chemical groups. For example, an “alkyl substituted by halo” refers to a halogenated alkyl.

[0100] The term “halogen” includes fluorine, chlorine, bromine and iodine. “Halogenated” used as a prefix of a group, means one or more hydrogens on the group are replaced with one or more halogens.

[0101] The term “amine” or “amino” used alone or as a suffix or prefix, refers to radicals of the general formula - NRxRy, wherein Rxand Ryare independently selected from hydrogen or a hydrocarbon radical. Where a group -N(RX)2is indicated, each Rxis independently selected from the respective hydrocarbon groups indicated.

[0102] When any variable (e.g., R1, R4, etc.) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-3 R1, then said group can optionally be substituted with 0, 1 , 2 or 3 R1groups and R1at each occurrence is selected independently from the definition of R1. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0103] A variety of compounds in the present invention can exist in particular geometric or stereoisomeric forms. The present invention encompasses all such compounds unless explicitly stated otherwise, including cisand trans isomers, R- and S- enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof. Additional asymmetric carbon atoms can be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention. The compounds herein described can have asymmetric centers. Compounds of the present invention containing an asymmetrically substituted atom can be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. When required, separation of the racemic material can be achieved by methods known in the art. Many isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Insofar as applicable, cis and trans isomers of the compounds of the present invention are described and can be isolated as a mixture of isomers or as separated isomeric forms. All chiral, diastereomeric, racemic forms and all other isomeric forms of a structure are intended to be covered, unless the specific stereochemistry or isomeric form is specifically indicated.

[0104] As used herein, “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0105] As used herein, “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, maleic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

[0106] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosure of which is hereby incorporated by reference.

[0107] Technical terms are used by their common sense or meaning to the person skilled in the art. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

[0108] As surprisingly discovered by the inventors of the present disclosure, domain swapping in GT-B type glycosyltransferases (exchange of acceptor and donor domains between different types of GT-B type glycosyltransferases) allows for the generation of xanthan derivatives that differ from native xanthan in terms of structure of the side chains of the polysaccharide backbone, particularly in terms of the type of terminal monosaccharide unit of the side chains.

[0109] The present invention relates to a polypeptide, said polypeptide comprising an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C-terminal donor binding site domain. In the context of the present invention, it is generally envisaged that, due to domain swapping, at least the N-terminal acceptor binding site domain and the C-terminal donor binding site domain originate from different GT-B type glycosyltransferases, or at least the N-terminal acceptor binding site domain and at least part of the C-terminal donor binding site domain, such as at least 80 %, preferably at least 85 % or at least 90 %, such as about 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 % of the C-terminal donor binding site domain, originate from different GT-B type glycosyltransferases.

[0110] In the context of the present invention, the term “GT-B type glycosyltransferase” refers to a specific type of glycosyltransferases, also referred to as GT-B fold glycosyltransferases, that catalyze the transfer of a monosaccharide moiety or monosaccharide derivative moiety from donor molecules, typically nucleotidesugar or lipid-phospho-sugar donors, to a wide range of acceptor substrates, including lipids, proteins, and (oligo)saccharides. GT-B type glycosyltransferases are characterized by their tertiary structure and presence and arrangement of enzyme domains, namely two “Rossmann-fold” domains, separated by a linker forming a deep cleft that includes the catalytic center. A “Rossmann-fold” domain contains two sets of p-a-p-a-p units (654123 topology), together forming a single parallel sheet flanked by a-helices. A long loop, or cross-over, frequently between strands 3 and 4, creates a natural cavity that participates in the binding of the donor. It is generally accepted that in GT-B enzymes, the donors bind to the C-terminal domain of the protein, whereas the N-terminal domain is involved in acceptor substrate recognition. Among GT-B type glycosyltransferases, the C-terminal domains show higher similarities since they recognize the same or similar donors, whereas the N-terminal domain shows lower similarity due to the greater variety of acceptor molecules. Ligand binding is associated with conformational changes in the relative orientation of the domains involved. In contrast to GT-A enzymes, structural and kinetic evidence indicate that divalent cations are not essential for enzymatic activity but may accelerate conversion rates. GT-B type glycosyltransferases are able to permanently or temporarily associate to a cell’s phospholipid bilayer by a combination of hydrophobic and electrostatic interactions. In the context of the present invention, the term GT-B type glycosyltransferases encompasses both inverting and retaining GTs, i.e., GTs that can proceed with either inversion or retention of the anomeric configuration with respect to the reaction substrates and products.

[0111] In the context of the present invention, the term “different GT-B type glycosyltransferases” or expressions such as “glycosyltransferase distinct from another glycosyltransferase” refers to GT-B type glycosyltransferases differing at least in terms of the type of donor and / or acceptor that is recognized and enzymatically converted by the respective enzyme. The combining of segments or domains of at least two different GT-B type glycosyltransferases, i.e., so as to generate the chimeric GT-B type glycosyltransferase of the present invention, is also referred to as “domain swapping”. In some embodiments, particularly, the term “different GT-B type glycosyltransferases” refers to GT-B type glycosyltransferases that differ at least in terms of amino acid sequence of their respective C-terminal domain, i.e., donor binding site. This may mean that the sequence identity between their C-terminal donor binding sites is less than 100 %. Alternatively, or additionally, this term may mean that they differ at least in terms of amino acid sequence of their respective N-terminal domain, i.e., acceptor binding site. Again, this may mean that the sequence identity between the N-terminal domains is less than 100 %. In some embodiments, the term “different GT- B type glycosyltransferases” refers to GT-B type glycosyltransferases that differ in both, the amino acid sequence of their respective C-terminal domain, i.e., donor binding site, and in terms of amino acid sequence of their respective N-terminal domain, i.e., acceptor binding site. In various embodiments, different GT-B type glycosyltransferases recognize and enzymatically convert different types of donor molecules and / or different acceptor molecules. In some instances, it may be preferred that the different GT- B type glycosyltransferases differ in their recognition and use of donor molecules in that the donor molecules preferably bound and added are different monosaccharides or monosaccharide derivatives. In the chimeric enzymes of the present invention, the linker loop linking the N-terminal acceptor binding site and the C-terminal donor binding site of the chimeric GT-B type glycosyltransferase of the present invention may originate from either one of the at least two different GT-B type glycosyltransferases used for domain swapping or may originate from a third type of different GT-B type glycosyltransferase or may be artificially designed. Non-limiting examples of linker loop amino acid sequences are provided herein below.

[0112] In the context of the present invention, the type of GT-B type glycosyltransferases used for domain swapping, as herein described and defined, is not particularly limited, which means that any GT-B type glycosyltransferases known in the art or discovered in the future may be used for designing the chimeric GT-B type glycosyltransferases of the present invention. Non-limiting examples of GT-B type glycosyltransferases known in the art are GT-B type glycosyltransferases of the GT 1 , GT3, GT4, GT5, GT9, GT10, GT20, GT23, GT28, GT30, GT35, GT41 , GT52, GT63, GT65, GT68, GT70, GT72, and GT80 family, such as, for instance but without limitation, membrane-associated GT-B type glycosyltransferases like Alg13 (UDP-GlcNAc:Dol-PP-GlcNAc A / -acetylglucosaminyltransferase) / Alg14, Ugt2b7 (UDP-GlcA:p- glucuronosyltransferase 2B7), AviGT4 (eurekanate-attachment enzyme), CGT (cholesterol a- glucosyltransferase), PimA (GDP-Man:phosphatidylinositol mannosyltransferase), PimB' (GDP-Man: Phosphatidylinositolmannose mannosyltransferase), SUS1 (sucrose synthase), WaaG (UDP-Glc:l-glycero- d-manno-heptose II -1 ,3-glucosyltransferase I), WsaF (TDP-p-L-Rha; S-Layer glycoprotein p-1 ,2- rhamnosyltransferase), WbaZ (putative mannosyltransferase), WaaC (LPS heptosyltransferase I), WaaF (LPS heptosyltransferase II), Vpar_0760 (putative heptosyltransferase), FucT (a-1 ,3-fucosyltransferase), FUT8 ( / V-acetyl-d-glucosaminide-1 ,6-l-fucosyltransferase), MurG (UDP-GIcNAc: / V-acetylmuramyl- (pentapeptide)-PP-C55 / V-acetylglucosaminyltransferase), WaaA (CMP-p-KDO: a-3-deoxy-d-manno-2- octulosonic-acid (KDO) transferase), NST (CMP-Neu: (LOS) p-galactosamide a-2,3-sialyltransferase), GumK (UDP-GIcA: (xanthan) a-Man-(1 ,3)-p-Glc-(1 ,4)-a-Glc-PP-polyisoprenyl p-1 ,2- glucuronosyltransferase), PmST1 (CMP-NeuAc: a-2,3 / 2,6-sialyltransferase 1), ST (CMP-NeuAc: a- / p- galactoside a-2,3-sialyltransferase), and Pst6-224 (CMP-NeuAc: p-galactoside a-2,6-sialyltransferase), and further non-membrane-associated GT-B type glycosyltransferases like CalG1 (calicheamicin GT 1) / CalG2 (calicheamicin GT 2) / CalG4 (calicheamicin GT 4), CalG3 (calicheamicin GT 3), EryCIII (TDP- desosamine:a-mycarosyl erythronolide B desosaminyltransferase), GtfA (dTDP-p-L-4-epi- epivancosamine:epivancosaminyltransferase), GtfB (TDP / UDP-glucose:aglycosyl-vancomycin glucosyltransferase), GtfD (UDP-p-l-4-epi-vancosamine:vancomycin-pseudoaglycone vancosaminyltransferase), OGT / NGT (UDP-Glc:sinapoyl-alcohol-,2,5-DHBA-,3,4-DHBA- glucosyltransferase), OleD (oleandomycin GT ) / Olel (oleandomycin GT), SpnG (TDP-p-l-Rha:spynosin 9- O-a-l-rhamnosyltransferase), Ufgt (UDP-GIc: Anthocyanidin 3-O-glucosyltransferase), UGT71 G1 (UDP- Glc:flavonoid p-glucosyltransferase), UGT78G1 (UDP-glucose:flavonoid p-glucosyltransferase), UGT85H2 (UDP-glucose:(iso)flavonoid p-glucosyltransferase), UrdGT2 (urdamycin A GT II), Gsy2 (glycogen synthase), BshA (UDP-GIcNAc: l-malate a- / V-acetylglucosaminyltransferase), MshA (UDP- GlcNAc:inositol-P / V-acetylglucosaminyltransferase), NY2A_B736L (putative mannosyltransferase), SpsA (sucrose phosphate synthase), TreT (trehalose synthase), A / GIgA (glycogen synthase), EcGIgA (glycogen synthase), PaGIgA (glycogen synthase), OsGBSSI (rice granule bound starch synthase), / 7vSSI (barley starch synthase I), OtsA (a,a-trehalose-phosphate synthase), NodZ (a-1 ,6-l-fucosyltransferase), GP (glycogen phosphorylase) (liver / muscle), Gph1 (glycogen phosphorylase), MalP (maltodextrin phosphorylase), SP (starch phosphorylase), / 7sOGT (human OGT), XcOGT Xanthomonas campestris OGT), HMW1 C, BGT (UDP-GIc: DNA p-glucosyltransferase), POFUT1 (Protein O-Fucosyltransferase 1), POFUT2 (Protein O-Fucosyltransferase 2), and AGT (UDP-GIc: DNA a-glucosyltransferase), \Nxc (a- rhamnosyltransferase), \NxcC (a-rhamnosyltransferase), GT2 (Glucosyltransferase), AceQ (Glucosyltransferase).

