New process for the synthesis of oligosaccharide phosphate or polysaccharide phosphate

Abstract

The invention relates to the field of biotechnology. More particularly, the invention relates to an enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1), comprising providing a preparation comprising at least one glycoside phosphorylase enzyme (10a) that does not catalyze the formation of 1-1 glycosidic bonds; providing a composition comprising at least one sugar-phosphate substrate of said enzyme (10b), said composition substantially not comprising any unphosphorylated sugar acceptor substrate of said at least one glycoside phosphorylase enzyme; reacting (20) the preparation comprising the at least one glycoside phosphorylase with the composition comprising at least one sugar-phosphate substrate of said enzyme; and obtaining (30) a mixture comprising at least one oligosaccharide phosphate or at least one polysaccharide phosphate. Figure to be published with the abstract: Figure 2

Description

Title of the invention: New process for the synthesis of phosphate oligosaccharides or phosphate polysaccharides Technical field

[0001] The invention relates to the field of biotechnology. More particularly, the invention relates to enzymes active on carbohydrates, and a new process for the in vitro enzymatic synthesis of phosphate polysaccharides and / or phosphate oligosaccharides, without reducing ends. Prior art

[0002] Oligo- and polysaccharides represent a very diverse class of biomolecules consisting of linear or branched chains of glycosylated units linked together by different types of α or [3-osidic bonds. Their structural diversity results from both the nature of the monomers and the position and anomerism of their covalent bonds (α- or [3-). These molecules play diverse and important roles in a multitude of cellular processes. Due to their very diverse structures and biological functions, oligosaccharides are used in a wide range of applications in the food industry, in the health industry, in cosmetics, in the chemical industry.

[0003] The use of chemical synthesis processes to produce them on an industrial scale presents high costs and risks of contamination by undesirable chemical compounds or synthetic intermediates. These processes can also pose problems of pollution and reprocessing of the numerous solvents generated.

[0004] Enzymatic synthesis is a particularly interesting alternative to chemical synthesis because it is less energy-intensive, more environmentally friendly than chemical processes, and particularly specific with regard to the synthesized products. The development of glycomics and analytical methods allow the development of new tools for the purpose of identifying and exploring the activities of enzymes active on carbohydrates.

[0005] Thus, the functional annotation of the numerous carbohydrate-active enzymes has enabled the development of the Carbohydrate-Active Enzymes database (CAZy; http: / / www.cazy.org), listing nearly 300 families of catalytic and auxiliary modules which are accessible online and contain more than 100,000 non-redundant entries.

[0006] Among the enzymes active on carbohydrates, we distinguish 3 large families, glycoside hydrolases (GH), glycoside transferases (GT) and glycoside phos- phorylases (GPs). GPs are valuable catalysts for white biotechnology due to their ability to efficiently synthesize oligo- and polysaccharides without the need for expensive activated sugars as substrates. The reversibility of the phosphorolysis reaction makes them attractive tools for the diversification of glycosylation reactions.

[0007] Thus, GPs are known in the art and used either to produce phosphate monosaccharides from oligosaccharides or polysaccharides, or to synthesize oligosaccharides from phosphate monosaccharides and an acceptor glycoside (WO 2021 / 229185; Li et al; 2022).

[0008] Quite unexpectedly, it was discovered that it is possible to use these enzymes to synthesize oligo- and polysaccharide phosphates: These enzymes can in fact use a sugar-phosphate both as a sugar donor and as a sugar acceptor for the synthesis of oligosaccharide phosphates ([Fig.2]), this reaction is hereinafter referred to as the "sugar phosphate synthesis reaction".

[0009] The process of the invention thus makes it possible to produce oligo- and polysaccharides without a reducing end because the latter is involved in the bond with the phosphate group. This property is of particular interest for many applications because it can avoid Maillard reactions when the sugars are used in formulations comprising peptides or proteins.

[0010] Furthermore, as mentioned above, these phosphate oligo- or polysaccharides derived from this process have multiple applications, for example in the field of nutraceuticals and food supplements, medicine, in particular vaccinology (sugars being an essential component of the wall of certain pathogenic microorganisms), drug development, pharmaceutical and cosmetological formulation (Wang et al, 2022; Budhathoki et al, 2022).

[0011] There is a strong need for an environmentally friendly, reliable and high-yield alternative for the synthesis of these compounds. The method of the present invention fulfills this need. Summary of the invention

[0012] The invention therefore relates to the discovery by the inventors of a new enzymatic activity of the glycoside phosphorylase for the synthesis of phosphate sugars which, implemented in the process of the invention, makes it possible to produce non-reducing phosphate oligosaccharides and phosphate polysaccharides. These molecules have multiple applications, since oligosaccharides and polysaccharides are used in many technological fields.

[0013] An object of the present invention is therefore an enzymatic process for the synthesis in vitro of an oligosaccharide phosphate and / or polysaccharide phosphate comprising the following steps: - provision of a preparation comprising at least one glycoside phosphorylase enzyme which does not catalyze the formation of a 1-1 osidic bond, - providing a composition comprising at least one sugar-phosphate substrate of said enzyme, said composition substantially not comprising any non-phosphorylated sugar acceptor substrate of said at least one glycoside phosphorylase enzyme, - reacting the preparation comprising at least one glycoside phosphorylase with the composition comprising at least one sugar-phosphate substrate of said enzyme, - obtaining a mixture comprising at least one phosphate oligosaccharide or one phosphate polysaccharide.

[0014] As mentioned, such a method allows the simple and specific production of non-reducing phosphate oligosaccharides or phosphate polysaccharides without resorting to chemical synthesis methods which have the aforementioned disadvantages of pollution, low or unsatisfactory yield and / or specificity.

[0015] According to other optional features of the method, the latter may optionally include one or more of the following features, alone or in combination: - the at least one glycoside phosphorylase enzyme belongs to a family of enzymes with carbohydrate activity referenced in the CAZY.org database in the following families: GH3, GH13_18, GH65, GH94, GH112, GH130, GH149, GH161, GT35, GT108 and GH131. Indeed, the GPs identified by the inventors are part of these CAZy classification groups; - the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: AAQ05801.1, AUG44408.1, 2207198A, AAD40317.1, AAN24362.1, AAN58596.1, AAO21868.1, AAO33821.1, AAO84039.1, AAX33736.1, ABS59292.1, ADL69407.1, CCA61958.1, BAN03569.1, AGK37834.1, BAA14344.1, BAF62433.1, CAA30846.1, CAA80424.1, ADP98617.1, ADH62582.1, AEJ61152.1, AAC74391.2, AAV43670.1, ADH99560.1, BAC54904.1, BAD97810.1, CAA11905.1, AAO80764.1, ABX42243.1, ABX43667.1, ABX43668.1, AAE30762.1, ABP66077.1, ADQ05832.1, AAC74398.1, ACL68803.1, ADC90669.1, SHM33122.1, ABX41399.1, ADI00307.1, AAB95491.2, AAC45510.1, AAD36910.1, AAL67138.1, AAQ20920.1, ABD80580.1, ABN51514.1, ADU20744.1, BAA25846.1, BAA28631.1, CAB16926.1, ADU22883.1, BAB71818.1, ABN54185.1, AAC45511.1, BAJ10826.1, ABX81345.1, AAG23740.1, BAC87867.1, CAC97070.1, ABX41081.1, AAM43298.1, ABD80168.1, EAA28929.1, 6HQ6, ABX81671.1, ACL75793.1, ACL76454.1, ACL76688.1, ACL77700.1, AFH48717.1, AHC20114.1, AIQ57809.1, AIQ60507.1, QCO92799.1, QCO92836.1, QCO92991.1, QCO92946.1, QCO92990.1, QCO92945.1, QCO92835.1, ACB74662.1, ABX42289.1, AAO07997.1, ABX40964.1, ABX43387.1, EEG94248.1, ACV29689.1, ZP_05748149.1, ACZ00636.1, ABG83511.1, BAH10636.1, ADL32981.1, AEN95946.1, CAC96089.1, ABY93074.1, ABY93073.1, CAZ94304.1, AAS 19693.1, ADU21379.1, ADU20661.1, CAH06518.1, VCV21228.1, AAO76140.1, VCV21229.1, AAD36300.1, WP_026485574.1, WP_026486530.1, ABK05267.1, AUO30192.1, QCO92814.1, WP_019688419.1, ACJ76363.1, BAU78234.1, CBZ24448.1, CBZ24449.1, CBZ24451.1, CBZ24452.1, CAH1385118.1, CAH1385117.1, CAH1385115.1, CAH1385116.1, ADD61463.1, BAD80751.1, ABP51432.1, AAL81659.1, AAD28735.1, ABN51595.1, AAD03471.1, AAC06896.1, AAC00218.1, AAM24997.1, AAM52219.1, BAB98701.1, BAB99480.1, AAC76453.1, AAC76442.1, BAA19592.1, AAD53957.1, AAN59210.1, AAL26558.1, AGL50099.1, BAB11741.1, CAC93400.1, AAB46846.1, ABB88567.1, CAA44069.1, AAA33211.1, AAD46887.1, AAN17338.1, AAL23578..1, AAP33020.1, AAK69600.1, AAB60395.1, CAA75517.1, AAC17451.1, BAK00834.1, AAA63271.1, AAK01137.1, AAL23577.1, AAD30476.1, AAG00588.1, ACJ76617.1, AAK15695.1, BAB92854.1, AAV87308.1, AAB68800.1, AAA41252.1, AAH70901.1, AAA41253.1, AAB68057.1, AAA33809.1, BAA00407.1, CAA52036.1, CAA59464.1, AAL23579.1, AAF82787.1, CAA84494.1 or CAA85354.1 or sequence SEQ ID NO 1; . The inventors have notably identified one or more donor phosphate sugars for each of these enzymes, as follows: the sugar-phosphate substrate is [3-glucose-1P and Pat least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: AAQ05801.1, AUG44408.1, AAV43670.1, ADH99560.1, BAC54904.1, BAD97810.1, CAA11905.1, AAO80764.1, ABX42243.1, ABX43667.1, ABX43668.1, AAE30762.1, ABP66077.1, ADQ05832.1, AAC74398.1, ACL68803.1, ADC90669.1, SHM33122.1, ABX41399.1, or ADI00307.1; the sugar-phosphate substrate is α-mannose-1P and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: ADD61463.1, CAC96089.1, ABY93074.1, ABY93073.1, CBZ24449.1, CAH1385118.1, CAH1385117.1, BAJ10826.1, CAZ94304.1, AAS19693.1, ADU21379.1, ADU20661.1, CAH06518.1, VCV21228.1, AAO76140.1, VCV21229.1, AAD36300.1, WP_026485574.1, WP_026486530.1, ABK05267.1, CBZ24448.1, CBZ24451.1, CBZ24452.1, CAH1385115.1, or CAH1385116.1, or of sequence SEQ ID NO 1, as shown in the experimental part CAC96089.1, ABY93073.1, CBZ24449.1, CAH1385118.1 or CAH1385117.1 are particularly effective for synthesizing non-reducing phosphorylated oligo- or polysaccharide polymers of mannosyl residues at [3-1,2 or [3-1,3] linkages, and are therefore particularly preferred, the sugar-phosphate substrate is α-glucose-1P and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: 2207198A, AAD40317.1, AAN24362.1, AAN58596.1, AAO21868.1, AAO33821.1, AAO84039.1, AAX33736.1, ABS59292.1, ADL69407.1, CCA61958.1, BAN03569.1, AGK37834.1, BAA14344.1, BAF62433.1, CAA30846.1, CAA80424.1, ADP98617.1, ADH62582.1, AEJ61152.1, AAC74391.2, AAB95491.2, AAC45510.1, AAD36910.1, AAL67138.1, AAQ20920.1, ABD80580.1, ABN51514.1, ADU20744.1, BAA25846.1, BAA28631.1, CAB16926.1, ADU22883.1, BAB71818.1, ABN54185.1, AAC45511.1, BAJ10826.1, ABX81345.1, CAC97070.1, ABX41081.1, AAM43298.1, ABD80168.1, EAA28929.1, ACL75793.1, ACL76454.1, ACL76688.1, ACL77700.1, AFH48717.1, AHC20114.1, AIQ57809.1, AIQ60507.1, QCO92799.1, QCO92836.1, QCO92991.1, QCO92946.1, QCO92990.1, QCO92945.1, QCO92835.1, AUO30192.1, QCO92814.1, WP_019688419.1, ACJ76363.1, BAU78234.1, 6HQ6, ABP51432.1, AAL81659.1, AAD28735.1, ABN51595.1, AAD03471.1, AAC06896.1, AAC00218.1, AAM24997.1, AAM52219.1, BAB98701.1, BAB99480.1, AAC76453.1, AAC76442.1, BAA19592.1, AAD53957.1, AAN59210.1, AAL26558.1, AGL50099.1, BAB11741.1, CAC93400.1, AAB46846.1, ABB88567.1, CAA44069.1, AAA33211.1, AAD46887.1, AAN17338.1, AAL23578..1, AAP33020.1, AAK69600.1, AAB60395.1, CAA75517.1, AAC17451.1, BAK00834.1, AAA63271.1, AAK01137.1, AAL23577.1, AAD30476.1, AAG00588.1, ACJ76617.1, AAK15695.1, BAB92854.1, AAV87308.1, AAB68800.1, AAA41252.1, AAH70901.1, AAA41253.1, AAB68057.1, AAA33809.1, BAA00407.1, CAA52036.1, CAA59464.1, AAL23579.1, AAF82787.1, CAA84494.1 or CAA85354.1; . the sugar-phosphate substrate is aN-acetyl-D-glucosamine-lP and at least a glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: AAG23740.1, BAC87867.1 or ABX81671.1, or the sugar-phosphate substrate is [3-N-acetyl-D-glucosamine-1P and Pat least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the NCBI or UNIPROT accession number AAQ05801.1; the method further comprises a step of purification of the at least one oligosaccharide phosphate and / or of the at least one polysaccharide phosphate, which is of particular interest for facilitating subsequent uses of these products of the method according to the invention; in the method of the invention at least one glycoside phosphorylase is immobilized. In the case of the use of recombinant GPs, this has the advantage, in particular, of being able to reuse them indefinitely immobilized on their support, as long as they exhibit the new activity identified here at a satisfactory level. - the preparation comprising at least one glycoside phosphorylase enzyme is a culture of a microorganism such as a bacterium or a yeast or a fungus expressing the at least one glycoside phosphorylase enzyme. The direct use of the culture of the microorganism expressing the recombinant GP can advantageously make it possible to dispense with the steps of purification of the recombinant GP. - said reaction is carried out in solution, with a purified recombinant enzyme preparation in solution. The use of a purified preparation of recombinant GP can make it possible to effectively control the parameters of the new reaction and the activity of the GP; or - maintaining excess concentrations of at least one sugar-phosphate substrate of GP. This makes it possible in particular to shift the equilibrium of the reaction towards the production of oligo- and / or polysaccharide phosphates and thus to increase the yield and / or production of these. Brief Descriptions of the Figures

