Glucosyltransferase and its use in the production of water-insoluble glucan

Abstract

The present invention relates to a polypeptide comprising or consisting of an amino acid sequence having at least 80 %, more preferably at least 90 %, still more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide does not consist of any of the following amino acid sequences: the amino acid sequence set forth in SEQ ID NO: 13; the amino acid sequence set forth in SEQ ID NO: 13, wherein the leucine residue of position 441 of the amino acid sequence of SEQ ID NO: 13 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence set forth in SEQ ID NO: 14; wherein the leucine residue of position 400 of the amino acid sequence of SEQ ID NO: 14 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 15; the amino acid sequence set forth in SEQ ID NO: 15, wherein the residue at position 654 of the amino acid sequence of SEQ ID NO: 15 is replaced by any other amino acid. The invention further relates to a reaction solution comprising water, sucrose, and such a polypeptide; to a method for the production of a glucan polymer, the method comprising contacting such a polypeptide with sucrose to obtain the glucan polymer; and to a composition comprising a glucan polymer obtainable by such a method, wherein the composition is selected from foods and / or feeds, cosmetics, medicinal compositions, pharmaceutical compositions, inks and printing compositions, composites, such as thermoplastic and rubber composites, sizings for papers and / or textiles, drilling muds, concrete, coating, films, and fibers.

Description

[0001] Universitat Munster

[0002] Our ref.: U08370WO / LH

[0003] METHOD FOR PRODUCTION OF INSOLUBLE GLUCAN

[0004] TECHNICAL FIELD

[0005] The present invention relates to a polypeptide comprising or consisting of an amino acid sequence having at least 80 %, more preferably at least 90 %, still more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 , wherein the polypeptide does not consist of any of the following amino acid sequences: the amino acid sequence set forth in SEQ ID NO: 13; the amino acid sequence set forth in SEQ ID NO: 13, wherein the leucine residue of position 441 of the amino acid sequence of SEQ ID NO: 13 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence set forth in SEQ ID NO: 14; wherein the leucine residue of position 400 of the amino acid sequence of SEQ ID NO: 14 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 15; the amino acid sequence set forth in SEQ ID NO: 15, wherein the residue at position 654 of the amino acid sequence of SEQ ID NO: 15 is replaced by any other amino acid. The invention further relates to a reaction solution comprising water, sucrose, and such a polypeptide; to a method for the production of a polysaccharide, e.g., glucan polymer, the method comprising contacting such a polypeptide with sucrose to obtain the glucan polymer; and to a composition comprising a polysaccharide, e.g., glucan polymer obtainable by such a method, wherein the composition is selected from foods and / or feeds, cosmetics, medicinal compositions, pharmaceutical compositions, inks and printing compositions, composites, such as thermoplastic and rubber composites, sizings for papers and / or textiles, drilling muds, concrete, coating, films, and fibers.

[0006] BACKGROUND OF THE INVENTION

[0007] Exopolysaccharides (EPSs) such as glucan represents interesting and valuable alternatives to conventional types of polymers commonly used in various applications ranging from the food and medicinal sector to composite structures and generally films and coatings in being biodegradable and producible from renewable sources.

[0008] Glucan is a polysaccharide derived from D-glucose monomers. The monomers are linked by glycosidic bonds. Glucans exist in two forms, namely as alpha glucans or beta glucans. Examples of alpha glucans include starch and glycogen, examples of beta glucan include cellulose and laminarin.

[0009] Most of the known glucans and many other exo polysaccharides are water-soluble. Only a few bacterial sources of water-insoluble alpha-glucan are known thus far, which mainly originate from the Streptococcal bacteria.

[0010] US 9284540 B2 discloses in vitro methods for the production of water-insoluble alpha-glucan, said method relying on the glucosyltransferase GtfJ from Streptococcus salivarius. S. salivarius is a pathogenic bacterium and thus its application as a whole-cell biocatalyst is limited due to complicated downstream purification processes for the removal of potential living cells or toxins, particularly if the end product in intended for use in foods, pharmaceutical or medicinal applications.

[0011] LH:KAThe inventors of the present invention have discovered a novel glucosyltransferase (termed Gtfc3) from the nonpathogenic bacterium Leuconostoc suionicum, which is able to generate a water-insoluble glucan polymer. Using the glucosyltransferase Gtfc3, production of the said glucan polymer can be realized both in vivo and in vitro. Recombinant expression of Gtfc3 can be effected in recombinant hosts, wherein employment of nonpathogenic hosts such as Escherichia coli or Paenibacillus polymyxa, which is generally recognized as a safe (GRAS) organism, allows for less intensive purification of the desired end product. Due to the water insolubility of the glucan polymer, the in vitro production method is overall more sustainable as well as time- and cost-efficient compared to state-of-the-art methods, particularly those disclosed in US 9284540 B2, since the glucan polymer can be separated from the cultivation broth by simple filtration.

[0012] SUMMARY OF THE INVENTION

[0013] In a first aspect, the present invention thus provides fora polypeptide comprising or consisting of an amino acid sequence having at least 80 %, more preferably at least 90 %, still more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 , wherein the polypeptide does not consist of any of the following amino acid sequences: the amino acid sequence set forth in SEQ ID NO: 13; the amino acid sequence set forth in SEQ ID NO: 13, wherein the leucine residue at position 441 of the amino acid sequence of SEQ ID NO: 13 is replaced by an amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence set forth in SEQ ID NO: 14; wherein the leucine residue at position 400 of the amino acid sequence of SEQ ID NO: 14 is replaced by an amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 15; the amino acid sequence set forth in SEQ ID NO: 15, wherein the residue at position 654 of the amino acid sequence of SEQ ID NO: 15 is replaced by any other amino acid.

[0014] In some embodiments, the polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the enzymatic glucosyltransferase activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 .

[0015] In some embodiments, the polypeptide is isolated.

[0016] In a further aspect, the present invention provides for a reaction solution comprising water, sucrose, and at least one polypeptide according to the present invention, i.e., as defined and disclosed herein.

[0017] In some embodiments, the reaction solution is not comprised within a bacterial cell and / or released from a bacterial cell.

[0018] In some embodiments, the reaction solution is characterized in that the polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the enzymatic glucosyltransferase activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 .

[0019] In a further aspect, the present invention provides for a method for the production of a polysaccharide,preferably a glucan polymer, the method comprising contacting at least one polypeptide comprising 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 with sucrose to obtain the polysaccharide, wherein the polypeptide does not consist of any of the following amino acid sequences: the amino acid sequence set forth in SEQ ID NO: 13; the amino acid sequence set forth in SEQ ID NO: 13, wherein the leucine residue of position 441 of the amino acid sequence of SEQ ID NO: 13 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence set forth in SEQ ID NO: 14; wherein the leucine residue of position 400 of the amino acid sequence of SEQ ID NO: 14 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 15; the amino acid sequence set forth in SEQ ID NO: 15, wherein the residue at position 654 of the amino acid sequence of SEQ ID NO: 15 is replaced by any amino acid.

[0020] In some embodiments, the method is characterized in that the polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the enzymatic glucosyltransferase activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1.

[0021] In some embodiments, the method is characterized in that the glucan polymer is water-insoluble.

[0022] In some embodiments, the method is characterized in that the method is an in vitro or an in vivo method.

[0023] In some embodiments, the method is characterized in that the method comprises a step of isolating the glucan polymer produced.

[0024] In some embodiments, the method is characterized in that the method comprises the recombinant expression of the glucosyltransferase in a suitable host cell.

[0025] In some embodiments, the method is characterized in that the method yields the glucan polymer in a concentration of at least about 10 g / L, preferably in about 48 hours, or in a concentration of at least about 12 g / L, preferably in about 48 hours, or in a concentration of at least about 14 g / L, preferably in about 48 hours.

[0026] In some embodiments, the method is characterized in that the host cell is selected from bacterial cells, for instance Gram-negative bacterial cells, such as Escherichia coli, preferably Gram-positive bacterial cells, preferably selected from Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, or Paenibacillus, such as Paenibacillus polymyxa.

[0027] In another aspect, the present invention provides for a nucleic acid molecule encoding the polypeptide according to the present invention, as herein defined and described.

[0028] In another aspect still, the present invention also provides for a vector comprising a nucleic acid molecule according to the present invention, wherein the vector preferably is a plasmid.In yet another aspect, the present invention provides for a host cell comprising a nucleic acid molecule according to the present invention or a vector according to the present invention, the host cell preferably being a prokaryotic host cell.

[0029] In some embodiments, the host cell is characterized in that it is not Leuconostoc suionicum.

[0030] In another aspect, provided herein is a method for the production of a polypeptide according to the present invention, as herein defined and described, comprising

[0031] (1) cultivating the host cell according to the present invention under conditions that allow the expression of the polypeptide according to the present invention;

[0032] (2) optionally isolating the expressed polypeptide from the host cell.

[0033] In some embodiments, the method for the production of a polypeptide of the present invention is characterized in that the polypeptide is heterologously expressed in said host cell.

[0034] In some embodiments, the method for the production of a polypeptide of the present invention is characterized in that the host cell is selected from bacterial cells, for instance Gram-negative bacterial cells, such as E. coli, preferably Gram-positive bacterial cells, preferably selected from Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, or Paenibacillus, such as Paenibacillus polymyxa, wherein the host cell is preferably not Leuconostoc suionicum.

[0035] Another aspect provided herein is the use of a polypeptide according to the present invention for the biotechnological production of a polysaccharide, preferably a glucan polymer.

[0036] In some embodiments of this aspect, the polysaccharide is water-insoluble, wherein preferably the polysaccharide is a glucan polymer.