[0113] In some embodiments, GT-B type glycosyltransferases useful for domain-swapping may be selected from the GT1 family, particularly GT1 CT, GumINT, and GumKCT.

[0114] In various embodiments, the polypeptide of the present invention is a chimeric GT-B type glycosyltransferase. Particularly, in some embodiments, the polypeptide of the present invention is characterized in that it catalyzes the transfer of a monosaccharide moiety or monosaccharide derivative moiety from a donor (e.g., glycosyl donor molecule), such as nucleotide-sugar or lipid-phospho-sugar donors, to an acceptor substrate (e.g., glycosyl acceptor molecule) selected from monosaccharides, disaccharides, polysaccharides, lipids and proteins, preferably polysaccharides, including modified variants thereof, such as (partially) acetylated and / or pyruvylated variants thereof.

[0115] The polypeptides of the present invention may be, in some embodiments, either isolated, cytosolic or membrane-bound.

[0116] Particularly, in some embodiments, the polypeptide of the present invention is capable of catalyzing the transfer of a monosaccharide moiety or monosaccharide derivative moiety from a donor, preferably a sugar nucleotide or lipid-phospho-sugar, to a polyprenyl phospho oligosaccharide.

[0117] Particularly, in some embodiments, the polypeptide of the present invention is capable of catalyzing the transfer of a monosaccharide moiety or monosaccharide derivative moiety from a donor, preferably a sugar nucleotide, to p-D-GlcA-(1 ^2)-a-D-Man-(1 ^3)-p-D-Glc-(1 ^4)-D-Glc, which preferably may be linked to a carrier molecule, such as an isoprenoid lipid carrier molecule, optionally through bound through a pyrophosphate linking group, such as in the form of p-D-GlcA-(1 ^2)-a-D-Man-(1 ^3)-p-D-Glc-(1 ^4)-D- Glc-1-diphospho-ditrans,octacis-undecaprenol.

[0118] Glycosyltransferases can tolerate modifications to the acceptor sugar, as long as the acceptor meets specific structural requirements, e.g., appropriate stereochemistry and availability of the reactive hydroxyl group involved in the glycosidic bond.

[0119] In some embodiments, the present invention relates to a polypeptide, said polypeptide comprising:

[0120] (i) an N-terminal amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 , wherein preferably said N-terminal amino acid sequence is an N-terminal acceptor binding site domain of a GT-B type glycosyltransferase; and

[0121] (ii) a C-terminal amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or a C-terminal amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, wherein preferably said C-terminal amino acid sequence is a C-terminal donor binding site domain of a GT-B type glycosyltransferase; and

[0122] (iii) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4, linking the N-terminal amino acid sequence and the C-terminal amino acid sequence through peptide bonds.

[0123] In some embodiments, the present invention relates to a polypeptide, said polypeptide comprising an N- terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C-terminal donor binding site domain, wherein

[0124] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and / or

[0125] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and / or

[0126] (iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or comprises or consists of an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and / or

[0127] (iv) the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a GT-B type glycosyltransferase distinct from the GT- B type glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same GT-B type glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence through peptide bonds.

[0128] In some embodiments, provided is a polypeptide, said polypeptide comprising:

[0129] (i) an N-terminal amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 , wherein preferably said N-terminal amino acid sequence is an N-terminal acceptor binding site domain of a GT-B type glycosyltransferase; and

[0130] (ii) a C-terminal amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 21 , wherein said C-terminal amino acid sequence is a chimeric C-terminal donor binding site domain of the GT-B glycosyltransferase type; and

[0131] (iii) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4, linking the N-terminal amino acid sequence and the C-terminal amino acid sequence through peptide bonds.

[0132] In some embodiments, the polypeptide of the present invention may be defined as comprising or consisting of:

[0133] (1) an N-terminal amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 , wherein preferably said N-terminal amino acid sequence is an N-terminal acceptor binding site domain of a GT-B type glycosyltransferase; and

[0134] (2) a C-terminal amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or a C- terminal amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, wherein preferably said C-terminal amino acid sequence is a C-terminal donor binding site domain of a GT- B type glycosyltransferase; or a C-terminal amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 21 , wherein said C-terminal amino acid sequence is a chimeric C-terminal donor binding site domain of the GT-B glycosyltransferase type; and

[0135] (3) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4, linking the N- terminal amino acid sequence and the C-terminal amino acid sequence through peptide bonds, i.e., amide type of covalent chemical bond linking two consecutive alpha-amino acids from C1 of one alpha-amino acid and N2 of another.

[0136] In other words, in some embodiments, the polypeptide of the present invention may be defined to be comprising or consisting of an amino acid sequence (1)-(3)-(2), wherein (1), (2), and (3) are as defined herein above, wherein indicates a peptide bond.

[0137] In some embodiments, the polypeptide comprises an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C- terminal donor binding site domain, wherein the N-terminal acceptor binding site domain and the C-terminal donor binding site domain originate from different GT-B type glycosyltransferases, wherein

[0138] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0139] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4.

[0140] In some embodiments, the polypeptide comprises an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C- terminal donor binding site domain, wherein the N-terminal acceptor binding site domain and at least part of the C-terminal donor binding site domain, such as about 50 %, 55 %, 60 %, 65 %, 70%, 75 %, 80 %, 85 % or 90 %, such as about 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, preferably about 80 to 90 % of the C-terminal donor binding site domain, originate from different GT-B type glycosyltransferases, wherein

[0141] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0142] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the linking of the N-terminal acceptor binding site domain and the C-terminal donor binding site domain through the linker loop is through peptide bonds, i.e., amide type of covalent chemical bond linking two consecutive alpha-amino acids from C1 of one alpha-amino acid and N2 of another.

[0143] In some embodiments, the polypeptide comprises an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C- terminal donor binding site domain, wherein the N-terminal acceptor binding site domain and the C-terminal donor binding site domain originate from different GT-B type glycosyltransferases, wherein

[0144] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0145] (ii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0146] In some embodiments, the polypeptide comprises an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C- terminal donor binding site domain, wherein the N-terminal acceptor binding site domain and the C-terminal donor binding site domain originate from different GT-B type glycosyltransferases, wherein

[0147] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0148] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and

[0149] (iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0150] In some embodiments, the polypeptide comprises an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C- terminal donor binding site domain, wherein the N-terminal acceptor binding site domain and at least part of the C-terminal donor binding site domain, such as about 50 %, 55 %, 60 %, 65 %, 70%, 75 %, 80 %, 85 % or 90 %, such as about 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, preferably about 80 to 90 % of the C-terminal donor binding site domain, originate from different GT-B type glycosyltransferases, wherein

[0151] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0152] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and

[0153] (iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0154] In some embodiments, the polypeptide comprises an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C- terminal donor binding site domain, wherein the N-terminal acceptor binding site domain and the C-terminal donor binding site domain originate from different GT-B type glycosyltransferases, wherein

[0155] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0156] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and

[0157] (iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0158] In some embodiments, the polypeptide comprises an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C- terminal donor binding site domain, wherein the N-terminal acceptor binding site domain and the C-terminal donor binding site domain originate from different GT-B type glycosyltransferases, wherein

[0159] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0160] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and

[0161] (iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the polypeptide comprises an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C- terminal donor binding site domain, wherein the N-terminal acceptor binding site domain and at least part of the C-terminal donor binding site domain, such as about 50 %, 55 %, 60 %, 65 %, 70%, 75 %, 80 %, 85 % or 90 %, such as about 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, preferably about 80 to 90 % of the C-terminal donor binding site domain, originate from different GT-B type glycosyltransferases, wherein

[0162] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0163] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and

[0164] (iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0165] In some embodiments, the polypeptide comprises an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C- terminal donor binding site domain, wherein the N-terminal acceptor binding site domain and at least part of the C-terminal donor binding site domain, such as about 50 %, 55 %, 60 %, 65 %, 70%, 75 %, 80 %, 85 % or 90 %, such as about 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, preferably about 80 to 90 % of the C-terminal donor binding site domain, originate from different GT-B type glycosyltransferases, wherein

[0166] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0167] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and

[0168] (iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0169] As outlined herein above, GT-B type glycosyltransferases are characterized in comprising a linker region linking the two “Rossmann-fold” domains, i.e., the N-terminal domain and the C-terminal domain. In the context of the present invention, said linker is referred to as the “linker loop”.