[0016] [Fig. 1] is a diagram representing the method according to the invention.

[0017] [Fig.2] represents the reaction catalyzed by glycoside phosphorylase in the process of the invention. The 2 sugar-phosphates are condensed to produce a phosphate oligosaccharide and an inorganic phosphate, according to direction 1 of the sugar-phosphate synthesis reaction.

[0018] [Fig.3] is the result of the analysis by anion exchange chromatography with pulsed amperometric detection (in English terminology High Performance Anion Exchange Chromatography with Pulsed Amperometric detection or "HPAEC-PAD") of the products obtained at the end of the process of the invention using the phosphate sugar synthesis reaction catalyzed by, in A: the GP [3-1,2 mannoside phosphorylases Teth514_1788 (GENBANK: ABY93073.1), LinO857 (GENBANK: CAC96089.1), Uhgb_MS (GENBANK: CAH1385118.1), MTP4 (GENBANK: CBZ24449.1), and in B: the GP [3-1,3 mannoside phosphorylase U7 (GENBANK: CAH1385117.1). For all these GP, the donor substrate is mannose-1-phosphate (Man-IP).

[0019] [Fig.4] includes a chromatogram obtained by exchange chromatography of anions coupled with dual detection by pulsed amperometry and mass spectrometry (HPAEC-PAD-MS). The products obtained at the end of the process of the invention using the phosphate sugar synthesis reaction catalyzed by GP MTP4 are separated and detected in the form of peaks with different retention times. The signals corresponding to the molecular masses of the phosphate oligosaccharides are extracted from the overall mass spectrometry (MS) signal and compared to the amperometric (PAD) signal. The MS detection range used makes it possible to highlight phosphate oligosaccharides consisting of 2 to 6 mannosyl units (DP2 to DP6). Note that, for this enzyme, the PAD signal makes it possible to detect non-reducing oligosaccharides whose size is estimated to extend up to 13 mannosyl units (DP13).

[0020] [Fig.5] includes a chromatogram obtained by HPAEC-PAD-MS. The The main products obtained from the process of the invention using the phosphate sugar synthesis reaction catalyzed by GP Tethl788 are made up of phosphate oligosaccharides made up of 2 to 6 mannosyl units (DP2 to DP6). Note that, for this enzyme, the PAD signal makes it possible to detect non-reducing oligosaccharides whose size is estimated to extend beyond 14 mannosyl units (>DP14).

[0021] [Fig.6] includes a chromatogram obtained by HPAEC-PAD-MS. The main products obtained at the end of the process of the invention using the phosphate sugar synthesis reaction catalyzed by GP Lin0857 are made up of phosphate oligosaccharides made up of 2 and 3 mannosyl units (DP2 and DP3).

[0022] [Fig.7] includes a chromatogram obtained by HPAEC-PAD-MS. The The main products obtained at the end of the process of the invention using the phosphate sugar synthesis reaction catalyzed by GP Uhgb_MS are made up of phosphate oligosaccharides made up of 2 and 3 mannosyl units (DP2 and DP3).

[0023] [Fig.8] includes a chromatogram obtained by HPAEC-PAD-MS. The main products obtained at the end of the process of the invention using the phosphate sugar synthesis reaction catalyzed by GP U7 are made up of phosphate oligosaccharides made up of 2 and 3 mannosyl units (DP2 and DP3).

[0024] [Fig.9] is the result of HPAEC-PAD analysis of the purification of phosphate oligosaccharides obtained by the process of the invention using the phosphate sugar synthesis reaction catalyzed by GP MTP4. Before purification, the phosphate oligosaccharides are mixed with normal oligosaccharides and mannose. After purification, only the phosphate oligosaccharides are detected. Description of the embodiments

[0025] The invention is described below in more detail and specified for certain embodiments. Definitions

[0026] For the purposes of the invention, an "enzymatic process for in vitro synthesis" of an oligosaccharide or polysaccharide phosphate means a process carried out using purified or partially purified enzyme preparations (such as culture supernatants and / or cell lysates) or prokaryotic or eukaryotic organisms modified and cultured so as to allow the implementation of said enzymatic process for in vitro synthesis of non-reducing oligo- and / or polysaccharide phosphates via adequate expression of these enzymes. Thus, in a particular embodiment, this process can be carried out entirely within a recombinant microorganism, i.e. genetically modified so as to carry out the process under adequate culture conditions. In another particular embodiment, a process according to the invention can be carried out in part, i.e. at least enzymatic reaction, within a living organism microorganism.In another particular embodiment, the method is carried out exclusively using purified or partially purified recombinant enzymes.

[0027] A glycoside phosphorylase (GP) is defined as an enzyme that catalyzes i) the degradation of glycosides using inorganic phosphate to break the osidic bonds (phosphorolysis) and / or ii) their synthesis using phosphate sugars as glycosylate donors (in English terminology, reverse phosphorolysis) (Lia et al, 2022). The two opposing reactions of phosphorolysis and reverse phosphorolysis therefore result in an equilibrium that depends on the reaction conditions. GPs share structural and mechanical characteristics with glycoside hydrolases and glycosyltransferases. Thus, an enzyme categorized, for example in the CAZy database, as a GH or a GT may very well have activities relevant to a GP.More specifically, enzymes with GP activities may exhibit sequence similarities (in English terminology "sequence similarity networks") that allow the identification of new GPs (as, for example, according to the methodology described by Lib et al (2022)). Thus, once an enzyme is identified as a GP, either by its enzymatic activities (Lia . et al, 2022) or by sequence analogies, it can easily be tested by those skilled in the art who can verify whether it exhibits a sugar phosphate synthesis activity as discovered by the inventors.