[0037] In another aspect, the present invention provides for a composition comprising a polysaccharide, preferably a glucan polymer, obtainable by a method for the production of a polysaccharide according to the present invention, wherein the composition is selected from foods and / or feeds, cosmetics, medicinal compositions, pharmaceutical compositions, inks and printing compositions, composites, such as thermoplastic and rubber composites, sizings for papers and / or textiles, drilling muds, concrete, coating, films, and fibers.

[0038] In a final aspect, the present invention also provides for the use of a polysaccharide, preferably glucan polymer, obtainable by a method for the production of polysaccharide according to the present invention as an ingredient in foods and / or feeds, cosmetics, medicinal and pharmaceutical formulations, inks and printing formulations, composites, such as thermoplastic and rubber composites, sizings for papers and / or textiles, drilling muds, concrete, coating, films or fibers.

[0039] In some embodiments of this aspect, the glucan polymer is used as a thickening and / or gelling agent and / or as a rheology modifier and / or fibrous component.

[0040] In a further aspect, the present invention also relates to a polysaccharide obtainable by a method for theproduction of polysaccharide according to the present invention, as herein described and defined, wherein preferably the polysaccharide is a glucan polymer.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows EPS production by engineered P. polymyxa and the effect of substrate concentration on recombinant glucan production.

[0043] Figure 2 shows characterization of the water-insoluble glucan produced in accordance with the present invention by (A) thermogravimetric (TGA) analysis and (B) its solubility range in sodium hydroxide.

[0044] Figure 3 shows production of the water-insoluble glucan by engineered E. coli carrying gtfc3 gene from L. suionicunr. (A) schematic overview of different protein variants created in this study, each variant having a unique combination of 6xHis-Tag and signal peptide; (B) production of water-insoluble glucan by E. coli strains carrying different protein variants.

[0045] Figure 4 shows a schematic overview of production steps of water soluble and water-insoluble EPS.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] Before describing in detail exemplary embodiments ofthe present invention, definitions which are important for understanding the present invention are given.

[0048] 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.

[0049] “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.

[0050] In the context ofthe 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 %.

[0051] 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.“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.

[0052] 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 ofthe invention additionally comprise the amino acid M on the N-terminus.

[0053] “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. “Isolated” may thus mean that said molecule is no longer cell membrane-bound.

[0054] Determination ofthe 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.

[0055] Also possible are sequence alignments using the computer program 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.

[0056] Such a comparison also allows determination ofthe similarity ofthe 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. aminoacids that are similar in regard to 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, the polypeptide of the present invention has, over its entire length, the sequence identity as herein defined. For instance, but without limitation, the polypeptide of the invention may be a shortened variant of SEQ ID NO: 1 that comprises at least 1201 continuous amino acids of the amino acid sequence of SEQ ID NO: 1 (equaling 80 % sequence identity).

[0057] In the context of the present invention, the feature that an amino acid position corresponds to a numerically defined position in a reference sequence means that the respective position correlates to the numerically defined position in said reference sequence in an alignment obtained as described above.

[0058] 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 preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.

[0059] 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).

[0060] In the context of the present invention, the term “glucan” refers to a polysaccharide that consists exclusively of D-glucose monomers linked together by glycosidic bonds. The nature of those bonds (a- or p-, and the carbon positions involved) determines the glucan’s three-dimensional structure, solubility, and biological function. For most glucans, usually the pyranose form of glucose is the single monomer. In glucans, generally glycosidic linkage types such as, a-1 ,4; a-1 ,6; p-1 ,3; p-1 ,4; p-1 ,6 exist. Based on the linkage types, a linear (cellulose, amylose, curdlan) or branched structure exists (amylopectin, glycogen, various p glucans and dextran). The molecular weight can differ from a few thousand daltons to several million daltons.

[0061] 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.

[0062] As surprisingly found by the present inventors, the nonpathogenic bacterium L. suionicum expresses a number of glucosyltransferases, one of which is the glucosyltransferase Gtfc3, which is capable of generating a water-insoluble exopolysaccharide of the glucan type.In the context of the present invention, the term “water-insoluble” means that the respective molecule or compound is not soluble in water at 25 °C. Thus, the term “water-insoluble glucan-type exopolysaccharide” or “water-insoluble glucan polymer” refers to a polysaccharide of the glucan type that is not soluble in water at 25 °C, i.e., that does not dissolve in water at 25 °C.

[0063] In a first aspect, the present invention thus provides a polypeptide comprising or consisting of an amino acid sequence having at least 80 %, such as at least 81 , 82, 83 or 84 %, more preferably at least 85 %, such as at least 86, 87, 88 or 89 %, still more preferably at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide does not consist of any of the following amino acid sequences: the amino acid sequence set forth in SEQ ID NO: 13; the amino acid sequence set forth in SEQ ID NO: 13, wherein the leucine residue at position 441 of the amino acid sequence of SEQ ID NO: 13 is replaced by an amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence set forth in SEQ ID NO: 14; wherein the leucine residue at position 400 of the amino acid sequence of SEQ ID NO: 14 is replaced by an amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 15; the amino acid sequence set forth in SEQ ID NO: 15, wherein the residue at position 654 of the amino acid sequence of SEQ ID NO: 15 is replaced by any other amino acid.

[0064] In the context of the present invention, phrases such as “any amino acid other than”, “an amino acid other than”, and “any other amino acid” are understood to refer to an amino acid substitution at a specified position within an amino acid sequence, such that the amino acid originally present at the specified position in the referenced amino acid sequence, i.e., the original amino acid sequence, is replaced by an amino acid that does not correspond to said original amino acid at the specified position within the original sequence. This means that the amino acid sequence resulting from such substitution does not match the original amino acid sequence at the specified position.

[0065] In various embodiment, the polypeptide of the present invention is characterized in that it has at least 85 %, such as at least 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93 or 94 %, more preferably at least 95 %, such as at least 96, 97, 98 or 99 %, most preferably at least 100 % of the enzymatic glucosyltransferase activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 . For instance, in some embodiments, enzymatic glucosyltransferase activity of a polypeptide of the present invention may be determined by quantifying the amount of water-insoluble glucan formed by the respective enzyme. Methods for quantification of polysaccharides are generally known to the skilled person. For instance, quantification of the water-insoluble glucan produced by the polypeptide of the present invention may be accomplished by pouring the reaction solution through a fine mesh sieve to collect the waterinsoluble glucan, then washing it with water to remove remaining impurities, and then drying it at 40 °C overnight in a vacuum oven; the weight of the dried water-insoluble glucan may be measured using an analytical balance. The determined amount may be compared with the amount formed by the glucosyltransferase having the amino acid sequence set forth in SEQ ID NO: 1. Formation of the waterinsoluble glycan is generally visible within the reaction vessel. The water-insoluble glycan may be collected, washed with water, and dried. In some embodiments, enzymatic glucosyltransferase activity of apolypeptide of the present invention may be determined by quantifying the decrease in the reaction substrate (sucrose), for example, using ion exclusion chromatography. The quantified decrease in the reaction substrate may be compared with the decrease in the reaction substrate determined for the glucosyltransferase having the amino acid sequence in SEQ ID NO: 1. In some embodiments, for ion exclusion chromatography, the HPLC is equipped with a Rezex™ ROA-organic acids H+column and coupled with a refractive index detector; the column temperature is set to 70 °C with a flow rate of 0.5 mL / min; the sample injection volume is 10 pL and 2.5 mM H2SO4 is used as the running buffer for the system; for sample preparation, 980 pL of a 1 % (w / v) glucan solution is mixed with 20 pL H2SO4 and incubated overnight at 90 °C; the sample is then centrifuged at 9,000 rpm for 5 min and the supernatant is filtered through a 0.22 pm PVDF filter. Quantifying the amount of the water-insoluble glucan may thus encompass optionally hydrolyzing the glucan and measuring the glucose monomers. In some embodiments, determination of enzymatic glucosyltransferase activity of a polypeptide of the present invention may include determining the weight of the purified (washed) and dried glucan and determining of glucose monomers via hydrolysis and HPLC (HILIC) measurement.

[0066] In some preferred embodiments, the polypeptide of the present invention comprises or consists of an amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide does not consist of any of the following amino acid sequences: the amino acid sequence set forth in SEQ ID NO: 13; the amino acid sequence set forth in SEQ ID NO: 13, wherein the leucine residue of position 441 of the amino acid sequence of SEQ ID NO: 13 is replaced by an amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence set forth in SEQ ID NO: 14; wherein the leucine residue of position 400 of the amino acid sequence of SEQ ID NO: 14 is replaced by an amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 15; the amino acid sequence set forth in SEQ ID NO: 15, wherein the residue at position 654 of the amino acid sequence of SEQ ID NO: 15 is replaced by any other amino acid.

[0067] In some preferred embodiments, the polypeptide of the present invention is characterized in comprising the amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, which is linked, preferably through a peptide bond, to an amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17, such that the amino acid sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 precedes the amino acid sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17 in the reading from the N-terminus to the C-terminus.

[0068] In particular embodiments, the polypeptide of the present invention is characterized in comprising the amino acid sequence having at least 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, which is linked, preferably through a peptide bond, to an amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly atlast 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0069] In some preferred embodiments, the polypeptide of the present invention is characterized in consisting of amino acid sequence having at least 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 and an amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17, wherein the amino acid sequence having at least 95 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 is linked, preferably through a peptide bond, to the amino acid sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0070] In some preferred embodiments, the polypeptide of the present invention is characterized in comprising the amino acid sequence forth in SEQ ID NO: 16, which is linked, preferably through a peptide bond, to an amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0071] In some preferred embodiments, the polypeptide of the present invention is characterized in consisting of the amino acid sequence forth in SEQ ID NO: 16 and an amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17, wherein the amino acid sequence of SEQ ID NO: 16 is linked, preferably through a peptide bond, to the amino acid sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0072] 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 or deletions 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.

