Compositions and methods for the production of beta-1,3-oligoglucans

Abstract

Described herein are composition and methods for the production of β-1,3-oligoglucans. Compositions may include a beta(β)-glucan phosphorylase (βGP) with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs:1 and 2; α-D-glucose 1-phosphate (α-G1P); and a primer molecule.

Description

COMPOSITIONS AND METHODS FOR THE PRODUCTION OF BETA-1, 3- OLIGOGLUCANSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of EP Patent Application No. 23216784.1, filed January 24, 2024, which is incorporated by reference herein in its entirety.REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the Sequence Listing XML file of the sequence listing named “PT-1776- WO-PCT.xml” which is 26,286 bytes in size created on January 10, 2025 and electronically submitted via Patent Center herewith the application is incorporated by reference in its entirety.BACKGROUND

[0003] The health and wellness industry has led to an increase in dietary awareness, and in particular to the consumption of healthy alternatives to conventional carbohydrates. Dietary fibers are one such alternative that receive significant attention due to their health-promoting benefits, including, but not to be limited to, cholesterol and blood glucose lowering effects, beneficial immunomodulatory effects, reducing the risk of cardiovascular disease, and potential uses as anticancer therapies. One type of dietary fiber includes the class of polysaccharides called P-glucans, and in particular P-1, 3 -oligoglucans. The P-l,3-oligoglucans are characterized in that they contain multiple D-glucose subunits linked together with P-l,3-glycosidic bonds to form glucose homopolymers.

[0004] The P-1, 3 -oligoglucans can be used in prepared foods as functional additives (e.g., fat replacers, thickening agents, gelling agents, etc.), prebiotics, skin conditioning agents, and the like. However, current production methods for the manufacture of P-glucans are limited to extraction from natural sources, chemical synthesis, or via fermentation. Theses current methods can be cost prohibitive for producers on a large-scale and can further result in products with variable yield and inconsistent purity. Further, these current methods can result in issues with P- glucan degradation and an off taste or odor when added to foods.

[0005] The P-1, 3 -oligoglucans are produced by various plant sources, such as seaweed, oats, and barley, and can be synthesized by various microbes including fungi, algae, and bacteria. In bacteria and other microbes, P-l,3-oligoglucans are degraded by a class of enzymes called P- glucan phosphorylases, where the P-glucan phosphorylases can degrade P-l,3-oligoglucans intovarious oligosaccharides and monosaccharides that can be further used as an energy source by the microbe.

[0006] The P-glucan phosphorylases are grouped into a number of families of enzymes possessing glycoside phosphorylase activity based on sequence similarity and substrate specificity. These families include, among others, the glycoside hydrolase family 94 (GH94), the glycoside hydrolase family 149 (GH149), and the glycoside hydrolase family 161 (GH161). The GH94 family of enzymes, also referred to as the laminaribiose phosphorylases, contain inverting glycoside phosphorylases that have a high activity toward glucose and can accomplish syntheses up to a degree of polymerization of 5 (i.e., DP5). The GH149 family of enzymes, also referred to as the P-l,3-oligoglucan phosphorylases, contain inverting glycoside phosphorylases that have a high activity toward glucose and can accomplish syntheses up to a DP20. The GH161 family of enzymes, also referred to as the P-l,3-glucan phosphorylases, have limited to no activity toward glucose and require laminaribiose or higher oligosaccharides as a priming substrate, and can accomplish syntheses of up to a DP70. However, many of the physicochemical properties of known enzymes of these families do not have the combined properties of stability at industrial temperatures (e.g., from 50-60 °C) and high activity towards glucose, and that can also achieve synthesis of products having a wide range of DP. These enzymes further have not been capable of meeting the demand for large-scale production of P-l,3-oligoglucans.

[0007] Thus, a need exists to produce P-glucans, and in particular P-l,3-oligoglucans, at large- scale production volumes that is economical on an industrial scale and that produces consistent product compositions.SUMMARY

[0008] The present disclosure provides methods for producing P-l,3-oligoglucans comprising contacting a-D-glucose 1 -phosphate (a-GlP) with a beta(P)-glucan phosphorylase (PGP) in the presence of a priming substrate to produce one or more P-l,3-oligoglucans, wherein the PGP has an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs: l and 2. The PGP a-GlP contacting step may be carried out at a pH between 5.0 and 8.5, between 5.5 and 8.0, or between 6.0 and 7.5; and / or a temperature between 35 and 80 °C, between 40 and 75 °C, between 45 and 70 °C. or between 50 and 65 °C. The a-D-glucose 1 -phosphate may be provided at a concentration of 50 mM to 2 M, and / or the primer molecule may be provided at a concentration of 0.001 mM to 1 M, or from 0.01 mM to 500 mM, or from 0.1 mM to 100 mM.

[0009] The method may additionally comprise the step of contacting a glucose donor with an alpha(a)-glucan-phosphorylase (aGP) or a sucrose phosphorylase (SP) in the presence of inorganic phosphate (e.g., sodium phosphate or potassium phosphate) to produce the a-GIP. The glucose donor may have a degree of polymerization equal to or greater than 4 and is contacted by an aGP. The glucose donor may have a partially hydrolyzed starch (e.g., maltodextrin) and is contacted by an aGP. The glucose donor may be sucrose and is contacted by an SP. The glucose donor aGP contacting step may be carried out at a pH between 5.0 and 8.5, between 5.5 and 8.0, or between 6.0 and 7.5; and / or a temperature between 35 and 80 °C, between 40 and 75 °C, between 45 and 70 °C. or between 50 and 65 °C. The SP may have an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:3; and / or the aGP has an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:4. Sucrose may be provided at a concentration of 50 mM to 5 M. The glucose donor may be a partially hydrolyzed starch (e.g., maltodextrin) with a degree of polymerization equal to or greater than 4 and is provided at a concentration of 1 wt% to 50 wt%, or from 5 wt% to 40 wt%, or from 10 wt% to 25 wt%.

[0010] The produced P-l,3-oligoglucans comprise at least 60%, at least 70%, at least 80%, or at least 90% by weight P-1, 3 -oligoglucans having a degree of polymerization (DP) of from 3 to 80. The ratio of a-GIP to priming substrate may be greater than 25: 1 and the P-l,3-oligoglucans comprise at least 60%, at least 70%, at least 80%, or at least 90% by weight P-1, 3 -oligoglucans having a degree of polymerization (DP) greater than 20 (>DP20). The ratio of a-GIP to priming substrate may be about 25: 1 and the P-1, 3 -oligoglucans comprise at least 60%, at least 70%, at least 80%, or at least 90% by weight P-1, 3 -oligoglucans having a degree of polymerization (DP) of 5-20 (DP5-DP20). The ratio of a-GIP to priming substrate may be less than 25: 1 and the P-1, 3- oligoglucans comprise at least 60%, at least 70%, at least 80%, or at least 90% by weight P-1, 3- oligoglucans having a degree of polymerization (DP) less than 10 (<DP10).

