New method for synthesising phosphate oligosaccharides or phosphate polysaccharides

Abstract

The invention relates to an enzymatic method for the in vitro synthesis of at least one phosphate oligosaccharide and / or at least one phosphate polysaccharide (1), the method comprising providing a preparation comprising at least one glycoside phosphorylase enzyme (10b) that does not catalyse the formation of 1-1 glycosidic bonds; providing a composition comprising at least one sugar-phosphate substrate of the enzyme (10c), the composition comprising substantially no unphosphorylated sugar acceptor substrate of the 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 the enzyme; obtaining (30) a mixture comprising at least one phosphate oligosaccharide or at least one phosphate polysaccharide.

Description

Description New process for the synthesis of oligosaccharide phosphate or polysaccharide phosphate technical field

[0001] The invention relates to the field of biotechnology. More particularly, the invention relates to carbohydrate-active enzymes, and a novel in vitro enzymatic synthesis process for phosphate polysaccharides and / or phosphate oligosaccharides, without reducing ends. Previous technique

[0002] Oligosaccharides and polysaccharides represent a highly diverse class of biomolecules composed of linear or branched chains of glycosylated units linked together by various types of α- or β-glycosidic bonds. Their structural diversity results from both the nature of the monomers and the position and anomericity of their covalent bonds (α- or β-). These molecules play diverse and important roles in a multitude of cellular processes. Due to their highly varied structures and biological functions, oligosaccharides are used in a wide range of applications in the food, healthcare, cosmetics, and chemical industries.

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

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

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

[0006] Among carbohydrate-active enzymes, three main families are distinguished: glycoside hydrolases (GH), glycoside transferases (GT), and glycoside phosphorylases (GP). GPs are valuable catalysts for white biotechnology due to their ability to efficiently synthesize oligo- and polysaccharides without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction makes them attractive tools for diversifying 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 has been discovered that it is possible to use these enzymes to synthesize oligo- and polysaccharide phosphates: These enzymes can indeed use a sugar-phosphate both as a sugar donor and as a sugar acceptor for the synthesis of oligosaccharide phosphates (Figure 2), this reaction is hereafter 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, since this end is involved in the bond with the phosphate group. This property is of particular interest for numerous applications because it can prevent Maillard reactions when sugars are used in formulations containing peptides or proteins.

[0010] Furthermore, as mentioned above, these oligo- or polysaccharide phosphates from this process have multiple applications, for example in the field of nutraceuticals and food supplements, medicine, particularly vaccinology (sugars being an essential component of the wall of certain pathogenic microorganisms), drug development, pharmaceutical and cosmetic 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 process of the present invention fulfills this need. Summary of the invention

[0012] The invention relates to the inventors' discovery of a new enzymatic activity of glycoside phosphorylase for the synthesis of phosphate sugars, which, when implemented in the process of the invention, allows the production of phosphate oligosaccharides and non-reducing phosphate polysaccharides. These molecules have multiple applications, as oligosaccharides and polysaccharides are used in numerous technological fields.

[0013] An object of the present invention is therefore an enzymatic process for the in vitro synthesis of an oligosaccharide phosphate and / or polysaccharide phosphate comprising the following steps: identification of at least one sugar-phosphate substrate of at least one glycoside phosphorylase enzyme capable of enabling the production of sugar-phosphate, the at least one glycoside phosphorylase enzyme not catalyzing the formation of 1-1 glycosidic bonds; provision of a preparation comprising the at least one glycoside phosphorylase enzyme; provision of a composition comprising the at least one sugar-phosphate substrate of said enzyme, said composition not substantially comprising any unphosphorylated sugar acceptor substrate of said at least one glycoside phosphorylase enzyme; reaction of the preparation comprising the at least one glycoside phosphorylase with the composition comprising the at least one sugar-phosphate substrate of said enzyme.obtaining a mixture comprising at least one oligosaccharide phosphate or at least one polysaccharide phosphate.

[0014] As mentioned, such a process allows for the simple and specific production of phosphate oligosaccharides or non-reducing phosphate polysaccharides without resorting to chemical synthesis processes, which present the aforementioned drawbacks of pollution, low or unsatisfactory yield and / or specificity. The identification step allows for the selection of the enzyme and its phosphate sugar substrate to implement the process of the invention.

[0015] According to other optional features of the process, the latter may optionally include one or more of the following features, alone or in combination: at least one glycoside phosphorylase enzyme belongs to a family of carbohydrate-active enzymes referenced in the CAZY.org database in the following families: GH3, GH13_18, GH65, GH94, GH112, GH130, GH149, GH161, GT35, GT108 and GH 131. Indeed, the GPs identified by the inventors are part of these CAZy classification groups; 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, BAJ 10826.1, 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, BAH 10636.1, 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, BAD80751.1, ABP51432.1, AAL81659.1, AAD28735.1, ABN51595.1, AAD03471.1, AAC06896.1, AAC00218.1, AAM24997.1, AAM 52219.1, 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, AAN 17338.1, 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, CAA85354.1, sequence SEQ ID NO: 1, sequence SEQ ID NO: 6 ; or the sequence SEQ ID NO: 7; The inventors have notably identified one or more donor phosphate sugars for each of these enzymes, as follows: • The sugar-phosphate substrate is p-glucose-1P and at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following GenBank 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 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, CAH 1385118.1, CAH 1385117.1, 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, CAH1385116.1, with SEQ ID NO: 1 or SEQ ID NO: 7 sequences, as shown in the experimental section, CAC96089.1, ABY93073.1, CBZ24449.1, CAH 1385118.1, CAH 1385117.1, CAH 1385115.1, ADD61463.1, AAD36300.1, ABY93074.1, or with SEQ ID NO: 7 sequences are particularly effective at synthesizing oligo- or non-reducing phosphorylated polysaccharide polymers of mannosyl residues with p-1,2 or p-1,3, or p-1,4 linkages and are therefore particularly preferred. • The sugar-phosphate substrate is α-glucose-1P and 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, BAJ 10826.1, 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 AAN 17338.1, 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 α-A-acetyl-D-glucosamine-1 P and at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI accession number: AAG23740.1, BAC87867.1 or ABX81671.1, the sugar-phosphate substrate is β-A-acetyl-D-glucosamine-1 P and 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 at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI accession number: ACB74662.1, ABX42289.1, AAO07997.1, ABX40964.1, ABX43387.1, EEG94248.1, ACV29689.1, ZP_05748149.1, ACZ00636.1, ABG83511.1, BAH 10636.1, ADL32981.1, or AEN95946.1, or SEQ ID NO: 6; the process further comprises a purification step of at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate, such This step is of particular interest for facilitating subsequent uses of these products of the process according to the invention; in the process of the invention, at least one glycoside phosphorylase is immobilized on an insoluble support. In the case of the use of recombinant glycosides, this has the advantage, in particular, of being able to reuse them indefinitely while immobilized on their support, as long as they exhibit the new activity identified herein at a satisfactory level. The preparation comprising at least one glycoside phosphorylase enzyme is a culture of a microorganism such as a bacterium, yeast, or fungus expressing at least one glycoside phosphorylase enzyme. The direct use of the culture of the microorganism expressing the recombinant glycoside can advantageously eliminate the need for the purification steps of the recombinant glycoside. Said reaction is carried out in solution, with a preparation of recombinant enzyme purified in solution.The use of a purified preparation of recombinant GP can allow for effective control of the parameters of the new reaction and the activity of the GP; or the maintenance of excess concentrations of at least one sugar-phosphate substrate of the GP. This makes it possible, in particular, to shift the reaction equilibrium towards the production of oligo- and / or polysaccharide phosphates and thus increase their yield and / or production. Brief descriptions of the Figures

[0016] Other features and advantages of the invention will be better understood from the description that follows and with reference to the attached drawings, given for illustrative purposes only and not for limitation.

[0017] Figure 1 is a diagram representing an embodiment of the process according to the invention. The steps shown by dotted lines may be optional.

[0018] Figure 2 shows the reaction catalyzed by glycoside phosphorylase in the process of the invention. The two sugar-phosphates are condensed to produce an oligosaccharide phosphate and an inorganic phosphate, according to direction 1 of the sugar-phosphate synthesis reaction.