[0170] The linker loop may, in the context of the present invention, originate from the same GT-B type glycosyltransferase as the N-terminal domain or as the C-terminal domain, or may originate from a third type of GT-B type glycosyltransferase that is different from both the GT-B type glycosyltransferase that the N-terminal originates from and the GT-B type glycosyltransferase that the C-terminal domain originates from. In the latter case, the resultant chimeric GT-B type glycosyltransferase comprises parts of three different types of GT-B type glycosyltransferases.

[0171] In some embodiments, for instance, the polypeptide according to the present invention is characterized in that, due to domain swapping, the N-terminal acceptor binding site domain and at least part of the C- terminal donor binding site domain originate from different GT-B type glycosyltransferases.

[0172] In some embodiments, the N-terminal domain (or N-terminal acceptor binding site) and the linker loop originate from different GT-B type glycosyltransferases.

[0173] In some other embodiments, the C-terminal domain (or C-terminal donor binding site) and the linker loop originate from different GT-B type glycosyltransferases.

[0174] In some further embodiments, the polypeptide according to the present invention is characterized in that, due to domain swapping, the N-terminal domain (or acceptor binding site) and the linker loop originate from different GT-B type glycosyltransferases and the C-terminal domain (or donor binding site) and the linker loop originate from different GT-B type glycosyltransferases.

[0175] In some other embodiments, the polypeptide according to the present invention is characterized in that, due to domain swapping, the N-terminal domain (or N-terminal acceptor binding site) and the linker loop originate from different GT-B type glycosyltransferases or the C-terminal domain (or C-terminal donor binding site) and the linker loop originate from different GT-B type glycosyltransferases.

[0176] In some embodiments, the N-terminal acceptor binding site domain and the linker loop originate from identical GT-B type glycosyltransferases and the C-terminal donor binding site domain originates from a different GT-B than the N-terminal acceptor binding site domain and the linker loop.

[0177] In some embodiments, the N-terminal acceptor binding site domain and the linker loop originate from identical GT-B type glycosyltransferases and at least part of the C-terminal donor binding site domain originates from a different GT-B glycosyltransferase than the N-terminal acceptor binding site domain and the linker loop.

[0178] In some embodiments, the polypeptide of the present invention is characterized in that the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a GT-B type glycosyltransferase distinct from the GT-B type glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same GT-B type glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence, preferably through peptide bonds, wherein optionally: the second amino acid sequence of the chimeric C-terminal donor binding site domain constitutes about 5 to 70 %, such as about 8 to 20 %, or 10 to 15 % or 10 to 30 %, or 20 to 40 %, or 30 to 50 %, or 40 to 60 %, or 50 to 70 %, of the entire length of the chimeric C-terminal donor binding site domain; and / or the first amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 13; and / or the second amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 14; and / or the N-terminal acceptor binding site and the linker loop originate from different GT-B type glycosyltransferases or the N-terminal acceptor binding site domain and the linker loop originate from identical GT-B type glycosyltransferases; and / or the polypeptide comprises or consists of amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0179] It is generally understood that such a polypeptide of the present invention may be defined as comprising or consisting of:

[0180] (1) an N-terminal amino acid sequence, wherein preferably said N-terminal amino acid sequence is an N-terminal acceptor binding site domain of a GT-B type glycosyltransferase; and

[0181] (2) a C-terminal amino acid sequence, wherein preferably said C-terminal amino acid sequence is a chimeric C-terminal donor binding site domain of a GT-B type glycosyltransferase; and

[0182] (3) an amino acid sequence linking the N-terminal amino acid sequence and the C-terminal amino acid sequence through peptide bonds, i.e., amide type of covalent chemical bond linking two consecutive alphaamino acids from C1 of one alpha-amino acid and N2 of another, such that, in other words, the polypeptide of the present invention may be defined to be comprising or consisting of an amino acid sequence (1)-(3)- (2), wherein (1), (2), and (3) are as defined herein above, wherein indicates a peptide bond.

[0183] As has been surprisingly discovered by the present inventors, the concept of “domain swapping” in the generation of chimeric glycosyltransferases according to the present invention can be further improved by employing chimeric C-terminal donor binding site domains of the GT-B glycosyltransferase type. In the context of the present invention, the term “chimeric C-terminal donor binding site domain” refers to a C- terminal donor binding site domain comprising at least a first and a second amino acid sequence, wherein the first amino acid sequence is derived from or originates from a first GT-B type glycosyltransferase and the second amino acid sequence is derived from or originates from a second GT-B type glycosyltransferase, wherein the first GT-B type glycosyltransferase and the second GT-B type glycosyltransferase are not identical. Without wishing to be bound by theory, it is believed that presence of the second amino acid in the chimeric C-terminal donor binding site domain, as herein defined, stabilizes the tertiary structure of the chimeric glycosyltransferase of the present invention. In various such embodiments, the polypeptide of the present invention is characterized in that the C- terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a GT-B type glycosyltransferase distinct from the GT-B type glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same GT-B type glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence, preferably through peptide bonds, wherein the second amino acid sequence of the chimeric C-terminal donor binding site domain constitutes about 5 to 15 %, such as about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 %, of the entire length of the chimeric C-terminal donor binding site domain; and the second amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 14; wherein optionally the N-terminal acceptor binding site and the linker loop originate from identical GT-B type glycosyltransferases.

[0184] In other embodiments, the polypeptide of the present invention is characterized in that the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a GT-B type glycosyltransferase distinct from the GT-B type glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same GT-B type glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence, preferably through peptide bonds, wherein the second amino acid sequence of the chimeric C-terminal donor binding site domain constitutes about 5 to 15 %, such as about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 %, of the entire length of the chimeric C-terminal donor binding site domain; and the first amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 13; wherein optionally the N-terminal acceptor binding site and the linker loop originate from identical GT-B type glycosyltransferases.

[0185] In other embodiments, the polypeptide of the present invention is characterized in that the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a GT-B type glycosyltransferase distinct from the GT-B type glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same GT-B type glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence, preferably through peptide bonds, wherein the second amino acid sequence of the chimeric C-terminal donor binding site domain constitutes about 5 to 15 %, such as about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 %, of the entire length of the chimeric C-terminal donor binding site domain; and the first amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 13; and the second amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 14; wherein optionally the N-terminal acceptor binding site and the linker loop originate from identical GT-B type glycosyltransferases.

[0186] In some embodiments, the polypeptide of the present invention is characterized in that the linker loop consists of about 28 to 36 amino acid residues, such as 28, 29, 30, 31 , 32, 33, 34, 35 or 36 amino acid residues, preferably of 29 to 36 amino acid residues, for instance, 30 to 35 amino acid residues. In various embodiments, the polypeptide of the present invention is characterized in that the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4.

[0187] In some embodiments, the polypeptide of the present invention is characterized in that the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a GT-B type glycosyltransferase distinct from the GT-B type glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same GT-B type glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence, preferably through peptide bonds, wherein

[0188] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0189] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4; wherein optionally: the second amino acid sequence of the chimeric C-terminal donor binding site domain constitutes about 5 to 15 %, such as about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 %, of the entire length of the chimeric C-terminal donor binding site domain; and / or the first amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 13; and / or the second amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 14.

[0190] In other embodiments, the polypeptide of the present invention is characterized in that the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a GT-B type glycosyltransferase distinct from the GT-B type glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same GT-B type glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence through a peptide bond, wherein

[0191] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0192] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and

[0193] (iii) the second amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 14; wherein optionally the second amino acid sequence of the chimeric C-terminal donor binding site domain constitutes about 5 to 15 %, such as about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 %, of the entire length of the chimeric C-terminal donor binding site domain; and / or the first amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 13.

[0194] In other embodiments, the polypeptide of the present invention is characterized in that the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a GT-B type glycosyltransferase distinct from the GT-B type glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same GT-B type glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence through a peptide bond, wherein

[0195] (i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and

[0196] (ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and

[0197] (iii) the second amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 14; wherein further the second amino acid sequence of the chimeric C-terminal donor binding site domain constitutes about 5 to 15 %, such as about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 %, of the entire length of the chimeric C-terminal donor binding site domain; and the first amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 13.

[0198] In various embodiments, the polypeptide of the present invention is characterized in that it comprises or consists of an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 5.

[0199] In various other embodiments, the polypeptide of the present invention is characterized in that it comprises or consists of an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

[0200] In some embodiments, the polypeptide comprises or consists of amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0201] It is furthermore contemplated that the polypeptides of the present invention may be modified, such as to facilitate purification, allow for detection, modify expression and / or secretion thereof, etc. The modifications may be of a minor nature, i.e., may be conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function.

[0202] Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Val, Ala / Glu, and Asp / Gly.

[0203] Alternatively or additionally, the polypeptides of the invention may comprise additional sequences that may include, among others, an affinity tag and / or a protease recognition and cleavage site as well as other proteins / polypeptides to which the chimeric GT-B type glycosyltransferases of the invention are fused, thus forming a fusion protein. Examples of such proteins to which the polypeptides of the invention may be fused include, without limitation, albumins and antibodies, as well as antibody fragments or antibody-like molecules and antibody derivatives. The term “affinity tag” as used herein relates to entities which are coupled to a molecule of interest and allow enrichment of the complex between the molecule of interest and the affinity tag using an affinity tag receptor. In certain embodiments affinity tags may be selected from the group consisting of the Strep-tag® or Strep-tag® II, the myc-tag, the FLAG-tag, the His-tag, the small ubiquitin-like modifier (SUMO) tag, the covalent yet dissociable NorpD peptide (CYD) tag, the heavy chain of protein C (HPC) tag, the calmodulin binding peptide (CBP) tag, or the HA-tag or proteins such as Streptavidin binding protein (SBP), maltose binding protein (MBP), and glutathione-S-transferase.