[0011] The disclosure also provides a composition comprising a glucose donor; inorganic phosphate (e.g., sodium phosphate or potassium phosphate); and an alpha(a)-glucan- phosphorylase (aGP) or a sucrose phosphorylase (SP). If the glucose donor is sucrose, the composition comprises an SP with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:3. If the glucose donor is a partially hydrolyzed starch (e.g., maltodextrin) with a degree of polymerization equal to or greater than 4, the composition comprises an aGP an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:4. Theglucose donor may be a partially hydrolyzed starch (e.g., maltodextrin) with a degree of polymerization equal to or greater than 4 and is provided at a concentration of 1 wt% to 50 wt%, or from 5 wt% to 40 wt%, or from 10 wt% to 25 wt%. Sucrose may be provided at a concentration of 50 mM to 5 M. The composition may additionally comprise a beta(P)-glucan phosphorylase (PGP) with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs: 1 and 2.

[0012] The disclosure also provides a composition comprising a beta(P)-glucan phosphorylase (PGP) with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs: l and 2; a-D-glucose 1-phosphate (a-GlP); and a primer molecule. The a-D-glucose 1-phosphate may be provided at a concentration of 50 mM to 2 M, and / or the primer molecule may be provided at a concentration of 0.001 mM to 1 M, or from 0.01 mM to 500 mM, or from 0.1 mM to 100 mM.

[0013] The disclosure herein further provides for compositions of P-l,3-oligoglucans produced using the composition and methods described herein.

[0014] The disclosure also provides use of a beta(P)-glucan phosphorylase (PGP) with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs: l and 2 for the production of one or more P-1, 3- oligoglucans.BRIEF DESCRIPTION OF THE FIGURES

[0015] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.

[0016] FIG. 1 shows the reaction scheme for the production of a P-glucan catalyzed by a PGP using a-GlP and a priming substrate.

[0017] FIG. 2 shows a reaction scheme for the production of P-l,3-glucans by a regioselective PGP, for example 4 / GP or A7GP, using a-GlP and a priming substrate.

[0018] FIG. 3 shows the reaction scheme for the production of a-GlP from a glucose donor, such as sucrose, in the presence of inorganic phosphate (Pi) and a sucrose phosphorylase (SP).

[0019] FIG. 4 shows the reaction scheme for coupled production of P-l,3-oligoglucans by the combination of a sugar phosphorylase (SP) and a regioselective PGP starting from a glucose donor through an a-GlP intermediate in the presence of a priming substrate.

[0020] FIG. 5 shows the reaction scheme for coupled production of P-l,3-oligoglucans by the combination of an a-glucan phosphorylase and a regioselective PGP starting from a partiallyhydrogenated starch (e.g., maltodextrin) through an a-GlP intermediate in the presence of a priming substrate.

[0021] FIG. 6 shows the temperature profile of A7GP and 4 / GP as outlined in Example 4.

[0022] FIG. 7 shows the pH profile of A7GP and 4 / GP as outlined in Example 4.

[0023] FIG. 8 shows the pH profile of 7baGP as outlined in Example 4.

[0024] FIG. 9 shows a spectrum obtained by high performance anion exchange chromatography of various P-l,3-oligoglucans as outlined in Example 5.

[0025] FIG. 10 shows spectrum obtained by high performance anion exchange chromatography of various P-l,3-oligoglucans as outlined in Example 6.

[0026] FIG. 11 shows spectrum obtained by high performance anion exchange chromatography of various P-l,3-oligoglucans as outlined in Example 6.

[0027] FIG. 12 shows spectrum obtained by high performance anion exchange chromatography of various P-l,3-oligoglucans as outlined in Example 7.

[0028] FIG. 13 shows spectrum obtained by high performance anion exchange chromatography of various P-l,3-oligoglucans as outlined in Example 7.

[0029] FIG. 14 shows spectrum obtained by high performance anion exchange chromatography of various P-1, 3 -oligoglucans obtained from reactions at 30 °C and 50 °C as outlined in Example 8.

[0030] FIG. 15 shows spectrum obtained by high performance anion exchange chromatography of various P-1, 3 -oligoglucans obtained at 24 hours from reactions as outlined in Example 9.

[0031] FIG. 16 shows spectrum obtained by high performance anion exchange chromatography of various P-1, 3 -oligoglucans obtained at 48 hours from reactions as outlined in Example 9.

[0032] FIG. 17 shows the docking site of the 4 / GP homology model with a docked laminaribiose, with the 8 residues selected for mutational analysis, the catalytic acid (D660), and their hydrogen bonding (dashed lines) shown.

[0033] FIG. 18 shows Michaelis-Menten curves for the 4 / GP variants outlined in Example 11.DETAILED DESCRIPTION

[0034] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosedsubject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0035] It should be understood that the definitions described herein apply to all aspects as described unless otherwise stated.

[0036] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference is to be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0037] Values expressed in a range format are to be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1 % to about 5 %” or “about 0.1 % to 5 %” is to be interpreted to include not just about 0.1 % to about 5 %, but also the individual values (e.g., 1 %, 2 %, 3 %, and 4 %) and the sub-ranges (e.g., 0.1 % to 0.5 %, 1.1 % to 2.2 %, 3.3 % to 4.4 %) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0038] Unless expressly stated, ppm (parts per million), percentage, and ratios are on a by weight basis. Percentage on a by weight basis (% w / w or w / w %) is also referred to as weight percent (wt%) or percent by weight (% wt.) herein.

[0039] As discussed above, there is a need to produce large-scale volumes of P-1, 3- oligoglucans that is cost effective and provides products having consistent composition. The methods herein provide for the production of P-1, 3 -oligoglucans using a novel enzymatic synthesis process, including one or more types of P-glucan phosphorylase (P-GP), a-D-glucose 1- phosphate (a-GlP), and a priming substrate. In general, compositions for making P-1, 3- oligoglucans include a-GlP, a primer molecule, and a P-glucan phosphorylase enzyme. Methods for making P-1, 3 -oligoglucans described herein include incubating the compositions including a-G1P, the primer molecule, and the PGP at a temperature and for a time sufficient to produce the P-l,3-oligoglucan. The disclosure also provides compositions for the synthesis of a-GlP from a glucose donor, for example, sucrose, or a partially hydrolyzed starch such as maltodextrin. These a-GlP synthesis compositions may include a glucose donor, phosphate, and an a-glucan phosphorylase (aGP) or a sugar phosphatase (SP). Methods for making a-GlP described herein include incubating the a-GlP synthesis compositions at a temperature and for a time sufficient to produce the a-GIP. The disclosure further provides compositions comprising the produced P-1, 3- oligoglucans.

[0040] As used herein, the term “enzyme” refers to a polypeptide that catalyzes a chemical reaction. The recitation of any particular enzyme, either independently or as part of a biosynthetic pathway is understood to include the co-factors, co-enzymes, and metals necessary for the enzyme to properly function. It will be appreciated that the enzymes herein can also be referred to as polypeptides or proteins. As used herein, the term “polypeptide” refers to a continuous chain of amino acids covalently linked by peptide bonds. A polypeptide can have a primary, secondary, tertiary, or quaternary structure that give it its unique function and properties. Peptides can refer to short chains of amino acids covalently bonded by peptide bonds, whereas polypeptides can refer to longer amino acid chains covalently bonded by peptide bonds. Generally, polypeptides can further be referred to as “proteins.”

[0041] A summary of the amino acids and their three and one letter symbols as understood in the art is presented in Table 1. The amino acid name, three letter symbol, and one letter symbol are used interchangeably herein.Table 1 : Amino Acid three and one letter symbols

[0042] Variants or sequences having substantial identity or homology with the polypeptides described herein can be utilized in the compositions and methods. Such sequences can be referred to as variants or modified sequences. That is, a polypeptide sequence can be modified yet still retain the ability to exhibit the desired activity. Generally, the variant or modified sequence may include greater than about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the wild type, naturally occurring polypeptide sequence, or with a variant polypeptide as described herein.