[0019] Figure 3 is the result of the analysis by anion-exchange chromatography with pulsed amperometry detection (in Anglo-Saxon terminology, High Performance). Anion Exchange Chromatography with Pulsed Amperometric Detection (or "HPAEC-PAD") of the products obtained from the process of the invention using the phosphate sugar synthesis reaction catalyzed by, in A: the GP p-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 p-1,3 mannoside phosphorylase U7 (GENBANK: CAH1385117.1). For all these GPs, the donor substrate is mannose-1-phosphate (Man-1 P).

[0020] Figure 4 shows a chromatogram obtained by anion-exchange chromatography coupled with dual detection by pulsed amperometry and mass spectrometry (HPAEC-PAD-MS). The products obtained from the process of the invention using the GP MTP4-catalyzed synthesis of phosphate sugars are separated and detected as 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 allows for the detection of phosphate oligosaccharides consisting of 2 to 6 mannosyl units (DP2 to DP6). It should be noted that, for this enzyme, the PAD signal can detect non-reducing oligosaccharides whose size is estimated to extend up to 13 mannosyl units (DP13).

[0021] Figure 5 shows a chromatogram obtained by HPAEC-PAD-MS. The main products obtained from the process of the invention using the synthesis of phosphate sugars catalyzed by GP Teth1788 consist of phosphate oligosaccharides composed of 2 to 6 mannosyl units (DP2 to DP6). It should be noted that, for this enzyme, the PAD signal allows the detection of non-reducing oligosaccharides whose size is estimated to extend beyond 14 mannosyl units (>DP14).

[0022] Figure 6 includes a chromatogram obtained by HPAEC-PAD-MS. The main products obtained from the process of the invention using the synthesis reaction of phosphate sugars catalyzed by GP Lin0857 are phosphate oligosaccharides consisting of 2 and 3 mannosyl units (DP2 and DP3).

[0023] Figure 7 includes a chromatogram obtained by HPAEC-PAD-MS. The main products obtained from the process of the invention using the synthesis reaction of phosphate sugars catalyzed by GP Uhgb_MS are phosphate oligosaccharides consisting of 2 and 3 mannosyl units (DP2 and DP3).

[0024] Figure 8 includes a chromatogram obtained by HPAEC-PAD-MS. The main products obtained from the process of the invention using the reaction of GP U7 catalyzed phosphate sugar synthesis consists of phosphate oligosaccharides made up of 2 and 3 mannosyl units (DP2 and DP3).

[0025] Figure 9 shows the result of HPAEC-PAD analysis of purified phosphate oligosaccharides obtained by the process of the invention using the GP MTP4-catalyzed phosphate sugar synthesis reaction. Before purification, the phosphate oligosaccharides are mixed with normal oligosaccharides and mannose. After purification, only the phosphate oligosaccharides are detected.

[0026] Figure 10 includes a chromatogram obtained by LC-MS analysis of the main products obtained at the end of the process of the invention using the synthesis reaction of phosphate sugars catalyzed by GP ChBP: they consist of phosphate oligosaccharides made up of 2 N-acetylglucosamine units linked by a p-1,4 bond (TIC signal: total ionic current).

[0027] Figure 11 includes a chromatogram obtained by LC-MS analysis of the main products obtained at the end of the process of the invention using the synthesis reaction of phosphate sugars catalyzed by the GP GH112-SM2: they consist of phosphate oligosaccharides made up of 2 galactosyl units (TIC signal: total ionic current).

[0028] Figure 12 includes a chromatogram obtained by LC-MS analysis of the main products obtained at the end of the process of the invention using the synthesis reaction of phosphate sugars catalyzed by the GP TM 1225: they consist of phosphate oligosaccharides made up of up to 6 mannosyl units linked by a p-1,4 bond (TIC signal: total ionic current).

[0029] Figure 13 includes a chromatogram obtained by LC-MS of the main products obtained at the end of the process of the invention using the synthesis reaction of phosphate sugars catalyzed by GP U7: they consist of phosphate oligosaccharides made up of up to 3 mannosyl units linked by a p-1,3 bond (TIC signal: total ionic current).

[0030] Figure 14 includes a chromatogram obtained by LC-MS analysis of the main products obtained at the end of the process of the invention using the synthesis reaction of phosphate sugars catalyzed by GP Teth1788: they consist of phosphate oligosaccharides made up of up to 7 mannosyl units linked by a p-1,2 bond (TIC signal: total ionic current).

[0031] Figure 15 includes a chromatogram obtained by LC-MS analysis of the main products obtained at the end of the process of the invention using the synthesis reaction of phosphate sugars catalyzed by GT MTP4: they consist of of phosphate oligosaccharides consisting of up to 7 mannosyl units linked by a p-1,2 bond (TIC signal: total ionic current).

[0032] Figure 16 shows spectra obtained by NMR analysis of the unphosphorylated Man-(P-1,2)-Man dimer (denoted p-1,2-MOS (DP2)) and the Man-(P-1,2)-Man-1 P dimer (denoted p-1,2-MOS-aPi (DP2)) of the MTP4 reaction products obtained according to the process of the invention. The dashed circles represent the signals corresponding to the proton associated with the anomeric carbon of the reducing or similar end. Description of the implementation methods

[0033] The invention is described in more detail below and specified for certain embodiments. Definitions

[0034] For the purposes of this invention, an "enzymatic process for the 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 modified prokaryotic or eukaryotic organisms cultured in such a way as to allow the implementation of said enzymatic process for the in vitro synthesis of non-reducing oligo- and / or polysaccharide phosphates via appropriate expression of these enzymes. Thus, in one particular embodiment, this process can be carried out entirely within a recombinant microorganism, that is to say, one genetically modified to perform the process under suitable culture conditions. In another particular embodiment, a process according to the invention can be carried out in part, that is to say, at least the enzymatic reaction, within a living microorganism.In another particular embodiment, the process is carried out exclusively using purified or partially purified recombinant enzymes.

[0035] A glycoside phosphorylase (GP) is defined as an enzyme that catalyzes i) the degradation of glycosides using inorganic phosphate to break glycosidic bonds (phosphorolysis) and / or ii) their synthesis using phosphate sugars as glycosylated donors (in Anglo-Saxon terminology, reverse phosphorolysis) (Li a et al, 2022). The two opposing reactions of phosphorolysis and reverse phosphorolysis thus result in an equilibrium that depends on the reaction conditions. GPs share structural and mechanistic characteristics with glycoside hydrolases and glycosyltransferases. Thus, an enzyme categorized, for example in the base of CAZy data, such as GH or GT, can very well have GP-like activities. More specifically, enzymes with GP activities can exhibit sequence similarities (in Anglo-Saxon terminology, "sequence similarity networks") allowing the identification of new GPs (as, for example, according to the methodology described by Li b et al (2022)). Thus, once an enzyme is identified as a GP, either by its enzymatic activities (Li a (et al, 2022) or by sequence analogies, it can easily be tested by a person skilled in the art who can verify whether it exhibits phosphate sugar synthesis activity as discovered by the inventors. Such enzymatic tests are known to those skilled in the art, and some are mentioned in particular in the experimental section.

[0036] Table 1 lists examples of glycoside phosphorylases; the Genbank number refers to the peptide sequence of the protein with a phosphate sugar substrate usable in the phosphate sugar synthesis reaction. Table 1

[0037] The GP named G1-8A: p-(1,3)-mannose phosphorylase (G1-8A_GL0038729 locus= scaffold17912_1:1303:2508) in Table 1 has the following protein sequence (SEQ ID NO: 1): MHKPIIRYSPEPVLEPKEGCIWADTMVLNPAIVKDPYSDTIHMLFRATGPWPAKNLKGKS DPYPIFLGYAKSDDRGETWQPDFGRPALAPALEYEMDKMYIRDIYDREWNYANGCVED PRIFEVEGELYLTTACRMFPPPGPYWEGDKRRDNIPDWAMEPDNPFGTTASRNDTTTVLF KLDLDKLKSREYERAFGYVCNLTDGNVTANRDVFLFPRKMKIDGKMRYVMLHRPENPDK FEAGKGIYKPAIFLAAAEKPEDFCTGKCMHKLLAKGIFDWEEERIGASFPPVEIESGKWLI SYHGKQMPDYGYTQSFMIVEEQEDDFPLIIHRCPERLMYARQKWELPDKFACPCIFTSG GIVMDEELVMSYGAADQKVGLARANLEEIVDYVSSFDENGNEIELE.