[0073] 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.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 polypeptides 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.

[0074] 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.

[0075] In some embodiments, the polypeptide 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.

[0076] In various embodiments, the polypeptide 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 molecularweights, is known as a means to alter pharmacokinetics of such compounds.

[0077] The polypeptide of the present invention may be an isolated polypeptide, as herein defined.

[0078] 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.

[0079] Thus, in some embodiments, a nucleic acid of the present invention is characterized in that it has at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, 98 or 99 %, more particularly 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 2.Exemplary nucleic acid sequences encoding exemplary polypeptides of the present invention are set forth in the following Table 1 :

[0080] Table 1: Amino acid sequences and corresponding nucleic acid sequences in accordance with the present invention

[0081]

[0082] If the polypeptide 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 aforementioned amino acid sequences.

[0083] 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.

[0084] 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. In some embodiments, the sacB operator system, including the sacB promoter, may be employed. In some embodiments, the nucleic acid may be in the form of linear nucleic acid molecules, such as linear DNA, single-stranded or double-stranded, which can be integrated into the respective host organism.

[0085] 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.In certain embodiments the nucleic acid molecules disclosed herein are comprised in a cloning vector.

[0086] 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.

[0087] 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, 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.

[0088] The above-described nucleic acid molecule of the present invention, comprising a nucleic acid sequence encoding for the polypeptide of the invention, 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.

[0089] In addition, the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. 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.

[0090] 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.

[0091] 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 forprokaryotic expression are, for example, the araBAD promoter, the tet-promoter, the lacUV5 promoter, the the tac promotor, the T7promoter, the lac promotor, or the sacB promoter. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter, the AOX1 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.

[0092] The vector may be effective for prokaryotic or eukaryotic protein expression. Suitable vectors are known to those skilled in the art.

[0093] The vectors of the present invention may be chosen from the group consisting of high, medium and low copy vectors.

[0094] 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.

[0095] Thus, in a further aspect, the present invention also relates to a host cell comprising a vector or nucleic acid molecule as disclosed herein.

[0096] 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 transfer of free DNA, conjugation, 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.

[0097] 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 order to 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).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 selected from bacterial cells, preferably Gram-positive bacterial cells, preferably selected from Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, or Paenibacillus, such as Paenibacillus polymyxa. In some embodiments, the host cell is a Gram-negative bacterium, such as Escherichia coli (E. coli). In certain embodiments, the host cell is E. coli. In other embodiments, the host cell is selected from Gram-positive bacteria, such as those selected from Bacillus, such as Bacillus subtilis, Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, or Paenibacillus, such as Paenibacillus polymyxa. In further embodiments, the host cell is selected from the group consisting of E. coli, Pseudomonas, Serratia marcescens, Salmonella, Shigella (and other enterobacteriaceae), Neisseria, Hemophilus, Klebsiella, Proteus, Enterobacter, Helicobacter, Acinetobacter, Moraxella, Stenotrophomonas, Bdellovibrio, Vibrio, Legionella, acetic acid bacteria, Bacilli, such as Bacillus subtilis, Corynebacterium, Clostridium, Listeria, Streptococcus, Staphylococcus, and Archaea cells. Suitable eukaryotic host cells are among others CHO cells, insect cells, fungi, such as Trichoderma and Aspergillus, yeast cells, e.g., Saccharomyces cerevisiae, S. pombe, Pichia pastoris, and the like.

[0098] Employment of non-pathogenic Gram-positive bacterial cells such as those selected from Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, or Paenibacillus, such as Paenibacillus polymyxa allow for less extensive and less complicated downstream processing, e.g., purification, of the polypeptide and / or the EPS produced thereby.

[0099] In some embodiments, the host cell is selected from the group consisting of E. coli, in particular E. coli BL21 (DE3), E. coli BL21 , E. coli K12, E. coli BLR, E. coli BL21 Al, E. coli BL21 pLysS, E. coli , E. coli DH5a, E. coli DH1, E. coli DM1, E. coli HB101, E. coli JmlOI-110, E. coli Rosetta(DE3)pLysS, E. co / / SURE, E. coli TOP10, E. coli XLI-Blue, E. coli XL2-Blue, and E. coli XLIO-Blue.

[0100] 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.

[0101] 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.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; 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.

[0102] 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.

[0103] 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.

[0104] 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, or from the cells or both. The recovery of the polypeptide may include various purification steps.

[0105] Generally, any known culture medium suitable for growth of the selected host may be employed in this method.

[0106] 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.

[0107] In several embodiments, the method may comprise the treatment of the 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.

[0108] 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.In a further aspect, the present invention thus relates to a method for the production of a polypeptide of the present invention, comprising

[0109] (1) cultivating the host cell of the present invention under conditions that allow the expression of the polypeptide;

[0110] (2) optionally isolating the expressed polypeptide from the host cell.

[0111] In various embodiments, the polypeptide is heterologously expressed in said host cell. Non-limiting examples of suitable host cells have been discussed herein above and include Gram-positive bacterial cells, preferably selected from Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, or Paenibacillus, such as Paenibacillus polymyxa; and Gram-negative bacteria, such as E. coli.

[0112] Using the polypeptide of the present invention, i.e., the glucosyltransferase Gtfc3 or a variant thereof, as herein defined and described, production of a water-insoluble glucan-type exopolysaccharide can be realized.

[0113] Therefore, in a further aspect, the present invention relates to the use of a polypeptide, as herein described and defined, i.e., the glucosyltransferase Gtfc3 or a variant thereof, for the biotechnological production of a polysaccharide, e.g., a glucan polymer.

[0114] In another aspect, the present invention relates to the polypeptide, as herein described and defined, i.e., the glucosyltransferase Gtfc3 or a variant thereof, for use in the biotechnological production of a polysaccharide, e.g., a glucan polymer.

[0115] In this context, the present invention further relates to a reaction solution comprising water, such as tap water or deionized water, and further sucrose and at least one polypeptide according to the present invention, i.e., as disclosed and defined herein, e.g., a polypeptide comprising an amino acid sequence having at least 80 %, more preferably at least 90 %, still more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide does not consist of any of the following amino acid sequences: the amino acid sequence set forth in SEQ ID NO: 13; the amino acid sequence set forth in SEQ ID NO: 13, wherein the leucine residue of position 441 of the amino acid sequence of SEQ ID NO: 13 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence set forth in SEQ ID NO: 14; wherein the leucine residue of position 400 of the amino acid sequence of SEQ ID NO: 14 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 15; the amino acid sequence set forth in SEQ ID NO: 15, wherein the residue at position 654 of the amino acid sequence of SEQ ID NO: 15 is replaced by any other amino acid.

[0116] In various embodiments, the reaction solution is not comprised within the organism Leuconostoc suionicum, or is not comprised within a bacterial cell of the genus Leuconostoc, or is not comprised within a bacterial cell of the family of Lactobacillaceae, or is not comprised within a Gram-positive bacterial cell, and / or is notreleased from the organism Leuconostoc suionicum or a bacterial cell of the genus Leuconostoc, or a bacterial cell of the family of Lactobacillaceae, or a Gram-positive bacterial cell, or a bacterial cell. In some embodiments, the reaction solution is not comprised within the organism Leuconostoc suionicum and / or is not released from the organism Leuconostoc suionicum. In some embodiments, the reaction solution is not comprised within a bacterial cell of the genus Leuconostoc and / or is not released from a bacterial cell of the genus Leuconostoc. In some embodiments, the reaction solution is not comprised within a bacterial cell of the family of Lactobacillaceae and / or is not released from a bacterial cell of the family of Lactobacillaceae. In some embodiments, the reaction solution is not comprised within a Gram-positive bacterial cell and / or is not released from a Gram-positive bacterial cell. In some embodiments, the reaction solution is not comprised within a bacterial cell and / or is not released from a bacterial cell. In this context, the term “released from” means that the composition of the reaction solution is not obtained by breaking of the cell membrane, such as by lysis, of the respective organism. In some embodiments, the reaction solution comprises, in addition to water, sucrose, and a polypeptide of the present invention, one or more divalent cations, such as Ca2+, Mg2+, and / or one or more monovalent ions such as phosphorus. In some embodiments, the reaction solution comprises, in addition to water, sucrose, and a polypeptide of the present invention, at least one buffer system, such as a buffer system including EDTA. In some embodiments, the buffer system is selected from the group consisting of a phosphate buffer system, a carbonate buffer system, an acetate buffer system, a citrate buffer system, an ammonium buffer system, a borate buffer system, a triethanolamine buffer system, and a tartrate buffer system, preferably from a phosphate buffer system. In some embodiments, the pH of such a composition may be, for instance but without limitation, within the range of about 4.5 to 10, such as about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10, preferably within the range of about 4.5 to 8, such as about 5 to 7, such as, for instance, between about 5 to 6.

[0117] In some embodiments, the reaction solution is characterized in that the polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the enzymatic glucosyltransferase activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 .