[0204] In some embodiments, the polypeptides of the present invention may comprise a protease (recognition and) cleavage site. The term “protease (recognition and) cleavage site” refers to a peptide sequence which can be cleaved by a selected protease thus allowing the separation of peptide or protein sequences which are interconnected by a protease cleavage site. In certain embodiments the protease cleavage site is selected from the group consisting of a Factor Xa, a tobacco edge virus (TEV) protease, a enterokinase, a SUMO Express protease, an Arg-C proteinase, an Asp-N endopeptidases, an Asp-N endopeptidase + N- terminal Glu, a caspase 1 , a caspase 2, a caspase 3, a caspase 4, a caspase 5, a caspase 6, a caspase 7, a caspase 8, a caspase 9, a caspase 10, a chymotrypsin-high specificity, a chymotrypsin-low specificity, a clostripain (Clostridiopeptidase B), a glutamyl endopeptidase, a granzyme B, a pepsin, a prolineendopeptidase, a proteinase K, Welqut protease, Clean Cut protease, a staphylococcal peptidase I, a Thrombin, a Trypsin, intein, and a Thermolysin cleavage site. It can be preferred, in some embodiments, to design the protease recognition site such that as few amino acids as possible of the recognition and cleavage site remain attached to the peptide or protein of interest.

[0205] In various embodiments, the polypeptide of the present invention may be derivatized or conjugated to another chemical moiety, said derivatization / conjugation including, amongst others, PEGylation, glycosylation. In particular, PEGylation, i.e., covalent coupling to polyethylene glycol of various molecular weights, is known as a means to alter pharmacokinetics of such compounds.

[0206] The invention further relates to the nucleic acid, in particular the isolated nucleic acid molecule, encoding the polypeptide of the present invention, as herein described above. Exemplary nucleic acid sequences encoding exemplary polypeptides of the present invention are set forth in the following Table 1 :

[0207] Table 1 : Amino acid sequences and corresponding nucleic acid sequences in accordance with the present invention

[0208] If the polypeptide of the invention comprises in addition to the amino acid sequence specified herein optional further amino acid sequences, all of these amino acid sequences are typically linked by peptide bonds and expressed as a single fusion protein. To facilitate said expression, the nucleic acid molecule comprises nucleotide sequences encoding all amino acid sequences, with said nucleotide sequences being operably linked to allow expression of the single fusion protein comprising all afore-mentioned amino acid sequences.

[0209] The term “operably linked” in the context of nucleic acid sequences means that a first nucleic acid sequence is linked to a second nucleic acid sequence such that the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter sequence is operably linked to a coding sequence of a heterologous gene if the promoter can initiate the transcription of the coding sequence. In a further context, a sequence encoding the polypeptide of the present invention is linked such to another amino acid sequence, that if the two sequences are translated a single peptide / protein chain is obtained.

[0210] In certain embodiments, the above defined nucleic acid molecules may be comprised in a vector, for example a cloning or expression vector. Generally, the nucleic acid molecules of the invention can also be part of a vector or any other kind of cloning vehicle, including, but not limited to a plasmid, a phagemid, a phage, a baculovirus, a cosmid, or an artificial chromosome. Generally, a nucleic acid molecule disclosed in this application may be “operably linked” to a regulatory sequence (or regulatory sequences) to allow expression of this nucleic acid molecule. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Non-limiting examples of regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.

[0211] Such cloning vehicles can include, besides the regulatory sequences described above and a nucleic acid sequence of the present invention, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art and are commercially available.

[0212] In certain embodiments the nucleic acid molecules disclosed herein are comprised in a cloning vector. Therefore, in a further aspect, the present invention also relates to a vector comprising a nucleic acid molecule according to the present invention, wherein preferably the vector is a plasmid.

[0213] In some embodiments the nucleic acid molecules disclosed herein are comprised in an expression vector. The vectors may comprise regulatory elements for replication and selection markers. In certain embodiments, the selection marker may be selected from the group consisting of genes conferring ampicillin, kanamycin, neomycin, polymyxin, chloramphenicol, tetracycline, blasticidin, spectinomycin, gentamicin, hygromycin, and zeocin resistance. In various other embodiments, the selection may be carried out using antibiotic-free systems, for example by using toxin / antitoxin systems, cer sequence, triclosan, auxotrophies or the like. Suitable methods are known to those skilled in the art.

[0214] The above-described nucleic acid molecule of the present invention, comprising a nucleic acid sequence encoding for the polypeptide of the invention, e.g., the chimeric GT-B type glycosyltransferase, if integrated in a vector, must be integrated such that the polypeptide can be expressed. Therefore, a vector of the present invention comprises sequence elements which contain information regarding to transcriptional and / or translational regulation, and such sequences are “operably linked” to the nucleotide sequence encoding the polypeptide. An operable linkage in this context is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression. The precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions comprise a promoter which, in prokaryotes, contains both the promoter per se, i.e., DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation. Such promoter regions normally include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35 / - 10 boxes and the Shine- Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5'- capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell.

[0215] In addition, the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination or polyadenylation. If, however, these termination sequences are not satisfactory functional in a particular host cell, then they may be substituted with signals functional in that cell.

[0216] In various embodiments, a vector comprising a nucleic acid molecule of the invention can therefore comprise a regulatory sequence, preferably a promoter sequence. In certain embodiments, the promoter is identical or homologous to promoter sequences of the host genome. In such cases endogenous polymerases may be capable to transcribe the nucleic acid molecule sequence comprised in the vector. In various embodiments, the promoter is selected from the group of weak, intermediate and strong promoters, preferably from weak to intermediate promoters.

[0217] In another preferred embodiment, a vector comprising a nucleic acid molecule of the present invention comprises a promoter sequence and a transcriptional termination sequence. Suitable promoters for prokaryotic expression are, for example, the araBAD promoter, the tet-promoter, the lacUV5 promoter, the CMV promo tor, the EF1 alpha promotor, the AOX1 promotor, the tac promotor, the T7promoter, the sacB promotor, or the lac promotor. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter. Furthermore, a nucleic acid molecule of the invention can comprise transcriptional regulatory elements, e.g., repressor elements, which allow regulated transcription and translation of coding sequences comprised in the nucleic acid molecule. Repressor element may be selected from the group consisting of the Lac-, AraC-, or MalR-repressor.

[0218] The vector may be effective for prokaryotic or eukaryotic protein expression. Suitable vectors are known to those skilled in the art.

[0219] The vectors of the present invention may be chosen from the group consisting of high, medium and low copy vectors.

[0220] The above-described vectors of the present invention may be used for the transformation or transfection of a host cell in order to achieve expression of a peptide or protein which is encoded by an above-described nucleic acid molecule and comprised in the vector DNA.

[0221] Thus, in a further aspect, the present invention also relates to a host cell comprising a vector or nucleic acid molecule as disclosed herein.

[0222] Also contemplated herein are host cells, which comprise a nucleic acid molecule as described herein integrated into their genomes. The skilled person is aware of suitable methods for achieving the nucleic acid molecule integration. For example, the molecule may be delivered into the host cells by means of liposome transfer or viral infection and afterwards the nucleic acid molecule may be integrated into the host genome by means of homologous recombination. In certain embodiments, the nucleic acid molecule is integrated at a site in the host genome, which mediates transcription of the peptide or protein of the invention encoded by the nucleic acid molecule. In various embodiments, the nucleic acid molecule further comprises elements which mediate transcription of the nucleic acid molecule once the molecule is integrated into the host genome and / or which serve as selection markers.

[0223] In certain embodiments, the nucleic acid molecule of the present invention is transcribed by a polymerase natively encoded in the host genome. In various embodiments, the nucleic acid molecule is transcribed by an RNA-polymerase which is non-native to the host genome. In such embodiments, the nucleic acid molecule of the present invention may further comprise a sequence encoding for a polymerase and / or the host genome may be engineered or the host cell may be infected to comprise a nucleic acid sequence encoding for an exogenous polymerase. The host cell may be specifically chosen as a host cell capable of expressing the gene. In addition or otherwise, in orderto produce the (isolated) polypeptide of the invention, the nucleic acid coding for it can be genetically engineered for expression in a suitable system. Transformation can be performed using standard techniques (Sambrook, J. et al. (2001), Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0224] Prokaryotic or eukaryotic host organisms comprising such a vector for recombinant expression of the polypeptide as described herein form also part of the present invention. Suitable host cells can be prokaryotic cells. In certain embodiments the host cells are selected from the group consisting of Gram positive and Gram-negative bacteria. In some embodiments, the host cell is a Gram-negative bacterium, such as Xanthomonas, Kozakia baliensis, Komagateibacter xylinum, or Escherichia. In certain embodiments, the host cell is Xanthomonas campestris, Kozakia baliensis, Vibrio natriegens, Bacillus subtilis, Bacillus licheniformis, or Escherichia coll (E. coli). In further embodiments, the host cell is selected from the group consisting of X. campestris, such as Xanthomonas campestris pv. campestris, Xanthomonas campestris pv. begoniae, Xanthomonas campestris pv. pelargonii, Xanthomonas campestris pv. hyacinthii, Xanthomonas campestris pv. phaseoli, Xanthomonas campestris pv. translucens, Xanthomonas campestris pv. juglandis, Xanthomonas campestris pv. citri, Xanthomonas campestris pv. malvacearum, Xanthomonas campestris pv. oryzae, Xanthomonas campestris pv. graminis, acetic acid bacteria such as K. baliensis, and E. coli, Pseudomonas, Serratia marcescens, Salmonella, Shigella (and other enterobacteriaceae), Neisseria, Hemophilus, Klebsiella, Proteus, Enterobacter, Helicobacter, Acinetobacter, Moraxella, Stenotrophomonas, Bdellovibrio, Vibrio, Legionella, Bacilli such as Bacillus subtilis, Paenibacilliu polmyxa and further ones Corynebacterium, Clostridium, Listeria, Streptococcus, Staphylococcus, and Archaea cells. Suitable eukaryotic host cells are among others CHO cells, insect cells, fungi, yeast cells, e.g., Saccharomyces cerevisiae, S. pombe, Pichia pastoris, and the like.

