METHOD FOR SELECTIVELY PREPARING D-TAGATOSE-1,6-BISPHOSPHATE OR D-TAGATOSE-1-PHOSPHATE, AND ITS USES, PARTICULARLY FOR PREPARING D-TAGATOSE

Abstract

The present invention relates to the use of an enzyme belonging to the aldolase group and comprising a polypeptide having at least 30% identity with an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:4, for the stereoselective preparation of d-tagatose-1,6-bisphosphate (TBP) or d-tagatose-1-phosphate. The present invention also relates to a method for preparing d-tagatose-1,6-bisphosphate (TBP) or d-tagatose-1-phosphate, comprising a reaction step, in a suitable reaction medium containing dihydroxyacetone phosphate (DHAP) or at least one of its isomers or analogs, and optionally d-glyceraldehyde, with an enzyme belonging to the aldolase group, said enzyme comprising a polypeptide having at least 30% identity with an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:4. The present invention also relates to a method for preparing d-tagatose. Figure 4

Description

Title of the invention: METHOD FOR SELECTIVELY PREPARING D-TAGATOSE-1,6-BISPHOSPHATE OR D-TAGATOSE-1-PHOSPHATE, AND ITS USES, IN PARTICULAR FOR PREPARING D-TAGATOSE. Technical field

[0001] The present invention relates to the use of an enzyme to selectively prepare d-tagatose-1,6-Z?zsphosphate (TBP) or d-tagatose-1-phosphate, to a method for preparing d-tagatose-1,6-Z?zsphosphate (TBP) or d-tagatose-1-phosphate (T1P), and to a process for preparing d-tagatose.

[0002] The present invention finds applications particularly in the food industry and the pharmaceutical industry.

[0003] In the description below, references in brackets ([ ]) refer to the list of references presented at the end of the text. State of the art

[0004] D-tagatose is a natural sweetener, still relatively uncommon, produced from milk sugar (lactose). The body's digestion of d-tagatose differs from that of sucrose and it has less of an effect on blood glucose and insulin levels. D-tagatose is therefore a true alternative to sugar, particularly for diabetics, as, despite its sweet taste, it has little effect on blood sugar levels.

[0005] One of the industrial production methods for D-tagatose relies on the use of lactose as a source of D-galactose, which is an aldehyde monosaccharide. This galactose is then converted into D-tagatose, and therefore a cheap source of galactose is necessary for this process. Lactose (a disaccharide consisting of one glucose unit and one galactose unit) is the most suitable source of galactose because it is a by-product of whey. α-Lactose monohydrate is therefore inexpensive and readily available.

[0006] The principle of this process consists of 2 steps: the first is the hydrolysis of lactose to release d-galactose and the second step is the isomerization of d-galactose into d-tagatose.

[0007] Different conditions are used to carry out the hydrolysis of lactose. This is either achieved by a chemical method under acidic conditions with hydrochloric or sulfuric acid, most often by heating to increase the reaction rate, or by enzymatic methods with lactases / [3-galactosidases]. Enzymatic hydrolysis has all the classic advantages of enzymatic methods. Compared to chemical methods, this method also has drawbacks. Depending on the initial lactose concentration, products other than glucose and galactose may be present in the medium. The lactases used for hydrolysis can generate byproducts, including oligosaccharides (3-D-galactose-(16)-D-glucose) or allolactose, 3-D-galactose-(16)-D-galactose or 6-galactobiose, and 3-D-galactose-(16)-lactose or 6'-galactosyl-lactose. High initial lactose concentrations increase the likelihood that the reaction products will remain in contact long enough to lead to these byproducts. Furthermore, D-galactose also acts as a competitive inhibitor, potentially prolonging the reaction time.

[0008] The second step can be carried out using rare earths or alkaline earths as catalysts for isomerization, but the availability of these catalysts is a problem. Similar to the first step, enzymatic isomerization methods have been developed, involving L-arabinose isomerases. The use of enzymatic isomerization of d-galactose is by far the most common, because the chemical conditions of isomerization involve significant degradation of d-galactose over time, making this step even more difficult.

[0009] Enzymatic isomerization yields are highly dependent on the substrate / product thermodynamic equilibrium. Furthermore, at the end of this step, purifications will be necessary to obtain the pure product. These treatments are often costly and laborious, drastically increasing production costs.

[0010] All of these factors mean that D-tagatose is still produced on a small scale and is not available at prices competitive with the cost of table sugar. Therefore, new methods are being sought to facilitate its production.

[0011] Thus, syntheses are being developed from inexpensive starting substrates in order to make this sugar with its multiple properties accessible. D-glucose meets these criteria, and researchers have developed syntheses of D-tagatose from it.

[0012] Document CN117305204 ([1]) describes a synthesis from a recombinant microorganism in which three enzymes are overexpressed: a d-tagatose-1,6-bisphosphate aldolase (TagA), a fructose-1-phosphate phosphatase, and a glucose-6-phosphate phosphatase. The production of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde-3-phosphate (D-G3P), necessary for the synthesis of TagA, is achieved via glucose metabolism, with some of the glucose removed, and D-tagatose was produced in a reported yield of 80%. However, this process does not allow for the removal of certain glucose analogs, such as fructose or its phosphorylated analogs.

[0013] The US-based company Bonumose has developed a method for synthesizing D-tagatose from bioresources, but also using a glucose analog. Their process involves the conversion of fructose-6-phosphate (F6P) to tagatose-6-phosphate (T6P) catalyzed by an epimerase. Dephosphorylation of T6P is carried out by a phosphatase. F6P can be obtained from glucose-6-phosphate using a phosphoglucose isomerase, itself obtained by the conversion of glucose-1-phosphate by a phosphoglucomutase (WO2017059278 ([2])). Various glucose sources have been described to enable the valorization of this process with the use of inexpensive renewable bio-based raw materials. Starch, cellulose or sucrose have been indicated as bioresources, which can lead to the production of D-tagatose with a yield of up to 99% by a fully enzymatic route.However, this synthetic route also has drawbacks. Indeed, this process involves a number of enzymatic steps (five enzymes are used starting from the glucose-1-phosphate / glucose mixture), and enzyme production represents a significant cost. Furthermore, the enzymes are not used in vivo but in isolation. This necessitates immobilizing them to reduce costs. All these conditions create difficulties to overcome and therefore additional costs which, despite the advertised 99% D-tagatose yield, make the development of alternative methods essential.

[0014] The TagA family, which cleaves or forms a C-C bond at positions 3 and 4 and has expected stereoselectivity (35,451) because it acts naturally on d-tagatose-1,6-Wsphosphate (TBP), is the least studied family of DHAP aldolases. Indeed, the TagAs described in the literature have not shown high stereoselectivity at carbon 4, leading to the kinetic formation of TBP, but subsequently rapidly forming D-fructose-C-Wsphosphate (FBP) with the configuration (35,47?). This stereoselectivity problem results in a mixture of products in different proportions (Fessner, W.-D. and Eyrisch, O. ([3])). Thus, since TBP is difficult to synthesize and is always formed as a mixture, it is expensive, and many suppliers have even stopped its production. Therefore, attempting to obtain this compound more easily is important.Currently, a whole range of compounds with particular properties is under-explored and under-exploited, but which can be opened up to chemists provided that effective methods and / or biocatalysts are found to break the various barriers that hinder the obtaining of these products with configuration 35,45.