[0118] A further aspect of the present invention pertains to a method for the production of a polysaccharide, e.g., a glucan polymer, the method comprising contacting at least one polypeptide according to the present invention, i.e., as defined and disclosed herein, e.g., a polypeptide comprising 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 with a suitable substrate to obtain the polysaccharide, e.g., the glucan polymer, wherein the polypeptide does not consist of any of the following amino acid sequences: the amino acid sequence set forth in SEQ ID NO: 13; the amino acid sequence set forth in SEQ ID NO: 13, wherein the leucine residue of position 441 of the amino acid sequence of SEQ ID NO: 13 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence set forth in SEQ ID NO: 14; wherein the leucine residue of position 400 of the amino acid sequence of SEQ ID NO: 14 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 15; the amino acid sequence set forth in SEQ ID NO: 15, wherein the residue at position 654 of the amino acid sequence of SEQ ID NO: 15 is replaced by any other amino acid.In various embodiments, the suitable substrate comprises or consists of sucrose, preferably consists of sucrose.

[0119] In some embodiments, the polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the enzymatic glucosyltransferase activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1.

[0120] In some embodiments, the glucan polymer is water-insoluble. In some embodiments, the glucan polymer produced by the polypeptide of the present invention is characterized in having a high proportion of a-1 ,3 linkages alongside a-1 ,6 linkages. In some embodiments, the 1 ,6 / 1 ,3 linkage ratio is in the range of about 1.2 to 2.2, preferably in the range of about 1.4 to about 2.0, more preferably in the range of about 1.5 to 1.9, such as about 1 .5, 1.6, 1.7, 1.8, or 1.9. In some embodiments, said ratio is about 1.7. The ratio within the said range indicates a relatively high share of 1 ,3 linkages. Branching side chains appear at the 0-3 position, and show shorter side chains compared to soluble dextrans such as the most commonly known water soluble dextrans from L. mesenteroides NRRL B-512F and close relatives. In some embodiments, branching side chains appear exclusively at the 0-3 position. In some other embodiments, about 80 to 99 %, such as about 85 to 95 %, of the branching side chains appear at the 0-3 position.

[0121] In some embodiments, the method for the production of a glucan polymer is an in vitro or an in vivo method.

[0122] In some embodiments, a method for the production of a polysaccharide, e.g., glucan polymer, according to the present invention typically comprises the following steps:

[0123] (a) expressing a polypeptide according to the present invention in a suitable host cell; and

[0124] (b) contacting said polypeptide with a suitable substrate under conditions that allow the enzymatic production of a glucan polymer.

[0125] In some embodiments, the method comprises a step of isolating the glucan polymer produced.

[0126] In various embodiments, the method for the production of a polysaccharide, e.g., glucan polymer, is characterized in that the polypeptide is heterologously expressed in said host cell.

[0127] Preferably, the suitable substrate comprises or consists of sucrose.

[0128] In the context of the present invention, the term “suitable host cell” refers to a cell, for example a microbial cell, that is able to express a polypeptide of the present invention, as herein defined and described.

[0129] Non-limiting examples of suitable host cells include, in particular, bacterial cells selected from Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, Bacillus and Paenibacillus, such as Bacillus subtilis and Paenibacillus polymyxa, and E. coll, such as E. coll 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. coliTOP10, E. coli XLI-Blue, E. coli XL2-Blue, and E. coli XLIO-Blue. In some embodiments, the host cell is selected from E. coli.

[0130] In some embodiments, the host cell is selected from E. coli and the polypeptide of the present invention is characterized in comprising the amino acid sequence having at least 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, which is linked, preferably through a peptide bond, to an amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some preferred embodiments, the polypeptide of the present invention is characterized in consisting of amino acid sequence having at least 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 and an amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17, wherein the amino acid sequence having at least 95 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 is linked, preferably through a peptide bond, to the amino acid sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0131] The polypeptide of the present invention may be expressed in said host cell, as herein defined, by means of an expression plasmid. Alternatively or additionally, integration of the glucosyltransferase coding sequence into the host genome may occur.

[0132] In some embodiments, step a) includes a cultivation step. Depending on the type of host cell selected, the total culturing period may be in the range of about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3 or 2 hours or 1 hour or less, such as 30, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3 or minutes or 1 minute. In some embodiments, the culturing period is in the range of about 2 hours to 80 hours, such as about 5 hours to 48 hours.

[0133] The culturing times indicated may vary depending on the type and / or volume of culturing. The cultivation step may include one or more preculturing steps and one or more main culturing steps. For instance, but without limitation, it may be necessary to carry out a culture cascade, particularly with regard to very large fermentation volumes, such that at least one step of preculturing and at least one step of main culturing may be required. Additionally or alternatively, longer preculturing and / or main culturing times might be required.

[0134] It has been found helpful to provide for lower dissolved oxygen levels and / or lower pH levels, both of which, also in combination, were found to be beneficial for the production of recombinant glucan, particularly when employing Paenibacillus, such as Paenibacillus polymyxa, or E. coli as the host. In some embodiments, the host cell is E. coli.In some embodiments, the pH of the culture medium may be, for instance but without limitation, within the range of about 4 to 8, such as about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8, preferably within the range of about 5.5 to 7, such as about 5.8 to 6.8.

[0135] In some embodiments, the concentration of dissolved oxygen within the culture medium is in the range of about 2 to 40 %, preferably about 3 to 35 %, more preferably about 5 to 32 %, such as about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 %, particularly within the range of about 5 to 20 %, such as about 5 to 15 %, such as about 5, 6, 7, 8, 9 or 10 %.

[0136] In some embodiments, the method for the production of the polysaccharide according to the present invention yields the polysaccharide in a concentration of about 10 g / L, more preferably about 11 g / L, even more preferably about 12 g / L, still more preferably about 13 g / L, particularly about 14 g / L, preferably after a culturing period in the range of about 20 to 60 hours, preferably about 30 to 55 hours, more preferably about 30 to 50 hours, such as about 35 to 48 hours. In some embodiments, the host cell is selected from Paenibacillus, preferably Paenibacillus polymyxa, and the method yields the polysaccharide in a concentration of about 13 g / L, preferably about 14 g / L, more preferably about 18 g / l, still more preferably about 20 to 50 g / l, such as 20 to 30 g / l; optionally after a culturing period in the range of about 20 to 60 hours, preferably about 23 to 55 hours, more preferably about 30 to 50 hours, such as about 35 to 48 hours.

[0137] In some embodiments, step (b) is carried out using a crude cell extract of the host cells of step (a). Methods for obtaining a crude cell extract from a microbial culture are generally known in the art.

[0138] In some embodiments, in order to facilitate purification of the final product, i.e., the glucan-type polysaccharide, and / or improve overall performance of the polypeptide according to the present invention, the crude cell extract may be obtained by lysis of the cells to obtain a lysed cell composition and removal of any solid components from the lysed cell composition to obtain the crude cell extract, such as by filtration or centrifugation and decantation, preferably filtration and / or decantation.

[0139] In particular embodiments, the crude cell extract contains the polypeptide of the present invention, as herein defined and described, for example in such an amount that it exhibits a specific activity (Vmax) of at least 60.000 units per litre culture medium. For instance, in some embodiments, enzymatic glucosyltransferase activity of a polypeptide of the present invention may be determined by quantifying the amount of waterinsoluble glucan formed by the respective enzyme. Methods for quantification of polysaccharides are generally known to the skilled person. For instance, quantification may be accomplished by pouring the reaction solution through a fine mesh sieve to collect the water-insoluble glucan, then washing it with water to remove remaining impurities, and then drying it at 40 °C overnight in a vacuum oven; the weight of the dried water-insoluble glucan may be measured using an analytical balance. The determined amount may be compared with the amount formed by the glucosyltransferase having the amino acid sequence set forth in SEQ ID NO: 1. Formation of the water-insoluble glycan is generally visible within the reaction vessel. The water-insoluble glycan may be collected, washed with water, and dried. In some embodiments, additionally, enzymatic glucosyltransferase activity of a polypeptide of the present invention may be further determined by quantifying the decrease in the reaction substrate (sucrose), for example, using ion exclusionchromatography. The quantified decrease in the reaction substrate may be compared with the decrease in the reaction substrate determined for the glucosyltransferase having the amino acid sequence in SEQ ID NO: 1. In some embodiments, for ion exclusion chromatography, the HPLC is equipped with a Rezex™ ROA-organic acids H+column and coupled with a refractive index detector; the column temperature is set to 70 °C with a flow rate of 0.5 mL / min; the sample injection volume is 10 pL and 2.5 mM H2SO4 is used as the running buffer for the system; for sample preparation, 980 pL of a 1 % (w / v) glucan solution is mixed with 20 pL H2SO4 and incubated overnight at 90 °C; the sample is then centrifuged at 9,000 rpm for 5 min and the supernatant is filtered through a 0.22 pm PVDF filter. Quantifying the amount of the water-insoluble glucan may thus encompass optionally hydrolyzing the glucan and measuring the glucose monomers. In some embodiments, determination of enzymatic glucosyltransferase activity of a polypeptide of the present invention may include determining the weight of the purified (washed) and dried glucan and determining of glucose monomers via hydrolysis and HPLC (HILIC) measurement.

[0140] Due to such high enzymatic activity, the resultant crude cell extract can be used as is, i.e., without further purification of the polypeptide of the present invention.

[0141] For the generation of the polysaccharide, e.g., the glucan-type polymer, said crude cell extract is typically combined with a composition comprising a suitable substrate, e.g., sucrose, typically in form of an aqueous solution. In various embodiments, such substrate solutions may contain sucrose in an amount in the range of about 10 to about 200 g / L, such as about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 g / L, preferably about 30 to about 100 g / L, more preferably about 50 to about 90 g / L sucrose, for instance about 75 g / L sucrose.