[0043] As used herein, the phrases “% sequence identity,” “% identity,” and “percent identity,” are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods of amino acid and nucleic acid sequence alignment are well-known. Sequence alignment and generation of sequence identity include global alignments and local alignments which are carried out using computational approaches. An alignment can be performed using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters. Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for shortinput sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected. Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only. A sequence having an identity score of XX% (for example, 80%) with regard to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical or, equivalently, have XX% sequence identity to the reference sequence.

[0044] Polypeptide or polynucleotide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0045] The polypeptides disclosed herein may include “variant” polypeptides, “mutants,” and “derivatives thereof.” As used herein the term “wild-type” is a term of the art understood by skilled persons and means the form of a polypeptide as it occurs in nature as distinguished from variant or mutant forms. As used herein, a “variant, “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule.

[0046] The amino acid sequences of the polypeptide variants, mutants, derivatives, or fragments as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule. “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutionssubstantially conserve the structure and the function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge and / or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulk of the side chain.

[0047] Compositions for the synthesis of P-1, 3 -oligoglucans include (i) a-GlP, a primer molecule, and a P-glucan phosphorylase described herein; (2) a glucose donor (e.g., sucrose), inorganic phosphate, a P-glucan phosphorylase as described herein, and a sugar phosphorylase as described herein; or (3) a de-branched partially hydrolyzed starch (e.g., maltodextrin), inorganic phosphate, a P-glucan phosphorylase as described herein, and an a-glucan phosphorylase as described herein. Inorganic phosphate may be provided in the composition in the form of potassium phosphate, sodium phosphate, and the like, at a concentration between 1 mM and 250 mM, between 2 mM and 100 mM, between 5 mM and 80 mM, or between 10 and 50 mM. The composition may additionally include a buffer and / or reducing agent. Suitable buffers may include, but are not limited to, a phosphate buffer (e.g., a potassium phosphate buffer, a sodium phosphate buffer, or preferably a citric acid Na2HPO4 buffer system pH 5.5-7.6) a 2-(N- morpholino)ethanesulfonic acid (MES) buffer, and the like. Suitable reducing agents included in the reactions may include, but are not limited to, dithiothreitol (DTT), tris(2- carboxyethyl)phosphine (TCEP), ascorbic acid, cysteine, sodium bisulfite, SO2, and combinations thereof. The composition may have a pH from 5.0 to 8.5, from 5.5 to 8.0, from 6.0 and 7.5, or from 6.5 to 7.0.

[0048] As used herein, the term “0-1,3-oligoglucan” can be used interchangeably with the term “0-1,3-glucan” and refers to glucose polymers covalently linked together by 0-1,3-glycosidic bonds. The 0-1, 3 -oligoglucans produced by the methods herein further can be branched such that they include additional glucose molecules covalently bonded by 0-1,6-linkages. The 0-1,3- oligoglucans produced by the methods herein can have a degree of polymerization (DP) of from 3 to 80 (i.e., DP3 to DP80), or above 80 (DP80+). The compositions formed by the methods herein can include mixtures of 0-1, 3 -oligoglucans having a degree of polymerization of from DP3 to DP80+, including mixtures in the range of DP20 to DP80+, DP3 to DP20, DP25 to DP35, DP3 to DP 10, DP 10 to DP20, DP20 to DP35, and the like. Further, the degree of polymerization of the 0-1,3-oligoglucans produced by the methods herein can include those 0-1, 3 -oligoglucans having a DP of DP3, DP4, DP5, DP6, DP7, DP8, DP9, DP10, DP11, DP12, DP13, DP14, DP15, DP16, DP17, DP18, DP19, DP20, DP21, DP22, DP23, DP24, DP25, DP26, DP27, DP28, DP29, DP30,DP31, DP32, DP33, DP34, DP35, DP36, DP37, DP38, DP39, DP40, DP41, DP42, DP43, DP44, DP45, DP46, DP47, DP48, DP49, DP50, and so on up to DP80+ or can include a range from within any of the forgoing degrees of polymerization. The P-l,3-oligoglucans may be soluble, with a degree of polymerization between DP3 and DP20. In general, as the degree of polymerization increases, solubility of the P-1, 3 -oligoglucans decreases. The P-1, 3 -oligoglucans may be insoluble, with a degree of polymerization above DP25 (e.g., above DP25, above DP35, above DP50, or DP80+).

[0049] As used herein, “PGP,” “beta-GP,” “beta-glucan phosphorylase,” and “P-glucan- phosphorylase” are used interchangeably and refer to an enzyme that reversibly catalyzes the phosphorolysis of glycosidic linkages in P-glucans to form a-D-glucose 1 -phosphate. The reverse reaction catalyzes the synthesis of P-glucans through phosphorolytic transfer of glucose from a- D-glucose 1 -phosphate to an acceptor primer molecule (e.g., glucose, sophorose, laminaribiose, cellobiose, and the like). FIG. 1 shows a reaction scheme for the production of a P-glucan catalyzed by a PGP using a-GlP and a priming substrate.

[0050] The PGP enzyme may be regioselective, for example, selectively catalyzing linkages at the P-1,3 position of glycose in the primer molecule. FIG. 2 shows a reaction scheme for the production of P-l,3-glucans by a regioselective PGP, for example^zGP or A7GP, using a-GlP and a priming substrate. As used herein, enzyme that catalyze regioselective addition of glucose at the P-1,3 position of the primer molecule from a-GlP are said to have “P-l,3-glucan phosphorylase activity.” As used herein “P-l,3-glucan phosphorylase” refers to a PGP enzyme with P-l,3-glucan phosphorylase activity. Suitable enzymes with P-l,3-glucan phosphorylase activity include, but are not limited to the P-glucan phosphorylase from Anaerolinea sp. or the P- glucan phosphorylase from Spirochaeta sp. For example, a suitable P-glucan phosphorylase includes the Anaerolinea thermophila P-glucan phosphorylase (4 / GP) and the Spirochaeta thermophila P-glucan phosphorylase (A' / GP). The PGP with P-l,3-glucan phosphorylase activity may have a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 1 and 2. Suitable P-l,3-glucan phosphorylases include those that have an amount of a-D-glucose 1 -phosphate hydrolytic side activity to produce glucose from a-D-glucose 1 -phosphate.

[0051] The PGP with P-l,3-glucan phosphorylase activity may be, or may be derived from, the Anaerolinea thermophila glucan phosphorylase of SEQ ID NO: 1. The PGP with P-1, 3 -glucan phosphorylase activity may have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 1. The PGP withP-l,3-phosphorylase activity may have a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 1 and comprise amino acid residues tyrosine 208 (Y208), arginine 452 (R452), tryptophan 454 (W454), asparagine 455 (N), arginine 948 (R948), glutamate 956 (E956), tyrosine 296 (Y296), and glutamine 1028 (QI 028) relative to SEQ ID NO: 1.

[0052] The PGP with |3-l,3-glucan phosphorylase activity may be, or may be derived from, the Spirochaeta thermophila glucan phosphorylase of SEQ ID NO:2. The PGP with [3-1,3-glucan phosphorylase activity may have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO:2.