[0015] There is therefore a real need for solutions that overcome these defects, drawbacks and obstacles of the prior art, allowing for a simpler and / or more efficient synthesis at a lower cost of d-tagatose starting from renewable, available and low-cost raw materials and its possible precursors.

[0016] Description of the invention

[0017] The present invention is specifically designed to meet these needs and drawbacks by providing methods and biocatalysts for stereoselectively synthesizing D-tagatose-1,6-bisphosphate (TBP), d-tagatose-1-phosphate (T1P) and D-tagatose.

[0018] The inventors have indeed succeeded, through extensive research, in identifying TagAs that are far more stereoselective than those described in the literature up to that point. They have thus succeeded in stereoselectively synthesizing TBP and T1P, intermediates which subsequently enabled the synthesis of d-tagatose.

[0019] Advantageously, the proposed methods allow the use of various starting products such as glucose, starch or glycerol.

[0020] Moreover, the methods of the invention allow for fewer by-products than in the prior art.

[0021] Thus, a first object of the invention relates to the use of an enzyme belonging to the aldolase group and comprising a polypeptide having at least 30% identity with an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:4, to stereoselectively prepare TBP or T1P.

[0022] Another object of the invention relates to a method for preparing TBP or Tl P, comprising a reaction, in a suitable reaction medium containing dihydroxyacetone phosphate (DHAP), or at least one of its isomers or analogues, and optionally d-glyceraldehyde, with an enzyme belonging to the aldolase group, said enzyme comprising a polypeptide having at least 30% identity with an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:4.

[0023] Another object of the invention relates to a process for preparing D-tagatose, comprising the steps of: a. Preparation of the TBP or T1P by implementing the method defined above, and b. Dephosphorylation of the TBP or T1P obtained in step (a).

[0024] For the purposes of this invention, "aldolase" means a lyase capable of catalyzing in vitro or in vivo the asymmetric addition of DHAP to D-glyceraldehyde 3-phosphate (d-G3P) to give TBP and / or FBP. Advantageously, aldolase can also reverse aldolize TBP and / or FBP.

[0025] Aldolase activity can be evaluated in vitro or in vivo by contacting the enzyme with substrates, for example DHAP as a nucleophile and d-G3P as an electrophile, under conditions conducive to enzymatic activity and the detection of TBP or FBP formation. The in vitro conditions for evaluating The potential aldolase activity of an enzyme can be assessed by contacting the enzyme with substrates, possibly in the presence of a divalent metal cation, for example, selected from Mg, Zn, Co, and Mn, and preferably Co or Zn. Aldolase activity can also be evaluated in vitro by contacting the enzyme with TBP or FBP and detecting the release of DHAP (reverse aldol condensation). This can be assessed by adding glycerol phosphate dehydrogenase (GPDH), and the disappearance of NADH is measured, for example, by spectrophotometry, as shown below. One unit (U) for aldolase is defined as the amount of enzyme that converts 1 micromole of substrate per minute under standard conditions. For example, the enzymatic reaction can be carried out in a buffer at a pH between 6.5 and 8, and at a temperature between 18°C ​​and 25°C.Appropriate methods are described in more detail in the examples below, as well as in the document (Paulat et al. (

[11] )).

[0026] Suitable enzymes having aldolase activity as defined above may be isolated from or obtained from a microorganism. They may be obtained, for example, by expressing the enzyme in a suitable wild-type or recombinant expression system known to those skilled in the art. Methods for introducing a foreign gene and inducing its expression in a host cell are well known in the prior art. Alternatively, the enzyme may be identified from organisms, including animals or microorganisms, having TagA or FruA activity, such as bacteria and fungi.It may be, for example, a microorganism belonging to a genus chosen from among the Enterobacteriaceae such as Escherichia, Shigella, Edwardsiella, Enterobacter, Salmonella, Pectobacterium, Serratia, Silicibacter and Providencia, the Enterobacterales, such as Citrobacter, Yersinia, or Photorhabdus, the Vibrionales such as Vibrio or Photobacterium, the Aeromonadales such as Aeromonas, the Firmicutes such as Clostridia, the Anaerotruncus, the Fusobacterium and the Bacillus. The enzyme can be identified, obtained and isolated from microorganisms isolated in nature, for example, soil, composts, water, etc. The enzyme can also be obtained by screening genomic DNA, a cDNA library from microorganisms, a mixed DNA sample or DNA samples obtained directly from samples of natural materials as mentioned above for example.

[0027] The sequence SEQ ID NO: 1 is as follows: MPLVTTTEFLKKAQDGHYAVGAFNVENLEMAQAVIQAATELNAPVIIQTTS STVKYASLSVYRAIVAALAESAPVPVAMHLDHGSSYELCEQAACAGYTSVM IDGSKLSFEENIAVSAKTAAMAKQRGIPTEAELGTVGGKEDDHVVLDADAM YTNPEKAREFVERTGVGSLAVAIGTAHGFYKGEPRLDFDRLAEIRKVVSVP LVLHGASGVPDEAVQKTIGLGICKVNFATELRDAYTKAVRPVLANDASVFD PKVYGKAGRAAVVELVKHKITVCGSQGKA

[0028] The SEQ ID NO sequence: 1 currently corresponds to the protein sequence of the enzyme B0P6N8 (identified in the Uniprot database as KB), annotated in Uniprot as belonging to the family of FBP aldolases class II (EC 4.1.2.13), and originating from the organism Anaerotruncus colihominis.

[0029] The SEQ ID NO: 2 sequence is as follows: MLVSTRQLLLDAQKRKYAVPAFNVHNMETIQTVIEAAAELKS PIIVAATPGTMKYAGAEFFIKLVEICSEKYDIPIAMHLDHHESYDEIVNAI DIGTKSVMIDASHLSFEENIAKVKKVVDYAHKFDVEGELGILGGQEDDL IRDDKDSKYTNPAQAREYVERTGIDSLAVAIGTAHGVYKEEPKLDFERLAE IRSVVDIPLVLHGASGVPADQVKKAIDIGITKVNIATELKMPFAETLRQVL VNNPNESDPRKYFGPAKESMKKVAIEKILMCGSNGKA.

[0030] The SEQ ID NO sequence: 2 currently corresponds to the protein sequence of the enzyme C6JL87 (identified in the Uniprot database as KB), annotated in Uniprot as belonging to the TBP aldolase family (EC 4.1.2.40), and originating from the organism Fusobacterium varium.

[0031] The sequence SEQ ID NO: 3 is as follows: MFIISTKTMLKKAQREGYAVPAFNI HNLETLQVVVETAAEMRAPLIVAGTPGTFSYAGVSNIVAIASELAKTWNHP LAIHLDHHEKTMDIEEKVRAGVRSVMIDGSHLPFADNIALVKNVTDYCHRY DVSVEAELGRLGGQEDDLIVDGKDALYTHPEQAQEFVAKTGIDSLAVAIGT AHGLYTAEPKLDFERLAEIRQHVDIPLVLHGASGLPGSDIRKAISLGICKV NVATELKIAFSDALKNYLRTHSDASDPRHYMVPAKAAMKDVVRKVITDCGC EGRL.

[0032] The SEQ ID NO sequence: 3 currently corresponds to the protein sequence of the enzyme A0A3R9CKN0 (Uniprot database name KB), annotated in Uniprot as belonging to the TBP aldolase class II family (EC 4.1.2.40), and originating from the organism Citrobacter koseri.

