SUGAR PRODUCTION BY PERIPLASMIC ALDOLIZATION

Abstract

The invention relates to a polypeptide comprising a bacterial membrane anchoring peptide and an aldolase capable of catalyzing an aldol condensation reaction between two selected compounds to form a sugar. The aldolase may be dependent on dihydroxyacetone phosphate (DHAP) or dihydroxyacetone (DHA). The invention also relates to a bacterium genetically modified to produce sugars, particularly rare sugars, phosphorylated or not, and a method for producing and secreting these sugars by culturing said bacterium in a suitable culture medium M1 comprising two compounds capable of forming a sugar by an aldol condensation reaction.

Description

Title of the invention: PRODUCTION OF SUGARS BY PERIPLASMIC ALDOLIZATION Technical field of the invention

[0001] The present invention belongs to the field of the production of molecules of interest by genetically modified microorganisms, and more particularly concerns the production of sugars by biocatalysis.

[0002] It relates to a polypeptide capable of catalyzing an aldol reaction in the periplasm of a bacterium, from substrates chosen to form a sugar by condensation. The invention also relates to a method for producing and secreting sugars, in particular rare sugars, phosphorylated or not. Technological background

[0003] Rare sugars are sugars that are very rarely found in nature (less than 1% of total sugars). They have multiple properties and can be used in different fields. They can be used as sweeteners in food but also serve as a basis for the synthesis of molecules of therapeutic interest. Some sugars are expected to have anti-cancer, antiviral or anti-diabetic activity. However, due to their rarity, the supply by extraction from plant sources is absolutely insufficient. It is certainly possible to synthesize many rare sugars by isomerization or epimerization of more common sugars, but some of them are not suitable for this, as is the case for example with L-fucose. It is then interesting to turn to the enzymatic route, and more particularly to the use of aldolases which are capable of forming a sugar by condensation of suitable substrates.

[0004] Phosphorylated sugars are also products of the metabolism of living beings. Their potential is promising but difficult to evaluate due to the obstacles existing in obtaining large quantities. However, this class of compounds is of major interest for the synthesis of new molecules, including rare sugars, having original chemical properties usable in pharmaceutical applications or the production of biomaterials. Different methods for producing phosphorylated sugars have been explored, without giving complete satisfaction. Indeed, these molecules are particularly difficult to produce chemically because their structure generally includes one or more centers of asymmetry, which is certainly an essential interest, but poses problems of stereospecificity of the reactions and separation of the isomers. In addition, the chemical intermediates to be phosphorylated are expensive.

[0005] In vitro enzymatic catalysis of sugars has the advantage of greater steric specificity. One possible scheme for the synthesis of phosphorylated sugars, the most used currently but also very expensive, is phosphorylation by a sugar kinase. Another reaction scheme that can be used to obtain rare sugars, whether phosphorylated or not, is based on the condensation of an acceptor compound such as an aldehyde with a donor compound, which can be a phosphorylated compound or not, the reaction being catalyzed by an aldolase. Aldolases are enzymes capable of condensing two chemical molecules by creating a carbon-carbon bond, which could be used in many industrial processes (Windle CL et al. (2014); Curr Opin Chem Biol; 19: 25-33). For example, document CN111041018 describes the synthesis of D-psicose from D-glyceraldehyde and dihydroxyacetone phosphate (DHAP), under the action of an aldolase and a phosphorylase.Enzymatic aldol reactions are also known that use a non-phosphorylated donor, such as dihydroxyacetone (DHA), hydroxyacetone or hydroxybutanone (Windle CL et 0011.). However, these techniques require the production of specific enzymes and cofactors. Here too, the basic equipment is very expensive.

[0006] More recently, research has turned to microbial production. In this case, the aim is to genetically modify a microorganism so that it produces the molecule of interest and secretes it into the medium. It is known that certain microbial aldolases can condense DHAP with an aldehyde to form different phosphorylated sugars in position 1 (Li A. et al. (2017); Carbohydr Res; 452: 108-115). However, phosphorylated sugars cannot be secreted by the microorganism, because the negative charge of the cell membrane repels the negative charge of the sugar's phosphate. To overcome this problem, it has been proposed to transform E. coli cells so that they co-express an aldolase and a phosphatase. The DHAP formed in the cell then reacts rapidly with an aldehyde and the resulting aldol is dephosphorylated. The lightly charged sugar molecules can then pass through the cell membrane (Aimin Li et al.(2017); Carbohydr Res; 452: 108-115). This technique makes it possible to direct cellular metabolism towards the production of dephosphorylated sugar, but in quantities limited by the cellular production of the substrates DHAP and aldehyde, especially since aldehydes are toxic to the cell. It is limited to the production of rare sugars but cannot be extended to the production of phosphorylated sugars.

[0007] It thus appears that no satisfactory technique has been developed to date which makes it possible to produce rare sugars, phosphorylated or not, accessible in large quantities and at an affordable price.