[0053] In general, suitable [3-1,3-glucan phosphorylases exhibit very high activity toward glucose, having a catalytic efficiency (z. e. , kcat / Km) of at least 20 times (20x) the catalytic efficiency for such a reaction using known enzymes. For example, the [3-1,3-glucan phosphorylase from Anaerolinea thermophila can have a catalytic efficiency kcat / Km) of greater than 10 s^mM'1.

[0054] The [3-1,3-glucan phosphorylases described for use in the methods herein exhibit catalytic activity when performed at temperatures ranging from 30 °C to 75 °C, or from 45 °C to 70 °C, or from 50 °C to 65 °C. For example, the [3-1,3-glucan phosphorylase from Anaerolinea thermophila can retain from 55% to 65% residual catalytic activity after 48 hours at 50 °C.

[0055] Suitable [3-1,3-glucan phosphorylases include those that have an amount of a-D-glucose 1 -phosphate hydrolytic side activity that catalyzes the production of glucose from a-D-glucose 1- phosphate. Glucose produced by a-D-glucose 1 -phosphate hydrolytic side activity can act as a priming substrate for generating the one or more [3-1,3-oligoglucans. For example, the Q-1,3- glucan phosphorylases may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 1, having both [3-1,3-glucan phosphorylase activity and a-D-glucose 1 -phosphate hydrolytic activity.

[0056] As used herein, “primer molecule” refers to a mono-, di-, or polysaccharide containing D-glucose. The primer molecule may be, but is not limited to, D-glucose, sophorose, laminaribiose, cellobiose, gentiobiose, and combinations thereof. Suitable primers are known and described in the art. See, for example, Ubiparip, et al. (“P-glucan phosphorylases in carbohydrate synthesis,” Applied Microbiology and Biotechnology, 2021, 105:4073-4087). Without wishing to be bound by any particular theory or mode of action, it is believed that the primer molecule acts as an initial scaffold upon which additional glucose molecules are added to produce the Q-1,3- oligoglucan. The primer molecule may also be referred to in the art as an acceptor molecule. Accordingly, the growing P-1, 3 -oligoglucan molecule itself becomes the acceptor molecule as thereaction progresses. The concentration of the priming substrate can include a range of from 0.001 mM to 1 M, or from 0.01 mM to 500 mM, or from 0.1 mM to 100 mM. For example, the priming substrate includes glucose at a concentration of from 0.001 mM to 1 M. Without being bound to any particular theory or mode of action, the concentrations of the primer molecule and the a-GlP may be tailored to produce a P-l,2-oligoglucan of a particular degree of polymerization. By providing a concentration of priming substrate of from 0.001 mM to 1 M, the distribution of P- 1,3 -oligoglucan products with various degrees of polymerization can be tailored within a range of from DP3 to DP80+. For example, as demonstrated in Example 6 below, the combination of a low concentration of the primer molecule and higher concentration of a-GlP (e.g., a-GIP / primer ratio greater than 250 / 10 mM) may be used to generate a P-l,3-oligoglucan with a high degree of polymerization (e.g., >DP20), such that at least 60%, at least 70%, at least 80%, or at least 90% of the generated P-1, 3 -oligoglucans have a DP greater than 20. Higher concentrations of the primer molecule may be used to generate a P-l,3-oligoglucan with a lower degree of polymerization (e.g., <DP13).

[0057] As used herein, the terms “a-D-glucose 1 -phosphate” and “a-GlP” are used interchangeably and refer to a glucose molecule with a glucosyl phosphate moiety covalently attached to the 1’ carbon. The a-D-glucose 1 -phosphate is provided in the reaction composition described herein at a concentration of from 50 mM to 2 M. Without wishing to be bound by any particular theory, it is understood from the process provided herein that use of higher concentrations of a-D-glucose 1 -phosphate can lead to more product per reaction volume, and as such, the concentration of a-D-glucose 1 -phosphate can be varied within the range of 50 mM to 2 M to control yield of P-l,3-oligoglucans.

[0058] The a-D-glucose 1 -phosphate used in the methods of making P-1, 3 -oligoglucans can be provided as a reactant or can be generated in situ as part of a coupled reaction in accordance with the methods herein. Inexpensive sources of D-glucose as a glucose donor, such as sucrose, can be used to produce D-glucose 1 -phosphate via the reaction of inorganic phosphate and the glucose donor in the presence of a sucrose phosphorylase enzyme. FIG. 3 shows a reaction scheme for the production of a-GlP from a glucose donor, such as sucrose, in the presence of inorganic phosphate (Pi) and a sucrose phosphorylase (SP) and FIG. 4 shows a reaction scheme for the coupled reaction for producing a-GlP from a glucose donor and subsequent production of P-l,3-oligoglucans. In a coupled reaction, the reaction composition may include a glucose donor (e.g., sucrose) at a concentration from 50 mM to 5 M.

[0059] For example, the sucrose phosphorylase enzymes can include the sucrose phosphorylase from Bifidobacterium adolescentis (Ba P,' SEQ ID NO:3). The SP with sucrose phosphorylase activity may have a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:3.

[0060] Partially hydrolyzed starch, e.g., maltodextrin, can also serve as a glucose donor. In general, the partially hydrolyzed starch will have a DP greater than 4. When a starch is used as the glucose donor, an a-glucan phosphorylase can be used to produce a-D-glucose 1 -phosphate. FIG. 5 shows a reaction scheme for coupled production of P-1, 3 -oligoglucans by the combination of an aGP and a regioselective PGP starting from a glucose donor through an a-GlP intermediate in the presence of a priming substrate. For example, the a-glucan phosphorylase enzyme can include the a-glucan phosphorylase from Thermosipho africanus (SEQ ID NO:4). The a-glucan phosphorylase enzyme may have a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:4. In a coupled reaction including an aGP, the glucose donor includes a partially hydrolyzed starch, such as maltodextrin, at a concentration of from 1 wt% to 50 wt%, or from 5 wt% to 40 wt%, or from 10 wt% to 25 wt%. Additional details of and support for production of a-GlP using an aGP is found in PCT Application No. PCT / US2023 / 073127, filed August 30, 2023, which is incorporated herein by reference in its entirety.

[0061] The present disclosure provides for various methods for the production of P-1, 3- oligoglucans using the compositions described herein. The method includes incubating a composition including a-D-glucose 1 -phosphate (a-GlP), a primer molecule, and a PGP with P- 1,3-glucan phosphorylase activity for a time and under conditions sufficient to produce P-1, 3- oligoglucans. Based on the disclosure herein, a skilled artisan will understand the time and conditions suitable to produce the P-l,3-oligoglucans. The composition may be incubated at a temperature between 35 °C and 85 °C, between 40 °C and 80 °C, between 45 °C and 75 °C, between 50 °C and 70 °C, or between 55 °C and 60 °C. The composition may be incubated at a temperature of about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, about 80 °C, or about 85 °C. The composition may have a pH between 5.5 and 7.5. The composition may have a pH from 5.0 to 8.5, from 5.5 to 8.0, from 6.0 and 7.5, or from 6.5 to 7.0. The composition may be incubated for at least 30 minutes, at least 1 hour, at least 5 hours, at least 10 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, or at least 44 hours. The composition may be incubated with shaking or agitation.