[0038] The GP named GH112_SM2: GalNAc / GIcNAc phosphorylase in Table 1 has the following protein sequence (SEQ ID NO: 6): MSKGRVTIPTDANFVDGTKKLLQYWGADAVRDCDGVSLPADVRQFGCDVYKAYFIVRE DHEYALAHKEYWQNVALTTDRITAKSDALDVDLLANTFRESLEVNTLDYKKYWQVIDRTD GTIHADWQYLGNNIVRITNCKLYHEYTVNFFAVNTWDPVQIYNYHVNNWHVHKDIDLDPV YPEALAHMLSRMESWLKSNPDVTWRFTTFFYNFFIVNVTGLKQRIWDWHCYAMTASPA MFELFSAETGREITLEDIVDGGYYSNRFRIPNPTMRAYVDFVQRKCADWAKLFVDLCHKY GKKAIMFDGDHRIGVEPYSPYFANIGLDGVVGAPSSAIYVQQIANMQGIDFTEGRLNPYFF PNECPGDERGTQILLNCWNSMRRGMLKKPIDRIGFGGYLKQVAEYPTFVKAVRQVCDEF REIRTNVGKTSCKSKGKIAVLSYWGRRDSWMLNGTFVDDAGQGACYYWSILAALAVLPF DVDFLSFDDVLEGKVDGYDIWSCGLTGTSFHGDKCWANPDLLTAIRRFVDNGGGFVGV GEPSGYQRQGRFIQLSDVLGVERECDFRHFEKRDDFDIVPEHWITQGVDMSLVGFNRFV RNIYPVGATVLAADYDWEYPKGSQNAGNVHFAVNEYGNGRSVYLSGITANNQTIRLLYR AFLWAMRGEDKTKYNYAESADVDVFFYEQSGKYALFNDSDNAVETTYYDVDGNKRSVT LGAKEIKWL.

[0039] La GP nommée U8 (P-1 ,2-mannoside phosphorylase) dans le tableau 1 a pour séquence protéique (SEQ ID NO: 7) : MVHVKREGVILERTSLEFENEGVMNPAVIAEGNTVHMFYRAVKKGNYSSIGYCRLEGPLT WERDVAPLLFSEFEYECQGVEDPRIVKIEDLYYLTYTAYDGYSAVGALAVSSDLRHFEK KGIITSQITYPQFREQLRTQQAIISKYFRSGNKREATNEKGHPLYMMDKNWFFPRKINGK FYFMHRIRPDIQYVAVERFDMLTKEFWKRYYQNFAKRILLAPYHDHESSYIGAGCPPIETK AGWLLIYHSVYDTPSGYVYSACAALLDIDNPAIEIARLPYPLFTPEAEYELSGVVNRVCFPT GTALFGDTLYI YYGAADKYVACVSVQLTELLDELI KQI K.

[0040] 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-1 P, α-glucose-1 P, α-mannose-1 P, α-N-acetyl-D-glucosamine-1 P, p-glucose-1 P, pN-acetyl-D-glucosamine-1 P.

[0041] "Polynucleotide(s)", "oligonucleotide(s)", "nucleic acid(s)", "Nucleotide(s)," "polynucleic acid(s)," or any equivalent term 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-natural or synthetic nucleotides, as well as nucleotide analogs. Nucleic acid sequences and vectors, for example, expression vectors, can be introduced into a cell, for example, by transfection, transformation, or transduction.The nucleotide sequence encoding a GP, as listed in Table 1 above, can be optimized depending on the host in which the recombinant GP is produced and, for example, on its genetic code and codon usage specific to that host. This nucleotide sequence can be either integrated into an expression vector or integrated into the host organism's genome. GP expression. Recombinant expression can be either inducible or constitutive. Preferably, recombinant GP expression is inducible.

[0042] “Expression vector” means any expression vector known to the person skilled in the art to be suitable for the expression of protein-coding genes in microorganisms and, more specifically, suitable for the overproduction and possible 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 be able to select the appropriate vector, culture conditions, and purification system based on the recombinant GP produced and the host microorganism. 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 those listed in online catalogs or described at the following websites: - https: / / france.promega.com / products / vectors / protein-expression-vectors, https: / / www.thermofisher.com / search / browse / category / fr / fr / 85801 / Bacterial%20Expre ssion%20Vectors, - https: / / www.embl.de / pepcore / pepcore_services / strains_vectors / vectors / bacterial_expre ssion_vectors / popup_bacterial_expression_vectors / index.html, - http: / / babel.ucmp.umu.se / cpep / web_content / Pages / CPEP_09_vectors.html, ou - https: / / www.embl.de / pepcore / pepcore_services / strains_vectors / vectors / gateway_vecto rs / index.html.

[0043] This could be, for example, the expression vector described in document WO 83 / 004261. It could be, for example, the pWKS130 plasmid described by Wang and Kushner (1991); the plasmid marketed by Thermo Fisher under the trade name pBAD-HisA; the plasmid marketed by Thermo Fisher under the trade name pTRC. It could be, for example, a plasmid chosen from the group including pBAD-HisA, pTRC, pMX, pUC19, pHP45-CmR, pcDNA3.1 (+), pcDNA3.3-TOPO, pcDNA3.4-TOPO, pFastBad, pET100 / D-TOPO, pET151 / D-T0P0, pRSET A, pYes2.1V5-His TOPO, pDONR221, pGEX-3X; pBluescript, pET, pGEX, pLys, pRARE, pDEST, pETG, preferably the pBAD-HisA plasmid

[0044] As mentioned, the vector can be any suitable yeast artificial chromosome (YAC) known to those skilled in the art and / or commercially available. For example, it could be a yeast artificial chromosome chosen from the group including pYAC-RC and pYAC3. The vector can also be any A bacterial artificial chromosome (BAC) adapted for expression in bacteria, known to those skilled in the art and / or commercially available. For example, it could be a bacterial artificial chromosome chosen from the group including pUvBBAC, pCCI BAC, and pBAC 108L.

[0045] The promoter can be any promoter known to a person skilled in the art that allows the expression of recombinant proteins in a microorganism such as a bacterium, yeast, or fungus, or even a cell of a higher eukaryote, preferably a bacterium. It could be, for example, a constitutive promoter or an inducible promoter. For example, it could be a constitutive promoter chosen from the group including the CMV, EF1A, or SV40 promoters. It could be, for example, a constitutive promoter listed on the webpage http: / / parts.igem.org / Promoters / Catalog / Constitutive, or any promoter of the E. coli K-12 strain known to a person skilled in the art, for example, a promoter chosen from among the 3908 E. coli K-12 strain promoters listed on the biocyc.org website: https: / / biocyc.org / group?id=:ALL-PROMOTERS&orgid=ECOLI. This could be, for example, an inducible promoter, such as pBAD or pLac.This could be, for example, an inducible promoter described and / or mentioned on the page http: / / parts.igem.org / Promoters / Catalog. A person skilled in the art will be able to choose the promoter based on the nucleic acid sequence to be expressed and / or the host cell, and more generally, based on the chosen system and expression conditions.

[0046] Recombinant protein production is now a common technology in the field of biotechnology and a routine operation for those skilled in the art who know how to determine production conditions (such as the production organism (like a bacterium, yeast, fungus, or even a higher eukaryote), genetic sequence optimization, culture conditions, purification conditions, and the choice and placement of a possible tag) based on the enzyme in question. A synthesis of the different possible strategies is reported by Tripathi and Shrivastava (2019). Typically, as illustrated by experimental examples, the GP expression host is E. coli BL21, and the expression vector is a plasmid of the pBAD or pET type, inducible by arabinose and lactose (and its derivatives such as IPTG or allolactose), respectively.The purification tag is most often a 6His tag, which can be positioned at the C-terminus and / or N-terminus of the recombinant protein. This tag can be introduced directly into the synthetic gene sequence or be present initially in the expression vector. Recombinant enzyme purification is typically performed by cobalt affinity chromatography (e.g., TALON® Metal Affinity Resin, Takara).