[0028] Table 1 lists examples of glycoside phosphorylases, the Genbank number refers to the peptide sequence of the protein with a substrate sugar phosphate usable in the reaction of synthesis of sugar phosphate(s). [Tables 1] Famille CAZy N° NCBI ou UNIPROT ou numéro de séquence Nom de la protéine Sucre-phosp hâte substrat GH 130 CAC96089.1 [3-(l,2)-oligomannan phosphorylase (Lin0857) a-mannose-1 P GH 130 ABY93074.1 [3-(l,2)-oligomannan phosphorylase (Teth514_1789) a-mannose-1 P GH 130 ABY93073.1 [3-(l,2)-oligomannan phosphorylase (Teth514_1788) a-mannose-1 P GT108 CBZ24449.1 [3-(l,2)-oligomannan phosphorylase / mannosyltransferase MTP4 (LMXM_10_1260) a-mannose-1 P GH 130 CAH1385118.1 [3-1,2-oligomannan phosphorylase (Uhgb_mS) a-mannose-1 P GH 130 CAH1385117.1 U7 :[3-l,3-oligomannan phosphorylase (U7; 340101.Vvad_PD3074) a-mannose-1 P GH3 AAQ05801.1 bifunctional N-acetyl-[3- glucosa-minidase / [3-glucosidase (Nag3) [3-N-acetyl-D-glucosamine-1P GH3 AUG44408.1 [3-glucoside phosphorylase (BglP) [3-glucose-lP GH13_18 2207198A sucrose phosphorylase (SPase) a-glucose-lP GH13_18 AAD40317.1 sucrose phosphorylase a-glucose-lP GH13_18 AAN24362.1 sucrose phosphorylase (Spl;BL0536) a-glucose-lP GH13_18 AAN58596.1 sucrose phosphorylase (GtfA;SMU.881) a-glucose-lP GH13_18 AAO21868.1 sucrose phosphorylase (LaSP;GtfA2;LBA1437) a-glucose-lP GH13_18 AAO33821.1 sucrose phosphorylase (SucP;SP;BaSP) a-glucose-lP GH13_18 AAO84039.1 sucrose phosphorylase (SplP) a-glucose-lP GH13_18 AAX33736.1 sucrose phosphorylase (LmSPl) a-glucose-lP . GH13_18 ABS59292.1 sucrose phosphorylase (742sp) a-glucose-lP GH13_18 ADL69407.1 6F-P-sucrose phosphorylase (SPP;TtSPP;Tthe_1921) a-glucose-lP GH13_18 CCA61958.1 6F-P-sucrose phosphorylase (sucP) a-glucose-lP GH13_18 BAN03569.1 sucrose 6(F)-phosphate phosphorylase (YM304_32550) a-glucose-lP GH13_18 AGK37834.1 sucrose phosphorylase (ScrP) a-glucose-lP GH13_18 BAA14344.1 sucrose phosphorylase (LmSPase) a-glucose-lP GH13_18 BAF62433.1 sucrose phosphorylase(Spl) a-glucose-lP GH13_18 CAA30846.1 sucrose phosphorylase (GftA;SmSP) a-glucose-lP GH13_18 ADP98617.1 glucosylglycerol phosphorylase (HP15_2853) a-glucose-lP GH13_18 ADH62582.1 glucosylglycerate phosphorylase (Mesil_0665) a-glucose-lP GH13_18 AEJ61152.1 glucosylglycerate phosphorylase (Spith_0877) a-glucose-lP GH13_18 AAC74391.2 glucosylglycerate phosphorylase (YcjM) a-glucose-lP GH65 AAV43670.1 maltose phosphorylase (MalP;LBA1870) [3-glucose-lP GH65 ADH99560.1 maltose phosphorylase (Bsel_2056) [3-glucose-lP GH65 BAC54904.1 maltose phosphorylase(MPase) [3-glucose-lP GH65 BAD97810.1 maltose phosphorylase(MapA) [3-glucose-lP GH65 CAA11905.1 maltose phosphorylase [3-glucose-lP GH65 AAO80764.1 maltose phosphorylase (MalP;EF0957) [3-glucose-lP GH65 ABX42243.1 nigerose phosphorylase (Cphy_1874) [3-glucose-lP GH65 ABX43667.1 nigerose phosphorylase (Cphy_3313) [3-glucose-lP GH65 ABX43668.1 nigerose phosphorylase (Cphy_3314) [3-glucose-lP GH65 AAE30762.1 kojibiose phosphorylase (KojP;KPase) [3-glucose-lP GH65 ABP66077.1 kojibiose phosphorylase [3-glucose-lP . (CsKP;Csac_0439) GH65 ADQ05832.1 1,3-a-oligoglucan phosphorylase (ChGP) [3-glucose-lP GH65 AAC74398.1 kojibiose phosphorylase (YcjT) [3-glucose-lP GH65 ACL68803.1 kojibiose phosphorylase (HoGP) [3-glucose-lP GH65 ADC90669.1 Trisaccharide a-glucan phosphorylase (MiGP) [3-glucose-lP GH65 SHM33122.1 nigerose phosphorylase (AmNP) [3-glucose-lP GH65 ABX41399.1 3-O-a-glucopyranosyl-L- rhamnose phosphorylase (Cphy_1019) [3-glucose-lP GH65 ADI00307.1 1,2-alpha-glucosylglycerol phosphorylase (Bsel_2816) [3-glucose-lP GH94 AAB95491.2 cellobiose phosphorylase(CbpA) a-glucose-lP GH94 AAC45510.1 cellobiose phosphorylase(CepA) a-glucose-lP GH94 AAD36910.1 cellobiose phosphorylase (CepA;TM1848) a-glucose-lP GH94 AAL67138.1 cellobiose phosphorylase(Cbp) a-glucose-lP GH94 ABD80580.1 cellobiose phosphorylase 94A (Cbp;SdCBP;Sde_1318;Cep94A) a-glucose-lP GH94 ABN51514.1 cellobiose phosphorylase (Cbp;CtCBP;Cthe_0275) a-glucose-lP GH94 ADU20744.1 cellobiose phosphorylase (CBP;RaCBP;Rumal_0187) a-glucose-lP GH94 BAA25846.1 cellobiose-phosphorylase (Cbp) a-glucose-lP GH94 BAA28631.1 cellobiose phosphorylase (Cbp;CgCBP) a-glucose-lP GH94 CAB 16926.1 cellobiose phosphorylase (CepA) a-glucose-lP GH94 ADU22883.1 cellodextrin phosphorylase (CDP;RaCDP;Rumal_2403) a-glucose-lP GH94 BAB71818.1 cellobiose phosphorylase (Cdp-ym4) a-glucose-lP GH94 ABN54185.1 cellodextrin-phosphorylase (Cdp;CtCDP;Cthe_2989) a-glucose-lP GH94 AAC45511.1 cellodextrin phosphorylase a-glucose-lP . (CepB;CsCdP) GH94 BAJ 10826.1 laminaribiose phosphorylase (LbpA) a-glucose-lP GH94 ABX81345.1 laminaribiose phosphorylase (ACL_0729;ACL0729) a-glucose-lP GH94 AAG23740.1 diacetylchitobiose phosphorylase (ChbP) a-N-acetyl-D -glucosamine-1P GH94 BAC87867.1 chitobiose phosphorylase (ChbP;VpChBP) a-N-acetyl-D -glucosamine-1P GH94 CAC97070.1 [3-(l,2)-oligoglucan phosphorylase (LiSOGP;Linl839) a-glucose-lP GH94 ABX41081.1 [3-(l,2)-oligoglucan phosphorylase (LpSOGP;Cphy_0694) a-glucose-lP GH94 AAM43298.1 cellobionic acid phosphorylase (CelAP;NdvB;XCC4077) a-glucose-lP GH94 ABD80168.1 cellobionic acid phosphorylase 94B (Cep94B ;CB AP;SdCB AP) a-glucose-lP GH94 EAA28929.1 cellobionic acid phosphorylase (CelAP;NdvB;NCU09425) a-glucose-lP GH94 ABX81671.1 acholetin ([3-1,3-N-acetylglucosaminide) phosphorylase (AchP) a-N-acetyl-D -glucosamine-1P GH94 ACL75793.1 cellodextrin phosphorylase (CdpA) a-glucose-lP GH94 ACL76688.1 cellodextrin phosphorylase (CdpB) a-glucose-lP GH94 ACL77700.1 cellodextrin phosphorylase (CdpC) a-glucose-lP GH94 AFH48717.1 4-O-[3-D-glucosyl-N-acetyl-D-glucosa mine phosphorylase (laGP) a-glucose-lP GH94 AHC20114.1 solabiose phosphorylase (GGalP) a-glucose-lP GH94 AIQ57809.1 3-O-[3-D-glucopyranosyl [3-D-glucuronide phosphorylase (PBOR_13355) a-glucose-lP GH94 AIQ60507.1 solabiose phosphorylase (PBOR_28850) a-glucose-lP . GH94 QCO92799.1 cellobiose phosphorylase (GH94_E) a-glucose-lP GH94 QCO92836.1 cellobiose phosphorylase (GH94_G) a-glucose-lP GH94 QCO92991.1 cellodextrin phosphorylase (GH94_A) a-glucose-lP GH94 QCO92946.1 cellodextrin phosphorylase (GH94_D) a-glucose-lP GH94 QCO92990.1 laminaribiose phosphorylase (GH94_B) a-glucose-lP GH94 QCO92945.1 laminaribiose phosphorylase (GH94_C) a-glucose-lP GH94 QCO92835.1 cellodextrin phosphorylase (GH94_F) a-glucose-lP GH112 ACB74662.1 D-galactosyl-[3-( 1,4)-L-rhamnose phosphorylase (GalRhaP) a-galactose-1 P GH112 ABX42289.1 D-galactosyl-( 1,4)-L-rhamnose phosphorylase (Cphy_1920) a-galactose-1 P GH112 AAO07997.1 [3-(l,3)-galactosyl-N- acetyl-hexosamine phosphorylase (GalGlyNAcP) a-galactose-1 P GH112 ABX40964.1 [3-(l,3)-galactosyl-N- acetyl-hexosamine phosphorylase (Cphy_0577) a-galactose-1 P GH112 ABX43387.1 [3-(l,3)-galactosyl-N- acetyl-hexosamine phosphorylase (Cphy_3030) a-galactose-1 P GH112 EEG94248.1 [3-1,3-galactosyl-N-acetylhexosamine phosphorylase (RiGLnbpll2) a-galactose-1 P GH112 ACV29689.1 [3-(l,3)-galactosyl-N- acetyl-hexosamine phosphorylase / galacto-N-biose / lacto-N-biose I phosphorylase (GLNBP;Apre_1669) a-galactose-1 P GH112 ZP_05748149.1 [3-(l,3)-galactosyl-N- acetyl-hexosamine phosphorylase / galacto-N-biose phosphorylase (GNBP;HMPREF0357_1319) a-galactose-1 P . GH112 ACZ00636.1 [3-(l,3)-galactosyl-N- acetyl-hexosamine phosphorylase / galacto-N-biose phosphorylase (GNBP;Smon_0146) a-galactose-1 P GH112 ABG83511.1 galacto-N-biose phosphorylase (CPF_0553) a-galactose-1 P GH112 BAH10636.1 [3-(l,3)-galactosyl-N- acetyl-hexosamine phosphorylase (GnpA) a-galactose-1 P GH112 ADL32981.1 [3-galactoside phosphorylase (bpr_I0232) a-galactose-1 P GH112 AEN95946.1 [3-1,3-galactosyl-N-acetylhexosamine phosphorylase (RhGLnbpll2) a-galactose-1 P GH112 BAD80751.1 galacto-N-biose / lacto-N-biose phosphorylase (LnpAl;LnbP;GLNBP;BLLJ_1623) a-galactose-1 P GH 130 CAZ94304.1 [3-(l,3)-mannooligosaccharide phosphorylase (zobellia_231) a-mannose-1 P GH 130 AAS 19693.1 [3-(l,4)-mannosylglucose phosphorylase (Unkl) a-mannose-1 P GH 130 ADU21379.1 [3-(l,4)-mannosylglucose phosphorylase (RaMPl ;RaMGP;Rumal_0852) a-mannose-1 P GH 130 ADU21379.1 [3-(l,4)-mannosylglucose phosphorylase (RaMPl ;RaMGP;Rumal_0852) a-mannose-1 P GH 130 ADU20661.1 [3-(l,4)-mannooligosaccharide phosphorylase (RaMP2;Rumal_0099) a-mannose-1 P GH 130 CAH06518.1 [3-(l,4)-mannosylglucose phosphorylase (MGP;BF0772) a-mannose-1 P GH 130 VCV21228.1 [3-1,4-mannosylglucose phosphorylase (RiGH130_l; ROSINTL182_05475) a-mannose-1 P GH 130 AAO76140.1 [3-(l,4)-mannosyl-N-acetyl-glucosami ne phosphorylase (BT1033) a-mannose-1 P GH 130 VCV21229.1 [3-1,4-mannooligosaccharide phos- a-mannose-1 . phorylase (Ril30_2; ROSINTL182_05474) P GH 130 AAD36300.1 P-(l,4)-mannooligosaccharide phos-phory lase(TM 1225) a-mannose-1 P GH 130 WP_026485574.1 CalpoDRAFT_0075 a-mannose-1 P GH 130 WP_026486530.1 CalpoDRAFT_1209 a-mannose-1 P GH 130 ABK05267.1 [3-mannoside phosphorylase (Arth_3892) a-mannose-1 P GH149 6HQ6 ou 6HQ8 [3-(l,3)-glucan phosphorylase (Pro_7066) a-glucose-lP GH149 AUO30192.1 [3-(l,3)-glucan phosphorylase(EgPl) a-glucose-lP GH149 QCO92814.1 [3-1,3-oligoglucan phosphorylase (GH149_H) a-glucose-lP GH161 WP_019688419.1 [3-(l,3)-glucan phosphorylase (PapP) a-glucose-lP GH161 ACJ76363.1 [3-(l,3)-glucan phosphorylase (TaCDP) a-glucose-lP GH161 BAU78234.1 [3-(l,3)-glucan phosphorylase (OdBGP) a-glucose-lP GT108 CBZ24448.1 [3-(l,2)-oligomannan phosphorylase / mannosyltransferase MTP3 (LMXM_10_1250) a-mannose-1 P GT108 CBZ24451.1 [3-(l,2)-oligomannan phosphorylase / mannosyltransferase MTP6 (LMXM_10_1280) a-mannose-1 P GT108 CBZ24452.1 [3-(l,2)-oligomannan phosphorylase / mannosyltransferase MTP7 (LMXM_10_1290) a-mannose-1 P GH 130 SEQ ID NO 1 G1-8A : [3-(l,3)-mannose phosphorylase (Gl-8A_GL0038729 locus= scaffoldl7912_l:1303:2508) a-mannose-1 P GH 130 CAH1385115.1 [3-1,4-mannosylglucose phosphorylase (Ul; MH0431_GL0150624) a-mannose-1 P . GH 130 CAH1385116.1 P-l,4-mannooligosaccharide phos-phorylase (U3; MH0373_GL0093988) a-mannose-1 P GH 130 ADD61463.1 (3-(1,4)-mannopyranosyl-[N- glycan] phosphorylase / (3-(1,4)- manno-pyrano s y 1-chitobio se pho sphory lase (Uhgb_MP) a-mannose-1 P GT35 ABP51432.1 glucan / maltodextrin phosphorylase (GlgP;PyglgP;Pars_1881) a-glucose-lP GT35 AAL81659.1 a-glucan / maltodextrin phosphorylase (PF1535) a-glucose-lP GT35 AAD28735.1 maltodextrin phosphorylase (MalP) a-glucose-lP GT35 ABN51595.1 a-glucan phosphorylase (a-GP;Cthe_0357) a-glucose-lP GT35 AAD03471.1 glycogen phosphorylase(GlgP) a-glucose-lP GT35 AAC06896.1 glycogen phosphorylase (GlgP;Aq_717) a-glucose-lP GT35 AAC00218.1 glycogen phosphorylase (GlgP;BSU30940) a-glucose-lP GT35 AAM24997.1 a-glucan phosphorylase (GlgP;TTE1805) a-glucose-lP GT35 AAM52219.1 glycogen phosphorylase a-glucose-lP GT35 BAB98701.1 maltodextrin / gly cogen pho sphorylase (MalP;GlgPl;cgl479) a-glucose-lP GT35 BAB99480.1 glycogen phosphorylase (GlgP;GlgP2;cg2289) a-glucose-lP GT35 AAC76453.1 glycogen phosphorylase (GlgP;b3428) a-glucose-lP GT35 AAC76442.1 maltodextrin phosphorylase (MalP;b3417) a-glucose-lP GT35 BAA19592.1 glycogen phosphorylase (GlgP) a-glucose-lP GT35 AAN59210.1 maltodextrin phosphorylase (GlgP;SMU.1564) a-glucose-lP . GT35 AAL26558.1 glycogen phosphorylase (Glg) a-glucose-lP GT35 AGL50099.1 a-glucan phosphorylase (AgpA;Tmari_l 175) a-glucose-lP GT35 BAB11741.1 a-glucan phosphorylase (GlgP) a-glucose-lP GT35 CAC93400.1 glycogen phosphorylase (glgP,YPO3938) a-glucose-lP GT35 AAB46846.1 glycogen phosphorylase (my opho sphory lase) a-glucose-lP GT35 ABB88567.1 plastidial starch phosphorylase (PhoB;Sta4) a-glucose-lP GT35 CAA44069.1 glycogen phosphorylase 1 (GlpV;GPl) a-glucose-lP GT35 AAA33211.1 glycogen phosphorylase 2 (GlpD;GP2) a-glucose-lP GT35 AAD46887.1 glycogen phosphorylase (GlyP;GLYP;CG7254;Dmel_CG7254 ) a-glucose-lP GT35 AAN17338.1 glycogen phosphorylase-2 a-glucose-lP GT35 AAB60395.1 glycogen phosphorylase (brain) (bGP) a-glucose-lP GT35 CAA75517.1 glycogen phosphorylase a-glucose-lP GT35 AAC17451.1 glycogen phosphorylase (PigM) a-glucose-lP GT35 BAK00834.1 plastidial a-(l,4)-glucan phosphorylase (Phol;HvPhol) a-glucose-lP GT35 AAA63271.1 a-glucan phosphorylase L a-glucose-lP GT35 AAK01137.1 starch phosphorylase a-glucose-lP GT35 AAD30476.1 glycogen phosphorylase (PygM) a-glucose-lP GT35 ACJ76617.1 glycogen phosphorylase (PygM) a-glucose-lP GT35 AAK15695.1 a-(l,4)-glucan phosphorylase L a-glucose-lP GT35 BAB92854.1 a-(l,4)-glucan phosphorylase (0s01g0851700) a-glucose-lP GT35 AAV873O8.1 glycogen phosphorylase (PYGB) a-glucose-lP GT35 AAB688OO.1 glycogen phosphorylase a-glucose-lP . GT35 AAA41252.1 glycogen phosphorylase a-glucose-lP GT35 AAB68057.1 glycogen phosphorylase (Gphl;YPR160w) a-glucose-lP GT35 AAA33809.1 a-glucan phosphorylase H a-glucose-lP GT35 BAA00407.1 a-glucan phosphorylase L1 a-glucose-lP GT35 CAA52036.1 a-glucan phosphorylase L2 a-glucose-lP GT35 CAA59464.1 a-glucan phosphorylase a-glucose-lP GT35 CAA84494.1 a-glucan phosphorylase (Pho2;VfPho2) a-glucose-lP The GP named G1-8A: [3-(l,3)-mannose phosphorylase (Gl-8A_GL0038729 locus= scaffoldl7912_l:1303:2508) in Table 1 has the protein sequence (SEQ ID NO 1): MHKPIIRYSPEPVLEPKEGCIWADTMVLNPAIVKDPYSDTIHMLFRATGPW-PAKNLKGKSDPYPIFLGYAKSDDRGETWQPDFGRPALAPALEYEMDKMYIRDI YDREVVNYANGCVEDPRIFEVEGELYLTTACRMFPPGPYWEGDKRRDNIPDW AMEPDNPFGTTASRNDTTTVLFKLDLDKLKSREYERAFGYVCNLTDGNVTAN RDVFLFPRKMKIDGKMRYVMLHRPENPDKFEAGKGIYKPAIFLAAAEKPEDFC TGKCMHKLLAKGIFDWEEERIGASFPPVEIESGKWLISYHGKQMPDYGYTQSF MIVEEQEDDFPLIIHRCPERLMYARQKWELPDKFACPCIFTSGGIVMDEELVMS YGAADQKVGLARANLEEIVDYVSSFDENGNEIELE.