[0142] For instance, but without limitation, said composition comprising sucrose, in addition to a base such as water, for instance tap water or deionized water, may comprise one or more additional components, such as, for instance but without limitation, buffers and stabilizing agents, antioxidants, additional saccharides, such as maltose, etc. The pH of such a composition may be, for instance but without limitation, within the range of about 4.5 to 10, such as about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10, preferably within the range of about 4.5 to 8, such as about 5 to 7, such as, for instance, between about 5 to 6.

[0143] Thus, in various embodiments, the substrate comprises or consists of sucrose.

[0144] Purification and / or of the polysaccharide produced by the polypeptide of the present invention may be performed. The polysaccharide, e.g., the glucan-type polymer, can be isolated and purified by means generally known in the art, such as in a preparatory way using, for example, fine mesh sieves, chromatography on activated carbon powder and / or ion-exchange chromatography.

[0145] Analysis of the product formed in accordance with the present invention can be carried out for example by GPC, NMR, or FTIR after isolation. Alternatively or additionally, analysis of the product formed can be carried out by HILIC-chromatography, employing an amino (NH2) column, using for instance a mixture of water and acetonitrile as the eluent.In a further aspect, the present invention also relates to a polysaccharide, wherein the polysaccharide is a glucan polymer and is characterized in:

[0146] being water-insoluble; and

[0147] having a ratio of a-1 ,6 linkages to a-1 ,3 linkages in the range of about 1 .2 to 2.2, preferably in the range of about 1.4 to about 2.0, more preferably in the range of about 1.5 to 1.9, such as about 1.5, 1.6, 1.7, 1.8, or 1 .9, particularly of about 1 .7; and

[0148] featuring branching side chains at the 0-3 position, wherein preferably about 80 to 99 %, such as about 85 to 95 %, of the branching side chains appear at the 0-3 position or wherein branching side chains appear exclusively at the 0-3 position.

[0149] In a further aspect, the present invention also relates to a polysaccharide obtainable by a method of producing a polysaccharide, e.g., glucan polymer, as herein described and defined above.

[0150] In various embodiments, the polysaccharide is water-insoluble. In some embodiments, the polysaccharide of the present invention is characterized in having a high proportion of a-1 ,3 linkages alongside a-1 ,6 linkages. In some embodiments, the 1 ,6 / 1 ,3 linkage ratio is in the range of about 1.2 to 2.2, preferably in the range of about 1.4 to about 2.0, more preferably in the range of about 1.5 to 1.9, such as about 1.5, 1.6, 1 .7, 1.8, or 1.9. In some embodiments, said ratio is about 1.7. The ratio within the said range indicates a relatively high share of 1 ,3 linkages. Branching side chains appear at the 0-3 position, and show shorter side chains compared to soluble dextrans such as the most commonly known dextran from L. mesenteroides NRRL B-512F and close relatives. In some embodiments, branching side chains appear exclusively at the 0-3 position. In some other embodiments, about 80 to 99 %, such as about 85 to 95 %, of the branching side chains appear at the 0-3 position.

[0151] In a further aspect, the present invention relates to the of use a polysaccharide, e.g., glucan polymer, obtainable by a production method as herein described and defined as an ingredient in foods and / or feeds, cosmetics, medicinal and pharmaceutical formulations, inks and printing formulations, composites, such as thermoplastic and rubber composites, sizings for papers and / or textiles, drilling muds, concrete, coating, films, or fibers.

[0152] In various embodiments, the polysaccharide of the present invention, at a concentration of about 5 wt.-%, is at least partially soluble in an aqueous solution of sodium hydroxide having a concentration of NaOH of at least about 2 wt.-%, such as about 2, 4, or 6 wt.-%, at a temperature of about 20 - 80 °C, wherein wt.-% of the polysaccharide is based on the total weight of the aqueous solution comprising NaOH and wherein wt.-% of NaOH is based on the total weight of the aqueous solution not comprising the polysaccharide. In various embodiments, at least 50 %, preferably at least 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 wt.-% of the total weight of the polysaccharide of the present invention is soluble in an aqueous solution of sodium hydroxide having a concentration of NaOH of at least about 2 wt.-% at a temperature of about 20 - 80 °C.

[0153] An exemplary method for determining the proportion of a water-insoluble polysaccharide, e.g., glucan, that is partially solubilized under alkaline conditions, preferably in an aqueous solution of 0.5-2.0 M NaOH, isthe following: The glucan sample of known dry mass is contacted with an aqueous solution of sodium hydroxide. The mixture is maintained at a controlled temperature, preferably about 20 - 80 °C, and under agitation, preferably about 50 - 1500 rpm, for a time sufficient to permit dissolution, for instance for about 15 - 25 h. Subsequently, the mixture is separated into a liquid phase containing dissolved glucan and a solid residue. The liquid phase is neutralized and the dissolved glucan is recovered and dried, while the solid residue is washed to neutrality and dried. The alkaline solubility of the glucan is determined as the mass percentage of the recovered dissolved fraction relative to the initial dry sample mass. By that, the amount of insoluble glucan can be quantified.

[0154] In various embodiments, the polysaccharide of the present invention, at a concentration of about 2 wt.-%, is completely soluble in an aqueous solution of sodium hydroxide having a concentration of NaOH of at least about 2 wt.-%, such as about 2, 4, or 6 wt.-%, at a temperature of about 20 - 80 °C, wherein wt.-% of the polysaccharide is based on the total weight of the aqueous solution comprising NaOH and wherein wt.-% of NaOH is based on the total weight of the aqueous solution not comprising the polysaccharide.

[0155] In various embodiments, the polysaccharide of the present invention, at a concentration of about 5 wt.-%, is completely soluble in an aqueous solution of sodium hydroxide having a concentration of NaOH of at least about 4 wt.-%, such as about 4 wt.-% or 6 wt.-%, at a temperature of about 20 - 80 °C, wherein wt.-% of the polysaccharide is based on the total weight of the aqueous solution comprising NaOH and wherein wt.-% of NaOH is based on the total weight of the aqueous solution not comprising the polysaccharide.

[0156] In various embodiments, the polysaccharide of the present invention, at a concentration of about 10 wt.-%, is completely soluble in an aqueous solution of sodium hydroxide having a concentration of NaOH of at least about 4 wt.-%, such as about 4 wt.-%, preferably less than 6 wt.-%, at a temperature of about 20 -80 °C, wherein wt.-% of the polysaccharide is based on the total weight of the aqueous solution comprising NaOH and wherein wt.-% of NaOH is based on the total weight of the aqueous solution not comprising the polysaccharide.

[0157] In various embodiments, the polysaccharide of the present invention, at a concentration of about 10 wt.-%, is partially soluble in an aqueous solution of sodium hydroxide having a concentration of NaOH of at least about 6 wt.-%, such as about 6 wt.-%, at a temperature of about 20 - 80 °C, wherein wt.-% of the polysaccharide is based on the total weight of the aqueous solution comprising NaOH and wherein wt.-% of NaOH is based on the total weight of the aqueous solution not comprising the polysaccharide.

[0158] In yet another aspect, the present invention relates to a composition comprising a polysaccharide, e.g., glucan polymer, obtainable by a production method as herein described as defined, wherein the composition is selected from foods and / or feeds, cosmetics, medicinal compositions, pharmaceutical compositions, inks and printing compositions, composites, such as thermoplastic and rubber composites, sizings for papers and / or textiles, drilling muds, concrete, coating, films or fibers. Such a composition may be obtainable by providing at least one polysaccharide, e.g., glucan polymer, obtainable by a production method according to the present invention and combining it with at least one further component, such as, without limitation, one or more auxiliaries or excipients. Also possible are diluents or solvents. The type andamount of such additional components may depend on the planned application or use and can be adapted accordingly. Formulation techniques and methods for different types of compositions envisaged herein, for instance cosmetic, pharmaceutical, medicinal, or agricultural compositions, are well known to those skilled in the art and routinely practiced.

[0159] All embodiments pertaining to the polypeptide and related aspects, the method of production thereof, use thereof, the method for the preparation of a polysaccharide, e.g., glucan polymer, according to the present invention, likewise and correspondingly apply to the products obtainable by said methods and respectively related aspects, such as uses thereof and compositions comprising the same, and vice versa.Embodiments

[0160] Embodiment 1 : Polypeptide comprising or consisting of an amino acid sequence having at least 80 %, such as at least 81 , 82, 83 or 84 %, more preferably at least 85 %, such as at least 86, 87, 88 or 89 %, still more preferably at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 , wherein the polypeptide does not consist of any of the following amino acid sequences: the amino acid sequence set forth in SEQ ID NO: 13; the amino acid sequence set forth in SEQ ID NO: 13, wherein the leucine residue of position 441 of the amino acid sequence of SEQ ID NO: 13 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 14; the amino acid sequence set forth in SEQ ID NO: 14; wherein the leucine residue of position 400 of the amino acid sequence of SEQ ID NO: 14 is replaced by any amino acid other than leucine; the amino acid sequence set forth in SEQ ID NO: 15; the amino acid sequence set forth in SEQ ID NO: 15, wherein the residue at position 654 of the amino acid sequence of SEQ ID NO: 15 is replaced by any other amino acid.

[0161] Embodiment 2: The polypeptide according to embodiment 1 , wherein the polypeptide

[0162] has at least 85 %, such as at least 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93 or 94 %, more preferably at least 95 %, such as at least 96, 97, 98 or 99 %, most preferably at least 100 % of the enzymatic glucosyltransferase activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 ; and / or

[0163] is characterized in comprising the amino acid sequence having at least 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, which is linked, preferably through a peptide bond, to an amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0164] Embodiment 3: The polypeptide according to embodiment 1 or embodiment 2, wherein the polypeptide is isolated.