[0047] A "polysaccharide" or "oligosaccharide" is defined as a linear or branched polymer of monosaccharide units linked together by α- or β-glycosidic bonds. By convention, in the art, an oligosaccharide comprises between 2 and 10 glycosylated units, and a polysaccharide is defined as a polymer of glycosylated units comprising more than 10 units. More specifically, in connection with the new activity identified by the inventors, a phosphate polysaccharide or phosphate oligosaccharide is defined as a polymer of glycosylated units linked together by one or more α- or β-glycosidic bonds, and whose reducing end is linked to an inorganic phosphate by an α- or β-glycosidic bond. Thus, such a phosphate polysaccharide or phosphate oligosaccharide does not have a reducing end.In other words, the phosphate polysaccharide or phosphate oligosaccharide produced by the process according to the invention does not contain a free hemiacetal group, as the hydroxyl group of the latter is involved in a bond with a phosphate group. As demonstrated in the experimental part, the process of the invention can result in the production of a mixture of phosphate polysaccharides or phosphate oligosaccharides that differ in the number of monosaccharide units they contain.

[0048] A "1-1 glycosidic bond" is a bond involving the hemiacetal hydroxyl residues of two sugars. Clearly, a GP catalyzing the formation of 1-1 glycosidic bonds will not be able to produce, during the sugar-phosphate synthesis reaction identified by the inventors, polysaccharide phosphate from a sugar-phosphate preparation, since the reducing ends of both sugars are involved in the glycosidic bond. Such activity can easily be identified using the methods described, for example, in the experimental section, or from the data recorded in the CAZy database (<CAZY.org> ).

[0049] As mentioned, the invention lies in the characterization of an activity hitherto unknown for GPs, which is the condensation of sugar-phosphates, to synthesize a non-reducing oligosaccharide phosphate or polysaccharide phosphate, as illustrated more particularly in Figure 2.

[0050] An object of the invention therefore lies in an enzymatic process for the in vitro synthesis of an oligosaccharide phosphate or polysaccharide phosphate 1 as illustrated in Figure 1, comprising the steps of: Provision of a preparation comprising at least one GP 10b enzyme that does not catalyze the formation of 1-1 glycosidic bonds:

[0051] The preparation may comprise or consist of a protein preparation enriched in glycoside phosphorylase. More specifically, the preparation comprising at least one glycoside phosphorylase is obtained by purifying the glycoside phosphorylase from a culture lysate or culture supernatant of a microorganism expressing the glycoside phosphorylase. Thus, in one embodiment, the preparation comprising at least one glycoside phosphorylase enzyme is a preparation in which the at least one glycoside phosphorylase represents at least 80%, at least 85%, and preferably at least 90% of the protein fraction of the preparation. This can be readily determined by measuring the specific activity of the glycoside phosphorylase in the preparation.

[0052] Alternatively, the preparation may consist of culturing a microorganism expressing the GP. The gene encoding the GP may be inserted into the host organism's genome to achieve its overexpression, either in the microorganism's chromosome or in an expression vector as described above. The microorganism may be a bacterium, a yeast, or a fungus, according to preferences determined by a person skilled in the art. However, in this embodiment, bacteria are particularly preferred.

[0053] The preparation may also consist of an unpurified culture supernatant. Alternatively, the preparation may consist of an unpurified cell lysate.

[0054] In another embodiment, the preparation may include the enzyme immobilized on a support. This offers the advantage of allowing the enzyme to be reused after the reaction is complete by recovering the insolubilized enzyme, for example, by filtration, sedimentation (passive, or, for example, by applying a magnetic field in the case of using a magnetic support for the enzyme), or centrifugation of the reaction mix, or even by continuous use. To implement this strategy, one can refer, for example, to the review by Sheldon et al. (2021) or to Marian Lujan Ferreira's manual "Biocatalyst immobilization: foundations and applications" (Elsevier, 2022), which lists the various immobilization strategies for enzymatic processes. One can also refer to Zhong et al. (2020), who focus specifically on GP immobilization.

[0055] This preparation can also include several different GPs, i.e., 2, 3, or more. This allows for the production of phosphate sugars linking different glycosylated units, or mixtures of phosphate sugars after the process has been implemented.

[0056] Thus, in a particular embodiment, the preparation of at least one glycoside enzyme GP comprises at least two GPs selected from the enzymes referenced under NCBI or UNIPROT accession numbers 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, BAH 10636.1, 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, CAH 1385118.1, CAH 1385117.1, CAH 1385115.1, CAH1385116.1, ADD61463.1, BAD80751.1, ABP51432.1, AAL81659.1, AAD28735.1, ABN51595.1, AAD03471.1, AAC06896.1, AAC00218.1, AAM24997.1, AAM 52219.1, 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, AAN 17338.1, 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, CAA85354.1, SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO: 7.

[0057] In one embodiment, at least one glycoside enzyme GP is selected from the enzymes referenced under 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, BAJ 10826.1, 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, BAH 10636.1, 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, CAH 1385118.1, CAH 1385117.1, CAH 1385115.1, CAH 1385116.1, ADD61463.1, or BAD80751.1 of sequence SEQ ID: NO 1, of sequence SEQ ID NO: 6 or of sequence SEQ ID NO: 7.In a particular embodiment, the preparation of at least one glycoside enzyme GP 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, AAQ84039.1, 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, AAQ80764.1, 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, BAJ 10826.1, 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, BAH 10636.1, 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 of sequence SEQ ID NO: 1, of sequence SEQ ID NO: 6 or of sequence SEQ ID NO: 7.

[0058] In another embodiment, at least one glycoside enzyme GP 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, AAQ84039.1, 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, BAJ 10826.1, 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, BAH 10636.1, 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 SEQ ID NO: 1. In a particular embodiment, the preparation of at least one glycoside enzyme GP comprises at least two GPs selected from the enzymes referenced under The number of accessions NCBI or UNIPROT: 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, BAH 10636.1, 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, CAH 1385118.1 , CAH 1385117.1, CAH 1385115.1 , 6HQ6, or CAH1385116.1 or SEQ ID NO: 1.

[0059] In another particular embodiment, the preparation of at least one glycoside enzyme GP comprises at least two glycoside phosphorylase enzymes, each belonging to a different family of carbohydrate-active enzymes referenced in the CAZY.org database and selected from: GH3, GH13_18, GH65, GH94, GH112, GH130, GH149, GH161, GT35, GT108 and GH131.

[0060] In another particular embodiment, the preparation of at least one glycoside enzyme GP comprises at least one glycoside phosphorylase enzyme belonging to a family of carbohydrate-active enzymes referenced in the CAZY.org database in one of the following families: GH94, GT108, GH112, or GH130. In another particular embodiment, the preparation of at least one glycoside enzyme GP comprises at least two glycoside phosphorylase enzymes, each belonging to a different family of carbohydrate-active enzymes referenced in the CAZY.org database and selected from: GH94, GT108, GH112, or GH130. • Supply of a composition comprising at least one sugar-phosphate substrate of said enzyme 10c, said composition not substantially comprising any unphosphorylated sugar acceptor substrate of said at least one glycoside phosphorylase enzyme.

[0061] Sugar-phosphate substrate refers to an aldose or ketose to which a phosphate group is attached on the hemiacetal function. In a particular embodiment, sugar-phosphate substrate refers to an oligo- or polysaccharide phosphate. 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 it comprises (excluding the hydroxyl group of the hemiacetal function); for example, and without limitation, 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 oligo- or polysaccharide phosphates bearing these functionalizations.