[0033] The SEQ ID NO: 4 sequence is as follows: MLTNTKKMLLDAQANGYAVPAFNIHNLETVQAVTEAAEALRS PVILAATPGTFSYAGRDYIQAIAETAAARCSIPVALHLDHHEEIDDIRESL SLGTKSVMIDASHHPFAENTAITASVVQYARQKDATVEAELGRLGGQEDDL IVDEADAYYTDPDAAKEFVEKTGIDSLAVAIGTAHGLYSKEPNIDFERLAA IQKRVSVPLVLHGASGISEQDVRKCIKLGCAKVNIATELKIPFSDKLRRYL HEHADASDPRKYMAPAKEAMKKIVQEKILMCMSNDRY

[0034] The sequence SEQ ID NO: 4 currently corresponds to the protein sequence of the enzyme Q65EY6 (Uniprot database name KB), annotated in Uniprot as belonging to the TBP aldolase class II family (EC 4.1.2.40), and originating from the organism Bacillus licheniformis.

[0035] “At least 30% identity” means a percentage of sequence identity between two amino acid sequences with 30% identity, or at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity. For example, the percentage of identity may be at least 40%, or even 45% identity with an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:4. For the purposes of the present invention, the "percentage of identity" between two amino acid sequences (A) and (B) is determined by comparing the two optimally aligned sequences through a comparison window. This sequence alignment can be performed by known methods, for example, using the Needleman-Wunsch global alignment algorithm. To compare two amino acid sequences, one can use, for example, the "Emboss needle" tool for pairwise alignment of protein sequences provided by EMBL-EBI and available at www.ebi.ac.uk / Tools / services / web / toolform.ebi?tool=emboss_needle&context=protein.

[0036] Advantageously, the enzyme may comprise or consist of a polypeptide having a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:4. In the case where the enzyme "consists of" a polypeptide having a sequence SEQ ID NO:X, the enzyme sequence has 100% identity with the sequence SEQ ID NO:X and does not include any additional sequence. When the enzyme "comprises" a polypeptide having a sequence SEQ ID NO:X, the latter may include one or more additional sequences, that is, sequences outside the sequence SEQ ID NO:X.

[0037] For example, the enzyme may be a hybrid polypeptide, meaning that the enzyme comprises a first polypeptide having the enzymatic activity of interest, which is fused or conjugated to another chemical or biochemical entity. The chemical or biochemical entity may be fused or conjugated to the N- or C-terminal region of the first polypeptide.

[0038] Thus, the hybrid enzyme may comprise a first polypeptide having the enzymatic activity of interest, which is fused to an additional polypeptide. This additional polypeptide may be selected to improve the stability of the enzyme, to promote the secretion (such as an N-terminal hydrophobic signal peptide) of the hybrid enzyme from a cell (such as a bacterial or yeast cell), or to aid in the purification of the hybrid enzyme. More specifically, the additional region may be a useful tag for the purification or immobilization of the hybrid enzyme. Such tags are known to those skilled in the art, for example, a His tag (HiSe), a FLAG tag, an HA tag (hemagglutinin-derived epitope, a maltose-binding protein). (MPB), a MYC tag (human proto-oncoprotein MYC derived epitope), streptavidin or avidin, or a GST tag (small glutathione-S-transferase).

[0039] When the polypeptide is conjugated, it may be a polypeptide whose amino acid sequence has been chemically conjugated to at least one chemical or biochemical entity. The techniques for conjugating an amino acid sequence to another chemical or biochemical entity are known to those skilled in the art. The additional entity and the polypeptide having the enzymatic activity of interest may be covalently linked to each other, either directly or via a spacer. The spacer may be a standard linker commonly used for the preparation of polypeptide constructs. In some cases, the spacer is a polypeptide comprising from 1 to 50 amino acid residues. Preferred examples are Gly-Ser linkers such as tetraglycyl-seryl-triglycyl-serine peptides or polyalanine linkers. The additional chemical or biochemical entities may be of any type.For example, additional or biochemical entities can be a useful way to immobilize the enzyme, for example a biotin or a reactive functional group, a means of detecting the enzyme, a tag, etc.

[0040] Alternatively or in addition, the enzyme may be a variant of an enzyme as defined above. Thus, it may be a polypeptide sequence that differs from that of a parental polypeptide sequence, namely the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, due to at least one amino acid modification. Generally, a variant comprises from 1 to 250 amino acid modifications, preferably from 1 to 200 amino acid modifications. In particular, the variant may have from 1 to 150, or from 1 to 100, or from 1 to 50, or from 1 to 25 amino acid modifications, compared to the parental sequence. The sequence of a variant may include one or more amino acid substitutions, and / or one or more amino acid insertions, and / or one or more amino acid deletions compared to the sequence of its parent.In some embodiments, the amino acid modifications are substitutions, preferably conservative ones. In other words, the amino acid modifications present in the variant do not significantly change its properties compared to those of its parent. Conservative substitutions and the corresponding rules are well described in the prior art. Examples of conservative substitutions are found in the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine), by . For example, common substitutions include: Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Ala, Ala / Glu, and Asp / Gly. Amino acid modifications can also alter the physicochemical properties of polypeptides. For example, amino acid modifications can improve the thermal stability of the polypeptide, modify substrate specificity, change the optimal pH, etc. The essential amino acids of a polypeptide can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis.The active site of the enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with amino acid mutation of the putative contact site. The identity of essential amino acids can also be inferred from alignment with a related polypeptide. Substitutions, deletions, and / or insertions of one or more amino acids can be performed and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by an appropriate screening procedure. Other methods can be used, including PCR with a risk of error, phage display, and site-directed mutagenesis.

[0041] For the purposes of this invention, "stereoselective preparation" means the synthesis, using the method of the invention, of TBP or T1P in a predominant form, in particular with a relative proportion of TBP or T1P of at least 50%, preferably more than 60%, 70%, 80%, 90%, 95%, or 98%, or between approximately 80% and approximately 98%, or even 100%, relative to FBP or F1P, respectively. Preferably, the synthesis of TBP or T1P is carried out without obtaining FBP or F1P.

[0042] For the purposes of the present invention, the enzyme can be selected from the group comprising a purified enzyme, present in free form or immobilized on a support, and an enzyme produced in situ by a cell expressing said enzyme.

[0043] Indeed, after being isolated and possibly purified, the enzyme of interest can be immobilized, for example by forming cross-linked enzyme aggregates, or on a support by any suitable method described in the prior art, for example by covalent bonding, adsorption, trapping, or membrane confinement. A wide variety of supports can be used to immobilize the enzyme. Practical supports include, but are not limited to, plastic, metallic, and inorganic supports such as glass, silica, alumina, bentonite, hydroxyapatite, nickel / nickel oxide, titanium, zirconia, metal-organic networks, polymer supports, and others. The support can be in the form of a surface, a powder, or micro- or nanobeads, a gel, a matrix or a gel swelling the solvent or water, a crosslinked matrix or gel, a membrane, a fibrous support, a porous support, etc. In a particular embodiment, the support is chosen from inorganic matrices and polymer matrices.For example, supports useful to the invention include resins or matrices comprising or consisting of a polysaccharide such as cellulose, carboxymethylcellulose, diethylaminocellulose (DEAE), dextran, crosslinked dextran such as Sephadex®, agarose, crosslinked agarose such as Sepharose®, starches, alginate, chitosan, a synthetic polymer such as polyamino acids, polyacrylamides, acrylic acid-based polymers and copolymers and their derivatives, polyamides, polystyrene, organopolysiloxanes, polyacrylate, polyvinyls, polyacriline, inorganic compounds such as hydroxyapatite, silica or bentonite, and so on. Such supports are commercially available.