[0029] A GP can accept 1, 2 or even more different sugar-phosphates as possible substrates. In a particular embodiment, this or these are selected from: α-galactose-lP, α-glucose-lP, α-mannose-lP, αN-acetyl-D-glucosamine-lP, [3-glucose-IP, [3-N-acetyl-D-glucosamine-IP.

[0030] “Polynucleotide(s)”, “oligonucleotide(s)”, “nucleic acid(s)”,"nucleotide(s)", "polynucleic acid(s)" or any equivalent used refers to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers to the primary structure of the molecule. It also includes modified forms, for example by methylation and / or capping, and unmodified forms of the polynucleotide. The term also includes polymers of nucleotides or nucleic acids comprising non-naturally occurring or synthetic nucleotides as well as nucleotide analogues. Nucleic acid sequences and vectors, for example expression vectors, may be introduced into a cell, for example by transfection, transformation or transduction.The nucleotide sequence encoding a GP as listed in Table 1 above may be optimized depending on the host in which the recombinant GP is produced and, for example, depending on its genetic code and . of the codon usage specific to that host. This nucleotide sequence can either be integrated into an expression vector or integrated into the genome of the host organism. Expression of the recombinant GP can be either inducible or constitutive. Preferably, expression of the recombinant GP is inducible.

[0031] “Expression vector” means any expression vector known to the person of the art to be suitable for the expression of genes encoding proteins in microorganisms and more particularly suitable for the overproduction and possibly the purification of recombinant GPs such as those listed in Table I. Many recombinant protein expression systems are known and commercially available. The person skilled in the art will know, depending on the recombinant GP produced and the host microorganism, how to choose the appropriate vector, culture conditions and purification system. The vector may be a plasmid, a yeast artificial chromosome (YAC) or a bacterial artificial chromosome (BAC). It may be, for example, any vector selected from the vectors listed in the online catalogs or described at the following sites:

[0032] - https: / / france.promega.com / products / vectors / protein-expression-vectors,

[0033] - https : / / www. thermofisher.com / search / browse / category / fr / fr / 8580 l / Bacterial%20Expre s sion%20V ectors,

[0034] - https: / / www.embl.de / pepcore / pepcore_services / strains_vectors / vectors / bacterial_expre ssion_vectors / popup_bacterial_expression_vectors / index.html,

[0035] - http: / / babel.ucmp.umu.se / cpep / web_content / Pages / CPEP_09_vectors.html, ou

[0036] - https: / / www.embl.de / pepcore / pepcore_services / strains_vectors / vectors / gateway_vecto rs / index.htm.

[0037] It may be, for example, the expression vector described in document WO 83 / 004261. It may be, for example, the plasmid pWKS130 described by Wang and Kushner (1991); the plasmid marketed by the company Thermofisher under the commercial reference pBAD-HisA, the plasmid marketed by the company Thermofisher under the commercial reference pTRC. It may be, for example, a plasmid chosen from the group comprising pBAD-HisA, pTRC, pMX, pUC19, pHP45-CmR, pcDNA3.1(+), pcDNA3.3-TOPO, pcDNA3.4-TOPO, pFastBacl, pET100 / D-TOPO, pET151 / D-TOPO, pRSET A, pYes2.1V5-His TOPO, pDONR221, pGEX-3X; pBluescript, pET, pGEX, pLys, pRARE, pDEST, pETG, preferably plasmid pBAD-HisA

[0038] As mentioned, the vector may be any suitable yeast artificial chromosome (YAC) known to those skilled in the art and / or commercially available. It may be, for example, a yeast artificial chromosome selected from the group comprising pYAC-RC and pYAC3. The vector may also be any bacterial artificial chromosome (BAC) suitable for expression in bacteria, known to those skilled in the art and / or commercially available. It may be, for example, a bacterial artificial chromosome selected from the group comprising pUvBBAC, pCCIBAC and pBAC 108L.

[0039] The promoter may be any promoter known to the person skilled in the art suitable for enabling expression of recombinant proteins in a microorganism such as a bacterium, a yeast or a fungus, or even a cell of a higher eukaryote, preferably in a bacterium. It may be, for example, a constitutive promoter or an inducible promoter. It may be, for example, a constitutive promoter chosen from the group comprising the CMV, EF1A, or SV40 promoter. This may be, for example, a constitutive promoter mentioned on the internet page http: / / parts.igem.org / Promoters / Catalog / Constitutive, any promoter of the E. coli K-12 strain known to the person skilled in the art, for example a promoter chosen from the 3908 promoters of the E. coli K-12 strain listed on the biocyc.org website: https: / / biocyc.org / group?id=:ALL-PROMOTERS&orgid=ECOLI. It may be, for example, an inducible promoter, for example pBAD or pLac.This may be, for example, an inducible promoter described and / or mentioned on the page: http: / / parts.igem.org / Promoters / Catalog. The person skilled in the art will be able to choose the promoter according to the nucleic sequence to be expressed and / or the host cell, more generally according to the system and the expression conditions chosen.

[0040] The production of recombinant proteins is today a common technology in the field of biotechnologies which is part of the routine operations for the person skilled in the art, who knows how to determine the production conditions (such as the production organism (such as a bacterium, a yeast, a fungus, or even a higher eukaryote), optimization of the genetic sequence, culture conditions, purification conditions, the choice and location of a possible Tag) depending on the enzyme concerned. A summary of the different possible strategies is reported by Tripathi and Shrivastava (2019). Typically, as illustrated by the experimental examples, the GP expression host is E. coli BL21 and the expression vector is a plasmid of the pBAD or pET type, inducible respectively to arabinose and lactose (and its derivatives such as 1TPTG or allolactose).The purification tag is most often a His tag that can be positioned at the C-ter and / or N-ter of the recombinant protein, which can be introduced directly into the synthetic gene sequence or initially present in the expression vector. Purification of recombinant enzymes is typically carried out by affinity chromatography on cobalt resin (for example: TALON® Metal Affinity Resin, Takara).

[0041] A “polysaccharide” or an “oligosaccharide” means a linear or branched polymer of monosaccharide units linked together by α- or [3-osidic bonds. By convention, in the art, an oligosaccharide comprises a number of glycosylated units of between 2 and 10 and a polysaccharide means a polymer of glycosylated units comprising more than 10 units. More particularly, in connection with the new activity identified by the inventors, a phosphate polysaccharide or a phosphate oligosaccharide means a polymer of glycosylated units linked together by one or more α- or [3-osidic bonds and the reducing end of which is linked to an inorganic phosphate by an α- or [3- bond. Thus, such a phosphate polysaccharide or phosphate oligosaccharide does not comprise a reducing end.In other words, the phosphate polysaccharide or phosphate oligosaccharide produced from the process according to the invention does not comprise a free hemiacetalic function, the hydroxyl of the latter being involved in a bond with a phosphate group.

[0042] A "1-1 sugar bond" is a bond involving the hemiaketal hydroxyl residues of two sugars. Obviously, a GP catalyzing the formation of 1-1 sugar bonds will not be able to produce, during the sugar phosphate synthesis reaction identified by the inventors, a phosphate polysaccharide from a sugar-phosphate preparation since the reducing ends of both sugars are engaged in the sugar bond. Such activity can easily be identified using the methods described, for example, in the experimental part, or from the data recorded in the CAZy database (<CAZY.org> ).

[0043] As mentioned, the invention lies in the characterization of an activity hitherto unknown for GPs, which is the condensation of sugars-phosphate, to synthesize a non-reducing phosphate oligosaccharide or phosphate polysaccharide, as illustrated more particularly in [Fig.2].

[0044] An object of the invention therefore lies in an enzymatic process for the in vitro synthesis of an oligosaccharide phosphate or polysaccharide phosphate 1 comprising the steps of: • Provision of a preparation comprising at least one GP enzyme (10a) not catalyzing the formation of 1-1 osidic bond:

[0045] Said preparation may comprise or consist of a protein preparation enriched in glycoside phosphorylase. More particularly, said preparation comprising at least one GP is derived from a step of purification of said GP from a culture lysate or a culture supernatant of a microorganism expressing said GP. Thus in one embodiment, said preparation comprising at least one glycoside phosphorylase enzyme is a preparation in which the at least one GP represents at least 80%, at least 85%, preferably at least 90% of the protein fraction of said preparation. This can easily be determined by measuring a specific GP activity in the preparation.

[0046] Alternatively, said preparation may consist of a culture of a microorganism expressing said GP. The gene coding for the GP may be inserted into the genome of the host organism to obtain its overexpression, in the chromosome of the microorganism or in an expression vector as described above. The microorganism may be a bacterium, a yeast or a fungus, according to preferences determined by the person skilled in the art. However, in this embodiment, bacteria are particularly preferred.

[0047] Also, said preparation may consist of an unpurified culture supernatant. Also, said preparation may consist of an unpurified cell lysate.

[0048] In another embodiment, said preparation may comprise the enzyme immobilized on a support. This has the advantage of possible reuse of the enzyme, once the reaction is complete by recovering the insolubilized enzyme, for example, by filtration, sedimentation (passive, or for example, by application of a magnetic field magnetization in the case of the use of a magnetic support for the enzyme), or centrifugation of the reaction mix, or even continuous use. To implement this strategy, one can for example refer to the review by Sheldon et al (2021) or to the manual by Marian Lujan Ferreira “Biocatalyst immobilization: foundations and applications” (Elsevier, 2022) which lists the different immobilization strategies for enzymatic processes. One can also refer to Zhong et al (2020) which is specifically interested in the immobilization of GP.

[0049] Also, said preparation can comprise several different GPs, i.e. 2, 3 GPs or more. This can make it possible to obtain sugar phosphates linking different glycosylated units, or to obtain mixtures of sugar phosphates at the end of the implementation of the process.