[0062] Preferably, said composition does not substantially comprise, nor does the enzyme preparation itself, any unphosphorylated sugar substrate acceptor of at least one GP; this ensures maximum yield of non-reducing phosphorylated oligosaccharide or polysaccharide. Thus, preferably, said composition comprising at least one sugar-phosphate substrate of said enzyme (10c) does not substantially comprise any unphosphorylated sugar substrate acceptor of at least one GP. Said composition may comprise one unphosphorylated sugar substrate acceptor of the GP; however, in this case, in said composition, the at least one sugar-phosphate substrate of the GP may represent at least 70% of the GP substrate sugars present in the composition, preferably at least 80%, even more preferably at least 90%, and particularly preferably at least 95% of the GP substrate sugars present in the composition.Of course, the composition may include without restriction other non-GP substrate sugars, phosphorylated or not.

[0063] In one particular embodiment, the composition comprising at least one sugar-phosphate substrate includes two, three, or even more sugar-phosphate substrates. Indeed, although most often very specific, some glycosidic compounds (GPs) can accept different sugar-phosphate substrates. That is to say, an enzyme can catalyze the reaction using one, two, or even more different sugar-phosphate substrates, resulting, for example, after carrying out the process according to this particular embodiment, in the production of a mixture of different oligo- or polysaccharide phosphates.

[0064] In a particular reaction mode, the sugar-phosphate can be selected by mi: α-mannose-1 P, pN-acetyl-D-glucosamine-1 P, p-glucose-1 P, α-glucose-1 P, α-N- acetyl-D-glucosamine-1P, α-galactose-1P, or mixtures thereof. In another particular reaction mode, the sugar-phosphate can be selected from: α-mannose-1P, α-acetyl-D-glucosamine-1P, α-galactose-1P, or mixtures thereof. • Reaction 20 of the preparation comprising at least one glycoside phosphorylase with the composition comprising at least one sugar-phosphate substrate of said enzyme,

[0065] "Reaction" means the introduction of at least one GP and at least one sugar-phosphate substrate of said GP and the provision of conditions favorable to the sugar-phosphate synthesis reaction as described in Figure 2.

[0066] In one particular embodiment, the sugar-phosphate substrate is α-mannose-1 P and 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, CAH 1385118.1, CAH 1385117.1, BAJ 10826.1, 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, CAH 1385115.1, or CAH1385116.1.

[0067] In one particular embodiment, the sugar-phosphate substrate is α-mannose-1 P and 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, CAH 1385118.1, CAH 1385117.1, BAJ 10826.1, 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, CAH 1385115.1 , or CAH1385116.1, ADD61463.1, or SEQ ID NO: 7.

[0068] In another particular embodiment, the sugar-phosphate substrate is α-galactose-1 P and 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, BAH 10636.1, ADL32981.1 or AEN95946.1.

[0069] In another particular embodiment, the sugar-phosphate substrate is α-galactose-1 P and 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, BAH 10636.1, ADL32981.1, AEN95946.1, or of SEQ ID NO: 6.

[0070] In another particular embodiment, the sugar-phosphate substrate is α-glucose-1P and 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, or BAU78234.1.

[0071] In another particular embodiment, the sugar-phosphate substrate is α-glucose-1P and 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, AAM 52219.1, 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, AAN 17338.1, 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 setting also includes the supply of an acceptor oligo- or polysaccharide of formula maltodextrin [(1,4)-aD-glucosyl]n.

[0072] A skilled person will know how to optimize the reaction conditions (such as temperature, salt concentration, pH, and the concentration of the sugar-phosphate substrate in the glucose solution) to ensure optimal yield of the sugar-phosphate synthesis reaction. For example, the reaction buffer used can be chosen from the list of buffers available on the following websites: https: / / hamptonresearch.com / uploads / cg_pdf / Hampton_Research_CG101_Buffer_Table.pdf, or https: / / www.sigmaaldrich.com / FR / fr / support / calculators-and-apps / buffer-calculator, which allows selection based on the desired optimum reaction pH, which may vary depending on the glucose solution used.

[0073] 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.

[0074] 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.

[0075] The reaction can be carried out in a closed container, for example, in a closed reactor, without exchange with the external environment, in which the two compositions react. This can be done in a stirred tank reactor, using, for example, a propeller homogenizer to maintain a homogeneous mixture of the enzyme and the reaction mixture. Any other method for maintaining the homogeneity of the mixture of the preparation and the composition can be used.

[0076] Advantageously, when the GP is immobilized, and to prevent degradation of the immobilized enzyme due to shear forces associated with agitation, the immobilized enzyme can be attached to a support through which or over which a flow of reaction mix (here, for example, the reaction buffer and the composition including the sugar-phosphate substrate of the GP) is carried. Alternatively, still in this embodiment of a flow reactor, the immobilized enzyme is not attached to 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), flows.

[0077] The reactor can 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, greater than or equal to 10000 L.

[0078] Examples of bioreactor design applicable here are provided by Sheldon et al (2021).

[0079] Advantageously, in a particular embodiment, the container in which the reaction takes place is equipped with a means for ensuring that the reaction equilibrium shifts in favor of the synthesis of oligo- or polysaccharide phosphate(s). This can be achieved, for example, by ensuring the removal of the free inorganic phosphate generated by the reaction (e.g., by using filtration means to remove the inorganic phosphate from the reaction medium or means to precipitate the inorganic phosphate formed) and / or the oligo- or polysaccharides produced, or by ensuring a high excess of the sugar-phosphate substrate(s) of at least one GP in the reaction vessel, for example, by continuously supplementing the reactor with the sugar-phosphate substrate(s) of at least one GP. In a particular embodiment, the inorganic phosphate is precipitated using, for example, calcium salts. • Obtaining 30 of a mixture comprising at least one oligosaccharide phosphate and / or at least one polysaccharide phosphate.

[0080] As illustrated in Figure 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 linked to an inorganic phosphate, and inorganic phosphate as a reaction product. • Optional purification step 40 of at least one oligosaccharide phosphate or at least one polysaccharide phosphate.

[0081] In a particular embodiment, said enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate or at least one polysaccharide phosphate 1 may further comprise a purification step of the oligo- or polysaccharide phosphate produced by the process of the invention. During this step, the oligo- or polysaccharide phosphate produced may be purified of inorganic phosphate, unphosphorylated oligosaccharides, and monosaccharides by calcium precipitation followed by ethanol precipitation, the result of which is illustrated in Figure 9. By For example, the introduction of an excess of Ca2+ ions into the reaction medium (e.g., 5 mol of Ca2+ / 1 mol of Man-1 P, the initial substrate of the reaction) causes the inorganic phosphate to precipitate 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 that can be easily removed afterward. 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 unphosphorylated oligosaccharides and monosaccharides remain soluble in the solution. After centrifugation, the pellet is washed with 85% ethanol and then dried under a stream of nitrogen. The pellet is then rehydrated and dried again in the rotary evaporator.Finally, the reaction products are lyophilized for optimal preservation and stability. Techniques for separating oligo- and / or polysaccharide phosphates can be applied to select one or more oligo- and / or polysaccharide phosphates with a specific degree of polymerization. • Optional step of sugar-phosphate substrate production of at least one GP.

[0082] In another particular embodiment, said enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate or at least one polysaccharide phosphate may further comprise a step of producing sugar-phosphate substrate for 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).

[0083] Preferably, the sugar-phosphate substrate of at least one GP is synthesized enzymatically, which has the advantages already listed. Thus, the sugar-phosphate substrate of 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 p-glycosides) in the presence of inorganic phosphate; by 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 which will catalyze the phosphorolysis of a disaccharide to produce a sugar 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.