[0044] By way of illustration, the enzyme can be trapped in a polymer matrix, for example, an alginate or chitosan matrix. The enzyme can also be covalently bound to the support. Generally, the support can contain functional groups capable of reacting directly, or after activation, with an amino acid present in the enzyme so as to create a covalent bond. Another solution is to adsorb the enzyme onto the support. The interactions between the support and the enzyme can then be stabilized by crosslinking with a bifunctional agent such as glutaraldehyde.

[0045] Once prepared, the support containing the immobilized enzyme with the enzymatic activity of interest can be used directly in the reaction medium. In other words, the support with the immobilized enzyme can simply be added to the reaction medium. When the support is solvent-inflatable, the reaction solvent can be chosen to ensure appropriate swelling of the support to make the immobilized enzyme accessible without impairing the enzyme's catalytic activity.

[0046] For the purposes of the invention, the enzyme of the invention can be produced in situ, namely in the reaction medium, by a cell or organism capable of expressing said enzyme, as described above.

[0047] In other embodiments, the enzyme may be expressed by in vitro protein expression (also known as in vitro translation, cell-free protein expression, cell-free translation, or cell-free protein synthesis). In vitro protein expression systems based on E. coli, RRL (rabbit reticulose lysate), wheat germ extracts, and insect cells may be used.

[0048] For the purposes of this invention, "suitable reaction medium" means any medium that allows the enzyme to be active. Thus, the reaction medium contains The medium contains the substrates for the enzyme, and possibly nutrients, triose phosphate isomerase (TPI), which catalyzes the reversible isomerization of DHAP to d-G3P, d-glyceraldehyde for the synthesis of T1P, and possibly at least one divalent metal cation, for example, selected from Mg2+, Zn2+, Co2+, and Mn2+, and preferably Co2+ or Zn2+. The various substances in the medium can be introduced as such or generated in situ using methods known to those skilled in the art. The pH of the reaction medium can be controlled using a buffer solution, for example, between approximately 5 and approximately 9, and the temperature can be between approximately 2°C and approximately 40°C. Those skilled in the art know how to adapt the medium and reaction conditions according to the substrates and the enzyme used.For example, when the enzyme is used in its free state, the pH, temperature, and solvent are chosen to promote enzymatic activity. When the enzyme is expressed in situ by a cell or host cell, the operating conditions are favorable for the expression of the enzyme by said cell or host cell.

[0049] Advantageously, in the method of the invention, the reaction can be carried out at a temperature between approximately 2°C and approximately 40°C, preferably approximately 10°C for the synthesis of TBP, and at room temperature, i.e., between 18°C ​​and 40°C, for the synthesis of T1P. Carrying out the reaction at room temperature represents a significant advantage in terms of industrialization. Advantageously, the synthesis of TBP can be carried out at 10°C using a polypeptide having at least 30% identity with the amino acid sequence SEQ ID NO:1.

[0050] The DHAP isomer can be any isomer, such as d- glyceraldehyde-3-phosphate (d-G3P).

[0051] The DHAP analogue can be any potentially dephosphorylated analogue, such as dihydroxyacetone (DH A). Advantageously, when DH A is used instead of DHAP, an anion capable of mimicking phosphate, such as borate, arsenate or vanadate, a phosphonate or methyl phosphate, is added to the reaction medium.

[0052] Advantageously, DHAP, and / or its isomers or analogs, can be produced in situ, for example by a host cell capable of (over)producing DHAP, and / or one of its isomers or analogs. Several microorganisms are known to, or have been modified to, overproduce DHAP. By way of illustration, see M. Wei et al. (

[13] ). In some other embodiments, the host cell produces, or has been modified to produce, an additional enzyme of interest, depending on the desired end product. This enzyme can be selected from among phosphatases, aldose isomerases, or dehydrogenases.

[0053] Advantageously, the method of the invention may further comprise a step of recovering and / or purifying TBP or d-tagatose-1-phosphate. The purification may be carried out by any purification method known in the prior art, such as precipitation, filtration, extraction, preparative chromatography, recrystallization, and combinations thereof. In certain particular embodiments, chiral chromatography may be performed to remove unwanted stereoisomers.

[0054] The method of the invention may also optionally include one or more steps before the reaction step of DHAP or at least one of its isomers or analogues with the enzyme, such as for example a step of generating DHAP in situ, for example from DHA and / or a step of generating d-glyceraldehyde in situ.

[0055] In the process for preparing d-tagatose, the dephosphorylation step of TBP or T1P can be carried out by any means known to those skilled in the art, for example by contacting TBP or T1P with a phosphatase. This could be, for example, a sugar phosphatase (EC 3.1.3.23), an acid phosphatase (EC 3.1.3.2), or an alkaline phosphatase (EC 3.1.3.1). Preferably, it is an acid phosphatase. In some embodiments, the phosphatase may be a natural phosphatase isolated from any type of organism, such as E. coli, potato, wheat germ, calf intestine, bovine, porcine, human, sweet potato, shrimp, guinea pig, or possibly a variant thereof. The phosphatase may be used in any form known to those skilled in the art.As such, phosphatase can be supplied in its free state, for example, as an isolated substance, as an enriched substance, as a purified substance, or as a semi-purified substance. For example, phosphatase may be present in a supernatant or in a supernatant extract recovered from a culture. Phosphatase can also be formulated as a composition. In some embodiments, the phosphatase is immobilized on a support as described above for the enzyme. It is understood that step (b) is carried out under conditions favorable to phosphatase activity, for example, at an acidic pH.

[0056] Other advantages may become apparent to a person skilled in the art upon reading the examples below, illustrated by the accompanying figures, which are given by way of illustration. Brief description of the drawings

[0057] Fig. 1 represents the screening method for potential TagA by successive DHAP / FBP / TBP assays.

[0058] Fig. 2 represents the principle of measuring retroaldol activity on the TBP of putative TagA.

[0059] Fig. 3 represents the reaction conditions for non-stereoselective synthesis of TBP catalyzed by TagA at 20°C.

[0060] Fig. 4 represents the reaction conditions for stereoselective synthesis of TBP catalyzed by TagA at 10°C.

[0061] Fig. 5 represents the stereoselective synthesis scheme of T1P at room temperature.

[0062] Fig. 6 represents the synthesis of TBP carried out with the enzymes B0P6N8, C6JL87, A0A3R9CKN0, Q65EY6, P0AB74, A0AGT7, C0D851 and A0A805ZVK9 at 20°C.

[0063] Fig. 7 represents the synthesis of TBP carried out with the enzymes B0P6N8, Q65EY6 (and P0AB74 as a control) at 10°C.

[0064] Examples

[0065] Example 1: Genomic exploration and construction of a TagA collection

[0066] In the literature, few class II TagAs have been described in detail, particularly with regard to their synthetic capacity. To build a TagA collection, two class II TagAs from E. coli (P0AB74 kbaY gene and P0C8J6 gatY gene) were used to construct a reference set, as their TagA activity has been demonstrated in the literature. A protein sequence homology search was performed using the BLAST (Basic Local Alignment Search Tool) software program. The chosen BLAST parameters allowed the identification of all proteins in the Uniprot database (https: / / www.uniprot.org / ) homologous to at least one of the enzymes in the reference set by more than 35% sequence identity over at least 80% of their length. This study identified 10,904 proteins homologous to the proteins in the reference set. To reduce complexity, all proteins with more than 70% sequence identity were grouped into the same protein cluster.In the end, we obtained 391 clusters of more than two proteins and 483 singlets (clusters of a single protein). Among these clusters, 245 were selected for studying the activities of these enzymes. The genes encoding these enzymes entered the cloning process, and 183 were successfully cloned. The corresponding proteins (= Collection of potential TagAs) were then produced in E. coli, and cell-free lysates were prepared for testing on different molecules to evaluate their activity.