[0050] Thus, in a particular embodiment, the preparation of at least one GP glycoside enzyme comprises at least two GPs chosen from the enzymes referenced under the NCBI or UNIPROT accession number AAQ05801.1, AUG44408.1, 2207198A, AAD40317.1, AAN24362.1, AAN58596.1, AAO21868.1, AAO33821.1, AAO84039.1, AAX33736.1, ABS59292.1, ADL69407.1, CCA61958.1, BAN03569.1, AGK37834.1, BAA14344.1, BAF62433.1, CAA30846.1, CAA80424.1, ADP98617.1, ADH62582.1, AEJ61152.1, AAC74391.2, AAV43670.1, ADH99560.1, BAC54904.1, BAD97810.1, CAA11905.1, AAO80764.1, ABX42243.1, ABX43667.1, ABX43668.1, AAE30762.1, ABP66077.1, ADQ05832.1, AAC74398.1, ACL68803.1, ADC90669.1, SHM33122.1, ABX41399.1, ADI00307.1, AAB95491.2, AAC45510.1, AAD36910.1, AAL67138.1, AAQ20920.1, ABD80580.1, ABN51514.1, ADU20744.1, BAA25846.1, BAA28631.1, CAB16926.1, ADU22883.1, BAB71818.1, ABN54185.1, AAC45511.1, BAJ10826.1, ABX81345.1, AAG23740.1, BAC87867.1, CAC97070.1, ABX41081.1, AAM43298.1, ABD80168.1, EAA28929.1, ABX81671.1, ACL75793.1, ACL76454.1, ACL76688.1, ACL77700.1, AFH48717.1, AHC20114.1, AIQ57809.1, AIQ60507.1, QCO92799.1, QCO92836.1, QCO92991.1, QCO92946.1, QCO92990.1, QCO92945.1, QCO92835.1, ACB74662.1, ABX42289.1, AAO07997.1, ABX40964.1, ABX43387.1, EEG94248.1, ACV29689.1, ZP_05748149.1, ACZ00636.1, ABG83511.1, BAH10636.1, ADL32981.1, AEN95946.1, CAC96089.1, ABY93074.1, ABY93073.1, CAZ94304.1, AAS 19693.1, ADU21379.1, ADU20661.1, CAH06518.1, VCV21228.1, AAO76140.1, VCV21229.1, AAD36300.1, WP_026485574.1, WP_026486530.1, ABK05267.1, AUO30192.1, QCO92814.1, WP_019688419.1, ACJ76363.1, BAU78234.1, CBZ24448.1, CBZ24449.1, CBZ24451.1, CBZ24452.1, CAH1385118.1, CAH1385117.1, CAH1385115.1, CAH1385116.1, ADD61463.1, BAD80751.1, ABP51432.1, AAL81659.1, AAD28735.1, ABN51595.1, AAD03471.1, AAC06896.1, AAC00218.1, AAM24997.1, AAM52219.1, BAB98701.1, BAB99480.1, AAC76453.1, AAC76442.1, BAA19592.1, AAD53957.1, AAN59210.1, AAL26558.1, AGL50099.1, BAB11741.1, CAC93400.1, AAB46846.1, ABB88567.1, CAA44069.1, AAA33211.1, AAD46887.1, AAN17338.1, AAL23578.1, AAP33020.1, AAK69600.1, AAB60395.1, CAA75517.1, AAC17451.1, BAK00834.1, AAA63271.1, AAK01137.1, AAL23577.1, AAD30476.1, AAG00588.1, ACJ76617.1, AAK15695.1, BAB92854.1, AAV87308.1, AAB68800.1, AAA41252.1, AAH70901.1, AAA41253.1, AAB68057.1, AAA33809.1, BAA00407.1, CAA52036.1, CAA59464.1, AAL23579.1, AAF82787.1, CAA84494.1, 6HQ6, or CAA85354.1 or sequence SEQ ID NO 1. .

[0051] In one embodiment, at least one GP glycoside enzyme is selected from the enzymes referenced under the NCBI or UNIPROT accession number: AAQ05801.1, AUG44408.1, 2207198A, AAD40317.1, AAN24362.1, AAN58596.1, AAO21868.1, AAO33821.1, AAO84039.1, AAX33736.1, ABS59292.1, ADL69407.1, CCA61958.1, BAN03569.1, AGK37834.1, BAA14344.1, BAF62433.1, CAA30846.1, CAA80424.1, ADP98617.1, ADH62582.1, AEJ61152.1, AAC74391.2, AAV43670.1, ADH99560.1, BAC54904.1, BAD97810.1, CAA11905.1, AAO80764.1, ABX42243.1, ABX43667.1, ABX43668.1, AAE30762.1, ABP66077.1, ADQ05832.1, AAC74398.1, ACL68803.1, ADC90669.1, SHM33122.1, ABX41399.1, ADI00307.1, AAB95491.2, AAC45510.1, AAD36910.1, AAL67138.1, AAQ20920.1, ABD80580.1, ABN51514.1, ADU20744.1, BAA25846.1, BAA28631.1, CAB16926.1, ADU22883.1, BAB71818.1, ABN54185.1, AAC45511.1, BAJ10826.1, ABX81345.1, AAG23740.1, BAC87867.1, CAC97070.1, ABX41081.1, AAM43298.1, ABD80168.1, EAA28929.1, 6HQ6, ABX81671.1, ACL75793.1, ACL76454.1, ACL76688.1, ACL77700.1, AFH48717.1, AHC20114.1, AIQ57809.1, AIQ60507.1, QCO92799.1, QCO92836.1, QCO92991.1, QCO92946.1, QCO92990.1, QCO92945.1, QCO92835.1, ACB74662.1, ABX42289.1, AAO07997.1, ABX40964.1, ABX43387.1, EEG94248.1, ACV29689.1, ZP_05748149.1, ACZ00636.1, ABG83511.1, BAH10636.1, ADL32981.1, AEN95946.1, CAC96089.1, ABY93074.1, ABY93073.1, CAZ94304.1, AAS19693.1, ADU21379.1, ADU20661.1, CAH06518.1, VCV21228.1, AAO76140.1, VCV21229.1, AAD36300.1, WP_026485574.1, WP_026486530.1, ABK05267.1, AUO30192.1, QCO92814.1, WP_019688419.1, ACJ76363.1, BAU78234.1, CBZ24448.1, CBZ24449.1, CBZ24451.1, CBZ24452.1, CAH1385118.1, CAH1385117.1, CAH1385115.1, CAH1385116.1, ADD61463.1, or BAD80751.1 or of sequence SEQ ID NO 1. In a particular embodiment, the preparation of at least one GP glycoside enzyme comprises at least two GPs selected from the enzymes referenced under the NCBI or UNIPROT accession number: AAQ05801.1, AUG44408.1, 2207198A, AAD40317.1, AAN24362.1, AAN58596.1, AAO21868.1, AAO33821.1, AAO84039.1, AAX33736.1, ABS59292.1, ADL69407.1, CCA61958.1 , BAN03569.1, AGK37834.1, BAA14344.1, BAF62433.1, CAA30846.1, CAA80424.1, ADP98617.1, ADH62582.1, AEJ61152.1, AAC74391.2, AAV43670.1, ADH99560.1, BAC54904.1, BAD97810.1, CAA11905.1, AAO80764.1, ABX42243.1, ABX43667.1, ABX43668.1, AAE30762.1, ABP66077.1, ADQ05832.1, AAC74398.1, ACL68803.1, ADC90669.1, SHM33122.1, ABX41399.1, ADI00307.1, AAB95491.2, AAC45510.1, AAD36910.1, AAL67138.1, AAQ20920.1, ABD80580.1, ABN51514.1, ADU20744.1, BAA25846.1, BAA28631.1, CAB16926.1, ADU22883.1, BAB71818.1, ABN54185.1, AAC45511.1, BAJ10826.1, ABX81345.1, AAG23740.1, BAC87867.1, CAC97070.1, ABX41081.1, AAM43298.1, ABD80168.1, EAA28929.1, ABX81671.1, ACL75793.1, ACL76454.1, ACL76688.1, ACL77700.1, AFH48717.1, AHC20114.1, AIQ57809.1, AIQ60507.1, QCO92799.1, QCO92836.1, QCO92991.1, QCO92946.1, QCO92990.1, QCO92945.1, QCO92835.1, ACB74662.1, ABX42289.1, AAO07997.1, ABX40964.1, ABX43387.1, EEG94248.1, ACV29689.1, ZP_05748149.1, ACZ00636.1, ABG83511.1, BAH10636.1, ADL32981.1, AEN95946.1, CAC96089.1, ABY93074.1, ABY93073.1, CAZ94304.1, AAS19693.1, ADU21379.1, ADU20661.1, CAH06518.1, VCV21228.1, AAO76140.1, VCV21229.1, AAD36300.1, WP_026485574.1, WP_026486530.1, ABK05267.1, AUO30192.1, QCO92814.1, WP_019688419.1, ACJ76363.1, BAU78234.1, CBZ24448.1, CBZ24449.1, CBZ24451.1, CBZ24452.1, CAH1385118.1, CAH1385117.1, CAH1385115.1, CAH1385116.1, ADD61463.1, 6HQ6 or BAD80751.1 or sequence SEQ ID NO 1.

[0052] In another embodiment the at least one glycoside enzyme GP is chosen among the enzymes referenced under the NCBI or UNIPROT accession number: AAQ05801.1, AUG44408.1, 2207198A, AAD40317.1, AAN24362.1, AAN58596.1, AAO21868.1, AAO33821.1, AAO84039.1, AAX33736.1, ABS59292.1, ADL69407.1, CCA61958.1, BAN03569.1, AGK37834.1, BAA14344.1, BAF62433.1, CAA30846.1, CAA80424.1, ADP98617.1, ADH62582.1, AEJ61152.1, AAC74391.2, AAV43670.1, ADH99560.1, BAC54904.1, BAD97810.1, CAA11905.1, AAO80764.1, ABX42243.1, ABX43667.1, ABX43668.1, AAE30762.1, ABP66077.1, ADQ05832.1, AAC74398.1, ACL68803.1, ADC90669.1, SHM33122.1, ABX41399.1, ADI00307.1, AAB95491.2, AAC45510.1, AAD36910.1, AAL67138.1, AAQ20920.1, ABD80580.1, ABN51514.1, ADU20744.1, BAA25846.1, BAA28631.1, CAB16926.1, ADU22883.1, BAB71818.1, ABN54185.1, AAC45511.1, BAJ10826.1, ABX81345.1, AAG23740.1, BAC87867.1, CAC97070.1, ABX41081.1, AAM43298.1, ABD80168.1, EAA28929.1, 6HQ6, ABX81671.1, ACL75793.1, ACL76454.1, ACL76688.1, ACL77700.1, AFH48717.1, AHC20114.1, AIQ57809.1, AIQ60507.1, QCO92799.1, QCO92836.1, QCO92991.1, QCO92946.1, QCO92990.1, QCO92945.1, QCO92835.1, ACB74662.1, ABX42289.1, AAO07997.1, ABX40964.1, ABX43387.1, EEG94248.1, ACV29689.1, ZP_05748149.1, ACZ00636.1, ABG83511.1, BAH10636.1, ADL32981.1, AEN95946.1, CAC96089.1, ABY93074.1, ABY93073.1, CAZ94304.1, AAS19693.1, ADU21379.1, ADU20661.1, CAH06518.1, VCV21228.1, AAO76140.1, VCV21229.1, AAD36300.1, WP_026485574.1, WP_026486530.1, ABK05267.1, AUO30192.1, QCO92814.1, WP_019688419.1, ACJ76363.1, BAU78234.1, CBZ24448.1, CBZ24449.1, CBZ24451.1, CBZ24452.1, . CAH1385118.1, CAH1385117.1, CAH1385115.1 or CAH1385116.1 or of sequence SEQ ID NO 1. In a particular embodiment, the preparation of at least one GP glycoside enzyme comprises at least two GPs chosen from the enzymes referenced under the NCBI or UNIPROT accession number: AAQ05801.1, AUG44408.1, 2207198A, AAD40317.1, AAN24362.1, AAN58596.1, AAO21868.1, AAO33821.1, AAO84039.1, AAX33736.1, ABS59292.1, ADL69407.1, CCA61958.1, BAN03569.1, AGK37834.1, BAA14344.1, BAF62433.1, CAA30846.1, CAA80424.1, ADP98617.1, ADH62582.1, AEJ61152.1, AAC74391.2, AAV43670.1, ADH99560.1, BAC54904.1, BAD97810.1, CAA 11905.1, AAO80764.1, ABX42243.1, ABX43667.1, ABX43668.1, AAE30762.1, ABP66077.1, ADQ05832.1, AAC74398.1, ACL68803.1, ADC90669.1, SHM33122.1, ABX41399.1, ADI00307.1, AAB95491.2, AAC45510.1, AAD36910.1, AAL67138.1, AAQ20920.1, ABD80580.1, ABN51514.1, ADU20744.1, BAA25846.1, BAA28631.1, CAB16926.1, ADU22883.1, BAB71818.1, ABN54185.1, AAC45511.1, BAJ10826.1, ABX81345.1, AAG23740.1, BAC87867.1, CAC97070.1, ABX41081.1, AAM43298.1, ABD80168.1, EAA28929.1, ABX81671.1, ACL75793.1, ACL76454.1, ACL76688.1, ACL77700.1, AFH48717.1, . AHC20114.1, AIQ57809.1, AIQ60507.1, QCO92799.1, QCO92836.1, QCO92991.1, QCO92946.1, QCO92990.1, QCO92945.1, QCO92835.1, ACB74662.1, ABX42289.1, AAO07997.1, ABX40964.1, ABX43387.1, EEG94248.1, ACV29689.1, ZP_05748149.1, ACZ00636.1, ABG83511.1, BAH10636.1, ADL32981.1, AEN95946.1, CAC96089.1, ABY93074.1, ABY93073.1, CAZ94304.1, AAS 19693.1, ADU21379.1, ADU20661.1, CAH06518.1, VCV21228.1, AAO76140.1, VCV21229.1, AAD36300.1, WP_026485574.1, WP_026486530.1, ABK05267.1, AUO30192.1, QCO92814.1, WP_019688419.1, ACJ76363.1, BAU78234.1, CBZ24448.1, CBZ24449.1, CBZ24451.1, CBZ24452.1, CAH1385118.1, CAH1385117.1, CAH1385115.1, 6HQ6, or CAH1385116.1 or of sequence SEQ ID NO 1. • Provision of a composition comprising at least one sugar-phosphate substrate of said enzyme 10b, said composition substantially not comprising any non-phosphorylated sugar acceptor substrate of said at least one glycoside phosphorylase enzyme.