[0084] This additional enzymatic step in the synthesis of the sugar-phosphate substrate of GP can be carried out externally using purified enzymes that will potentially catalyze successive reactions (as illustrated, for example, in Zhong et al. (2020)). Alternatively, a microorganism can be genetically modified to express the complementary enzyme(s) enabling it to produce, from a polysaccharide substrate, at least one desired oligosaccharide phosphate or at least one desired polysaccharide phosphate (as illustrated, for example, in application WO 2021 / 229185 or the article by Tian et al. (2020)). • Preliminary step of testing the synthesis activity of oligo- or polysaccharide phosphate

[0085] In a particular embodiment, said enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate or at least one polysaccharide phosphate 1 may further comprise an identification step 10a of the at least one sugar-phosphate substrate of at least one GP, said at least one GP not catalyzing the formation of 1-1 glycosidic bonds, capable of enabling the production of sugar-phosphate. Any test enabling the detection of one of the products of the reverse phosphorolysis reaction of the invention, as identified in Figure 2, is suitable and equivalent, insofar as it enables the identification of the activity identified by the inventors and underlying the invention.In other words, any method capable of detecting the production of inorganic phosphate and / or oligosaccharide phosphate or polysaccharide phosphate (e.g., any thin-layer chromatography technique) when the GP is brought into contact with a preparation containing only a sugar-1-phosphate as an enzyme substrate (i.e., no acceptor sugar, oligosaccharide, or polysaccharide) is suitable. Thus, any test known to those skilled in the art that detects the release of phosphate ions (inorganic phosphate) when a GP is brought into contact with a preparation of a sugar-phosphate substrate for said GP is suitable. For example, such a step, which may include a test like the one implemented by Macdonald et al. (2019), or illustrated in the experimental section, is particularly advantageous because it is quick and easy to perform. Therefore, in this embodiment, the enzymatic process may include... Prior to this, a test such as the molybdenum blue test detailed below is performed. This test, by contacting the GP with a preparation containing only a sugar-1-phosphate as a substrate, allows for the rapid detection of the release of inorganic phosphate resulting from the oligosaccharide phosphate or polysaccharide phosphate synthesis activity identified by the inventors, and thus for the identification of the sugar-phosphate substrate of the GP that enables this reaction. The tested enzyme exhibiting the reverse phosphorolysis activity identified by the inventors, and its sugar-phosphate substrate, are then selected to implement the process of the invention.

[0086] This preliminary testing step can also be carried out independently, i.e. separated in time from the subsequent steps of: supplying a preparation comprising at least one glycoside phosphorylase enzyme 10b; supplying a composition comprising at least one sugar-phosphate substrate of said enzyme 10c, reacting 20, and obtaining 30 a mixture comprising at least one oligosaccharide phosphate or at least one polysaccharide phosphate,

[0087] For example, in a particular embodiment the preliminary step is a high-throughput screening of enzyme preparations for the identification of glycoside phosphorylase enzyme possessing reverse phosphorolysis activity discovered by the inventors, the subsequent steps of the process of the invention being carried out using at least one previously identified glycoside phosphorylase enzyme.

[0088] Alternatively and / or in addition, the process of the invention may also include a GP characterization step as described below. • Optional step of GP characterization.

[0089] In another particular embodiment, said enzymatic process for the in vitro synthesis of at least one oligosaccharide phosphate or at least one polysaccharide phosphate may further comprise a step of characterizing the GP; for example, as mentioned above, it may be an enzyme for which no activity has been formally identified but which shares structural and mechanistic characteristics with glycoside hydrolases and glycosyltransferases, such as peptide sequence similarities. In other words, a step of identifying, for a given enzyme, the activities characteristic of a GP, namely the phosphorolysis of oligosaccharides and polysaccharides and / or reverse phosphorolysis as described above and detailed in Li et al. (2022). For example, such a step may include a test such as the one implemented by Macdonald et al. (2019). This characterization step may therefore include the identification of GP donor and acceptor sugars as explained in the experimental section. Experimental results i. Production of recombinant GPs Table 2: Recombinant glycoside phosphorylases produced and tested in vitro for their phosphate sugar synthesis activity. Table 2 Cloning

[0090] The gene encoding the Teth514-1788 enzyme of the Thermoanaerobacter sp. X-514 strain (Teth514-1788, Genbank accession number ABY93073.1) belonging to the GH 130 family of the CAZy classification (http: / / www.cazy.org / ) was synthesized and cloned into the pBAD HisA vector by Biomatik (Biomatik, Ontario, Canada) between the Ncol and Xhol restriction sites.

[0091] The gene encoding the Lin0857 enzyme of the Listeria innocua strain Clip 11262, (Genbank accession number CAC96089.1) belonging to the GH 130 family of the CAZy classification was synthesized by TWIST Bioscience (TWIST, San Francisco, USA) and cloned between the Ncol and Xhol restriction sites, in the pBAD-HisA plasmid.

[0092] The gene encoding the Uhgb_mS enzyme (Genbank accession number CAH1385118.1), belonging to the GH130 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 pET-23a (+) plasmid with a 6 (His) tag at the N- and C-terminal ends.

[0093] The gene encoding the U7 enzyme (Genbank accession number CAH 1385117.1) 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 Ndel and Xhol restriction sites in the pET-23a (+) plasmid with a 6 (His) tag at the N- and C-terminal ends.

[0094] The gene encoding the MTP4 enzyme from 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 Integrated DNA Technologies (Munich Airport, Germany) and cloned by the "in fusion" method into the pET-28 plasmid with a 6 (His) tag at the C-terminus using these primers to amplify the MTP4 gene and the pET-28 plasmid respectively: Fw: SEQ ID NO: 2: 5'-AGGAGATATACCATGAAGCAGACTAAGGCGTCCTTTGAGGCTAACAAACG-3'. Rev: SEQ ID NO: 3: 5'-GTGGTGGCTGCTGCCCAGGGATGAGGCCATCGGGAAATTCAGGTCTC-3' Fw: SEQ ID NO: 4: 5'-GGCAGCAGCCACCACCACCACCACCACTGAGATC-3' Rev: SEQ ID NO: 5: 5'-CATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAG-3' Equivalent cloning methods were used for the other GPs tested. Expression

[0095] The recombinant glycoside phosphorylases of the CAZy family, GH 130 and GT108, listed in Table 2, were produced in E. coli BL21. Enzymes placed on pET vectors were produced in E. coli BL21 (star), while those on pBADs were produced in BL21-AI. The production protocol was then identical for all five glycoside phosphorylases. A 50 pL volume of previously rendered competent cells was transformed with 1 pL of plasmid DNA containing the nucleotide sequence of the enzyme to be produced. After incubating the cells with the plasmid for 30 min, the transformation was carried out by heat shock at 42°C for 30 sec. After 1 hour of phenotypic expression in SOC medium at 37°C with 200 rpm of agitation, the cells were used to seed 20 mL of LB medium supplemented with the antibiotic of selection and then incubated overnight at 37°C at 200 rpm.The following day, the preculture was used to inoculate 200 mL of ZYM self-inducible medium supplemented with the selection antibiotic at an OD of 0.05. For the pET vectors, the ZYM medium contained lactose for plasmid induction, while it contained arabinose for the induction of genes carried by the pBAD vector. The cells were incubated for 24 h at 21 °C at 160 rpm. At the end of the culture, the cells were recovered by centrifugation (15 min, 10,000 g).

[0096] Any other organism and / or common technique for the production and purification of recombinant proteins may be used. Purification The purification of the protein proteins (PPs) was performed according to the same protocol. Cell pellets corresponding to 100 mL of culture were resuspended at OD = 100 in Tris-HCl (50 mM) + NaCl (350 mM) buffer at pH = 7.5. The cells were then lysed under cold conditions by sonication. The total protein fractions obtained were centrifuged under cold conditions for 30 min at 12,000 g, and only the soluble protein fractions were retained. The enzymes Tagged 6His were purified from other bacterial proteins by cobalt affinity chromatography (TALON® Metal Affinity Resin, Takara). The total fractions were incubated with the cobalt resin for 1 h at 4°C. Final elution was performed in Tris-HCl (20 mM) + NaCl (100 mM) + Imidazole (300 mM) buffer equilibrated to pH 7.5. The recombinant glycoproteins were then desalted on a PD-10 desalting column (GE Healthcare) according to the manufacturer's protocol. Final elution was performed with Tris-HCl (20 mM) + NaCl (150 mM) buffer equilibrated to pH 7.5. ii. Identification of the donor and recipient sugars

[0097] CAZy data (<http: / / www.cazy.org> ) identify the substrates of the enzymes identified in this database.

[0098] The donor and acceptor substrate sugars for a putative glycoside phosphorylase can easily be identified using, for example, chromogenic tests that detect the presence of inorganic phosphate released by the donor sugar during the oligosaccharide synthesis reaction by the glycoside phosphorylase.

[0099] An example is the molybdenum blue test (also known as the molybdate test).