[0225] In some embodiments, the host cell is selected from the group consisting of X. campestris, K. baliensis, E. coli, Vibrio natriegens, Bacillus sp. For instance E. coli BL21 (DE3), E. coli BL21 , E. coli K12, E. coli MG1655, E. coli BLR, E. coli BL21 Al, E. coli BL21 pLysS, E. coli X , E. coli DH5a, E. coli DH1 , E. coli DM1 , E. coli HB101 , E. coli JmlOI-110, E. coli Rosetta(DE3)pLysS, E. coli SURE, E. coli TOP10, E. coli XLI-Blue, E. coli XL2-Blue, and E. coli XLIO-Blue. In some embodiments, the host cell is Xanthomonas campestris. In some embodiments, the host cell is and Xanthomonas campestris with or without deletion of the terminal mannose transferring GTB Guml.

[0226] The transformed host cells are cultured under conditions suitable for expression of the nucleotide sequence encoding the polypeptide of the invention. In certain embodiments, the cells are cultured under conditions suitable for expression of the nucleotide sequence encoding a polypeptide of the invention and, optionally, its secretion.

[0227] For producing the polypeptide of the present invention, a vector of the invention can be introduced into a suitable prokaryotic or eukaryotic host organism by means of recombinant DNA technology (as already outlined above). For this purpose, the host cell is first transformed with a vector comprising a nucleic acid molecule according to the present invention using established standard methods (Sambrook, J. et al. (2001), supra). The host cell is then cultured under conditions, which allow expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide. Subsequently, the polypeptide is recovered either from the cell or from the cultivation medium.

[0228] For expression of the polypeptides of the present invention several suitable protocols are known to the skilled person. The method for expression of a recombinant polypeptide of the present invention may be achieved by the following method comprising: (a) introducing a nucleic acid molecule or vector of the invention into a host cell, wherein the nucleic acid molecule or vector encodes the polypeptide of the present invention, e.g., the chimeric GT-B type glycosyltransferase; and (b) cultivating the host cell in a culture medium under conditions that allow expression of the polypeptide of the present invention, and optionally secretion of the polypeptide into the culture medium.

[0229] Step (a) may be carried out by using suitable transformation and transfection techniques known to those skilled in the art. These techniques are usually selected based on the type of host cell into which the nucleic acid is to be introduced. In some embodiments, the transformation may be achieved using electroporation or heat shock treatment of the host cell.

[0230] Step (b) may include a cultivation step that allows growth of the host cells. Alternatively, such step allowing growth of the host cells and a step that allows expression of the polypeptide may be performed separately in that the cells are first cultivated such that they grow to a desired density and then they are cultivated under conditions that allow expression of the polypeptide. The expression step can however still allow growth of the cells.

[0231] The method may further include a step of recovering the expressed polypeptide. The polypeptide may be recovered from the growth medium, if it is secreted, orfrom the cells or both. The recovery ofthe polypeptide may include various purification steps.

[0232] Generally, any known culture medium suitable for growth of the selected host may be employed in this method.

[0233] In various embodiments, the method also encompasses the purification the polypeptide, wherein the polypeptide is purified using a method selected from affinity chromatography, ion exchange chromatography, reverse phase chromatography, size exclusion chromatography, and combinations thereof.

[0234] In several embodiments, the method may comprise the treatment ofthe polypeptide with a protease suitable for cleavage of a protease cleavage site within the polypeptide. In some embodiments, the polypeptide is purified prior to proteolytic cleavage using one or more methods disclosed above. Also after cleavage of peptide or protein, the method may comprise a further purification step as defined above. Thus, in some embodiments, the polypeptide is purified, subjected to proteolytic cleavage and the resulting polypeptide is further purified. In other embodiments, the protease may be co-expressed or added to the cultivation medium or expressed by co-cultivated microorganisms, such that cleavage occurs before purification.

[0235] In a further aspect, the present invention relates to the use of a vector or nucleic acid molecule as disclosed herein for the expression of a polypeptide according to the present invention. In some embodiments, the vector is used for the expression and optionally secretion of the polypeptide. The expression or expression and secretion may be achieved using the method described herein.

[0236] Therefore, in a further aspect, the present invention relates to a method for the recombinant production of a polypeptide of the present invention, comprising (1) cultivating the host cell of the present invention under conditions that allow the expression of the polypeptide, e.g., the chimeric GT-B type glycosyltransferase;

[0237] (2) optionally isolating the expressed polypeptide, e.g., chimeric GT-B type glycosyltransferase, from the host cell.

[0238] In a further aspect, the present invention relates to the use of a polypeptide, i.e., the chimeric GT-B type glycosyltransferase, as herein described and defined, for the production, i.e., biotechnological production, of a polysaccharide.

[0239] In yet another aspect, the present invention thus also relates to a method for the production of a polysaccharide using the polypeptide, i.e., the chimeric GT-B type glycosyltransferase, according to the present invention.

[0240] A method for the production of a polysaccharide according to the present invention typically comprises the following steps:

[0241] (a) optionally expressing a polypeptide, i.e., a chimeric GT-B type glycosyltransferase, according to the present invention in a suitable host cell; and

[0242] (b) contacting said polypeptide, i.e., the chimeric GT-B type glycosyltransferase of the present invention, with at least one suitable donor substrate (i.e., glycosyl donor molecule) and at least one suitable acceptor substrate (i.e., glycosyl acceptor molecule) under conditions that allow the enzymatic production of the polysaccharide, wherein preferably the polysaccharide is xanthan or a xanthan derivative.

[0243] In the context of the present invention, the term “xanthan derivative” refers to a polysaccharide that differs from the molecular structure of naturally occurring xanthan at least in not comprising the terminal mannose moiety in the polysaccharide side chain, preferably wherein the remainder of the molecule of the xanthan derivative is identical to naturally occurring xanthan. Thus, in other words, said term refers to polysaccharides featuring, instead of the terminal side chain D-mannosyl moiety of xanthan, a different monosaccharide moiety in said position. The precise structure of a xanthan derivative of the present invention therefore depends on the respective type of GT that is used for C-terminal donor binding site domain swapping, in accordance with the present invention.

[0244] In some embodiments, the method comprises a step of isolating the produced polysaccharide.

[0245] The polysaccharide may be produced in vitro in cell-free systems, which may be generally prepared by lysing cells of the host cell, preferably in the presence of a suitable buffer, such as a buffer including EDTA, and obtaining the polypeptide, i.e., a chimeric GT-B type glycosyltransferase, which may subsequently be subjected to step (b) of the method for the production of the polysaccharide according to the present invention, which may generally encompass incubating the chimeric GT-B type glycosyltransferase with one or more suitable donor substrates and one or more suitable acceptor substrates. Cell lysis may be achieved by means and methods known in the art, including chemical or physical means or combinations thereof, including, without limitation, detergent treatment, enzymatic treatment, sonication, French pressure cell press, and combinations thereof. Advantageously, one type of donor substrate and one type of acceptor substrate is used so as to produce only one type of particular polysaccharide. The choice of donor and acceptor substrates depends on the type of desired polysaccharide end product. The skilled person further readily appreciates that the exact composition of donor and acceptor substrates may be controlled by applying methods and means for chemical and / or enzymatical depletion of host cell endogenous substrates.

[0246] In some embodiments, the donor substrate is a nucleotide-sugar or lipid-phospho-sugar. Non-limiting examples of donor substrates include UDP-glucose, UDP-galactose, UDP-xylose, UDP-N-acetyl- glucosamine, UDP-N-acetyl- galactosamine, UDP-galactofuranose, UDP-glucuronic acid, GDP-mannose, GDP-fucose, TDP-rhamnose, CMP-N-acetylneuraminic acid, and CMP-2-keto-3-deoxy-mannooctanoic acid.

[0247] Typically, the acceptor substrate is a polysaccharide, preferably an oligosaccharide, more preferably p-D- GlcA-(1 ^2)-a-D-Man-(1 ^3)-p-D-Glc-(1 ^4)-D-Glc, which preferably is in the form of a polyprenyl phospho oligosaccharide, such as p-D-GlcA-(1 — >2)-a-D-Man-(1 — >3)-p-D-Glc-(1 — >4)-D-Glc coupled to a carrier molecule, such as a isoprenoid lipid carrier molecule, more preferably, the acceptor substrate is p-D-GIcA- (1 -^2)-a-D-Man-(1 -^3)-p-D-Glc-(1 — >4)-D-Glc-1 -diphospho-c / / fr‘ans,ocfac / s-undecaprenol.

[0248] Additionally, for the purpose of obtaining acetylated and / or pyruvylated polysaccharides, acetyl-CoA, and phosphoenylpyruvate may be considered to represent additional donor substrates, i.e., to be used in addition to the at least one glycosyl donor molecule. Thus, the person skilled in the field readily appreciates that a non-acetylated polysaccharide may be obtained by omitting acetyl-CoA as a substrate and that a non-pyruvylated polysaccharide may be obtained by omitting phosphoenolpyruvate as a substrate, as well as by the genomic deletion of the corresponding acetyl- and pyruvyl transferases.

[0249] Since the biosynthesis of xanthan has been found to encompass involvement of lipid carriers, particularly pyrophosporyl-linked lipid carriers, the method for production of a polysaccharide of the present invention may further, in some embodiments, encompass presence of one or more lipid carrier molecules or precursors thereof, for instance isoprenoid pyrophosphate.

[0250] The acceptor substrate, the donor substrate, any additional donor substrate, and / or lipid carrier molecules may be generated by the host cell and / or may be added to the cell-free in vitro production system.