[0062] The methods provide for an uncoupled reaction process, for example, as shown in FIG. 2. The reaction composition comprises a-GlP, a primer molecule, and a P-glucan phosphorylase described herein, for example d / GP or A7GP. The a-GlP can be provided at a concentration from 50 mM to 2M as described herein. The primer molecule can be provided at a concentration from 0.001 mM to IM as described herein. Suitable primer molecules and various concentrations of a- G1P and the primer molecules are described herein.

[0063] The methods also provide for a coupled reaction where a-GlP is produced in situ using an SP enzyme, for example, as shown in FIG. 4. The reaction composition comprises a glucose donor (e.g., sucrose), inorganic phosphate, a P-glucan phosphorylase as described herein (e.g., 4 / GP or 57GP), and a sugar phosphorylase as described herein (e.g., aSP). In general, this reaction will proceed through an a-GlP intermediate, so the reaction composition can also be considered to include a-GlP produced by the reaction itself. The glucose donor can be provided at a concentration of 50 mM to 5M as described herein. The inorganic phosphate may be provided at a concentration of 1 mM - 250 mM, 2 mM - 100 mM, 5 mM - 80 mM, or 10 - 50 mM as described herein. Suitable glucose donors and various concentrations of said glucose donors and inorganic phosphate are described herein.

[0064] The methods also provide for a coupled reaction where a-GlP is produced in situ using an aGP enzyme, for example, as shown in FIG. 5. The reaction composition comprises a partially hydrolyzed starch glucose donor (e.g., maltodextrin), inorganic phosphate, a P-glucan phosphorylase as described herein (e.g., 4 / GP or 57GP), and an a-glucan phosphorylase as described herein (e.g., 7baGP). In general, this reaction will proceed through an a-GlP intermediate, so the reaction composition can also be considered to include a-GlP produced by the reaction itself. The partially hydrolyzed starch glucose donor can be provided at a concentration of 1 wt% to 50 wt%.

[0065] All of the methods for producing P-l,3-oligoglucans herein can include a further step of drying the P-l,3-oligoglucans. Suitable methods for drying are known and described in the art. For example, the P-1, 3 -oligoglucans produced by the methods can be dried at 60 °C to a concentration of 35% dry matter.Compositions of B-l,3-oligoglucans

[0066] The present disclosure also provides for compositions including the P-l,3-oligoglucans produced by the methods herein. The compositions can include one or more P-1, 3 -oligoglucans having a degree of polymerization of from DP3 to DP80+. The compositions formed by themethods herein can include mixtures of P-l,3-oligoglucans having a degree of polymerization of from DP3 to DP80+, including mixtures in the range of DP20 to DP80+, DP3 to DP20, DP25 to DP35, DP3 to DP 10, DP 10 to DP20, DP20 to DP35, and the like. Thus, the degree of polymerization of the P-1, 3 -oligoglucans produced by the methods herein can include those -1,3- oligoglucans having a DP of DP3, DP4, DP5, DP6, DP7, DP8, DP9, DP10, DP11, DP12, DP13, DP14, DP15, DP16, DP17, DP18, DP19, DP20, DP21, DP22, DP23, DP24, DP25, DP26, DP27, DP28, DP29, DP30, DP31, DP32, DP33, DP34, DP35, DP36, DP37, DP38, DP39, DP40, DP41, DP42, DP43, DP44, DP45, DP46, DP47, DP48, DP49, DP50, up to DP80+, or can include a range from within any of the forgoing degrees of polymerization.

[0067] The P-1, 3 -oligoglucans produced by the methods herein can be used in the preparation of a food product, a beverage product, an animal feed product, a person care product (e.g., hand gels, moisturizing creams), a cosmetic product, a supplement product, and the like. For example, the P-l,3-oligoglucans may be used a replacement for fiber. The P-l,3-oligoglucans may also be used in industrial applications, for example in oil drilling.EXAMPLES

[0068] Various aspects of the present disclosure can be better understood by reference to the following Examples, which are offered by way of illustration. The present disclosure is not limited to the Examples given herein.Example 1 : Gene Cloning and Transformation

[0069] The Examples herein utilize a P-l,3-oligoglucan phosphorylase expressed from a gene from Anaerolinea thermophila or Spirochaeta thermophila and optimized for expression in E. coli.

[0070] Briefly, the gene encoding the amino acid sequence for the putative Anaerolinea thermophila 0-1,3-oligoglucan phosphorylase (4 / GP, SEQ ID NO: 1, UniProt identifier E8N5S1) and the gene encoding the amino acid sequence for the Spirochaeta thermophila [3- 1 ,3 -oligoglucan phosphorylase CS7GP, SEQ ID NO:2) were codon optimized for E. coli, synthesized (Life Technologies, Merelbeke, Belgium), and were subsequently subcloned into separate pET21 vector at the Nhel and Xhol restriction sites, consequently introducing a C-terminal His6-tag. The plasmids were transformed in E. coli BL21(DE3) agp- electrocompetent cells.Example 2: Protein Expression and Purification

[0071] Anaerolinea thermophila P-1, 3 -oligoglucan phosphorylase and Spirochaeta thermophila P-1, 3 -oligoglucan phosphorylase were expressed and purified from cultures of E. coli BL21(DE3).

[0072] An overnight culture was inoculated (2 %) in 500 mL LB-Lennox medium containing 100 pg / mL ampicillin in a 2-L shake flask and incubated at 37 °C with continuous shaking at 200 rpm. The cultures were grown to ODeoo 0.6, and expression of both enzymes (in pET21a) was induced by adding isopropyl P-d-l-thio-galactopyranoside to a final concentration of 0.1 mM. Gene expression ofd / GP and A7GP took place for 16 h at 30 °C. The cultures were then centrifuged (15 min, 9000 rpm), and the cell pellets were frozen at -20 °C for at least 4 h.

[0073] To extract the proteins, the cell pellets were thawed and dissolved in 10 mL lysis buffer consisting of 0.1 mM phenylmethyl sulfonyl fluoride (PMSF), 1 mg / mL lysozyme, 10 mM imidazole and 50 mM phosphate buffered saline (PBS), pH 7.4. This suspension was incubated on ice for 30 min and sonicated three times for 3 min (Branson Sonifier 250, level 3, 50 % duty cycle). The cell debris was removed by centrifugation at 9000 rpm for 2 x 30 min at 4 °C. Next, the clear protein solution was filtered over a sterile 0.22 pm o PES membrane filter (Millex® Syringe Filters 0.22 pm polyethersulfone, Merck-Millipore) to remove any suspended solids.

[0074] The resulting cell extract was further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography as described by the supplier (MCLab, San Francisco, USA), after which the buffer was exchanged to 50 mM 2-morpholinoethanesulfonic acid (MES, pH 6.5) in a 30-kDa Amicon Ultra centrifugal filter (Merck-Millipore, Burlington, Massachusetts, Verenigde Staten).

[0075] The a-glucan phosphorylase from Thermosipho africanus (TaaGP SEQ ID NO:3), as well as the gene encoding the amino acid sequence for Euglena gracilis P-1, 3 -oligoglucan phosphorylase (EgPl, SEQ ID NO: 5), and the gene encoding the amino acid sequence for the Pro_7066 P-l,3-oligoglucan phosphorylase (Pro_7066, SEQ ID NO:6) were expressed and purified using the same methods as those described above for 4 / GP and A7GP.