[0165] Embodiment 4: The polypeptide according to any one of embodiments 1-3, wherein the polypeptide is modified, wherein optionally the modification comprises or consists of one or more modifications selected from conservative amino acid substitutions and / or insertions, preferably 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.

[0166] Embodiment 5: The polypeptide according to any one of embodiments 1-4, wherein the polypeptide comprises one or more additional amino acid sequences, wherein said one or more additional amino acid sequences are selected from an affinity tag; a protease recognition and cleavage site; polypeptides to which the polypeptide is fused to form a fusion protein, preferably selected from albumins, antibodies, antibody fragments, antibody-like molecules, and antibody derivatives.Embodiment 6: The polypeptide according to any one or embodiments 1-5, wherein the polypeptide is derivatized or conjugated to another chemical moiety, said derivatization / preferably being selected from PEGylation and glycosylation.

[0167] Embodiment 7: Reaction solution comprising water, sucrose, and a polypeptide according to any one of embodiments 1 to 6.

[0168] Embodiment 8: The reaction solution according to embodiment 8, wherein the reaction solution is not comprised within the organism Leuconostoc suionicum and / or is not released from the organism Leuconostoc suionicum; or the reaction solution is not comprised within a bacterial cell of the genus Leuconostoc and / or is not released from a bacterial cell of the genus Leuconostoc; or is not comprised within a bacterial cell of the family of Lactobacillaceae and / or is not released from a bacterial cell of the family of Lactobacillaceae; or is not comprised within a Gram-positive bacterial cell and / or is not released from a Gram-positive bacterial cell; or is not comprised within a bacterial cell and / or is not released from a bacterial cell.

[0169] Embodiment 9: The reaction solution according to embodiment 7 or 8, wherein the reaction solution comprises, in addition to water, sucrose, and a polypeptide comprising or consisting of an amino acid sequence having at least 70 %, preferably at least 80 %, more preferably at least 90 %, still more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, one or more divalent cations, such as Ca2+, Mg2+, and / or one or more monovalent ions such as phosphorus.

[0170] Embodiment 10: The reaction solution according to any one of embodiments 7 to 9, wherein the reaction solution comprises, in addition to water, sucrose, and a polypeptide comprising or consisting of an amino acid sequence having at least 70 %, preferably at least 80 %, more preferably at least 90 %, still more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 , at least one buffer system, such as a buffer system including EDTA, wherein preferably the buffer system is selected from the group consisting of a phosphate buffer system, a carbonate buffer system, an acetate buffer system, a citrate buffer system, an ammonium buffer system, a borate buffer system, a triethanolamine buffer system, and a tartrate buffer system, more preferably from a phosphate buffer system.

[0171] Embodiment 11 : The reaction solution according to any one of embodiments 7 to 10, wherein the pH the reaction solution is within the range of about 4.5 to 10, such as about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10, preferably within the range of about 4.5 to 8, such as about 5 to 7, such as, for instance, between about 5 to 6.

[0172] Embodiment 12: The reaction solution according to any one of embodiments 7 to 11 , wherein the polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % ofthe enzymatic glucosyltransferase activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 .

[0173] Embodiment 13: Method for the production of a polysaccharide, e.g., glucan polymer, the method comprising contacting at least one polypeptide according to any one of embodiments 1-6 with a suitablesubstrate, said substrate preferably comprising or consisting of sucrose, to obtain the polysaccharide, e.g., glucan polymer.

[0174] Embodiment 14: The method according to embodiment 13, wherein the polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the enzymatic glucosyltransferase activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 .

[0175] Embodiment 15: The method according to embodiment 13 or embodiment 14, wherein

[0176] the polysaccharide, e.g., glucan polymer, is water-insoluble; and / or

[0177] the polysaccharide has a ratio of a-1 ,6 linkages to a-1 ,3 linkages in the range of about 1.2 to 2.2, preferably in the range of about 1.4 to about 2.0, more preferably in the range of about 1.5 to 1 .9, such as about 1 .5, 1 .6, 1.7, 1.8, or 1 .9, particularly of about 1.7; and / or

[0178] the polysaccharide features branching side chains at the 0-3 position, wherein preferably about 80 to 99 %, such as about 85 to 95 %, of the branching side chains appear at the 0-3 position or wherein branching side chains appear exclusively at the 0-3 position.

[0179] Embodiment 16: The method according to any one of embodiments 13-15, wherein the method is an in vitro or an in vivo method.

[0180] Embodiment 17: The method according to any one of embodiments 13-16, wherein the method comprises the recombinant expression of the glucosyltransferase in a suitable host cell.

[0181] Embodiment 18: The method according to any one of embodiments 13-17, comprising the following steps:

[0182] (a) expressing a polypeptide according to any one of embodiments 1 to 6 in a suitable host cell; and (b) contacting said polypeptide with a suitable substrate, said substrate preferably comprising or consisting of sucrose, under conditions that allow the enzymatic production of a glucan polymer.

[0183] Embodiment 19: The method according to embodiment 18, wherein

[0184] the host cell is selected from bacterial cells, for instance Gram-negative bacterial cells, such as E. coli, for instance such as E. coli BL21 (DE3), E. coli BL21 , E. coli K12, E. coli BLR, E. coli BL21 Al, E. coli BL21 pLysS, E. coli XL1 , E. coli DH5a, E. coli DH1 , E. coli DM 1 , E. coli HB101 , E. coli JmlOl- 110, E. coli Rosetta(DE3)pLysS, E. co / / SURE, E. coli TOP10, E. coli XLI-Blue, E. coli XL2-Blue, and E. coli XLIO-Blue, preferably Gram-positive bacterial cells, more preferably selected from Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, or Bacillus and Paenibacillus, such as Bacillus subtilis and Paenibacillus polymyxa; and / or

[0185] the host cell is selected from Gram-negative bacterial cells, preferably from E. coli, more preferably from E. coli BL21 (DE3), E. coli BL21 , E. coli K12, E. coli BLR, E. coli BL21 Al, E. coli BL21 pLysS,E. con XL1 , E. con 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, and the polypeptide of the present invention is characterized in comprising the amino acid sequence having at least 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 , which is linked, preferably through a peptide bond, to an amino acid sequence having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, or 99 %, more particularly 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0186] Embodiment 20: The method according to embodiment 18 or embodiment 19, wherein step a) includes a cultivation step, wherein optionally the total culturing period may be in the range of about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 hours or 1 hour or less, such as 30, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3 or minutes or 1 minute, such as in the range of about 2 hours to 80 hours, such as about 5 hours to 48 hours.

[0187] Embodiment 21 : The method according to embodiment 20, wherein the cultivation step may include one or more preculturing steps and one or more main culturing steps.

[0188] Embodiment 22: The method according to embodiment 20 or embodiment 21 , wherein the pH of the culture medium may be, for instance but without limitation, within the range of about 4 to 8, such as about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8, preferably within the range of about 5.5 to 7, such as about 5.8 to 6.8.

[0189] Embodiment 23: The method according to any one of embodiments 20-22, wherein the concentration of dissolved oxygen within the culture medium is in the range of about 2 to 40 %, preferably about 3 to 35 %, more preferably about 5 to 32 %, such as about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 %, particularly within the range of about 5 to 20 %, such as about 5 to 15 %, such as about 5, 6, 7, 8, 9 or 10 %.

[0190] Embodiment 24: The method according to any one of embodiments 18-23, wherein step (b) is carried out using a crude cell extract of the host cells of step (a), wherein optionally the crude cell extract is obtained by lysis of the cells to obtain a lysed cell composition and removal of any solid components from the lysed cell composition to obtain the crude cell extract, such as by filtration or centrifugation and decantation, preferably filtration and / or decantation.

[0191] Embodiment 25: The method according to embodiment 24, wherein the crude cell extract contains the polypeptide in such an amount that it exhibits a specific activity (Vmax) of at least 60.000 units per litre culture medium.

[0192] Embodiment 26: The method according to any one of embodiments 13-25, wherein the method comprises a step of isolating the polysaccharide, e.g., glucan polymer, produced.

[0193] Embodiment 27: The method according to any one of embodiments 24-26, wherein step b) comprisescontacting the crude cell extract with a composition comprising a suitable substrate, e.g., sucrose, typically in form of an aqueous solution, wherein optionally the aqueous substrate solution contains sucrose in an amount in the range of about 10 to about 200 g / L, such as about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 g / L, preferably about 30 to about 100 g / L, more preferably about 50 to about 90 g / L sucrose, for instance about 75 g / L sucrose.

[0194] Embodiment 28: The method according to embodiment 27, wherein said composition comprising sucrose, in addition to a base such as water, for instance tap water or deionized water, comprises one or more additional components, preferably selected from buffers and stabilizing agents, antioxidants, additional saccharides, such as maltose.

[0195] Embodiment 29: The method according to embodiment 27 or embodiment 28, wherein the pH of the composition comprising sucrose is within the range of about 4.5 to 10, such as about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10, preferably within the range of about 4.5 to 8, such as about 5 to 7, such as between about 5 to 6.

[0196] Embodiment 30: The method according to any one of embodiments 13-26, wherein the method yields the glucan polymer in a concentration of at least about 10 g / L, more preferably about 11 g / L, even more preferably about 12 g / L, still more preferably about 13 g / L, particularly about 14 g / L, preferably after a culturing period in the range of about 20 to 60 hours, preferably about 30 to 55 hours, more preferably about 30 to 50 hours, such as about 35 to 48 hours, wherein optionally the host cell is selected from Paenibacillus, preferably Paenibacillus polymyxa, and the method yields the polysaccharide in a concentration of about 13 g / L, preferably about 14 g / L, more preferably about 18 g / l, still more preferably about 20 to 50 g / L, such as 20 to 30 g / l; optionally after a culturing period in the range of about 20 to 60 hours, preferably about 23 to 55 hours, more preferably about 30 to 50 hours, such as about 35 to 48 hours.