[0053] Sugar-phosphate substrate means an aldose or a ketose on which a group phosphate is attached to the hemiacetal function. In a particular embodiment, the sugar-phosphate substrate is understood to be a phosphate oligo- or polysaccharide. It is understood that the sugar-phosphate substrate may be partially functionalized by means of functional groups attached to at least one of the hydroxyl groups that it comprises (except the hydroxyl of the hemiacetal function); for example, and in a non-limiting manner, it may be methylated, acylated, acetylated, N-acetylated, dehydroxylated, carboxylated, or dehydroxylated. This results in the formation, at the end of the process of the invention, of phosphate oligo- or polysaccharides bearing these functionalizations.

[0054] Preferably, said composition does not substantially comprise, as does obviously the enzymatic preparation, non-phosphorylated sugar acceptor substrate of at least one GP; this makes it possible to ensure a maximum yield of non-reducing phosphorylated oligosaccharide or polysaccharide. Thus, preferably, said composition comprising at least one sugar-phosphate substrate of said enzyme (10b), does not substantially comprise non-phosphorylated sugar acceptor substrate of at least one GP. Said composition may comprise a non-phosphorylated sugar acceptor substrate of the GP, nevertheless in this hypothesis, in said composition, the at least one sugar-phosphate substrate of the GP may represent at least 70% of the sugar substrates of the GP present in the composition, preferably at least 80%, even more preferably at least 90% and particularly preferably at least 95% of the sugar substrates of the GP present in the composition.Of course, the composition may include without restriction other non-substrate sugars of GP. phosphorylated or not.

[0055] In a particular embodiment, the composition comprising at least one sugar-phosphate substrate comprises 2, 3, or even more, sugar-phosphate substrates. Indeed, although most of the time very specific, certain GPs can accept different sugar-phosphate substrates. That is to say that an enzyme can catalyze said reaction from 1, 2, or even more, different sugar-phosphate substrates, resulting, for example, at the end of the implementation of the method according to this particular embodiment, in the production of a mixture of different oligo- or polysaccharide phosphates. • Reaction 20 of the preparation comprising at least one glycoside phosphorylase with the composition comprising at least one sugar-phosphate substrate of said enzyme,

[0056] “Reaction” means bringing together at least one GP and the at least one sugar-phosphate substrate of said GP and the provision of conditions conducive to the sugar-phosphate synthesis reaction as described in [Fig.2],

[0057] In a particular embodiment, the sugar-phosphate substrate is α-mannose-1P and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: CAC96089.1, ABY93074.1, ABY93073.1, CBZ24449.1, CAH1385118.1, CAH1385117.1, BAJ10826.1, CAZ94304.1, AAS19693.1, ADU21379.1, ADU20661.1, CAH06518.1, VCV21228.1, AAO76140.1, VCV21229.1, AAD36300.1, WP_026485574.1, WP_026486530.1, ABK05267.1, CBZ24448.1, CBZ24451.1, CBZ24452.1, CAH1385115.1, or CAH1385116.1.

[0058] In another particular embodiment, the sugar-phosphate substrate is α-galactose-1P and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following Genbank number: ACB74662.1, ABX42289.1, AAO07997.1, ABX40964.1, ABX43387.1, EEG94248.1, ACV29689.1, ZP_05748149.1, ACZ00636.1, ABG83511.1, BAH10636.1, ADL32981.1 or AEN95946.1.

[0059] In another particular embodiment, the sugar-phosphate substrate is α-glucose-1P and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: 2207198A, AAD40317.1, AAN24362.1, AAN58596.1, AAO21868.1, AAO33821.1, AAO84039.1, AAX33736.1, ABS59292.1, ADL69407.1, CCA61958.1, BAN03569.1, AGK37834.1, BAA14344.1, BAF62433.1, CAA30846.1, CAA80424.1, ADP98617.1, ADH62582.1, AEJ61152.1, AAC74391.2, AAB95491.2, AAC45510.1, AAD36910.1, AAL67138.1, AAQ20920.1, ABD80580.1, ABN51514.1, ADU20744.1, BAA25846.1, BAA28631.1, CAB16926.1, ADU22883.1, BAB71818.1, ABN54185.1, AAC45511.1, BAJ10826.1, ABX81345.1, CAC97070.1, ABX41081.1, AAM43298.1, ABD80168.1, EAA28929.1, ACL75793.1, ACL76454.1, ACL76688.1, ACL77700.1, AFH48717.1, AHC20114.1, AIQ57809.1, AIQ60507.1, QCO92799.1, QCO92836.1, QCO92991.1, QCO92946.1, QCO92990.1, QCO92945.1, QCO92835.1, AUO30192.1, QCO92814.1, WP_019688419.1, ACJ76363.1, 6HQ6, ou BAU78234.1

[0060] In another particular embodiment, the sugar-phosphate substrate is α-glucose-1P and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following Genbank number: ABP51432.1, AAL81659.1, AAD28735.1, ABN51595.1, AAD03471.1, AAC06896.1, AAC00218.1, AAM24997.1, AAM52219.1, BAB98701.1, BAB99480.1, AAC76453.1, AAC76442.1, BAA19592.1, AAD53957.1, AAN59210.1, AAL26558.1, AGL50099.1, BAB11741.1, CAC93400.1, AAB46846.1, ABB88567.1, CAA44069.1, AAA33211.1, AAD46887.1, AAN17338.1, AAL23578..1, AAP33020.1, AAK69600.1, AAB60395.1, CAA75517.1, AAC17451.1, BAK00834.1, AAA63271.1, AAK01137.1, AAL23577.1, AAD30476.1, AAG00588.1, ACJ76617.1, AAK15695.1, BAB92854.1, AAV87308.1, AAB68800.1, AAA41252.1, AAH70901.1, AAA41253.1, AAB68057.1, AAA33809.1, BAA00407.1, CAA52036.1, CAA59464.1, AAL23579.1, AAF82787.1, CAA84494.1 or CAA85354.1, and the reaction also comprises providing an acceptor oligo- or polysaccharide of formula maltodextrin [(l,4)-aD-glucosyl]n. .

[0061] The person skilled in the art will know how to optimize the reaction conditions (such as temperature, salt concentration, pH, sugar-phosphate substrate concentration of the GP) to ensure optimal yield of the sugar-phosphate synthesis reaction. For example, the reaction buffer used can be chosen from the list of buffers offered on the websites:

[0062] https: / / hamptonresearch.com / uploads / cg_pdf / Hampton_Research_CG101_Buffer_Ta ble.pdf,

[0063] or

[0064] https: / / www.sigmaaldrich.com / FR / fr / support / calculators-and-apps / buffer-calculator, which allows you to choose according to the desired optimum reaction pH which can vary depending on the GP used.

[0065] The reaction temperature can be between 5°C and 90°C, 10°C and 80°C, 15°C and 70°C, 20°C and 60°C, 25°C and 50°C, preferably between 30°C and 40°C; typically, the reaction temperature is 37°C but the optimal temperature of the enzyme can vary greatly depending on the enzyme considered and / or the production conditions and will be easily determined by in vitro tests.

[0066] The duration of the reaction can be from 1 h to 48 h, preferably from 5 h to 40 h, from 10 h to 30 h, or even from 15 h to 20 h depending on the conditions of implementation of the process.

[0067] The reaction may be carried out in a closed container, for example in a closed reactor, without exchange with the external environment, in which the two compositions are then reacted. This may be in a stirred tank reactor, for example using a propeller homogenizer, to maintain a homogeneous mixture of the enzyme and the reaction medium. Any other means aimed at maintaining the homogeneity of the mixture of the preparation and the composition may be used.

[0068] Advantageously, when the GP is immobilized, and to avoid degradation of the immobilized enzyme due to the shear forces associated with stirring, the immobilized enzyme can be associated with a support through which or on which a flow of reaction mix (here for example the reaction buffer and the composition comprising the sugar-phosphate substrate of the GP) is operated. Alternatively, still in this embodiment of flow reactor, the immobilized enzyme is not associated with a support but is kept in suspension in a compartment of the reactor through which the reaction mix and in particular the sugar-phosphate(s) circulate.

[0069] The reactor may have a volume greater than or equal to 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or even greater than or equal to 1 L, 2 L, 3 L, 4 L, 5 L, 10 L, 20 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, or even greater than or equal to 100 L, 200 L, 300 L, 500 L, 1000 L, 2000 L, 3000 L, or greater than or equal to 10000 L.

[0070] Examples of bioreactor design applicable herein are provided by Sheldon et al (2021).

[0071] Advantageously, in a particular embodiment, the container in which the reaction takes place is provided with a means for ensuring the shift in the equilibrium of the reaction in favor of the synthesis of phosphate oligo- or polysaccharide(s). This can be done, for example, by ensuring the removal of the free inorganic phosphate generated by the reaction (for example by using filtration means for removing the inorganic phosphate from the reaction medium or means for precipitating the inorganic phosphate formed) and / or the oligo- or polysaccharides produced, or by ensuring a high excess of sugar-phosphate substrate(s) of the at least one GP in the reaction chamber, for example by continuously supplementing the reactor with the sugar-phosphate substrate(s) of the at least one GP. In a particular embodiment, the inorganic phosphate is precipitated using, for example, calcium salts. Obtaining 30 a mixture comprising at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate.

[0072] As illustrated in [Fig.2], the product of the phosphate sugar synthesis reaction as discovered by the inventors comprises at least one phosphate oligosaccharide or at least one phosphate polysaccharide whose hemiacetal end is covalently bonded to an inorganic phosphate, and inorganic phosphate as the reaction product.

[0073] • Optional step of purification of at least one oligosaccharide phosphate or at least one polysaccharide phosphate.

[0074] In a particular embodiment, said enzymatic method for the in vitro synthesis of at least one phosphate oligosaccharide or at least one phosphate polysaccharide 1 may further comprise a step of purification of the phosphate oligosaccharide(s) or polysaccharide(s) produced by the method of the invention. During this step, the phosphate oligosaccharide(s) or polysaccharide(s) produced may be purified from the inorganic phosphate, the non-phosphorylated oligosaccharides and the monosaccharides by calcium precipitation followed by ethanol precipitation, the result of which is illustrated [Fig.9]. For example, the introduction of excess Ca2+ ions into the reaction medium (for example, 5 mol of Ca2+ / 1 mol Man-IP initial substrate of the reaction) makes it possible to precipitate the inorganic phosphate because these two chemical species combine to form an insoluble precipitate.It is preferable to supply the Ca2+ ions via a salt that has little effect on the overall physicochemical behavior of the reaction medium and can be easily removed later. Calcium acetate is a preferred salt. After centrifugation, the supernatant is collected, filtered and mixed with ethanol (e.g., 85% final concentration) to precipitate the phosphate oligo- and / or polysaccharides. The non-phosphorylated oligosaccharides and monosaccharides remain soluble in the solution. After centrifugation, the pellet is washed with 85% ethanol and then dried under nitrogen flow. The pellet is then rehydrated and dried again on a rotary evaporator. Finally, the reaction products are lyophilized for optimal preservation and stability. • Optional step of production of sugar-phosphate substrate of at least one GP.

[0075] In another particular embodiment, said enzymatic method for the in vitro synthesis of at least one oligosaccharide phosphate or at least one polysaccharide phosphate 1 may further comprise a step of producing sugar-phosphate substrate of the at least one GP. This may be carried out enzymatically or chemically, the enzymatic route being particularly preferred. The chemical route may be, for example, as described by Degani and Halmann (1971).