[0251] Consequently, in some embodiments, the method, which may be an in vitro method or an in vivo method, of the present invention may be further characterized in that, in addition to the polypeptide of the present invention, i.e., the chimeric GT-B type glycosyltransferase, one or more additional enzymes may be expressed in the host cell, wherein said one or more additional enzymes are capable of generating one or more of acceptor substrates, donor substrates, additional donor substrates, and lipid carrier molecules for the polypeptide of the present invention, and in some embodiments one or more precursor molecules, such as one or more precursor molecules to the acceptor substrate for the polysaccharide of the present invention. This is particularly useful for whole-cell in vivo systems. In otherwords, the method of the present invention may, in some embodiments, additionally comprise expression of one or more enzymes that are capable of catalyzing one or more of sugar addition steps l-IV of the biosynthesis of xanthan as schematically outlined in Fig. 1.

[0252] In some embodiments, the method of the present invention comprises employment of one or more enzymes playing crucial roles in cell sugar metabolism and for generating complex carbohydrates (e.g., nucleotide sugars), such as one or more isomerases, for instance one or more epimerases or dehydrogenases, preferably a UDP-GIc-Epimerase. Presence of such an enzyme in methods for production of a polysaccharide of the present invention can improve overall activity and efficiency of the polypeptide of the present invention by generating an abundance of UDP-galactose from UDP-glucose, resulting in further improvement of yields and terminal side chain saturation with the terminal side chain sugar moiety, as herein described, particularly galactose and / or glucuronic acid, of the polysaccharide of the present invention. In some embodiments, the method of the present invention comprises employment of a UDP- GIc-Epimerase, preferably of a UDP-GIc-Epimerase comprising or consisting of the amino acid sequence having at least 80 %, preferably at least 85 %, more preferably at least 90 %, such as 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the method of the present invention comprises employment of a UDP-GIc-Epimerase, preferably of a UDP-GIc-Epimerase comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence having at least 80 %, preferably at least 85 %, more preferably at least 90 %, such as 100 % sequence identity to the nucleic sequence set forth in SEQ ID NO: 20.

[0253] In various embodiments, the method for the production of a polysaccharide is characterized in that the polypeptide of the present invention is heterologously expressed in said host cell. Alternatively or additionally, in some embodiments, the one or more additional enzymes may be heterologously or homologously expressed in the host cell. It is of course also possible to add the said one or more additional enzymes to the cell-free in vitro system for production of the polysaccharide of the present invention.

[0254] Insofar a host cell is naturally capable of producing xanthan, particularly capable of catalyzing addition steps V of the pathway schematically outlined in Fig. 1 , deletion of the gene coding for the respective enzyme may be useful such as to overall improve production efficacy of the desired polysaccharide and / or avoid production of mixed types of polysaccharides, wherein said mixed types of polysaccharide may differ in terms of terminal side chain sugar moiety of the polysaccharides thusly produced.

[0255] As a non-limiting example of this concept, Xanthomonas campestris as host cell in a production method of the present invention may be genetically engineered to not only include the nucleic acid molecule or vector encoding the polypeptide of the present invention, but further by deletion of the gene coding forthe enzyme capable of catalyzing step V of the xanthan production pathway schematically outlined in Fig. 1 , i.e., deletion of the gene coding for the membrane-associated mannosyltransferase Guml that catalyzes the transfer of the terminal mannose in the xanthan side chain.

[0256] In some embodiments, Xanthomonas campestris as host cell in a production method of the present invention may be genetically engineered to not only include the nucleic acid molecule or vector encoding the polypeptide of the present invention, but further by deletion of the gene coding for the enzyme capable of catalyzing step V of the xanthan production pathway schematically outlined in Fig. 1 , i.e., deletion of the gene coding for the membrane-associated mannosyltransferase Guml that catalyzes the transfer of the terminal mannose in the xanthan side chain, and further still by incorporation of the gene coding for a UDP- Glc-Epimerase, preferably a UDP-GIc-Epimerase comprising or consisting of the amino acid sequence having at least 80 %, preferably at least 85 %, more preferably at least 90 %, such as 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, a corresponding method of the present invention comprises employment of a UDP-GIc-Epimerase, preferably of a UDP-GIc- Epimerase comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence having at least 80 %, preferably at least 85 %, more preferably at least 90 %, such as 100 % sequence identity to the nucleic sequence set forth in SEQ ID NO: 20.

[0257] Non-limiting examples of suitable host cells include Xanthomonas campestris, in particular, Xanthomonas campestris pv. campestris, Xanthomonas campestris pv. begoniae, Xanthomonas campestris pv. pelargonii, Xanthomonas campestris pv. hyacinthii, Xanthomonas campestris pv. phaseoli, Xanthomonas campestris pv. translucens, Xanthomonas campestris pv. juglandis, Xanthomonas campestris pv. citri, Xanthomonas campestris pv. malvacearum, Xanthomonas campestris pv. oryzae, Xanthomonas campestris pv. graminis, acetic acid bacteria such as Kozakia sp., such as Kozakia baliensis, Komagataeibacter sp., such as Komagateibacter xylinus, E. coli, for instance for instance E. coli BL21 (DE3), E. coli BL21 , E. coli K12, E. coli BLR, E. coli BL21 Al, E. coli BL21 pLysS, E. coliXL , E. coli DH5a, E. coli DH1 , E. coli DM1 , E. coli HB101 , E. coli JmlOI-110, E. coli Rosetta(DE3)pLysS, E. coli SURE, E. coli TOP10, E. coli XLI-Blue, E. coli XL2-Blue, and E. coli XLIO-Blue, Pseudomonas, Serratia marcescens, Salmonella, Shigella (and other enterobacteriaceae), Neisseria, Hemophilus, Klebsiella, Proteus, Enterobacter, Helicobacter, Acinetobacter, Moraxella, Stenotrophomonas, Bdellovibrio, Vibrio, Legionella, Bacilli, Corynebacterium, Clostridium, Listeria, Streptococcus, Staphylococcus, and Archaea cells. In some embodiments, the host cell is Xanthomonas campestris.

[0258] The polypeptide of the present invention and / or optionally the one or more additional enzymes may be expressed in said host cell, as herein defined, by means of an expression plasmid. Alternatively or additionally, integration of the chimeric GT-B type glycosyltransferase coding sequence and / or the coding sequences of the one or more additional enzymes into the host genome may occur.

[0259] Instead of a cell-free in vitro system, the method for production of a polysaccharide according to the present invention may be an in vivo method. Therefore, in some embodiments, step (a) and / or step (b) of the method for the production of a polysaccharide of the present invention take place within whole cells. Similarly to the cell-free system, substrates and / or additional enzymes may be added to / provided for the host cell or instead be generated by the host cell, in which case the host cell may, insofar necessary, be subjected to gene alteration to allow for expression of the respective gene products, e.g., enzymes, within the host cell. In a further aspect, the present invention relates to a polysaccharide obtainable by a method for the production of a polysaccharide according to the present invention, as herein defined and described above, preferably xanthan or a xanthan derivative.

[0260] In various embodiments, the polysaccharide obtainable by the production method according to the present invention is characterized in not comprising a p-[1 ,4]-linked D-mannose moiety or a p-[1 ,4]-linked D- mannose moiety containing a ketal-linked pyruvic acid at the 4,6 position at the terminal position in its side chains ( / .e., the oligosaccharide side chains alpha, 1->3 linked (a-[1 ,3]-linked) to the polysaccharide backbone, which is formed by beta, 1->4-linked (p-[1 ,4]-linked) D-glucose moieties, said oligosaccharide side chains extending from alternate glucose moieties of the backbone); in other words, in some preferred embodiments, said polysaccharide is not naturally occurring xanthan.

[0261] In various embodiments, the polysaccharide obtainable by a method for the production of a polysaccharide according to the present invention is characterized in having an average molecular weight between about 1.0 • 104and 1.0 • 109Da, preferably between about 1.0 • 105and 1.0 • 108Da, more preferably between about 1.0 • 106and 9.0 • 107Da, such as between about 2.0 • 106and 5.0 • 107Da. Methods for determination of average molecular weights of polysaccharides are generally known in the art. For instance, the molecular mass can be determined from the elution profile by comparison to a pullulan standard (Mp 49.9-1020 kDa) elution profile with the calculations performed by the KNAUER ClarityChrom software. As a non-limiting example of a method for determination of the average molecularweight, the following protocol may be applied: A solution of 1 mg / mL of the polysaccharide in NaNCh 0.2 M and N32HPO4 0.05 M containing 200 ppm NaNs can be prepared, filtered through a 0.22 pm membrane filter. Elution was performed according to the following protocol: Knauer Aura high-pressure size exclusion chromatograph (HPSEC) equipped with a Knauer Azura 2.1 L refractive index detector (RID). Two gel-permeation columns from AppliChrom (1x ABOA SuperOH-P-450 50x8 mm and 2 x ABOA SuperOH-P-450) were used in series with a separation range from 50 kDa to 10,000 kDa; with analyses being performed at 38 °C using 0.2 M NaNOs and 0.05 M N32HPO4 containing 200 ppm NaNs as eluent with a flow rate of 0.5 mL / min.

[0262] In various embodiments, the polysaccharide obtainable by a method for the production of a polysaccharide according to the present invention is characterized in having a k value (viscosity) in the range of about 10 Panto about 35 Pan, preferably in the range of about 12 Panto about 30 Pan, more preferably in the range of about 12 Panto about 28 Pan, such as about 12, 15, 17, 20, 23, 25 or 27 Pan, as measured using a 1 % (w / v) polysacchairde sample in ultra pure water, analyzed with a Modular Compact Rheometer 302 from Anton Paar equipped with a CP 50-1 cone-and-plate measuring system, 50 mm diameter, 1 ° cone angle and 50 pm cone truncation (Anton Paar GmbH, Austria) and a Peltier controlled TEK 150 P temperature unit (Anton Paar GmbH, Austria), at 20 °C (samples preferably incubated in the measuring system at 20 °C for 5 min prior to the measurement).