[0076] The codon-optimized genes encoding the amino acid sequence of 4 / GP (SEQ ID NO: 1) was used as a template for mutagenesis using the primers outlined in Table 2. Mutated plasmids were extracted from E. coli DH5a cells, sequence verified, and transformed into E. coli BL21(DE3) agp- electrocompetent cells. The 4 / GP variant P-l,3-oligoglucan phosphorylase enzymes were expressed and purified from cultures of E. coli BL21 (DE3) cells. Overnight cultures were inoculated (2 %) in 500 mL LB-Lennox medium containing 100 pg / mL ampicillin in a 2-L shake flask and incubated at 37 °C with continuous shaking at 200 rpm. The cultures were grown to ODeoo 0.6, and enzyme expression (in pET21a) was induced by adding isopropyl P-d-l-thio-galactopyranoside to a final concentration of 0.1 mM. Gene expression of d / GP took place for 16 h at 30 °C. The cultures were then centrifuged (15 min, 9000 rpm), and the cell pellets were frozen at -20 °C for at least 4 h. To extract the 4 / GP enzymes, the cell pellets were thawed and dissolved in 10 mL lysis buffer consisting of 0.1 mM phenylmethyl sulfonyl fluoride (PMSF), 1 mg / mL lysozyme, 10 mM imidazole and 50 mM phosphate buffered saline (PBS), pH 7.4. This suspension was incubated on ice for 30 min and sonicated three times for 3 min (Branson Sonifier 250, level 3, 50 % duty cycle). The cell debris was removed by centrifugation at 9000 rpm for 2 x 30 min at 4 °C. Next, the clear protein solution was filtered over a sterile 0.22 pm o PES membrane filter (Millex® Syringe Filters 0.22 pm polyethersulfone, Merck-Millipore) to remove any suspended solids. The resulting cell extract was further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography as described by the supplier (MCLab, San Francisco, USA), after which the buffer was exchanged to 50 mM 2-morpholinoethanesulfonic acid (MES, pH 6.5) in a 30-kDa Amicon Ultra centrifugal filter (Merck-Millipore, Burlington, Massachusetts, United States).Table 2.Example 3 : Protein Purification for aSP

[0077] The thermostable variant of the sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) was expressed using the methods described above but was purified using heat purification. A 3 g cell pellet was suspended in 10 mL lysis buffer (10 mM imidazole, 1 mg / mL lysozyme, 0.1 mM phenylmethylsulfonylfluoride (PMSF), diluted in 50 mM MES pH 6.5) incubated at room temperature for 30 minutes. Following incubation, the solution was sonicated 3 times for 3 minutes each at level 3 with a 50 % duty cycle (Branson sonifier). Following sonication, the solution was centrifuged for 30 minutes at 9,000 rpm and the supernatant was transferred to a new falcon and heated for 1 hour at 60 °C. The supernatant was removed after centrifugation for 30 minutes at 9,000 rpm and stored at -20 °C.Example 4: Enzyme Characterization

[0078] Gawronski Phosphate Release assays were performed to characterize the 4 / GP and 57GP enzymes. The Gawronski assay is a colorimetric assay that detects inorganic phosphate (Pi) that is released from a-GIP. Ammonium molybdate reaction with Pi forming phosphomolybdate, which is reduced to molybdenum blue in the presence of ascorbic acid. Molybdenum blue can then be measured by a spectrophotometer at a wavelength of 655 nm. Reaction conditions for these tests are outline in Table 3. The necessary reagents for the Gawronski assay, ammonium molybdate and L-ascorbic acid in IM HCL were added to the reaction following the addition of a stop solution that included sodium citrate tribasic and acetic acid.Table 3.

[0079] To assay temperature profiles, reactions were prepared as outlined in Table 3. Reactions were run at 40, 45, 50, 55, 60, 65, 70, and 75 °C for 57GP and 4 / GP. Results are shown in FIG. 6.The results show that both enzymes had a temperature optimum of 65 °C under the conditions tested, but also that they retain activity as low as 40 °C and as high as 75 °C. Accordingly, the reaction to produce P-1, 3 -oligoglucans can be expected to work in this temperature range as well, even if activity is reduced.

[0080] To assay pH profiles, reactions were prepared as outlined in Table 3. Reactions were done at 50 °C in each of the following buffers: MES at pH 5, 6, and 6.5, 3-(N-morpholino)propane sulfonic acid (MOPS) at pH 7, and tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) at pH 7.5 and 8. Results are shown in FIG. 7. The results show that the optimum pH for A7GP is 7.0 and the optimum pH for 4 / GP is 6.5. However, both enzymes retained greater than 60% relative activity across the entire pH range tested. Accordingly, the reaction to produce P-1, 3- oligoglucans can be expected to work across this pH range.

[0081] To determine specific activities of 4 / GP, A7GP, E Pl, and Pro_7066, reactions were done with 50 mM MES buffer at pH 6.5, 25 mM D-glucose, 25 mM a-D-glucose 1-phosphate. For 4 / GP and A7GP, reactions were performed at 50 °C with 0.4 pg / mL and 0.5 pg / mL enzyme, respectively. For E Pl and Pro_7066 reactions were performed at 30 °C with 2 pg / mL and 8 pg / mL enzyme, respectively. Samples of 50 pL were taken at t = 0, 1, 2, 3, 4, 5, 6, and 7 min, and analyzed with the Gawronski Phosphate Release assay described above. As such, measurement of the specific activity within a linear range was assured. One unit (U) of enzyme activity is expressed as the amount of enzyme producing 1 pmol of phosphate per minute.

[0082] The a-D-glucose 1-phosphate assay was used to characterize the 7baGP enzyme. This assay utilizes a phosphoglucomutase, to convert a-GlP into glucose-6-phosphate, and a glucose- 6-phosphate dehydrogenase, to convert the glucose-6-phosphate into glucono- 1,5 -lactone while converting NAD+to NADH. The NADH concentration is then measured using a spectrophotometer at a wavelength of 340 nM. Reagents necessary for the a-D-glucose 1- phsophate assay, ethylenediamineteaacetic acid, MgSCh, NAD+, glucose 1,6-diphosphate, phosphoglucomutase, and glucose-6-phosphate dehydrogenase, were added to the reaction together with NaOH after 40 minutes.

[0083] To assay the pH profile of 7baGP, reactions were prepared with 5 wt% maltodextrin (MDX 01995, from Cargill, Incorporated), 100 mM buffer, and 30 mM phosphate. Reactions were done at 50 °C in each of the following buffers: MES at pH 5.5, 6, and 6.5, MOPS at pH 7, and Tris-HCL at pH 7.5 and 8. Results are shown in FIG. 8. The results show that the optimum pH for TaaGP is in the range of 6.0 to 7.0. However, the enzyme retained at least 35% relative activity across the entire pH range tested. Accordingly, a reaction to produce a-GlP from a partiallyhydrolyzed starch ush as maltodextrin and / or a coupled reaction to produce [3-1,3-oligoglucans can be expected to work across this pH range.Example 5: Uncoupled Production of Insoluble B-L3-glucans having a Degree of Polymerization ofDP25-DP35

[0084] To ensure the synthesis of [3-1,3-glucans with a maximal molecular weight, a 50 mL- reaction was set up with 2 U / mL 4 / GP and 1 M of a-GlP as substrate. As a result of a-GlP hydrolytic side-activity by A / GP, glucose would inherently be released and could consequently serve as priming substrate. The reaction was performed at 50 °C, 50 mM MES pH 6.5 and proceeded for 96 hours. By measuring the a-GlP concentration at the beginning and end of the reaction, a conversion of 76 % a-GlP was confirmed.