[0076] Preferably, the sugar-phosphate substrate of the at least one GP is synthesized enzymatically, which has the advantages already listed. Thus, the sugar-phosphate substrate of the at least one GP can be produced: - by using a GP under reaction conditions favoring the phosphorolysis reaction, i.e. lysis of an oligo- or polysaccharide (a- or [3-glycosides] in the presence of inorganic phosphate, - using a Glucose Hydrolase (GH) which will catalyze the hydrolysis of an oligosaccharide or polysaccharide to produce a monosaccharide not phosphorylated at its hemiacetal end, which is then 'phosphorylated' using, for example, a kinase in the presence of adenosine triphosphate as a phosphate donor (Liu et al (2015). - using a GP that will catalyze the phosphorolysis of a disaccharide to produce a non-phosphorylated sugar at its hemiacetal end, which is then 'phosphorylated' using, for example, a kinase in the presence of adenosine triphosphate as a phosphate donor.

[0077] This additional enzymatic step of synthesis of the sugar-phosphate substrate of the GP can be carried out extemporaneously from purified enzymes which will possibly catalyze successive reactions (as for example illustrated in Zhong et al (2020)). Alternatively, a microorganism can be genetically modified so as to express the complementary enzyme(s) making it possible to produce from a polysaccharide substrate at least one desired phosphate oligosaccharide or at least one desired phosphate polysaccharide, by said microorganism (as for example illustrated in application WO 2021 / 229185 or the article by Tian et al (2020)).

[0078] In a particular embodiment, said enzymatic method for in vitro synthesis of at least one oligosaccharide phosphate or at least one polysaccharide phosphate 1 may further comprise a step of identifying the at least one sugar-phosphate substrate of the GP. For example, such a step may comprise a test such as that implemented by Macdonald et al (2019), or illustrated in the experimental part, is particularly advantageous because it is rapid and simple to implement. • Optional GP characterization step.

[0079] In another particular embodiment, said enzymatic method for in vitro synthesis of at least one oligosaccharide phosphate or at least one polysaccharide phosphate 1 may further comprise a step of characterization of the GP; in other words a step of identification for a given enzyme of the characteristic activities of a GP, namely the phosphorolysis of oligo- and polysaccharides and / or reverse phosphorolysis as described above and detailed in Li et al (2022). For example, such a step may comprise a test such as that implemented by Macdonald et al (2019). This characterization step may therefore comprise the identification of the donor and acceptor sugars of the GP as explained in the experimental part. Experimental results

[0080] i. Production of recombinant GPs

[0081] Table 2: Recombinant glycoside phosphorylases produced and tested in vitro for their phosphate sugar synthesis activity. [Tables 2] Family CAZy GENBANK No. Name Origin GH 130 ABY93073.1 Tethl788 Thermonobacter sp. X-514 GH 130 CAC96089.1 Lin0857 Listeria innocua Clip 11262 GH 130 CAH1385118.1 Uhgb_mS Mammalian microbiome bacteria GH 130 CAH1385117.1 U7 Mammalian microbiome bacteria GT108 CBZ24449.1 MTP4 Leishmania mexicana MNYC / BZ / 62 / M379 Cloning

[0082] The gene coding for the enzyme Teth514-1788 of the strain Thermoanaerobacter sp. X-514 (Teth514-1788, Genbank accession number ABY93073.1) belonging to the GH130 family of the CAZy classification (http: / / www.cazy.org / ) was synthesized and cloned into the pBAD HisA vector by the company Biomatik (Biomatik, Ontario, Canada) between the NcoI and XhoI restriction sites.

[0083] The gene coding for the enzyme Lin0857 of the strain Listeria innocua Clip 11262, (Genbank accession number CAC96089.1) belonging to the GH 130 family of the CAZy classification was synthesized by the company TWIST Bioscience (TWIST, San Francisco, United States) and cloned between the NcoI and XhoI restriction sites, in the plasmid pBAD-HisA.

[0084] The gene encoding the enzyme Uhgb_mS (Genbank accession number CAH1385118.1), belonging to the GH 130 family of the CAZy classification, was synthesized with codon optimization for expression in E. coli by Biomatik Limited (Cambridge, Ontario, Canada) and cloned into the plasmid pET-23a (+) with a 6 (His) tag at the N- and C-termini.

[0085] The gene encoding the U7 enzyme (Genbank accession number CAH1385117.1) belonging to the GH 130 family of the CAZy classification was synthesized with codon optimization for expression in E. coli by Biomatik Limited (Cambridge, Ontario, Canada) and cloned between the NdeI and XhoI restriction sites in the plasmid pET-23a (+) with a 6 (His) tag at the N- and C-termini.

[0086] The gene coding for the MTP4 enzyme of the Leishmania mexicana strain MNYC / BZ / 62 / M379, (Genbank accession number CBZ24449.1) belonging to the GT 108 family of the CAZy classification was synthesized by the company Integrated DNA Technologies (München-Flughafen, Germany) and cloned by the “in fusion” method into the plasmid pET-28 with a 6 (His) tag at the C-terminus using these primers to amplify the MTP4 gene and the plasmid pET 28 respectively:

[0087] Fw: SEQID NO 2:

[0088] 5'-AGGAGATATACCATGAAGCAGACTAAGGCGTCCTTTGAGGCTAACAA ACG-3'.

[0089] Rev : SEQ ID NO 3:

[0090] 5 ' -GTGGTGGCTGCTGCCCAGGGATGAGGCCATCGGGAAATTCAGGTCTC-3

[0091] Fw: SEQ ID NO 4 :

[0092] 5 ' -GGCAGCAGCCACCACCACCACCACCACTGAGATC-3 '

[0093] Rev : SEQ ID NO 5 :

[0094] 5 ' -CATGGTATATCTCCTTCTTAAAGTTA AACAAAATTATTTCTAG-3 '

[0095] Expression

[0096] The recombinant glycoside phosphorylases of the CAZy family GH130 and GT108 listed in Table 2 were produced in E. coli BL21. The enzymes placed on pET vector were produced in E. coli BL21(star) while those present on pBAD were produced in BL21-AI. The production protocol is then identical for the 5 GPs. A volume of 50 pL of cells, previously made competent, was transformed with 1 pL of plasmid DNA containing the nucleotide sequence of the enzyme to be produced. After incubation of the cells in the presence of the plasmid for 30 min, the transformation was carried out by heat shock at 42°C for 30 sec. After 1 h of phenotypic expression in SOC medium at 37°C at 200 rpm of agitation, the cells were used to inoculate 20 mL of LB medium supplemented with the selection antibiotic and then incubated overnight at 37°C at 200 rpm.The next day, the preculture was used to inoculate 200 mL of ZYM autoinducible medium supplemented with the selection antibiotic at an OD = 0.05. For pET vectors, ZYM medium contained lactose for plasmid induction, while it contained arabinose for induction of genes carried by the pBAD vector. Cells were incubated for 24 h at 21°C at 160 rpm. At the end of the culture, cells were collected by centrifugation (15 min, 10,000 g).

[0097] Any other organism and / or usual technique for the production and purification of recombinant proteins can be used.

[0098] Purification

[0099] The purification of the 5 GPs was carried out according to the same protocol. The cell pellets corresponding to the 100 mL of culture were taken up at OD = 100 in Tris-HC1 buffer (50 mM) + NaCl (350 mM) at pH = 7.5. The cells were then lysed cold by sonication. The total protein fractions obtained were centrifuged cold 30 min at 12,000g, then only the soluble protein fractions were retained. The 6His-tagged enzymes were purified from other bacterial proteins by affinity chromatography on cobalt resin (TALON® Metal Affinity Resin, Takara). Incubation of the total fractions with cobalt resin lasted 1 h at 4°C. The final elution was carried out in Tris-HCl buffer (20 mM) + NaCl (100 mM) + Imidazole (300 rnM) equilibrated to pH = 7.5. The recombinant GPs were finally desalted on a PD-10 desalting column (GE Healthcare) according to the protocol provided by the manufacturer. The final elution was carried out with Tris-HCl buffer (20 mM) + NaCl (150 mM) equilibrated to pH = 7.5.

[0100] ii. Identification of donor and recipient sugar

[0101] The CAZy data (<http: / / www.cazy.org> ) identify the substrates of the enzymes identified in this database.

[0102] The donor and acceptor substrate sugars for a putative glycoside phosphorylase can be readily identified using, for example, chromogenic assays that detect the presence of the inorganic phosphate released from the donor sugar during the oligosaccharide synthesis reaction by the glycoside phosphorylase.

[0103] An example is the molybdenum blue test.

[0104] Briefly, this test is based on the formation of phosphomolybdenum in the presence of the reagent molybdate. Under acidic conditions, phosphomolybdate is reduced to molybdenum, of which blue is the reactant of the reaction. The intensity of the blue color can be quantified by measuring the absorbance at 655 nm.

[0105] For example, the reaction medium may contain 45 μL of purified enzyme for a final concentration of 0.03 mg.ml1 in 255 μL of a solution containing 20 mM Tris / HCl (pH 7.0), 200 mM sodium molybdate, 10 mM monosaccharide-1-phosphate. and 10 mM of an acceptor monosaccharide (final concentration). The reaction is monitored by 50 μL samples transferred to a 96-well microplate containing 150 μL of a 0.24% (W / V) L-ascorbic acid solution in 0.1 N HCl per well. After incubation at room temperature for 5 minutes, 150 μl of stop solution [2% (v / v) acetic acid and 2% (w / v) tribasic sodium citrate dihydrate] is added to stop the reaction. Absorbance is measured at 655 nm using a microplate reader (InfiniteM200pro, TECAN). One unit of activity corresponds to the amount of enzyme that catalyzes the production of 1 pmol of inorganic phosphate per min-1 under the assay conditions.

[0106] This method is suitable for the detection of GP-catalyzed activity produced in crude E. coli cell extracts and detailed by Macdonald et al (2019), allowing the screening of a large number of extracts, donor and acceptor sugars.

[0107] iii. Testing the activity of synthesis of oligo- or polysaccharides phosphate

[0108] The synthesis reaction with sugar-1-phosphate as the sole substrate can be conducted under conditions determined by the physicochemical characteristics of the GP tested, in particular its optimal conditions of activity and stability (temperatures, pH, salinity, etc.).

[0109] By adapting the molybdate test, using only a sugar-1-phosphate as a substrate, it is possible to detect potential synthesis activity of phosphate oligosaccharides or phosphate polysaccharides. Confirmation by HPAEC-PAD can then be carried out to confirm the nature of the synthesized products.

[0110] The enzymes in Table 2 were tested under similar conditions. In this case, the reactions were carried out in a volume of 500 pL at 37°C under 900 rpm of stirring. 5 pM of enzymes were placed in the presence of 75 mM of mannose-1-phosphate (Man-IP) for 20 h. For the enzymes Lin0857, MTP4, Uhgb_MS and U7, the reaction medium was at pH = 7 in a concentrated Tris-HCl buffer at 100 mM. For the enzyme Tethl788, the reaction medium was at pH = 5 in a concentrated sodium acetate buffer at 100 mM. At the end of the reaction, the enzyme was inactivated by heating the reaction medium for 5 min at 95°C. After centrifugation to remove the precipitated enzyme, the reaction products were analyzed by HPAEC-PAD and HPAEC-PAD-MS.

[0111] iv. Reaction for the synthesis of oligo- or polysaccharide phosphates

[0112] The synthesis reactions with sugar-1-phosphate as the sole substrate were carried out at 37°C and pH = 7 with stirring for 20 h. The final times as well as a control containing only sugar-1-phosphate were analyzed by simple anion exchange chromatography (High Performance Anion Exchange Chromatography with Pulsed Amperometric detection “HPAEC-PAD”) or coupled to a mass spectrometer (Mass Spectrometer, “HPAEC-PAD-MS”).

[0113] v. Detection and characterization of synthesized phosphate oligo- and / or polysaccharides

[0114] The reaction products obtained with the 5 GPs were analyzed initially by anion exchange chromatography coupled with a pulsed amperometric detector (High Performance Anion Exchange Chromatography with Pulsed Amperometric detection “HPAEC-PAD”, [Fig.3]). The separation was carried out on a Dionex CarboPac PA100 2 mm IC column (Thermo Scientific). With a flow rate of 0.250 mL / min on the mobile phase, the following protocol was used: 1) 3 min isocratic phase at 75 mM NaOH, 2) 1 min gradient from 75 to 150 mM NaOH, 3) 1 min isocratic phase at 150 mM NaOH, 4) 1 min gradient from 0 to 50 mM sodium acetate, 5) 10 min gradient from 50 to 175 mM sodium acetate. Detection was performed with a Dionex ED40 module operating with a gold electrode and an Ag / AgCl pH reference.