[0100] In short, this test is based on the formation of phosphomolybdenum in the presence of the reagent molybdate. Under acidic conditions, phosphomolybdate is reduced to molybdenum, the blue color of which is the reactant in the reaction. The intensity of the blue color can be quantified by measuring the absorbance at 655 nm.

[0101] For example, the reaction medium may contain 45 pL of purified enzyme for a final concentration of 0.03 mg.ml' 1In 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 transferring 50 µL aliquots to 96-well microplates 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 a stop solution [2% (v / v) acetic acid and 2% (w / v) tribasic sodium citrate dihydrate] is added to halt 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 test conditions.

[0102] This method is suitable for detecting GP-catalyzed activity produced in crude E. coli cell extracts and detailed by Macdonald et al. (2019), allowing for the screening of a large number of extracts, donor sugars, and acceptors. iii. Testing of oligo- or polysaccharide phosphate synthesis activity

[0103] The synthesis reaction with sugar-1-phosphate as the sole substrate can be carried out under conditions determined by the physicochemical characteristics of the GP tested, in particular its optimal conditions of activity and stability (temperatures, pH, salinity, etc.).

[0104] By adapting the molybdate test, using only a sugar-1-phosphate as a substrate, it is possible to detect potential oligosaccharide phosphate or polysaccharide phosphate synthesis activity. Confirmation by HPAEC-PAD can then be performed to confirm the nature of the synthesized products. Of course, any other test that allows the detection of oligophosphate synthesis products as discovered by the inventors—namely, inorganic phosphate, oligosaccharide phosphate, or polysaccharide phosphate—is equivalent and can be used. A person skilled in the art will be able to identify any direct or indirect method for detecting enzymatic activity.

[0105] The enzymes in Table 2 were tested under similar conditions. Specifically, the reactions were carried out in a 500 pL volume at 37°C with 900 rpm stirring. 5 pM of each enzyme was incubated with 75 mM mannose-1-phosphate (Man-1 P) for 20 h. For the enzymes Lin0857, MTP4, Uhgb_MS, U7, Chbp, GH112_SM2, Uhgb_MP, TM1225, U1, U8, and Teth1789, the reaction medium was at pH 7 in 100 mM Tris-HCl buffer. For the enzyme Teth1788, the reaction medium was at pH 5 in 100 mM sodium acetate buffer. 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 or LC-MS. iv. Synthesis reaction of oligo- or polysaccharide phosphate

[0106] 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”) or LC-MS. v. Detection and characterization of synthesized oligo- and / or polysaccharide phosphates

[0107] Reaction products obtained with the tested GPs were initially analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD, Figure 3). 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 with 75 mM NaOH, 2) 1-min gradient from 75 to 150 mM NaOH, 3) 1-min isocratic phase with 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. The detection was carried out with a Dionex ED40 module operating with a gold electrode and an Ag / AgCl pH reference.

[0108] The reaction products obtained with these GPs were subsequently analyzed by anion-exchange chromatography coupled with a pulsed amperometry detector and a mass spectrometer (Mass Spectrometer, "HPAEC-PAD-MS", Figures 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 with 75 mM NaOH, 2) 1-min gradient from 75 to 150 mM NaOH, 3) 1-min isocratic phase with 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 with 125 mM sodium acetate. Amperometric detection was performed with a Dionex ED40 module using a gold electrode and an Ag / AgCl pH reference. Molar mass detection was performed with an ISO EC mass spectrometer (Thermo Scientific).Two Dionex AERES 500e 2mm desalination 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 as follows: vaporization temperature: 289°C, ion transfer tube temperature: 300°C, positive ion source voltage: 3000 V, negative ion source voltage: -2000 V, nitrogen pressure: 55.3 psig, polarity: negative, mass analyzed: 175 to 1250, analysis time: 0.2 sec, spectrum type: centroid, CID source voltage: 5. The molar masses extracted from the overall signal were smoothed using a 301-point Gaussian function.

[0109] Phosphate polysaccharides and / or phosphate oligosaccharides can also be detected and characterized by LC-MS analyses (Figures 10 to 15). These analyses were carried out according to the following arrangements: Chromatographic separation was performed on an Asahipak NH2P-50 column (Shodex) using a Dionex UltiMate-3000 high-performance liquid chromatography (HPLC) system with a column temperature set at 40°C.With a flow rate of 0.950 mL / min of mobile phase containing a constant proportion of 10% acetonitrile, the following protocol was used: 1) 1 min isocratic phase with 50 mM ammonium formate pH 4.4; 2) 5 min gradient from 50 to 100 mM ammonium formate pH 4.4; 3) 5 min gradient from 100 to 200 mM ammonium formate pH 4.4; 4) 10 min isocratic phase with 200 mM ammonium formate pH 4.4; 5) 5 min gradient from 200 to 260 mM ammonium formate pH 4.4; 6) 9 min isocratic phase with 260 mM ammonium formate pH 4.4; 7) 1 min gradient of 260 to 50 mM ammonium formate pH 4.4; 8) 5 min isocratic phase with 50 mM ammonium formate pH 4.4.Oligomer detection was performed using a mass spectrometer (ISQ EC Mass Spectrometer (Thermo Fisher)) configured as follows: vaporization temperature 340°C; negative / positive voltage source voltage -3000 / 3000 V; negative ionic polarization; mass-to-charge range (m / z) 200 - 1500; CID voltage source voltage 50 V. The mass-to-charge ratios (m / z) extracted for ionized mannosides-1-phosphate, glucosides-1-phosphate, and galactosides-1-phosphate are [MH]-: 259 (DP1); 421 (DP2); 583 (DP3); 745 (DP4); 907 (DP5); 1069 (DP6). Those extracted for ionized N-acetylglucoside-1-phosphate are [MH]-: 300 (DP1); 503 (DP2); 706 (DP3); 909 (DP4); 1112 (DP5).

[0110] As shown in Figure 3, for the enzymes from Table 2 tested, peaks with very high retention times, exceeding that of mannose-1-phosphate, are visible. These peaks cannot correspond to common p-mannosides because their retention times are well known and shorter than that of mannose-1-phosphate. Given the catalytic activity of these enzymes, the regularity of the peaks, and their reproducibility (particularly for p-1,2-mannoside phosphorylases), they most likely correspond to p-mannoside phosphates of varying degrees of polymerization. Mass spectrometry analyses should confirm this hypothesis.

[0111] HPAEC-PAD-MS analyses of the peaks produced by the enzymes MTP4 (Figure 4), Teth1788 (Figure 5), Lin0857 (Figure 6), Uhgb_MS (Figure 7), and U7 (Figure 8) show, after signal processing, that they have precisely the mass of Man-1P molecules polymerized with one or more mannosyl units. In other words, these peaks correspond well to mannose oligosaccharides containing a single phosphorylation, the phosphate group can only be at the reducing end of the chains, in position a. The GPs mentioned in Table 2 are therefore capable of using mannose-1-phosphate as a donor and acceptor to synthesize phosphate oligosaccharides and polysaccharides.

[0112] As shown in Figures 4 to 8, H₂PA EC-PAD-MS analysis makes it possible to characterize the degree of polymerization (DP) of the phosphate oligo- and polysaccharides produced down to at least 6 units. However, the amperometric signal for MTP4 and T eth1788 suggests that phosphate oligo- and polysaccharides with a higher DP are produced. MTP4 would synthesize oligosaccharides up to a DP of 13. T eth1788 would synthesize oligo- and polysaccharides beyond a DP of 14 under the tested conditions.

[0113] As shown in Figures 10 to 15, for the enzymes GH112-SM2, TM1225, U7, Teth1788, and MTP4, respectively, peaks with retention times shorter than that of the sugar-phosphate substrate of the tested enzyme are visible. The corresponding masses indicate that these peaks correspond to sugar-phosphates with different degrees of polymerization (DP). Results corresponding to those listed in Table 2 were obtained for the enzymes Uhgb_MP, U1, U8, and Teth1789.

[0114] An NMR analysis was also performed on the phosphorylated disaccharide (Figure 16: p-1,2-MOS-αPi (DP2), p-1,2-mannobiose-α-1-phosphate) obtained from the reverse phosphorolysis reaction according to the invention with MTP4. The NMR spectrum obtained was compared with that of the corresponding commercially available unphosphorylated mannan disaccharide (Figure 16: p-1,2-MOS (DP2), p-1,2-mannobiose) obtained under the same conditions.