[0085] The insoluble product was recovered by centrifugation, after which it was washed with 50 % EtOH in water to increase the removal of residual a-GlP and buffer components. Next, the product was dried on a 0.22 pm o PES membrane filter, as centrifugation did not yield a solid pellet but rather a viscous product with intermediate density. The product profile was visualized by high performance anion exchange chromatography (HPAEC) analysis. The HPAEC- chromatogram of the purified product obtained from the uncoupled reaction starting from 2 U / mL 4 / GP and 1 M a-GlP is shown in FIG. 9. The reaction products are mainly composed of P-1, 3- glucan with a DP between 25 and 35, and with minor amounts of shorter oligo’ s and residual a- G1P.Example 6: B-L3-glucan DP Influenced by a-GIP / Glucose Ratio

[0086] In order to evaluate the influence of the a-GIP / glucose ratio on the product profile, several reactions were performed starting from 250 mM a-GlP and 0, 1, 10, or 50 mM glucose. Enzyme dosage was set at 2 U / mL 4 / GP or A7GP, and the reactions took place at pH 6.5 (50 mM MES) and 50 °C for 48 hours. Results are shown in FIGS. 10 (4 / GP) and 11 S7GP). The results show that both 4 / GP and A7GP synthesize high MW (>DP20) [3-1,3-glucans when the ratio of a- GlP:Glc is 25:1 or higher (e.g., reaction of 250 / 0 mM, 250 / 1 mM, and 250 / 10 mM), while at lower ratios (e.g., 250 / 50 mM) only soluble [3-1,3-glucans up to about DP 13 are formed (FIGS. 10 and 11).

[0087] This indicates the flexibility of the product profile and the applicability of both enzymes in synthesizing diverse products with a tailored MW distribution.Example 7: Coupled Production of B-L3-ghicans having a Degree of Polymerization of DP3-DP35

[0088] To enable the synthesis of P-l,3-glucans from inexpensive and readily available bulk substrates, 4 / GP was coupled to the activity of a thermostable variant of sucrose phosphorylase from Bifidobacterium adolescentis ( aSP)(Cerdobbel et al., 2011). As such, a-GlP could be produced from sucrose in situ, enabling the convenient recycling of phosphate.

[0089] During optimization, the molecular weight of the P-l,3-oligoglucan product could be controlled by adjusting the glucose concentration, similarly to the uncoupled reaction with only 4 / GP. Starting from 500 mM sucrose, a conversion of 99 mol% was achieved with <0.5% residual glucose. While the glucose moiety of sucrose is incorporated in the synthesized P-1, 3 -oligoglucan product, the fructose moiety of sucrose is released as byproduct.

[0090] As proof of concept, a 500 mL scale reaction was set up, starting from 500 mM sucrose, 20 mM potassium phosphate, 30 L / niL d / GP and 10 U / mL BaSP, for 24 h at 50 °C with agitation. The nearly complete conversion of sucrose resulted in a product with a molecular weight distribution from DPI to ± DP35. The HPAEC-chromatogram of the reaction mixture after the coupled reaction starting from 30 U / mL 4 / GP, 10 U / mL BaSP, 500 mM sucrose and 20 mM potassium phosphate is shown at the top in FIG. 12. The reaction products are mainly composed of P-1, 3 -oligoglucans with a DP between 2 and 35 and fructose byproduct, and with minor amounts of residual sucrose and glucose.

[0091] Cooling to 4 °C and addition of 9 parts of EtOH resulted in the precipitation of P-1, 3- oligoglucans with DP range of mainly DP3-35, which were conveniently recovered and dried by filtration. The HPAEC-chromatogram of the obtained product fractions after EtOH-precipitation of the coupled reaction mixture is shown in FIG. 12. The obtained precipitated product (center, FIG. 12) is mainly composed of P-1, 3 -oligoglucans with a DP between 3 and 35. The supernatant (bottom, FIG. 12) contains increasing amounts of shorter P-1, 3 -oligoglucans <DP8, as well as residual glucose, fructose, and sucrose (not shown).

[0092] A production with a slightly lower amount of P-l,3-glucans with >DP20 could also be obtained by significantly decreasing the enzyme dosage to 5 U / mL / GSP and 10 U / mL 4 / GP, resulting in a sucrose conversion of 96 mol% after 24 h at 50 °C with agitation FIG. 13. This illustrates the potential to vary the enzyme dosage and ratio further, thereby tailoring the product profile and process economy. In addition, by increasing the sucrose concentration further to, for example, 1 M or higher, a product enriched in insoluble P-1, 3 -glucans with >DP20 could potentially be achieved. The opposite could be achieved by adding glucose to the reaction mixture,leading to the synthesis of solely soluble P-1, 3 -oligoglucans with, for example, DP3-10 or DP15 or 20.Example 8: Uncoupled Production of Insoluble B-E3-glucans having a High DP

[0093] To evaluate the high molecular weight P-l,3-glucans achievable with 4 / GP, a 1 mL- reaction was set up with an excess 4 / GP (3000 U / mL) and IM of a-GIP. As a result of a-GlP hydrolytic side-activity by 4 / GP, glucose would inherently be released and could consequently serve as priming substrate. The reaction was performed at 30 °C and 50 °C, 50 mM MES pH 6.5 and proceeded for 6 hours.

[0094] The insoluble product was diluted 200-fold in 100 mM NaOH, thereby solubilizing the high MW P-1, 3 -glucans. The product profile was subsequently visualized by high performance anion exchange chromatography (HPAEC) analysis, which indicates the presence of P-1, 3 -glucan with a DP of 75 and above. FIG. 14 shows the chromatogram of the reactions at 30 °C (bottom) and at 50 °C (top). The HPAEC methods used in this characterization are unable to separate P- glucans with a DP above DP80. Accordingly, the “rest <80” peak in FIG. 14 includes minor amounts of DP80 and larger oligoglucan, for example, DP85, DP90, DP95, DP100, etc,, that all eluted at the same time. This suggests that an even higher DP could be achieved by further increasing the enzyme dosage.Example 9: GH149 GP Comparison

[0095] 4 / GP and A7GP were compared to two known GH149 enzymes: Euglena gracilis P-1, 3- glucan phosphorylase (E Pl) and Pro_7066, both described in Kuhaudomlarp et al., “Identification of Euglena gracilis P-l,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149,” Journal of Biological Chemistry, 293(8):2865-2876, February 2019. Table 4 reports the sequence identity between these four enzymes. Assays were prepared as describe in Example 6 containing 250 mM aGlP and 1 mM glucose. Results for the 24-hour and 48-hour time points are shown in FIGS.15 and 16, respectively. The results show that EgPl only produces soluble P-1, 3 -oligoglucans with the majority being between DP10 and DP15, with only minor amounts of glucans up to DP20. Similarly, Pro_7066 produced P-1, 3- oligoglucans with a DP between 10 and 20 and minor amounts up to DP30. However, neither EgPl nor Pro_7066 can produce a significant portion of P-glucan products with a DP above 20. On the contrary, both 4 / GP and A7GP produce P-l,3-oligoglucans with the majority being above DP20 and significant amounts of insoluble glucans above DP25.Table 4.Example 10: ri / GP Enzyme Variant Characterization

[0096] Gawronski Phosphate Release assays were performed to characterize the ri / GP variant enzymes, as described in Example 4. Reaction conditions for these tests are outline in Table 5. All reactions included 20 mM aGIP. The necessary reagents for the Gawronski assay, ammonium molybdate and L-ascorbic acid in IM HCL were added to the reaction following the addition of a stop solution that included sodium citrate tribasic and acetic acid. Kinetic parameters were calculated by non-linear regression of the Michaelis-Menten equation and a molecular weight of 133 kDa.