[0263] In another aspect, the present invention also relates to a polysaccharide having the formula (I): — k- D-glucose — D-glucose - -

[0264] I | J n

[0265] D-mannose

[0266] D-glucuro Inic acid x (I). wherein the D-glucose moieties are linked in a p-[1 ,4] configuration; the D-mannose moiety, optionally acetylated at the 6-0 position, is linked in an a-[1 ,3] configuration to the D-glucose moiety; the D-glucuronic acid moiety is linked in a p-[1 ,2] configuration to the D-mannose moiety;

[0267] X is a monosaccharide moiety or a monosaccharide derivative moiety, wherein X may be optionally substituted and wherein further optionally one or more, preferably one or two, monosaccharide hydroxy groups may be replaced by halogen, alkyl, preferably Ci-Ce alkyl, more preferably methyl or ethyl, alkoxy, preferably methoxy, monocarboxylic acids having 2 to 8 carbon atoms, preferably acetyl, and dicarboxylic acids having 2 to 9 carbon atoms, preferably pyruvyl, wherein the dicarboxylic acid may optionally be ketal-linked to the monosaccharide, NH2, -NHR1, and -N(R1)2, wherein each R1is independently selected from alkyl, preferably C1-C6 alkyl, monocarboxylic acids having 2 to 8 carbon atoms, preferably acetyl, and dicarboxylic acids having 2 to 9 carbon atoms, preferably pyruvyl, wherein the dicarboxylic acid may optionally be ketal- linked to the monosaccharide; wherein each alkyl, alkoxy, monocarboxylic acid, and dicarboxylic acid group may be independently substituted with one or more substituents selected from halogen, OH, CH3, OCH3, SH, SCH3, NH2, NHCH3, N(CH3)2; n is an integer from 2 to 2000; with the proviso that X is not a p-[1 ,4]-linked D-mannose moiety or a p-[1 ,4]-linked D-mannose moiety containing a ketal-linked pyruvic acid at the 4,6 position; or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof.

[0268] It is to be understood that the polysaccharide of the present invention described by formula (I) above consists of repeating units arranged sequentially. It has free chain ends at both termini, meaning no covalent bonds connect these ends to other structures. The polymer's identity is defined by the repetition of its core structural unit as specified by formula (I), with n representing the number of these units in the polymer chain.

[0269] In some embodiments, X is p-linked. In other embodiments, X is a-linked.

[0270] In various embodiments, X is substituted with one or more substituents, wherein each substituent is independently selected from halogen, alkyl, preferably Ci-Ce alkyl, more preferably methyl or ethyl, alkoxy, preferably methoxy, monocarboxylic acids having 2 to 8 carbon atoms, preferably acetyl, and dicarboxylic acids having 2 to 9 carbon atoms, preferably pyruvyl, wherein the dicarboxylic acid may optionally be ketal- linked to the monosaccharide, NH2, -NHR1, and -N(R1)2, wherein each R1is independently selected from C1-C6 alkyl, monocarboxylic acids having 2 to 8 carbon atoms, preferably acetyl, and dicarboxylic acids having 2 to 9 carbon atoms, preferably pyruvyl, wherein the dicarboxylic acid may optionally be ketal-linked to the monosaccharide; wherein each alkyl, alkoxy, monocarboxylic acid, and dicarboxylic acid group may be independently substituted with one or more substituents selected from halogen, OH, CH3, OCH3, NH2, NHCH3, and N(CH3)2. In some embodiments, X is not substituted.

[0271] In some embodiments, the polysaccharide of the present invention is characterized in that X is selected from a pentose, a hexose, a heptose, or an octose, as well as from their respective uronic acids, ulosonic acids, aldonic acids and aldaric acids, as well as from their respective amino sugar derivatives, preferably selected from a pentose and a hexose or a uronic acid or aminosugar derived therefrom.

[0272] In various further embodiments, X is selected from ribose, arabinose, cladinose, xylose, lyxose, deoxyribose, ribulose, xylulose, allose, altrose, glucose, fructose, mannose, mannuronic acid, guluronic acid, glucuronic acid, sorbose, gulose, idose, iduronic acid, psicose, galactose, talose, tagatose, fuculose, fucose, rhamnose, qinovose, ketodeoxyoctulosonic acid, neuraminic acid, glucosamine, N-acetyl- glucosamine, muramic acid, N-acetylmuramic acid, galactosamine, quinovosamine, rhamnosamine, 2- deoxy-glucose, fluorodeoxyglucose, 6-deoxyfructose, 1 ,6-dichlorofructose, 3,6-anhydrogalactose, 1-0- methyl galactose, 6-0-methyl galactose, 1-0-methyl glucose, 1-0-methyl fructose, 3-0-methyl fructose, sedoheptulose, mannoheptulose, 2-keto-3-deoxy-mannooctanoic acid, and sialinic acids, such as N- acetylneuraminic acid and N-glycolylneuraminic acid.

[0273] In some embodiments, X is selected from glucose, galactose, xylose, / V-acetyl-glucosamine, / V-acetyl- galactosamine, glucuronic acid, galactofuranose, mannose, fucose, rhamnose, ketodeoxyoctulosonic acid, neuraminic acid, sialic acids such as / V-acetylneuraminic acid and / V-glycolylneuraminic acid, and 2-keto- 3-deoxy-mannooctanoic acid, mannuronic acid, glucuronid acid, guluronic acid or iduronic acid. In some embodiments, X Is not D-mannose. In some embodiments, X is not L-mannose. In some embodiments, X is not mannose. In some embodiments, X is galactose. In other embodiments, X is glucuronic acid.

[0274] In some embodiments, the polysaccharide of the present invention can be defined by the following formula (II):

[0275] wherein n is as defined herein above in the context of formula (I); X is as defined herein above in the context of formula (I) and wherein X is linked to the remainder of the molecule via a glycosidic bond, which may be either a p-glycosidic bond or an a-glycosidic bond; and R2is independently selected from OH and O(CO)CH3.

[0276] It is to be understood that the polysaccharide of the present invention described by formula (II) above consists of repeating units arranged sequentially. It has free chain ends at both termini, meaning no covalent bonds connect these ends to other structures. The polymer's identity is defined by the repetition of its core structural unit as specified by formula (II), with n representing the number of these units in the polymer chain.

[0277] Thus, the polysaccharide of the present invention may further be defined by the following formula (11.1): wherein n, X, and R2are as defined herein above. In various embodiments, the polysaccharide of formula (I) is characterized in having an average molecular weight between about 1 .0 • 104and 1 .0 • 109Da, preferably between about 1 .0 • 105and 1 .0 • 108Da, more preferably between about 1.0 • 106and 9.0 • 107Da, such as between about 2.0 • 106and 5.0 • 107Da. A non-limiting example of a method for determination of the average molecular weight has been described herein above.

[0278] In various embodiments, the polysaccharide of the present invention is characterized in having a k value (viscosity) in the range of about 10 Panto about 35 Pan, preferably in the range of about 12 Panto about 30 Pan, more preferably in the range of about 12 Panto about 28 Pan, such as about 12, 15, 17, 20, 23, 25 or 27 Pan,. A preferred method for determining the k value has been described herein above.

[0279] In another aspect, the present invention relates to the use of a polysaccharide as herein described and defined, i.e., as obtainable by a method of the present invention and / or as defined by chemical formula (I), as a thickening and / or gelling agent and / or as a rheology modifier, preferably in foods and / or feeds, cosmetics, medicinal and pharmaceutical formulations, inks and printing formulations, sizings for papers and / or textiles, drilling muds, or concrete, film compositions, or coating compositions.

[0280] In a further aspect, the present invention relates to a composition comprising the polysaccharide as herein described and defined, i.e., as obtainable by a method of the present invention and / or as defined by chemical formula (I), wherein preferably the composition is selected from a food composition, a feed composition, a cosmetic composition, a medicinal composition, a pharmaceutical composition, an ink composition, a printing composition, a paper sizing composition, a textile sizing composition, a drilling mud composition, or a concrete composition, a film composition, or a coating composition.

[0281] The uses and compositions of the present invention encompass the polysaccharide of the present invention preferably in amounts typical for the respective application. For instance, but without limitation, the polysaccharide of the present invention may be present in the compositions of the present invention in amounts of about 0.001 to about 70, 60, 50, 40, 30 or 20 wt.-%, such as in amounts of about 0.01 to 15 wt.-%, for example in amounts of about 0.1 to 10 wt.-%, about 0.5 to 8 wt.-%, or about 0.5 to 5 wt.-%, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, 2.0, 1 .5, 3.0, 3.5, 4.0, 4.5 or 5.0 wt.-%. The polysaccharide of the present invention may be present as a thickening agent, a gelling agent, viscosity regulator, and / or friction regulator.

[0282] Examples

[0283] In the working examples described herein after, the following protocols were followed, including methods of characterization of the polysaccharides thusly produced: Papagianni et al. ("Xanthan production by Xanthomonas campestris in batch cultures." Process Biochemistry 37.1 (2001): 73-80) and Faria et al. ("Characterization of xanthan gum produced from sugar cane broth." Carbohydrate polymers 86.2 (2011): 469-476). 1 % (w / v) EPS samples in ultra pure water were analyzed with a Modular Compact Rheometer 302 from Anton Paar equipped with a CP 50-1 cone-and-plate measuring system, 50 mm diameter, 1 ° cone angle and 50 pm cone truncation (Anton Paar GmbH, Austria) and a Peltier controlled TEK 150 P temperature unit (Anton Paar GmbH, Austria). All measurements were performed at 20 °C and all samples were incubated in the measuring system at 20 °C for 5 min prior to the measurement.

[0284] Determination of flow curves were performed at a logarithmically increasing shear rate from 10“3-103s-1by measuring 4 data points per decade with a decreasing measuring time of 100- 5s per data point.