[0115] Previously, the separation of p-1,2-MOS-1 P from the reverse phosphorolysis reaction according to the invention with MTP4 was carried out by High Performance Liquid Chromatography on an Asahipak NH2P-50 column (Shodex), coupled with refractometric detection (HPLC-RI) (system comprising: Dionex UltiMate 3000 pump; Dionex UltiMate 3000 sampler; Dionex UltiMate 3000 column compartment; Dionex UltiMate 3000 variable wavelength detector; IOTA 2 Refractive Index Detector (Precision Instruments); UCI-50 Universal Chromatography Interface (Thermo Fischer)) according to the following parameters: sampler temperature 15°C, column temperature 25°C, injection volume 100 pL, flow rate 2.5 mL / min. Twenty milliliters of the p-1,2-MOS-1 P synthesis reaction are concentrated by a factor of 20 in the rotary evaporator and then injected onto the column. The separation is carried out according to the program in Table 3 below: Table 3 Gradient used to separate B-1.2-MOS-1 P from ultrapure water (A) and 400 mM ammonium formate pH 4.4 (C) The p-1,2-MOS-1 P fractions from each degree of polymerization (DP) are collected separately, then dried using a rotary evaporator and resuspended in approximately 1 mL of ultrapure water. Residual ammonium formate is removed by ethanol precipitation. The fractions of interest are mixed with absolute ethanol (90% of the total volume) and then vortexed at room temperature. They are then centrifuged to pellet the sugar-1-phosphates. The liquid phase is removed, and the pellet is dried under compressed air. The dried fractions are resuspended in ultrapure water, frozen, and then lyophilized.

[0116] For NMR analysis, samples of p-1,2-MOS-aPi (DP2) and p-1,2-MOS (DP2) were prepared in 3 mm tubes by dissolving the lyophilized compound in 150 µl of D2O. Chemical shifts were referenced to acetonitrile 5(-CH3) at 2.07 ppm. 1 H spectra were recorded at 313 K on a Bruker Avance 11,500 MHz spectrometer (Bruker) with a 5 mm z-gradient TBI probe and using standard pulse programs from the Bruker library (zgpr). The data were processed using TopSpin 3.6 software (Bruker).

[0117] Figure 16 shows that the chemical shift signals of the reducing (H1; 5.29 and 4.98 ppm) and non-reducing (HT; 4.82 and 4.77 ppm) anomeric proton of P-1,2-mannobiose (P-1,2-MOS (DP2)) are no longer present in the analysis spectrum of p-1,2-mannobiose-α-1-phosphate (P-1,2-MOS-αPi (DP2)). Two doublets are observed: a split doublet with chemical shifts of 5.44 and 5.47 ppm and coupling constants 3JH1-P -8.40 Hz and 3JH1-H2 -2.00 Hz, and another doublet at 4.86 ppm with a coupling constant 3JHT-H2' -0.95 Hz. This confirms for p-1,2-mannobiose- a-1 -phosphate the linkage of the phosphate group by a glycosidic bond α to the anomeric carbon C1 of the biose which then loses its reducing character.

[0118] Table 4 summarizes the activities and oligosaccharides detected for the tested enzymes. Table 4 Gal: galactose; Man: mannose Bibliographie Ladevèze S, Tarquis L, Cecchini DA, Bercovici J, André I, Topham CM, Morel S, Laville E, Monsan P, Lombard V, Henrissat B, Potocki-Véronèse G. Role of glycoside phosphorylases in mannose foraging by human gut bacteria. J Biol Chem. 2013 Nov 8;288(45):32370–32383. There a , A.; Benkoulouche, M.; Ladeveze, S.; Durand, J.; Cioci, G.; Laville, E.; Potocki-Veronese, G. Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases. Int. J. Mol. Sci. 2022, 23, 3043. There b A, Laville E, Tarquis L, Lombard V, Ropartz D, Terrapon N, Henrissat B, Guieysse D, Esque J, Durand J, Morgavi DP, Potocki-Veronese G. Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function. Microb Genom. 2020 0ct;6(10):mgen000404. Liu Y, Nishimoto M, Kitaoka M. Facile enzymatic synthesis of sugar 1-phosphates as substrates for phosphorylases using anomeric kinases. Carbohydr Res. 2015 Jan 12;401 :1-4 Lujan Ferreira, M. (Editor) Biocatalyst Immobilization Foundations and Applications. Biocatalyst Immobilization Foundations and Applications 1st Edition - November 12, 2022. Macdonald SS, Armstrong Z, Morgan-Lang C, Osowiecka M, Robinson K, Hallam SJ, Withers SG. Development and Application of a High-Throughput Functional Metagenomic Screen for Glycoside Phosphorylases. Cell Chem Biol. 2019 Jul 18;26(7):1001-1012.e5. Sheldon RA, Basso A, Brady D. New frontiers in enzyme immobilisation: robust biocatalysts for a circular bio-based economy. Chem Soc Rev. 2021 May 21 ;50(10):5850-5862. Tian C, Yang J, Li Y, Zhang T, Li J, Ren C, Men Y, Chen P, You C, Sun Y, Ma Y. Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies. Metab Eng. 2020 Sep;61 :215-224. Tripathi NK, Shrivastava A. Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development. Front Bioeng Biotechnol. 2019 Dec 20;7:420. Wang N, Kong Y, Li J, Hu Y, Li X, Jiang S, Dong C. Synthesis and application of phosphorylated saccharides in researching carbohydrate-based drugs. Bioorg Med Chem. 2022 Aug 15;68:116806. Wang RF, Kushner SR. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene. 1991 Apr; 100: 195-9. Zhong C, Nidetzky B. Three-Enzyme Phosphorylase Cascade for Integrated Production of Short-Chain Cellodextrins. Biotechnol J. 2020 Mar; 15(3):e1900349.

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: identification (10a) of at least one sugar-phosphate substrate of at least one glycoside phosphorylase enzyme capable of allowing the production of sugar phosphate, the at least one glycoside phosphorylase enzyme not catalyzing the formation of 1-1 osidic bond, provision of a preparation comprising the at least one glycoside phosphorylase enzyme (10b), provision of a composition comprising the at least one sugar-phosphate substrate of said enzyme (10c), said composition substantially not comprising non-phosphorylated sugar acceptor substrate of said at least one glycoside phosphorylase enzyme, reaction (20) of 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, 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, BAJ 10826.1, 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, BAH 10636.1, 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, CAH 1385118.1, CAH 1385117.1, CAH 1385115.1, 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, AAN 17338.1, 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, CAA85354.1, sequence SEQ ID NO: 1, sequence SEQ ID NO: 6 or sequence SEQ ID NO:

7.

4. 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 p-glucose-1 P and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the Genbank number next: 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, CAH 1385118.1, CAH 1385117.1, BAJ 10826.1, 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, CAH 1385115.1, CAH 1385116.1, of sequence SEQ ID NO: 1 or of sequence SEQ ID NO: 7, the sugar-phosphate substrate is α-glucose-1 P 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, BAJ 10826.1, 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, AAM 52219.1, 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, AAN 17338.1, 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-1 P 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 pN-acetyl-D-glucosamine-1 P and the at least one glycoside phosphorylase enzyme corresponds to an enzyme referenced under the following NCBI or UNIPROT accession number AAQ05801.1 , or the sugar-phosphate substrate is α-galactose-1 P 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, BAH 10636.1, ADL32981.1, AEN95946.1 or sequence SEQ ID NO:

6.

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: AAD36300.1, ADD61463.1, BAC87867.1, CAC96089.1, ABY93074.1, ABY93073.1, CBZ24449.1, CAH 1385115.1, CAH 1385118.1, CAH1385117.1, of sequence SEQ ID NO: 6 or of sequence SEQ ID NO:

7.

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 at least one 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 wherein said reaction is carried out 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 wherein the reaction comprises maintaining excess concentrations of the at least one sugar-phosphate substrate of the glycoside phosphorylase.

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