[0097] The ri / GP variants tested are based on a homology model ofri / GP built using the crystal structure of Protein Data Base (PDB) ID 6HQ6, the only available crystal structure of a GH149 enzyme. PDB ID 6HQ6 shows the crystal structure of the P-1, 3 -oligoglucan phosphorylase Pro_7066 as described in Kuhaudomlarp et al., “The structure of a GH149 P-(l— >3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide-specific GH94 glucose-(l— >3)-glucose (laminaribiose) phosphorylase,” Proteins, 87(10):885-892, October 2019. The homology model with a docked substrate showed 25 amino acids within 4 A of the substrate docking site. A subset of these amino acids originating from the +1 subsite as well as its interfaces with subsites -1 and +2, were selected as the likely residues critical in the binding of glucose. FIG. 17 shows the docking site of the ri / GP homology model with a docked laminaribiose, with the 8 residues selected for mutational analysis, the catalytic acid (D660), and their hydrogen bonding (dashed lines) shown.

[0098] The kinetic parameter results shown in Table 5 indicate that substitution of residues R452 and E956 resulted in complete loss of activity. Given E956 forms a hydrogen bond (H-bond) with the C2-OH of glucose in subsite +1 (FIG. 17), its interaction can be considered crucial in acceptor substrate recognition. A dramatic loss in affinity is also seen for R948, which does not interact directly with the +1 glucose, but rather forms H-bonds with E956 (FIG. 17) and thecatalytic acid D660, presumably ensuring their correct orientation. Similarly, R452 does not interact directly with the substrate but forms the floor of the active site, and its substitution potentially disrupts the active site architecture, explaining the complete loss in activity. W454 is located on the interface of subsites +1 and +2, facilitating a stacking interaction in subsite +2. The indole ring can form hydrophobic interactions with the Cl (3.7 A) and C2 (4.0 A) of the +1 glucose, and its importance is shown by the near complete loss of affinity in W454A. The W454A variant still showed minor activity (0.5% of WT) albeit with no signs of saturation (FIG. 18), even at high glucose concentrations (2 M).Table 5.ND - not determined due to no sign of saturation up to 2 M glucoseNA - no activity detected at 100 mM glucose

Claims

CLAIMSWhat is claimed is:

1. A method for producing P-l,3-oligoglucans comprising: contacting a-D-glucose 1 -phosphate (a-GlP) with a beta(P)-glucan phosphorylase (PGP) in the presence of a priming substrate to produce one or more P-1, 3 -oligoglucans, wherein the PGP has an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs: 1 and 2.

2. The method of claim 1, wherein the PGP a-GlP contacting step is carried out at a pH between 5.0 and 8.5, between 5.5 and 8.0, or between 6.0 and 7.5; and / or a temperature between 35 and 80 °C, between 40 and 75 °C, between 45 and 70 °C. or between 50 and 65 °C.

3. The method of any preceding claim, additionally comprising the step of contacting a glucose donor with an alpha(a)-glucan-phosphorylase (aGP) or a sucrose phosphorylase (SP) in the presence of inorganic phosphate (e.g., sodium phosphate or potassium phosphate) to produce the a-GIP.

4. The method of claim 3, wherein the glucose donor has a degree of polymerization equal to or greater than 4 and is contacted by an aGP with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:4; wherein the glucose donor is a partially hydrolyzed starch (e.g., maltodextrin) and is contacted by an aGP with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:4; and / or wherein the glucose donor is sucrose and is contacted by an SP with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:3.

5. The method of any one of claims 3-4, wherein the glucose donor aGP contacting step is carried out at a pH between 5.0 and 8.5, between 5.5 and 8.0, or between 6.0 and 7.5;and / or a temperature between 35 and 80 °C, between 40 and 75 °C, between 45 and 70 °C. or between 50 and 65 °C.

6. The method of any one of claims 3-5, wherein the PGP enzyme comprises amino acid residues tyrosine 208 (Y208), arginine 452 (R452), tryptophan 454 (W454), asparagine 455 (N), arginine 948 (R948), glutamate 956 (E956), tyrosine 296 (Y296), and glutamine 1028 (QI 028) relative to SEQ ID NO: 1.

7. The method of any preceding claim, wherein the P-1, 3 -oligoglucans comprise at least 60%, at least 70%, at least 80%, or at least 90% by weight P-l,3-oligoglucans having a degree of polymerization (DP) of from 3 to 80.

8. The method of any preceding claim, wherein the ratio of a-GlP to priming substrate is greater than 25: 1 and the P-1, 3- oligoglucans comprise at least 60%, at least 70%, at least 80%, or at least 90% by weight P-l,3-oligoglucans having a degree of polymerization (DP) greater than 20 (>DP20); wherein the ratio of a-GlP to priming substrate is about 25: 1 and the P-1, 3- oligoglucans comprise at least 60%, at least 70%, at least 80%, or at least 90% by weight P-l,3-oligoglucans having a degree of polymerization (DP) of 5-20 (DP5-DP20); wherein the ratio of a-GlP to priming substrate is less than 25: 1 and the P-1, 3- oligoglucans comprise at least 60%, at least 70%, at least 80%, or at least 90% by weight P-l,3-oligoglucans having a degree of polymerization (DP) less than 10 (<DP10).

9. A composition comprising: i. a glucose donor; ii. inorganic phosphate (e.g., sodium phosphate or potassium phosphate); and iii. an alpha(a)-glucan-phosphorylase (aGP) or a sucrose phosphorylase (SP); wherein if the glucose donor is sucrose, the composition comprises an SP with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:3; wherein if the glucose donor is a partially hydrolyzed starch (e.g., maltodextrin) with a degree of polymerization equal to or greater than 4, the composition comprises an aGPan amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:4.

10. The composition of claim 9, wherein the composition additionally comprises a beta(P)- glucan phosphorylase (PGP) with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs: l and 2.

11. The method or composition of any one of claims 3-10, wherein sucrose is provided at a concentration of 50 mM to 5 M; and / or wherein the glucose donor is a partially hydrolyzed starch (e.g., maltodextrin) with a degree of polymerization equal to or greater than 4 and is provided at a concentration of1 wt% to 50 wt%, or from 5 wt% to 40 wt%, or from 10 wt% to 25 wt%12. A composition comprising: i. a beta(P)-glucan phosphorylase (PGP) with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs: 1 and 2; ii. a-D-glucose 1-phosphate (a-GlP); and iii. a primer molecule.

13. The method or composition of any one of claims 1-8 or 12, wherein the a-D-glucose 1-phosphate is provided at a concentration of 50 mM to2 M, and / or the primer molecule is provided at a concentration of 0.001 mM to 1 M, or from 0.01 mM to 500 mM, or from 0.1 mM to 100 mM.

14. A composition comprising the P-1, 3 -oligoglucans obtained by the method of any one of claims 1-8, 11, or 13.

15. The use of a beta(P)-glucan phosphorylase (PGP) with an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs: 1 and 2 for the production of one or more P-l,3-oligoglucans.