[0285] Frequency sweeps were performed in the linear viscoelastic region (LVE) at a logarithmically increasing frequency from 10“2- 101Hz.

[0286] Table 2: results of rheological analysis

[0287] The average molecular weight of xanthan ranges between 2.0 • 106and 5.0 • 107Da.

[0288] Table 3: Results of molecular weight analysis

[0289] Table 4: Results of monomer analysis (determined according to 1-phenyl-3-methyl-5-pyrazolone method; cf Fig. 2)

Claims

CLAIMS1. Polypeptide, comprising an N-terminal acceptor binding site domain, a C-terminal donor binding site domain, and a linker loop linking the N-terminal acceptor binding site domain and the C-terminal donor binding site domain, wherein(i) the N-terminal acceptor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; and / or(ii) the linker loop comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 4; and / or(iii) the C-terminal donor binding site domain comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and / or(iv) the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain comprising a first amino acid sequence derived from a GT-B type glycosyltransferase distinct from the GT- B type glycosyltransferase from which the N-terminal acceptor binding site domain is derived, and a second amino acid sequence derived from the same GT-B type glycosyltransferase as the N-terminal acceptor binding site domain, wherein the first amino acid sequence is linking the linker loop and the second amino acid sequence.

2. The polypeptide according to claim 1 , wherein the polypeptide is a polypeptide according to any one or more of items (i)-(iii) and wherein the N-terminal acceptor binding site domain and the C-terminal donor binding site domain originate from different GT-B type glycosyltransferases.

3. The polypeptide according to claim 1 , wherein(i) The polypeptide is a chimeric GT-B type glycosyltransferase; and / or(ii) the polypeptide is isolated, cytosolic or membrane-bound; and / or(iii) the N-terminal acceptor binding site domain and the linker loop originate from different GT-B type glycosyltransferases; and / or(iv) the C-terminal donor binding site domain and the linker loop originate from different GT-B type glycosyltransferases; and / or(v) the linker loop consists of 28 to 36 amino acid residues, preferably of 29 to 36 amino acid residues, for instance, 30 to 35 amino acid residues.

4. The polypeptide according to any one of the preceding claims, wherein the polypeptide is capable of catalyzing the transfer of a monosaccharide moiety from a donor to an acceptor substrate selected from monosaccharides, disaccharides, polysaccharides, lipids and proteins, preferably polysaccharides.

5. The polypeptide according to any one of the preceding claims, wherein the polypeptide is capable of catalyzing the transfer of a monosaccharide moiety from a donor, preferably a sugar nucleotide or lipid- phospho-sugar, to p-D-GlcA-(1 ^2)-a-D-Man-(1 ^3)-p-D-Glc-(1 ^4)-D-Glc, which preferably may be linked to a carrier molecule, such as an isoprenoid lipid carrier molecule, optionally bound through a pyrophosphate linking group, such as in the form of p-D-GlcA-(1 ^2)-a-D-Man-(1 ^3)-p-D-Glc-(1 ^4)-D- Glc-1-diphospho-ditrans,octacis-undecaprenol.

6. The polypeptide according to any one of the preceding claims, comprising or consisting of(i) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, or(ii) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

7. The polypeptide according to any one of claim 1 and claims 3-5, wherein the C-terminal donor binding site domain is a chimeric C-terminal donor binding site domain as defined in item (iv) of claim 1 and wherein(i) the second amino acid sequence of the chimeric C-terminal donor binding site domain constitutes about 5 to 70 %, such as about 8 to 20 %, or 10 to 15 % or 10 to 30 %, or 20 to 40 %, or 30 to 50 %, or 40 to 60 %, or 50 to 70 %, of the entire length of the chimeric C-terminal donor binding site domain; and / or(ii) the first amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 13; and / or(iii) the second amino acid sequence comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably at least 95 %, such as 100 % sequence identity to the amino acid set forth in SEQ ID NO: 14; and / or(iv) the polypeptide comprises or consists of amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98, 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

8. Nucleic acid encoding the polypeptide according to any one of claims 1 to 7.

9. Vector comprising a nucleic acid molecule according to claim 8, wherein the vector preferably is a plasmid.

10. Host cell comprising a nucleic acid molecule according to claim 8 or a vector according to claim 9, the host cell preferably being a prokaryotic host cell.

11. Method for the production of a polypeptide according to any one of claims 1 to 7, comprising(1) cultivating the host cell of claim 10 under conditions that allow the expression of the polypeptide;(2) optionally isolating the expressed polypeptide from the host cell.

12. Method for the production of a polysaccharide using the polypeptide according to any one of claims 1 to 7.

13. The method according to claim 12, comprising(a) optionally expressing a polypeptide of any one of claims 1 to 7 in a suitable host cell; and(b) contacting said polypeptide with at least one suitable donor substrate (glycosyl donor molecule) and at least one suitable acceptor substrate (glycosyl acceptor molecule) under conditions that allow the enzymatic production of a xanthan derivative, wherein optionally(i) the donor substrate is a nucleotide-sugar or lipid-phospho-sugar; and / or(ii) the acceptor substrate is a polysaccharide, preferably a polyprenyl phospho oligosaccharide, more preferably p-D-GlcA-(1 -^2)-a-D-Man-(1 -^3)-p-D-Glc-(1 — >4)-a-D-Glc-1 -diphospho-d / trans.octac / s- undecaprenol; and / or(iii) the method comprises a step of isolating the produced polysaccharide; and / or(iv) the host cell is Xanthomonas campestris.

14. Polysaccharide obtainable by a method according to claim 12 or claim 13.

15. Polysaccharide having the formula (I):— D-glucose — D-glucose - -I | J nD-mannoseD-glucuronic acidwherein the D-glucose moieties are linked in a p-[1 ,4] configuration; the D-mannose moiety, optionally acetylated at the 6-0 position, is linked in an a-[1 ,3] configuration to the D-glucose moiety; the D-glucuronic acid moiety is linked in a p-[1 ,2] configuration to the D-mannose moiety;X is a monosaccharide moiety or a monosaccharide derivative, wherein X may be optionally substituted and wherein further optionally one or more, preferably one or two, monosaccharide hydroxy groups may be replaced by halogen, alkyl, preferably Ci-Ce alkyl, more preferably methyl or ethyl, alkoxy, preferably methoxy, monocarboxylic acids having 2 to 8 carbon atoms, preferably acetyl, and dicarboxylic acids having 2 to 9 carbon atoms, preferably pyruvyl, wherein the dicarboxylic acid may optionally be ketal-linked to the monosaccharide, NH2, -NHR1, and -N(R1)2, wherein each R1is independently selected from alkyl, preferably C1-C6 alkyl, monocarboxylic acids having 2 to 8 carbon atoms, preferably acetyl, and dicarboxylic acids having 2 to 9 carbon atoms, preferably pyruvyl, wherein the dicarboxylic acid may optionally be ketal-linked to the monosaccharide; wherein each alkyl, alkoxy, monocarboxylic acid, and dicarboxylic acid group may be independently substituted with one or more substituents selected from halogen, OH, CH3, OCH3, SH, SCH3, NH2, NHCH3, N(CH3)2; n is an integer from 2 to 2000; with the proviso that X is not a p-[1 ,4]-li nked D-mannose moiety or a p-[1 ,4]-lin ked D-mannose moiety containing a ketal-linked pyruvic acid at the 4,6 position; or a stereoisomer, tautomer, pharmaceutically acceptable salt, or hydrate thereof, wherein optionallyX is selected from a pentose, a hexose, a heptose, or an octose, as well as from their respective uronic acids, ulosonic acids, aldonic acids and aldaric acids, as well as from their respective amino sugar derivatives, preferably selected from a pentose and a hexose or a uronic acid or aminosugar derived therefrom; and / orX is selected from ribose, arabinose, cladinose, xylose, lyxose, lactose, deoxyribose, ribulose, xylulose, allose, altrose, glucose, fructose, mannose, mannuronic acid, guluronic acid, glucuronic acid, sorbose, gulose, idose, iduronic acid, psicose, galactose, talose, tagatose, fuculose, fucose, rhamnose, qinovose, ketodeoxyoctulosonic acid, neuraminic acid, glucosamine, N-acetyl-glucosamine, muramic acid, N- acetylmuramic acid, galactosamine, quinovosamine, rhamnosamine, 2-deoxy-glucose, fluorodeoxyglucose, 6-deoxyfructose, 1 ,6-dichlorofructose, 3,6-anhydrogalactose, 1-O-methyl galactose, 6-O-methyl galactose, 1-O-methyl glucose, 1-O-methyl fructose, 3-O-methyl fructose, sedoheptulose, mannoheptulose, 2-keto-3-deoxy-mannooctanoic acid, and sialinic acids, such as N-acetylneuraminic acid and N-glycolylneuraminic acid, more preferably X is selected from glucose, galactose, xylose, / V-acetyl- glucosamine, / V-acetyl-galactosamine, glucuronic acid, galactofuranose, mannose, fucose, rhamnose, ketodeoxyoctulosonic acid, neuraminic acid, sialic acids such as / V-acetylneuraminic acid and N- glycolylneuraminic acid, and 2-keto-3-deoxy-mannooctanoic acid.

16. Use of a polysaccharide according to any one of claims 14 and 15 as a thickening and / or gelling agent and / or as a rheology modifier, preferably in foods and / or feeds, cosmetics, medicinal and pharmaceutical formulations, inks and printing formulations, sizings for papers and / or textiles, drilling muds, concrete, film compositions, or coating compositions.

17. Composition comprising the polysaccharide according to any one of claims 14 and 15, wherein preferably the composition is selected from a food composition, a feed composition, a cosmetic composition, a medicinal composition, a pharmaceutical composition, an ink composition, a printing composition, a paper sizing composition, a textile sizing composition, a drilling mud composition, or a concrete composition as well as filming compositions and coating compositions.

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