[0197] Embodiment 31 : Nucleic acid molecule encoding the polypeptide according to any one of embodiments 1-6.

[0198] Embodiment 32: The nucleic acid molecule according to embodiment 31 , wherein the nucleic acid molecule comprises or consists of a nucleic acid molecule having at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at last 95 %, such as at least 96, 97, 98 or 99 %, more particularly 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 2.

[0199] Embodiment 33: The nucleic acid molecule according to embodiment 31 or 32, wherein the nucleic acid molecule is comprised in a vector.

[0200] Embodiment 34: Vector comprising a nucleic acid molecule according to embodiment 31 or 32, wherein the vector is a cloning vector or an expression vector, more preferably a plasmid.

[0201] Embodiment 35: Host cell comprising a nucleic acid molecule according to any one of embodiments 31-33or a vector according to embodiment 34, the host cell preferably being a prokaryotic host cell.

[0202] Embodiment 36: The host cell according to embodiment 35, wherein the host cell is selected from the group consisting of Gram positive and Gram-negative bacteria, preferably from Gram-negative bacteria, such as Escherichia coli (E. coli), in particular E. coli BL21 (DE3), E. coli BL21 , E. coli K12, E. coli BLR, E. coli BL21 Al, E. coli BL21 pLysS, E. coli XL1 , E. coli DH5a, E. coli DH1 , E. coli DM1, E. coli HB101 , E. coli JmlOl-110, E. coli Rosetta(DE3)pLysS, E. co / / SURE, E. coli TOP10, E. coli XLI-Blue, E. coli XL2-Blue, and E. coli XLIO-Blue, or from Gram-positive bacteria, preferably from Bacillus, such as Bacillus subtilis; Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum; or Paenibacillus, such as Paenibacillus polymyxa.

[0203] Embodiment 37. Method for the production of a polypeptide according to any one of embodiments 1-6, comprising the steps of

[0204] (1) cultivating the host cell of embodiment 26 under conditions that allow the expression of the polypeptide; (2) optionally isolating the expressed polypeptide from the host cell.

[0205] Embodiment 38: The method according to embodiment 37, wherein the polypeptide is heterologously expressed in said host cell.

[0206] Embodiment 39: The method according to embodiment 37 or embodiment 38, wherein the host cell is selected from bacterial cells, for instance Gram-negative bacterial cels, such as E. coli, preferably grampositive bacterial cells, preferably selected from Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, or Paenibacillus, such as Paenibacillus polymyxa.

[0207] Embodiment 40: Use of a polypeptide according to any one of embodiments 1-6 for the biotechnological production of a glucan polymer.

[0208] Embodiment 41: Polysaccharide, e.g., glucan polymer, obtainable by a method according to any one of embodiments 13-30.

[0209] Embodiment 42: The polysaccharide according to embodiment 41 , wherein the polysaccharide is waterinsoluble.

[0210] Embodiment 43: The polysaccharide according to embodiment 41 or embodiment 42, wherein

[0211] the polysaccharide, e.g., glucan polymer, is water-insoluble; and / or

[0212] the polysaccharide has a ratio of a-1 ,6 linkages to a-1 ,3 linkages in the range of about 1.2 to 2.2, preferably in the range of about 1.4 to about 2.0, more preferably in the range of about 1.5 to 1 .9, such as about 1 .5, 1 .6, 1.7, 1.8, or 1 .9, particularly of about 1.7; and / or

[0213] the polysaccharide features branching side chains at the 0-3 position, wherein preferably about 80 to 99 %, such as about 85 to 95 %, of the branching side chains appear at the 0-3 position orwherein branching side chains appear exclusively at the 0-3 position.

[0214] Embodiment 44: Use of a polysaccharide, e.g., glucan polymer, obtainable by a method according to any one of embodiments 13-30 in foods and / or feeds, cosmetics, medicinal and pharmaceutical formulations, inks and printing formulations, composites, such as thermoplastic and rubber composites, sizings for papers and / or textiles, drilling muds, concrete, coating, films or fibers.

[0215] Embodiment 45: Composition comprising a polysaccharide, e.g., glucan polymer, obtainable by a method according to any one of claims 13-30, wherein the composition is selected from foods and / or feeds, cosmetics, medicinal compositions, pharmaceutical compositions, inks and printing compositions, composites, such as thermoplastic and rubber composites, sizings for papers and / or textiles, drilling muds, concrete, coating, films, and fibers.

[0216] Embodiment 46: The use according to embodiment 44 or the composition according to embodiment 45, wherein the polysaccharide is water-insoluble.

[0217] Embodiment 47: The use according to embodiment 44 or embodiment 46 or the composition according to embodiment 45 or embodiment 46, wherein

[0218] the polysaccharide, e.g., glucan polymer, is water-insoluble; and / or

[0219] the polysaccharide has a ratio of a-1 ,6 linkages to a-1 ,3 linkages in the range of about 1.2 to 2.2, preferably in the range of about 1.4 to about 2.0, more preferably in the range of about 1.5 to 1 .9, such as about 1 .5, 1 .6, 1.7, 1.8, or 1 .9, particularly of about 1.7; and / or

[0220] the polysaccharide features branching side chains at the 0-3 position, wherein preferably about 80 to 99 %, such as about 85 to 95 %, of the branching side chains appear at the 0-3 position or wherein branching side chains appear exclusively at the 0-3 position.

[0221] Embodiment 48: Polysaccharide, wherein the polysaccharide is a glucan polymer and is characterized in:

[0222] being water-insoluble; and

[0223] having a ratio of a-1 ,6 linkages to a-1 ,3 linkages in the range of about 1 .2 to 2.2, preferably in the range of about 1.4 to about 2.0, more preferably in the range of about 1.5 to 1.9, such as about 1.5, 1.6, 1.7, 1.8, or 1 .9, particularly of about 1 .7; and

[0224] featuring branching side chains at the 0-3 position, wherein preferably about 80 to 99 %, such as about 85 to 95 %, of the branching side chains appear at the 0-3 position or wherein branching side chains appear exclusively at the 0-3 position.Examples

[0225] Strains and Cultivation

[0226] P. polymyxa DSM 365 and L. suionicum DSM 20241 were obtained from the German Collection of Microorganisms and Cell Culture (DSMZ, Germany). E. coll Turbo (NEB, New England Biolabs) was routinely used for plasmid cloning, and E. coll S17-1 (ATCC 47055) was the donor for conjugation. P. polymyxa and E. coll strains were cultivated in LB media (10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCI) and 1.5 % agar was added to the plate media. Whenever necessary, 50 pg / mL neomycin and / or 40 pg / mL polymyxin were used as selective markers. Leuconostoc strain was cultivated in MRS media. P. polymyxa and Leuconostoc strain were cultivated at 30 °C and E. coll strains at 37 °C unless stated otherwise.

[0227] Plasmid Construction

[0228] All plasmids in this study were assembled by using isothermal assembly following the standard protocol (Gibson, D. G.; Young, L.; Chuang, R. Y.; Venter, J. C.; Hutchison, C. A.; Smith, H. O. Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases. Nat. Methods 2009, 6 (5), 343-345). The fragments required for the assembly were obtained by PCR amplification using Q5 DNA Polymerase (NEB). Gel purification and genomic DNA (gDNA) isolation were done by using the Monarch Gel Purification Kit (NEB) and Monarch Genomic DNA Purification Kit (NEB), respectively.

[0229] Genome editing of P. polymyxa was performed by employing the pCasPP plasmid (Rutering, M.; Cress, B. F.; Schilling, M.; Ruhmann, B.; Koffas, M. A. G.; Sieber, V.; Schmid, J. Tailor-Made Exo polysaccharides — CRISPR-Cas9 Mediated Genome Editing in Paenibacillus Polymyxa. Synth. Biol. 2017, 2 (1), 1-12). It is a one-plasmid system and contains the Cas9 of Streptococcus pyogenes as well as an sgRNA expression cassette. The 20-nt spacer sequences were identified within the targeted genes and located upstream of the NGG PAM sites. Approximately 1 kb of homologous regions upstream and downstream of the targeted regions were provided as the repair template. For gene integrations, the genes of interest were placed between the homologous flanks. The various parts of the final constructs were assembled via isothermal assembly (Gibson, D. G.; Young, L.; Chuang, R. Y.; Venter, J. C.; Hutchison, C. A.; Smith, H. O. Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases. Nat. Methods 2009, 6 (5), 343-345). Following the isothermal assembly, chemically competent E. co / / Turbo was transformed with the assembly mixture and plated on LB agar containing 50 pg / mL of neomycin.

[0230] The resulting colonies were screened with colony PCR using GoTaq polymerase (Promega) then plasmid isolation was performed using GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific). The plasmid was further checked by DNA sequencing to confirm for correct assembly. Subsequently, E. coli S17-1 was transformed with the plasmid. Primer synthesis and sequencing analysis were done by Microsynth AG (Switzerland). In silico primer design, cloning, and sequence alignment were performed by using SnapGene version 6.1.