[0067] 1. Screening of the TagA collection

[0068] The collection was screened by performing an aldol reaction on the natural substrates of these enzymes, DHAP and D-G3P, the latter being generated via isomerization of DHAP by a triose phosphate isomerase (TPI).

[0069] 1.1. Screening by successive DHAP / FBP / TBP assays

[0070] 1.1.1 Screening method by successive titrations

[0071] To identify the enzymes capable of forming D-TBP, the aldol condensation reaction of DHAP on D-G3P was therefore carried out ([Fig. 1]). 96-well plates, containing 10 rnM of DHAP (low concentration to limit potential inhibition), a metallic cofactor involved in class II mechanisms (here Zn2+) in aqueous solution at pH 7.5 in the presence of TPI (to generate D-G3P via DHAP), were prepared for screening. The enzymes, in the form of clarified extracts from E. coli strains, were used. ATPI coli (Paulat et al. (

[11] )) were then added to the mixture to initiate the aldol condensation reaction. After defined reaction times (2 h and 24 h), a volume of the reaction mixture was taken, and the aldol condensation reaction was stopped by the addition of perchloric acid, which denatured the enzymes at acidic pH.

[0072] The screening method consists of 3 successive enzymatic assays carried out on the previous acidified samples using UV-visible spectrophotometer monitoring at 340 nm.

[0073] This sample is analyzed by performing three successive enzymatic assays monitored with a UV-visible spectrophotometer at 340 nm. First, the unreacted DHAP is quantified using glycerol phosphate dehydrogenase (GPDH, commercial mixture: GPDH / TPI) in the presence of NADH, which absorbs at 340 nm, unlike its oxidized form NAD+. This assay will allow the extent of the reaction to be quantified (step 1 of [Fig. 1]).

[0074] Once the remaining DHAP has been measured, E. coli FruA is added to retroaldolize the D-FBP potentially present in the medium (step 2 of [Fig.1]) and reform the DHAP and D-G3P which will then be measured by the GPDH / TPI mixture present since step 1.

[0075] Finally, the D-TBP potentially present in the medium is in turn retroaldolized via the addition of E. coli TagA based on the same principle as in step 2 (step 3 of [Fig.1]).

[0076] These successive assays are made possible by the selectivity of the enzymes used, with the least selective being introduced last. Indeed, it has been shown that E. coli FruA (Uniprot number: P0AB71) allows for the retroaldol conversion of FBP but not TBP. In contrast, E. coli TagA (P0AB74) is capable of retroaldol conversion of both FBP and TBP. It is therefore strictly necessary to perform the test in this precise order.

[0077] Since TBP is described in the literature as a short-time observable kinetic product for E. coli TagA (Fessner, W.-D. and Eyrisch, O. ([3]), Eyrisch, O. et al. ([4])), it was chosen to perform a 24-hour assay.

[0078] 1.1.2 Results of screening by successive assays

[0079] If we consider the relative proportions of FBP and TBP measured for all the enzymes tested, FBP is the predominant of these two products. This result is consistent with the literature. Indeed, class II TagAs are described in the literature as kinetically yielding TBP (E. coli (Fessner, W.-D. and Eyrisch, O. ([3])) and ultimately leading to the thermodynamic product: FBP. It is therefore possible that at the arbitrarily chosen time, the reaction has already progressed towards FBP and that consequently little or no TBP is measured. Nevertheless, three enzymes showed a non-negligible proportion of TBP (approximately 50 / 50 FBP / TBP) at 24 h: B0P6N8 from Anaerotruncus colihominis (labeled FruA class II), C6JL87 from Fusobacterium varium (labeled TagA), and Q65EY6 from Bacillus licheniformis (also labeled TagA class II). The TagA from E.E. coli (P0C8J6) from the reference set, presented as a control on the screening plates, yielded only FBP.

[0080] By analyzing the data from the TagA collection screening, we can also observe that for many enzymes, there is a discrepancy between the amount of DHAP still present in the control wells at 24 h and the total amount of DHAP that we measure by combining the three assays. This discrepancy can be explained by the presence of products that would not be quantified according to our method.

[0081] In conclusion, this initial screening identified three potential TagA proteins that could be of interest in synthesis. However, as with many high-throughput screening methods that use common conditions to test all proteins in the collection, this one has biases or weaknesses, and that is why we wanted to supplement this screening with activity measurements by TBP back-aldol synthesis.

[0082] 1.2. Screening by measuring retroaldosterone activity on the TBP

[0083] 1.2.1 Screening method by measuring retroaldosterone activity on the TBP

[0084] Obtaining TBP by laboratory synthesis (described in Example 2 below) allowed us to perform back-aldol activity measurements with respect to TBP on the collection of enzymes from biodiversity. Two 96-well plates were prepared with 0.5 mM D-TBP, 0.2 mM ZnCl2, 1 MM NADH, 870 mU GPDH, and 8.6 U TPI, all in solution in 200 mM Glycylglycine buffer at pH 7.5. The enzymes were then added, and the specific activity was measured at 30°C by spectroscopic monitoring at 340 nm. More precisely, an initial absorbance measurement at 340 nm and 30°C was performed to verify that the absorbance values ​​were stable. After verification, 8 pL per well of the different enzymes from the collection are added to the plate and the absorbance at 340 nm is again measured over time at 30°C.The resulting slopes, characteristic of the retroaldol activity, are read and processed to give the specific enzymatic activity of each well ([Fig.2]).

[0085] For comparison, using the same method, we also measured the retroaldol activity on the FBP (this time at 20 mM).

[0086] 1.2.2 Screening results by measuring retroaldosterone activity on TBP and FBP

[0087] Specific activity is defined as the amount in moles of substrate converted per unit time and per mg of protein. It is most often expressed here in U / mg, which corresponds to pmol / min / mg of substrate converted. It is calculated as follows: _ / (&DO / 5Q y . / Where: Cl — y gx / X r X 1 v / / In - a is the specific activity in U / mg; - e is the molar extinction coefficient in L / mol / cm; - V the volume of the solution in L; - m the mass of protein in the solution in mg.

[0088] When the activity is carried out on lysates or clarified extracts whose protein concentration is unknown, the activity is reduced to a defined volume: for example the volume of a well.

[0089] Analysis of our results shows that the 3 enzymes selected during the first screening possess significant activity (from 4 to 10 mU / well) for TBP cleavage, thus validating our initial observations. However, other enzymes exhibited significant activities up to 25 mU / well. These were therefore selected (in addition to those already selected) for the continuation of our study, namely: A0A3R9CKN0 from Citrobacter koseri, A0AGT7 from Listeria welshimeri, A6BDQ4 from Dorea longicatena, C0D851 from Clostridium asparagiforme, M5DZ63 and M5E4H7 from Halanaerobium saccharolyticum, and Q046R5 from Lactobacillus gasseri.

[0090] 1.3. Summary of screenings on the TagA putative collection

[0091] A total of 10 enzymes were selected (Table 1). Of these ten enzymes, 2 These enzymes are coded by a lacD gene. These are the two of the three class I enzymes that we manually added to our collection (from an initial collection created in 2009) to maintain an overview of the synthetic potential of class I TagA.