[0115] The reaction products obtained with the 5 GPs were analyzed in a second time by anion exchange chromatography coupled with a pulsed amperometric detector and a mass spectrometer (Mass Spectrometer, “HPAEC-PAD-MS”, [Fig.4] to 8). The separation was performed on a Dionex CarboPac PA100 2 mm IC column (Thermo Scientific). With a flow rate of 0.250 mL / min on the mobile phase, the following protocol was used: 1) 3 min isocratic phase at 75 mM NaOH, 2) 1 min gradient from 75 to 150 mM NaOH, 3) 1 min isocratic phase at 150 mM NaOH, 4) 1 min gradient from 0 to 50 mM sodium acetate, 5) 16 min gradient from 50 to 125 mM sodium acetate, 6) 23 min isocratic phase at 125 mM sodium acetate. Amperometry detection was performed with a Dionex ED40 module operating with a gold electrode and an Ag / AgCl pH reference. Molar mass detection was performed with an ISQ EC mass spectrometer (Thermo Scientific). Two Dionex AERES 500e 2mm desalting modules (Thermo Scientific) were used with a water flow rate of 1 mL / min and a voltage of 50 and 100 mA.The parameters used were: vaporization temperature: 289°C, ion transfer tube temperature: 300°C, positive ion source voltage: 3000 V, negative ion source voltage: -2000V, nitrogen pressure: 55.3 psig, polarity: negative, analyzed mass: 175 to 1250, analysis time: 0.2 sec, spectrum type: centroid, CID source voltage: 5. The molar masses extracted from the global signal were smoothed using a 301-point Gaussian-type function.

[0116] As shown in [Fig. 3], for the enzymes tested in Table 2, peaks with very high retention times and higher than that of mannose-1-phosphate are visible. These peaks cannot correspond to usual [3-mannosides because their retention times are well known and lower than that of mannose-1-phosphate. Given the catalytic activity of these enzymes, the regularity of the peaks and their reproducibility (in particular for [3-1,2 mannosides phosphorylases), they most certainly correspond to [3-mannosides phosphate of different degrees of polymerization. Mass spectrometry analyses should confirm this hypothesis.

[0117] HPAEC-PAD-MS analyses of the peaks produced by the enzymes MTP4 ([Fig.4]), Tethl788 ([Fig.5]), Lin0857 ([Fig.6]), Uhgb_MS ([Fig.7]) and U7 ([Fig.8]) show, after signal processing, that they have exactly the mass of Man-IP molecules polymerized with one or more mannosyl units. In other words, these peaks correspond to mannose oligosaccharides with a single phosphorylation, the phosphate group being only able to be at the reducing end of the chains, in position a. The 5 GPs mentioned in Table 2 are therefore capable of using mannose-1-phosphate as a donor and acceptor to synthesize phosphate oligosaccharides and polysaccharides.

[0118] As shown in [Fig.4] to 8, HPAEC-PAD-MS analysis makes it possible to characterize the degree of polymerization (DP) of phosphate oligo- and polysaccharides produced up to at least 6 units. However, the amperometric signal for MTP4 and Tethl788 suggests that higher DP phosphate oligo- and polysaccharides are produced. MTP4 would synthesize oligosaccharides up to DP = 13. Tethl788 would synthesize oligo- and polysaccharides beyond DP = 14 under the conditions tested.

[0119] Table 3 summarizes the activities and oligosaccharides detected for the enzymes tested. [Tables 3] Name Substrate Product Maximum DP detected Tethl788 Man-IP [3-1,2-MOS-aPi >14 Lin0857 Man-IP [3-1,2-MOS-aPi 4 Uhgb_MS Man-IP [3-1,2-MOS-aPi 3 U7 Man-IP [3-1,2-MOS-aPi 4 MTP4 Man-IP [3-1,2-MOS-aPi 13 Bibliography

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Claims

Claims

1. Enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1) comprising the following steps: - providing a preparation comprising at least one glycoside phosphorylase enzyme (10a) not catalyzing the formation of a 1-1 osidic bond, - providing a composition comprising at least one sugar-phosphate substrate of said enzyme (10b), said composition substantially not comprising a non-phosphorylated sugar acceptor substrate of said at least one glycoside phosphorylase enzyme, - reacting (20) the preparation comprising the at least one glycoside phosphorylase with the composition comprising at least one sugar-phosphate substrate of said enzyme, - obtaining (30) a mixture comprising at least one oligosaccharide phosphate or at least one polysaccharide phosphate.

2. Enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1) according to the preceding claim, in which the at least one glycoside phosphorylase enzyme belongs to a family of enzymes with carbohydrate activity referenced in the CAZY.org database in the following families: GH3, GH13_18, GH65, GH94, GH112, GH130, GH149, GH161, GT35, GT108 and GH131.

3. Enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1) according to any one of the preceding claims, in which the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: AAQ05801.1, AUG44408.1, 2207198A, AAD40317.1, AAN24362.1, AAN58596.1, AAO21868.1, AAO33821.1, AAO84039.1, AAX33736.1, ABS59292.1, ADL69407.1, CCA61958.1, BAN03569.1, AGK37834.1, BAA14344.1, BAF62433.1, CAA30846.1, CAA80424.1, ADP98617.1, ADH62582.1, AEJ61152.1, AAC74391.2, AAV43670.1,

4. ADH99560.1, BAC54904.1, BAD97810.1, CAA11905.1, AAO80764.1, ABX42243.1, ABX43667.1, ABX43668.1, AAE30762.1, ABP66077.1, ADQ05832.1, AAC74398.1, ACL68803.1, ADC90669.1, SHM33122.1, ABX41399.1, ADI00307.1, AAB95491.2, AAC45510.1, AAD36910.1, AAL67138.1, AAQ20920.1, ABD80580.1, ABN51514.1, ADU20744.1, BAA25846.1, BAA28631.1, CAB16926.1, ADU22883.1, BAB71818.1, ABN54185.1, AAC45511.1, BAJ10826.1, ABX81345.1, AAG23740.1, BAC87867.1, CAC97070.1, ABX41081.1, AAM43298.1, ABD80168.1, EAA28929.1, 6HQ6, ABX81671.1, ACL75793.1, ACL76454.1, ACL76688.1, ACL77700.1, AFH48717.1, AHC20114.1, AIQ57809.1, AIQ60507.1, QCO92799.1, QCO92836.1, QCO92991.1, QCO92946.1, QCO92990.1, QCO92945.1, QCO92835.1, ACB74662.1, ABX42289.1, AAO07997.1, ABX40964.1, ABX43387.1, EEG94248.1, ACV29689.1, ZP_05748149.1, ACZ00636.1, ABG83511.1, BAH10636.1, ADL32981.1, AEN95946.1, CAC96089.1, ABY93074.1, ABY93073.1, CAZ94304.1, AAS19693.1, ADU21379.1, ADU20661.1, CAH06518.1, VCV21228.1, AAO76140.1, VCV21229.1, AAD36300.1, WP_026485574.1, WP_026486530.1, ABK05267.1, AUO30192.<h2 style=";text-align:left;direction:ltr">1, QCO92814.1, WP_019688419.1, ACJ76363.1, BAU78234.1, CBZ24448.1, CBZ24449.1, CBZ24451.1, CBZ24452.1, CAH1385118.1, CAH1385117.1, CAH1385115.1, CAH1385116.1, ADD61463.1, BAD80751.1, ABP51432.1, AAL81659.1, AAD28735.1, ABN51595.1, AAD03471.1, AAC06896.1, AAC00218.1, AAM24997.1, AAM52219.1, BAB98701.1, BAB99480.1, AAC76453.1, AAC76442.1, BAA19592.1, AAD53957.1, AAN59210.1, AAL26558.1, AGL50099.1, BAB11741.1, CAC93400.1, AAB46846.1, ABB88567.1, CAA44069.1, AAA33211.1, AAD46887.1, AAN17338.1, AAL23578.1, AAP33020.1, AAK69600.1, AAB60395.1, CAA75517.1, AAC17451.1, BAK00834.1, AAA63271.1, AAK01137.1, AAL23577.1, AAD30476.1, AAG00588.1, ACJ76617.1, AAK15695.1, BAB92854.1, AAV87308.1, AAB68800.1, AAA41252.1, AAH70901.1, AAA41253.1, AAB68057.1, AAA33809.1, BAA00407.1, CAA52036.1, CAA59464.1, AAL23579.1, AAF82787.1, CAA84494.1 or CAA85354.1, or sequence SEQ ID NO 1.<h2 style=";text-align:left;direction:ltr"> Enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1) according to one of claims 1 to 3, in which: the sugar-phosphate substrate is [3-glucose-1P] and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following Genbank number: AAQ05801.1, AUG44408.1, AAV43670.1, ADH99560.1, BAC54904.1, BAD97810.1, CAA11905.1, AAO80764.1, ABX42243.1, ABX43667.1, ABX43668.1, AAE30762.1, ABP66077.1, ADQ05832.1, AAC74398.1, ACL68803.1, ADC90669.1, SHM33122.1, ABX41399.1, or ADI00307.1, the sugar-phosphate substrate is α-mannose-lP and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: ADD61463.1, CAC96089.1, ABY93074.1, ABY93073.1, CBZ24449.1, CAH1385118.1, CAH1385117.1, BAJ10826.1, CAZ94304.1, AAS19693.1, ADU21379.1, ADU20661.1, CAH06518.1, VCV21228.1, AAO76140.1, VCV21229.1, AAD36300.1, WP_026485574.1, WP_026486530.1, ABK05267.1, CBZ24448.1, CBZ24451.1, CBZ24452.1, CAH1385115.1, or CAH1385116.1 or sequence SEQ ID NO1, the sugar-phosphate substrate is α-glucose-1P and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: 2207198A, AAD40317.1, AAN24362.1, AAN58596.1, AAO21868.1, AAO33821.1, AAO84039.1, AAX33736.1, ABS59292.1, ADL69407.1, CCA61958.1, BAN03569.1, AGK37834.1, BAA 14344.1, BAF62433.1, CAA30846.1, CAA80424.1, ADP98617.1, ADH62582.1, AEJ61152.1, AAC74391.2, AAB95491.2, AAC45510.1, AAD36910.1, AAL67138.1, AAQ20920.1, ABD80580.1, ABN51514.1, ADU20744.1, BAA25846.1, BAA28631.1, CAB16926.1, ADU22883.1, BAB71818.1, ABN54185.1, AAC45511.1, BAJ10826.1, ABX81345.1, CAC97070.1, ABX41081.1, AAM43298.1, ABD80168.1, EAA28929.1, ACL75793.1, ACL76454.1, ACL76688.1, ACL77700.1, AFH48717.1, AHC20114.1, AIQ57809.1, AIQ60507.1, QCO92799.1, QCO92836.1, QCO92991.1, QCO92946.1, QCO92990.1, QCO92945.1, QCO92835.1, AUO30192.1, QCO92814.1, WP_019688419.1, 6HQ6, ACJ76363.1, BAU78234.1, ABP51432.1, AAL81659.1, AAD28735.1, ABN51595.1, AAD03471.1, AAC06896.1, AAC00218.1, AAM24997.1, AAM52219.1, BAB98701.1, BAB99480.1, AAC76453.1, AAC76442.1, BAA19592.1, AAD53957.1, AAN59210.1, AAL26558.1, AGL50099.1, BAB11741.1, CAC93400.1, AAB46846.1, ABB88567.1, CAA44069.1, AAA33211.1, AAD46887.1, AAN17338.1, AAL23578..1, AAP33020.1, AAK69600.1, AAB60395.1, CAA75517.1, AAC17451.1, BAK00834.1, AAA63271.1, AAK01137.1, AAL23577.1, AAD30476.1, AAG00588.1, ACJ76617.1, AAK15695.1, BAB92854.1, AAV87308.1, AAB68800.1, AAA41252.1, AAH70901.1, AAA41253.1, AAB68057.1, AAA33809.1, BAA00407.1, CAA52036.1, CAA59464.1, AAL23579.1, AAF82787.1, CAA84494.1 or CAA85354.1, - the sugar-phosphate substrate is the a- N-acetyl-D-glucosamine-lP and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: AAG23740.1, BAC87867.1 or ABX81671.1, - the sugar-phosphate substrate is [3- N-acetyl-D-glucosamine-lP and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under NCBI accession number AAQ05801.1, or - the sugar-phosphate substrate is α-galactose-1P and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI accession number: BAD80751.1, ACB74662.1, ABX42289.1, AAO07997.1, ABX40964.1, ABX43387.1, EEG94248.1, ACV29689.1, ZP_05748149.1, ACZ00636.1, ABG83511.1, BAH10636.1, ADL32981.1 or AEN95946.

1.

5. Enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1) according to one of the preceding claims in which the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number: CAC96089.1, ABY93074.1, ABY93073.1, CBZ24449.1, CAH1385118.1 or CAH1385117.

1.

6. Enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1) according to one of the preceding claims, further comprising a step of purification (40) of the oligosaccharide phosphate and / or the polysaccharide phosphate.

7. Enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1) according to one of the preceding claims in which said glycoside phosphorylase is immobilized on an insoluble support.

8. Enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1) according to one of claims 1 to 6 wherein said preparation comprising at least one glycoside phosphorylase enzyme is a culture of a microorganism such as a bacterium, a yeast or a fungus expressing the at least one glycoside phosphorylase enzyme.

9. Enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1) according to one of claims 1 to 6 in which said reaction takes place in solution, with a purified recombinant enzyme preparation in solution.

10. Enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate (1) according to one of the preceding claims, in which the reaction comprises maintaining excess concentrations of the at least one sugar-phosphate substrate of the GP.

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