[0231] All strains, plasmids, oligonucleotides, and spacer sequences used in the context of the present invention are listed in Tables 1 and 2.Table 1 : List of plasmids and bacterial strains

[0232]

[0233] pET28a | 5 ,368 i Plasmid vector for recombinant protein i production in E. coli BL21 (DE3) pET28a-LsGtfc3-0His | 9,697 i Production of LsGtfc3 wild type protein

[0234]

[0235] 1: Rutering, M.; Cress, B. F.; Schilling, M.; Ruhmann, B.; Koffas, M. A. G.; Sieber, V.; Schmid, J. Tailor-Made Exopolysaccharides — CRISPR-Cas9 Mediated Genome Editing in Paenibacillus Polymyxa. Synth. Biol.

[0236] 2017, 2 (1), 1-12.

[0237] Meliawati, M.; Teckentrup, C.; Schmid, J. CRISPR-Cas9-Mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus Polymyxa. ACS Synth. Biol. 2022, 11 (1), 77-84.Table 2: List of oligonucleotides

[0238]

[0239] Conjugation

[0240] Single colonies of P. polymyxa and E. coli S17-1 strains harboring the plasmid of interest were inoculated in 3 mL LB media, with or without 50 pg / mL of neomycin, and cultivated overnight. The overnight culture was diluted 1 :100 in fresh media and then grown for 4 h at 37 °C and 250 rpm. Subsequently, 900 pL of P. polymyxa culture was incubated at 42 °C for 15 min and then mixed with 300 pl of the E. coli S17-1 culture. The mixture was centrifuged at 8,000 rpm for 2 min and the pellet was resuspended in 100 pL of LB media. The solution was dropped on an LB plate and incubated overnight at 30 °C. Afterward, the culture was scrapped off the plate and resuspended in 150 pL of LB media, Subsequently, the culture was plated on LB plates containing 50 pg / mL of neomycin and 40 pg / mL of polymyxin, followed by incubation for 48 h at 30 °C. If necessary, serial dilutions were performed before plating to obtain countable colonies on the plates. Subsequently, the colonies were checked with colony PCR to screen for the desired mutants. Colony PCR was performed using primers that bind outside of the homologous regions provided as the repair template. The resulting fragment was purified from the gel and sent for sequencing to confirm the genome modifications. Finally, the gDNA was isolated and again checked with PCR and sequencing to reconfirm the colony PCR results.

[0241] Glucan production by engineered P. polymyxa

[0242] For exopolysaccharide (EPS) production, P. polymyxa was cultivated in EPS-inducing media containing 30 g / L sucrose (unless stated otherwise), 5 g / L peptone, 1.33 g / L MgSC JFhO, 0.05 g / L CaCh, 1.67 g / LKH2PO4, 2 mL / L RPMI 1640 vitamin solution (Sigma-Aldrich), and 1 mL / L trace elements (2.5 g / L FeSO4.7H2O, 2.1 g / L C4H4Na2O6.2H2O, 1.8 g / L MnCI2.4H2O, 0.075 g / L CoCI2.6H20, 0.031 g / L CUSO4.7H2O, 0.258 g / L H3BO3, 0.023 g / L Na2Mo04, 0.021 g / L ZnCI2). On the other hand, Leuconostoc strains were cultivated in MRS media with sucrose. The strains for the production of water-soluble EPS were cultivated at 250 rpm, while the water-insoluble EPS-producing strain at 60 rpm. The results obtained for varying concentrations of sucrose are depicted in Fig. 1.

[0243] Glucan production by engineered E. coli

[0244] Different variants of the gtfc3 gene, with and without 6xHis-tag and signal peptide, were individually cloned into a pET28 plasmid by the isothermal assembly. E. coli BL21 (DE3) harboring the plasmid of interest was grown overnight in liquid LB media with 50 pg / mL kanamycin. Subsequently, 1% preculture was inoculated into a 250 mL shake flask filled with 50 mL EPSsuc media containing 50 pg / mL kanamycin and 0.5 mM IPTG. The culture was cultivated for 48 h at 30 °C and 60 rpm. Schemes of the variants used and respective results are depicted in Fig. 3. The water-insoluble glucan was then purified as described below.

[0245] EPS purification

[0246] The water-insoluble EPS was obtained by pouring the culture broth through a fine mesh sieve. The EPS collected on the sieve was washed three times with ddH2O and then dried in a vacuum oven as mentioned above.

[0247] Molecular weight determination

[0248] Gel permeation chromatography (GPC) analysis of the water-insoluble EPS was performed by using a Knauer Azura GPC / SEC Rl system and a SECcurity SLD7000 7-angle static light-scattering detector. The pullulan and xanthan standards were used to generate the calibration curve for the low and high Mw analysis, respectively. The GPC column as well as the Rl detector were set to 38 °C and the measurement was carried out at a flow rate of 0.5 mL / min for 60 min. The water-insoluble EPS was dissolved in DMSO to a final concentration of 0.5 % (w / v) with 100 mM LiCI and incubated overnight at 80 °C in a shaking heating block. The dissolved sample was then filtered through a PTFE filter.

[0249] Thermogravimetric analysis

[0250] TGA analysis was performed by using a LINSEIS STA PT 1600 instrument. The sample was heated from 30 °C to 600 °C with a heating rate of 10 °C / min with simultaneous air flow controlled at 10 mL / min. An empty AI2O3 crucible was placed in the measurement system and the zero curve was generated under an air atmosphere at a rate of 10 °C / min to 600 °C. Subsequently, approximately 15 mg sample was used for the analysis. Results are depicted in Fig. 2A.

[0251] Solubility profile in sodium hydroxide

[0252] Different concentrations of EPS were mixed with different sodium hydroxide concentrations and then incubated for 24 h at 80 °C and 1,000 rpm in a heating block. The EPS solubility was visually observed based on its degree of solubility and then categorized as soluble, partially soluble, and insoluble. Results are depicted in Fig. 2B.

Claims

CLAIMS1. Polypeptide comprising or consisting of an amino acid sequence having at leastt 80 %, more preferably at least 90 %, still more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 , wherein the polypeptide does not consist of any of the following amino acid sequences:the amino acid sequence set forth in SEQ ID NO: 13;the amino acid sequence set forth in SEQ ID NO: 13, wherein the leucine residue of position 441 of the amino acid sequence of SEQ ID NO: 13 is replaced by any amino acid other than leucine;the amino acid sequence set forth in SEQ ID NO: 14;the amino acid sequence set forth in SEQ ID NO: 14; wherein the leucine residue of position 400 of the amino acid sequence of SEQ ID NO: 14 is replaced by any amino acid other than leucine;the amino acid sequence set forth in SEQ ID NO: 15;the amino acid sequence set forth in SEQ ID NO: 15, wherein the residue at position 654 of the amino acid sequence of SEQ ID NO: 17 is replaced by any other amino acid.

2. The polypeptide according to claim 1 , wherein the polypeptidehas at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the enzymatic glucosyltransferase activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 ; and / oris isolated.

3. Reaction solution comprising water, sucrose, and a polypeptide as defined in claim 1 or claim 2, wherein optionally the glucan polymer is water-insoluble.

4. Method for the production of a glucan polymer, the method comprising contacting at least one polypeptide as defined in claim 1 or claim 2 with sucrose to obtain the glucan polymer, wherein optionally the glucan polymer is water-insoluble.

5. The method according to claim 4, whereinthe method is an in vitro or an in vivo method; and / orthe method comprises a step of isolating the glucan polymer produced; and / orthe method comprises the recombinant expression of the glucosyltransferase in a suitable host cell, wherein preferably wherein the host cell is selected from bacterial cells, for instance Gramnegative bacterial cells, such as E. coli, preferably Gram-positive bacterial cells, preferably selected from Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, or Paenibacillus, such as Paenibacillus polymyxa. and / orthe method yields the glucan polymer in a concentration of at least about 10 g / L, preferably in about 48 hours, or in a concentration of at least about 12 g / L, preferably in about 48 hours, or in a concentration of at least about 14g / L, preferably in about 48 hours.

6. Nucleic acid molecule encoding the polypeptide of claim 1 or claim 2.

7. Vector comprising a nucleic acid molecule according to claim 6, wherein the vector preferably is a plasmid.

8. Host cell comprising a nucleic acid molecule according to claim 6 or a vector according to claim 7, the host cell preferably being a prokaryotic host cell.

9. Method for the production of a polypeptide of claim 1 or claim 2, comprising(1) cultivating the host cell of claim 8 under conditions that allow the expression of the polypeptide;(2) optionally isolating the expressed polypeptide from the host cell.

10. The method according to claim 9, whereinthe polypeptide is heterologously expressed in said host cell; and / orthe host cell is selected from bacterial cells, for instance Gram-negative bacterial cels, such as E. coli, preferably gram-positive bacterial cells, preferably selected from Leuconostoc, such as Leuconostoc suionicum, Leuconostoc mesenteroides, or Leuconostoc dextranicum, or Paenibacillus, such as Paenibacillus polymyxa.

11. Use of a polypeptide of claim 1 or claim 2 for the biotechnological production of a polysaccharide, preferably a glucan polymer.

12. Use of a polysaccharide, preferably glucan polymer, obtainable by a method according to claim 4 or claim 5 in foods and / or feeds, cosmetics, medicinal and pharmaceutical formulations, inks and printing formulations, composites, such as thermoplastic and rubber composites, sizings for papers and / or textiles, drilling muds, concrete, coating, films or fibers.

13. Composition comprising a polysaccharide, preferably glucan polymer, obtainable by a method according to claim 4 or claim 5, wherein the composition is selected from foods and / or feeds, cosmetics, medicinal compositions, pharmaceutical compositions, inks and printing compositions, composites, such as thermoplastic and rubber composites, sizings for papers and / or textiles, drilling muds, concrete, coating, films, and fibers.

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