[0092] Table 1 lists the ten enzymes selected following the two screenings.

[0093] [Tables 1] Uniprot Annotations Uniprot Organisms Genes A0A3R9CKN0 TBP aldolase Citrobacter koseri gatY A0AGT7 TBP aldolase (EC 4.1.2.40 ) Listeria welshimeri lacD A6BDQ4 Cetose-bisphosphate aldolase Dorea longicatena / B0P6N8 FBP aldolase (Class II) (EC 4.1.2.13) Anaerotruncus colihominis fba C0D851 FBP aldolase (Class II) (EC 4.1.2.13) Clostridium asparagiforme fba C6JL87 TBP aldolase (EC 4.1.2.40 ) Fusobacterium varium kbaY M5DZ63 FBP aldolase (Class II) (EC 4.1.2.13) Halanaerobium saccharolyticu m / M5E4H7 TBP aldolase (EC 4.1.2.40 ) Halanaerobium saccharolyticu m / Q046R5 / A0A805ZVK9 TBP aldolase Lactobacillus gasseri lacD Q65EY6 TBP aldolase (Class II) (EC 4.1.2.40) Bacillus licheniformis /

[0094] Thus, both class I and II enzymes were revealed depending on the aldolization or retroaldolization approach chosen. Among these enzymes, some are annotated in Uniprot as FruA and others as TagA, and one of them only as a ketose-α-phosphate aldolase.

[0095] 2. Alignment and structure of the TagA

[0096] We performed a sequence alignment between the 8 selected class II enzymes, the two class II E. coli TagA enzymes (P0AB74 and P0C8J6), and the class II E. coli FruA enzyme (P0AB71). The E. coli enzymes were retained for comparison. The identity matrix shows percentages always strictly below 30% with P0AB71, whereas with the E. coli TagA enzymes, the identity rates range from 30% to 69%, seemingly confirming that they are TagA enzymes (Table 2).

[0097] [Tables2] P0AB 71 C0D85 1 A6BD Q4 M5DZ 63 B0P6 N8 M5E4 H7 C6JL 87 P0AB 74 Q65E Y6 A0A3 R9CK N0 P0C8 J6 P0AB71 100 25.87 23.67 23.1 25.7 24.32 26.15 23.94 24.73 24.65 26.06 C0D851 25.87 100 31.52 37.59 39.43 37.63 39.21 35.48 39.57 37.63 34.05 A6BDQ4 23.67 31.52 100 35.38 36.65 38.79 37.01 35.23 37.72 34.52 34.16 M5DZ63 23.1 37.59 35.38 100 41.52 47.65 46.93 41.88 45.13 41.52 38.99 B0P6N8 25.7 39.43 36.65 41.52 100 46.83 51.24 45.42 46.64 48.24 46.13 M5E4H7 24.32 37.63 38.79 47.65 46.83 100 57.6 53.5 58.66 57.04 53.17 C6JL87 26.15 39.21 37.01 46.93 51.24 57.6 100 51.59 59.01 56.54 53.71 P0AB74 23.94 35.48 35.23 41.88 45.42 53.5 51.59 100 55.48 59.51 54.58 Q65EY6 24.73 39.57 37.72 45.13 46.64 58.66 59.01 55.48 100 61.13 54.42 A0A3R9 CKN0 24.65 37.63 34.52 41.52 48.24 57.04 56.54 59.51 61.13 100 69.37 P0C8J6 26.06 34.05 34.16 38.99 46.13 53.17 53.71 54.58 54.42 69.37 100

[0098] Regarding the two class I TagAs selected (A0AGT7, A0A805ZVK9) during the screenings, they show a low percentage of identity (less than 16%) with the E. coli FruA (P0AB71) and the class II E. coli TagAs (P0AB74 and P0C8J6). Indeed, it is logical that the mechanistic differences between these two classes of enzymes imply low rates of identity (Table 3). In order to validate the belonging of these two enzymes to the TagA family rather than the FruA family, a class I FruA (characterized and crystallized) (B. Siebers et al. ([5]); E. Lorentzen ([6])) from Thermoproteus tena%(P58315) was chosen as a reference and aligned with the putative TagAs. It shows only low percentages of identity with the selected enzymes (approximately 20%).In contrast, A0AGT7 and A0A805ZVK9 show significantly higher percentages of identity (63% and 54%, respectively) with a class I TagA from Staphylococcus aureus (P0A010) that has been characterized (Bissett, DL and Anderson, RL ([7]); Anderson, RL; Bissett, DL ([8]); Bissett, DL et al. ([9])) and which was chosen as a reference (Table 3). This clearly indicates that the selected enzymes are more akin to class I TagAs than to class I FruAs.

[0099] [Tables3] P0AB7 1 PO AB 74 P0C8J6 P58315 A0A8 05ZV K9 A0AG T7 P0A0 10 P0AB71 100 23.94 26.41 12.09 16.2 14.24 15.17 P0AB74 23.94 100 54.58 15.23 13.7 14.46 12.1 P0C8J6 26.41 54.58 100 13.2 12.31 14.17 14.11 P58315 12.09 15.23 13.2 100 21.25 21.01 20.83 A0A805ZVK9 16.2 13.7 12.31 21.25 100 55.66 53.54 A0AGT7 14.24 14.46 14.17 21.01 55.66 100 63.47 P0A010 15.17 12.1 14.11 20.83 53.54 63.47 100

[0100] Example 2: Synthetic potential of enzymes for obtaining TBP

[0101] 1. Expression and purification of putative TagA

[0102] The ten selected enzymes, along with the two enzymes from the reference set, were produced and purified for initial investigation in TBP synthesis. The enzyme-encoding genes were cloned and overexpressed in E. coli (Atpi strains), with a 6-histidine tag introduced at the N-terminus to facilitate protein purification using a Ni2+-NTA-Agarose affinity resin. To this end, E. coli (expression strain) colonies containing the plasmids were cultured in 1 L of Luria-Bertani (LB) medium in the presence of a selection antibiotic (ampicillin 100 pg / mL) at 37°C with shaking. When the culture reached a ΔO₆₁₀m of 0.5, protein expression was induced with IPTG (0.5 mM) and the temperature was lowered to 30°C. The culture was incubated for an additional 12 hours.The cells were harvested by centrifugation, washed twice, and resuspended in buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). The cells were lysed by sonication, and the cell lysate was then centrifuged at 10,000 x g for 45 min. The clear supernatant was introduced onto a Ni2+-NTA-agarose column (Qiagen, h = 3 cm; 0 = 2.5 cm) pre-equilibrated with buffer A. The column was washed with buffer A and the retained proteins were eluted with the same buffer containing 125 mM imidazole (for A0AGT7, A0A3R9CKN0, A6BDQ4, B0P6N8, C0D851, C6JL87, M5DZ63, M5E4H7, A0A805ZVK9, P0AB74 and P0C8J6) or 500 mM imidazole (for Q65EY6).

[0103] After elution of the biocatalysts, the eluted fractions containing the pure proteins (detected by Bradford assay) were dialyzed with 3 M ammonium sulfate to remove NaCl and imidazole and precipitate the proteins (for A0AGT7, A6BDQ4, B0P6N8, C0D851, C6JL87, M5DZ63, M5E4H7, A0A805ZVK9, Q65EY6, and P0C8J6) or dialyzed with water for desalting and imidazole removal, then lyophilized (for A0A3R9CKN0 and P0AB74). The choice of protocol depends on the relative stability of these enzymes over time. Stable enzymes are preferentially obtained in powder form, i.e., in lyophilized form. For ammonium sulfate precipitation, the protein concentration was then determined by Bradford assay.

[0104] The quantities obtained for each enzyme and for 1 L of culture are reported in Table 4.

[0105] [Tables4] Uniprot mg protein / L of culture Storage A0A3R9CKN0 156 Powder at 4°C A0AGT7 21 Precipitation (NH4)2SO4 at 4°C A6BDQ4 10 Precipitation (NH4)2SO4 at 4°C B0P6N8 63 Precipitation (NH4)2SO4 at 4°C C0D851 339 Precipitation (NH4)2SO4 at 4°C C6JL87 35 Precipitation (NH4)2SO4 at 4°C M5DZ63 10 Precipitation (NH4)2SO4 at 4°C M5E4H7 10 Precipitation (NH4)2SO4 at 4°C A0A805ZVK9 103 Precipitation (NH4)2SO4 at 4°C Q65EY6 183 Precipitation (NH4)2SO4 at 4°C P0AB74 257 Powder at 4°C P0C8J6 2 Precipitation (NH4)2SO4 at 4°C

[0106] Among the enzymes, 4 were produced with poor yields, namely: A6BDQ4, M5DZ63, M5E4H7 and P0C8J6. 3 enzymes were produced with modest yields of between 20 and 65 mg of protein per liter of culture (A0AGT7, B0P6N8 and C6JL87) while all the other enzymes yielded 100 to 350 mg of protein per liter of culture.

[0107] 2. Synthesis of TBP with purified enzymes

[0108] The purified enzymes were studied and evaluated for their ability to synthesize stereoselectively, D-TBP was synthesized. Since an E. coli TagA has already been described as capable of oxidizing DHAP to HPP, each synthesis was performed under an inert atmosphere. The syntheses were carried out at 20°C (room temperature), under argon, with a TagA concentration of 0.5 mg / mL, in the presence of 0.5 mM Co2+, 174 U TPI, and at pH 7.5 (see [Fig. 3]). The mixture was titrated using a serial titration screening method. Specifically, 2.3 mL of 344 mM DHAP, 80 pL of 50 mM CoCl2 solution, and 5.6 mL of water were added to a flask. The pH of the mixture is then adjusted to 7.5 by adding 10 M sodium hydroxide solution. Three-necked flasks are filled with 1.5 mL of this solution, then placed under reduced pressure and argon. This operation is repeated three times to ensure the absence of air. Next, 0.75 mg of the different enzymes are dissolved in 45 µL of water and mixed with 5 µL of TPI (174 U), then the 50 µL... are injected into balloons which are agitated at 20°C. Samples are taken at different times and analyzed using the successive analysis method (see screening in Example 1 and [Fig.1]).

[0109] Each enzyme showed some activity, however minimal, on the synthesis of TBP or FBP under our reaction conditions, with the exception of A6BDQ4, M5DZ63, M5E4H7, and P0C8J6. These enzymes also gave poor production results (Table 4), so we did not continue our studies with them. We observed three trends among the eight remaining enzymes: - The enzymes that kinetically synthesize TBP in the majority, with a relative proportion of TBP ranging from 80% to 95%: B0P6N8, C6JL87, A0A3R9CKN0, Q65EY6 and P0AB74 (reference in E. coli): - Enzymes that synthesize TBP in a less stereoselective way with a relative proportion between 20% and 55%: A0AGT7 and A0A805ZVK9; - Enzymes that produced exclusively FBP: C0D851 (see [Fig.6]).

[0110] B0P6N8 from Anaerotruncus colihominis, although annotated FruA in the Uniprot database, was the most efficient and led to the production of TBP in 30 min with a diastereoisomeric excess of 92%. TBP was observed as the kinetic product and once 80% conversion of DHAP was reached (maximum conversion due to the equilibrium constant of the reaction (Fessner, W.-D. and Eyrisch, O. ([3]))), the composition of the mixture shifted towards FBP.

[0111] Three enzymes were chosen to study the effect of temperature on the stereoselectivity of the reaction (see [Fig.4]): B0P6N8 (which gave the best results), Q65EY6 (as a representative of C6JL87 and A0A3R9CKN0, these three enzymes having given the same results), and P0AB74 (as a reference).

[0112] To do this, 3.3 mL of a 153 mM DHAP solution, 50 pL of a 50 mM CoCl2 solution, and 1.7 mL of water are added to a flask. The pH of the mixture is then adjusted to 7.5 by adding 10 M sodium hydroxide solution. Three-necked flasks are filled with 1.5 mL of this solution, and the flasks are then placed under reduced pressure and argon. This operation is repeated three times to ensure the absence of air. 0.75 mg of the different enzymes are dissolved in 45 pL of water and mixed with 5 pL of TPI (174 U). The 50 pL of this mixture are then added to the flasks, and the flasks are shaken at 10°C. Samples are taken at different times and analyzed using the successive analysis method (see screening in Example 1 and [Fig.1]).

[0113] Lowering the temperature induced an improvement in stereoselectivity for each of the enzymes (Table 5 (ed: diastereomeric excess), [Fig.7]). For B0P6N8 at 10°C, before reaching the maximum conversion of 80%, FBP was not detected. It should be noted that the same behavior is observed for Q65EY6 but at 4°C, thus making this enzyme a little less interesting from a biocatalytic point of view.

[0114] [Tableaux5] Uniprot ed at 20°C (approximately 80% conversion) ed at 10°C (approximately 80% conversion) A0A3R9CKN0 70% (TBP) nd A0AGT7 26% (FBP) nd A6BDQ4 No activity nd B0P6N8 92% (TBP) >98% (TBP) C0D851 >98% (FBP) nd C6JL87 74% (TBP) nd M5DZ63 No activity nd M5E4H7 No activity nd A0A805ZVK9 4% (TBP) nd Q65EY6 80% (TBP) 80% (TBP) P0AB74 74% (TBP) 76% (TBP) P0C8J6 No activity nd

[0115] Since B0P6N8 is the most efficient enzyme, it was selected to synthesize TBP at 10°C. To do this, 1.5 mL of 166 mM DHAP, 20 pL of 50 mM CoCl2 solution, and 0.4 mL of water are added to a round-bottom flask. The pH of the mixture is then adjusted to 7.5 by adding sodium hydroxide solution. The flask is then placed under reduced pressure and filled with argon. This operation is repeated three times to ensure the absence of air. 1 mg of B0P6N8 is dissolved in 45 pL of water and mixed with 20 pL of TPI (696 U). The remaining 65 pL is then added to the flask, and the flask is shaken at 10°C. Samples were taken at different times and analyzed using the sequential titration method (see first screening and [Fig. 1]). After 57 min, the conversion was 72%, and the reaction was stopped by acidification to pH 3 with 37% hydrochloric acid. The pH of the mixture was readjusted to pH 7, and the mixture was lyophilized. The solid was reconstituted in 2 mL of water.Fractions of 770 pL are taken to optimize purification. The best purification conditions are as follows: 770 pL are purified on Dowex 1x8 anion exchange resin (as bicarbonate) and eluted with an ammonium bicarbonate gradient (0.2 M to 0.6 M). The fractions containing TBP (detected by UV spectrophotometric assay following the method described in [Fig. 1]) are evaporated and reconstituted in 2 mL of water. The solution is titrated to 17.5 mM of TBP, i.e. 3.5 x 105 mol of TBP corresponding to a yield of 70%.

[0116] The synthesis was also tested at 4°C with Q65EY6 for comparison. Under the same reaction conditions, at this temperature, 8.5 hours are required to form the TBP. The product was also purified following the same protocol, and 243 mg of TBP were obtained with a yield of 77%.

[0117] Finally, in the context of obtaining d-tagatose, it is important to note that with B0P6N8, d-tagatose-1-phosphate (T1P) was obtained with 98% conversion in 2 h by adding DHAP (50 mM) to d-glyceraldehyde (70 mM) at 20°C with an enzyme concentration of 1 mg / mL, according to the reaction scheme shown in [Fig. 5]. To do this, 2.3 mL of a 166 mM DHAP solution, 77 pL of a 50 mM CoCl2 solution, 50.4 mg of d-glyceraldehyde, and 5.3 mL of water were added to a flask. The pH of the mixture was then adjusted to 7.5 by adding a 10 M sodium hydroxide solution. The flask was then placed under reduced pressure and argon. This operation is repeated three times to ensure the absence of air. 7.7 mg of B0P6N8 are dissolved in 50 pL of water, then the 50 pL are added to the flask and it is shaken at 20°C. After 2 hours, the conversion is 98% and the reaction is stopped by acidification to pH 3 with 37% hydrochloric acid.The pH of the mixture is readjusted to pH 7 and the medium is lyophilized and analyzed by proton NMR and mass spectrometry.

[0118] This step offers further new perspectives on the synthesis of d-tagatose because, unlike TBP, it is not limited to a maximum conversion of 80% and can be carried out at room temperature, which represents a significant advantage. References

[0119] 1 :CN117305204.

[0120] 2: WO2017059278.

[0121] 3: Fessner, W.-D.; Eyrisch, O. One-Pot Synthesis of Tagatose 1,6-Bisphosphate by Diastereoselective Enzymatic Aldol Addition. Angew. Chem. Int. Ed. Engl. 1992, 31 (1), 56-58. https: / / doi.org / 10.1002 / anie.199200561.

[0122] 4: Eyrisch, O.; Sinerius, G.; Fessner, W.-D. Facile Enzymic de Novo Synthesis and NMR Spectroscopic Characterization of D-Tagatose 1,6-Bisphosphate. Carbohydr. Res. 1993, 238, 287-306. https: / / doi.org / 10.1016 / 0008-6215(93)87020-S.

[0123] 5 : B Siebers, H Brinkmann, C Dôrr, B Tjaden, H Lilie, J van der Oost, C H Verhees. Archaeal Fructose-1,6-Bisphosphate Aldolases Constitute a New Family of Archaeal Type Class I Aldolase. J. Biol. Chem. 2001, 276 (31). https: / / doi.org / 10.1074 / jbc .M103447200.

[0124] 6. Esben Lorentzen, Ehmke Pohl, Peter Zwart, Alexander Stark, Robert B Russell, Thomas Knura, Reinhard Hensel, Bettina Siebers. Crystal Structure of an Archaeal Class I Aldolase and the Evolution of (Betaalpha)8 Barrel Proteins. J. Biol. Chem. 2003, 278 (47). https: / / doi.org / 10.1074 / jbc.M305922200.

[0125] 7. Bissett, D. L.; Anderson, R. L. Lactose and D-Galactose Metabolism in Staphylococcus Aureus. IV. Isolation and Properties of a Class I D-Ketohexose-1,6-Diphosphate Aldolase That Catalyzes the Cleavage of D-Tagatose 1,6-Diphosphate. J. Biol. Chem. 1980, 255 (18), 8750-8755. https: / / doi.org / 10.1016 / S0021-9258(18)43564-8.

[0126] 8. Anderson, R. L.; Bissett, D. L. d-Tagatose-l,6-Bisphosphate Aldolase (Class I) from Staphylococcus Aureus. In Methods in Enzymology, Elsevier, 1982; Vol. 90, pp 228-232. https: / / doi.org / 10.1016 / S0076-6879(82)90130-6.

[0127] 9. Bissett, D. L.; Wenger, W. C.; Anderson, R. L. Lactose and D-Galactose Metabolism in Staphylococcus Aureus. IL Isomerization of D-Galactose 6-Phosphate to D-Tagatose 6-Phosphate by a Spécifie D-Galactose-6-Phosphate Isomerase. J. Biol. Chem. 1980, 255 (18), 8740-8744. https: / / doi.org / 10.1016 / S0021-9258(18)43562-4.

[0128] 10. M. Wei et al. ACS Catal. 2015, 5, 4060-4065.

[0129] 11. Léo Paulat, Victor Laurent, Dr. Virgil Hélaine, Mariline Théveniot, Jean-Louis Petit, Prof. Marielle Lemaire, Valérie Delmas, Dr. Madeleine Bouzon, Dr. Véronique De Berardinis, Dr. Christine Guérard-Hélaine. Insights on DHAP Aldolases' ability to convert dioxygen or a ketone as an electrophile: use of a strain depleted in triose phosphate isosomerase. ChemCatChem, 2024, Volume 16, Issue 11. doi.org / 10.1002 / cctc.202400202

Claims

Demands

1. Use of an enzyme belonging to the aldolase group and comprising a polypeptide having at least 30% identity with an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:4, to stereoselectively prepare d-tagatose-1,6-Wsphosphate (TBP) or d-tagatose-1-phosphate (T1P).

2. Method for preparing d-tagatose-1,6-εAphosphate (TBP) or d-tagatose-1-phosphate (T1P), comprising a reaction step, in a suitable reaction medium, containing dihydroxyacetone phosphate (DHAP) or at least one of its isomers or analogues, and optionally d-glyceraldehyde, with an enzyme belonging to the aldolase group, said enzyme comprising a polypeptide having at least 30% identity with an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:

4.

3. Method according to claim 2, wherein said enzyme comprises or consists of a polypeptide having a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:

4.

4. Method according to claim 2 or 3, wherein said enzyme is selected from the group comprising a purified enzyme, present in free form or immobilized on a support, and an enzyme produced in situ by a cell expressing said enzyme.

5. Method according to any one of claims 2 to 4, wherein said at least one isomer or analogue of DHAP is selected from dihydroxyacetone (DHA) and d-glyceraldehyde-3-phosphate (d-G3P).

6. Method according to claim 5, wherein when dihydroxyacetone (DHA) is used, an anion capable of mimicking phosphate such as borate, arsenate or vanadate, a phosphonate or methyl phosphate is added to the medium.

7. Method according to any one of claims 2 to 6, wherein said reaction medium contains triose phosphate isomerase (TPI).

8. A method according to any one of claims 2 to 7, wherein the reaction is carried out at a temperature between approximately 2°C and approximately 40°C, and preferably approximately 10°C. for TBP and at a temperature between 18°C ​​and 40°C for T1P.

9. A process for preparing d-tagatose, comprising the steps of: a. To prepare d-tagatose-1,6-Wsphosphate (TBP) or d-tagatose-1-phosphate by implementing the method defined in any one of claims 2 to 8 and b. Dephosphorylate the TBP or d-tagatose-1-phosphate obtained in step (a).

10. A method according to claim 9 wherein step (b) is carried out by contacting D-tagatose-1,6-Wsphosphate (TBP) or D-tagatose-1-phosphate with a phosphatase, preferably an acid phosphatase.