[0008] Objectives of the invention

[0009] The present invention proposes a novel approach consisting of synthesizing these sugars outside the cell itself, that is, outside the cytoplasm and more precisely in the periplasmic space, by involving cellular enzymatic tools to condense two extracellular compounds, namely an aldehyde and a possibly phosphorylated donor compound. However, since aldolases are produced in the cytoplasm of the cell, they cannot carry out the desired catalysis in their current state. The proposed solution consists of attaching to the membrane of a bacterium a functional aldolase, capable of catalyzing the aldolization reaction outside the cytoplasm. A polypeptide was thus developed consisting of the sequence of an aldolase and a signal peptide having an affinity for the membrane of a bacterium. A bacterial strain genetically transformed to express this polypeptide was also developed.Therefore, it is possible to achieve bacterial production of sugars of interest, in particular rare and / or phosphorylated sugars in the periplasm, which diffuse naturally into the culture medium and can be easily recovered.

[0010] The present invention is based on the discovery that a genetically transformed bacterial cell can produce an aldolase capable of being exported into the periplasmic space and of anchoring to the membrane, while retaining its catalytic capacities with respect to donor and acceptor compounds, despite the extracellular origin of the latter.

[0011] The approach adopted is all the more original since the production of the phosphorylated substrates necessary for obtaining phosphorylated sugars was until very recently particularly expensive, of the order of 1000 euros / gram. The development by the Applicant of a technology for producing these compounds by microbial means now makes it possible to have phosphorylated reagents, and in particular dihydroxyacetone phosphate (DHAP), at an affordable cost, thereby opening the way to the production of phosphorylated sugars in large quantities by microbial aldolization.

[0012] To enable the periplasmic aldol reaction, a synthetic polypeptide was designed. This comprises an aldolase fused to a cell membrane signal peptide. Surprisingly, it was found that this fusion protein is correctly addressed and anchored to the membrane, and in doing so, it is functional to catalyze aldolization from its specific reagents that it recognizes. Sugar compounds, which are normally synthesized in the cell and are produced in limited quantities due to the reduced availability of basic molecules, are difficult to extract. On the contrary, according to the present invention, the biochemical machinery of the cell is used, which is moved outside the bacterial cytoplasm so that the periplasmic space is the site of the synthesis of sugars which are then excreted to the culture medium, from where they can easily be extracted. Statement of the invention

[0013] This is why, according to a first aspect, the present invention relates to a polypeptide comprising an aldolase capable of catalyzing an aldolization reaction between two compounds, one acceptor and the other donor, and a bacterial membrane anchoring peptide.

[0014] An aldolization is an electrophilic condensation reaction during which a CC bond is formed. The reactants (substrates of the enzyme carrying out the catalysis) are compounds, one nucleophilic (electron donor) and the other electrophilic (electron acceptor), so designated because of the reaction mechanism involved. When the two reactants are different, the aldolization is called crossed or mixed. Generally speaking, in what follows, the acceptor compound will be an aldehyde. The nucleophile may or may not be a phosphorylated compound.

[0015] Different pairs of phosphorylated donor compounds - aldehyde can interact in an enzymatically catalyzed aldol reaction to form a phosphorylated sugar. Many examples are provided in the literature (see in particular the review by Windle CL et al. (2014); Curr Opin Chem Biol; 19: 25-33). Thus, AldoB, a liver aldolase, is able to use DHAP and glyceraldehyde to form D-fructose-1-phosphate. The same enzyme is also able to provide D-xylulose-1-phosphate from glycolaldehyde (Cam Y. et al., (2016); ACS Synth Biol 5(7): 607-618). Similarly, FbaB is able to condense DHAP with glyceraldehyde-3-phosphate to form fructose-1,6-biphosphate. Non-phosphorylated donor-aldehyde pairs can also interact in an aldol reaction to form a sugar.For example, the aldolase FSA is able to use DHA and lactaldehyde to generate fuculose, an isomer of L-fucose that is not very commercially available. Note that DHAP can also react via FucA with L-lactaldehyde to give L-fuculose-1-phosphate which, once dephosphorylated and isomerized, will give back L-fucose.

[0016] Thus, according to a preferred feature of the present invention, the polypeptide comprises a dihydroxyacetone-phosphate (DHAP)-dependent aldolase capable of catalyzing a condensation reaction between an aldehyde and DHAP or a dihydroxyacetone (DHA)-dependent aldolase capable of catalyzing a condensation reaction between an aldehyde and DHA. Generally, all kinds of aldolases can be used in the present invention, as long as they do not require particular cofactors. When the donor is DHAP, the sugars obtained are phosphorylated in position 1.

[0017] According to a particularly preferred embodiment of the present invention, the aldolase is chosen from AldoB, RhaD, YdjI, FSA or FbaB. These enzymes are capable to support DHAP or DHA.

[0018] Aldolase AldoB (or fructose-bisphosphate protein) is an aldolase capable of utilizing DHAP with different aldehydes. By varying the aldehyde, different phosphorylated sugars can be synthesized. Thus, from glyceraldehyde, D-fructose-1-phosphate is obtained, while from glycolaldehyde, D-xylulose-1P can be produced. Also, from D-glyceraldehyde-3-P, D-fructose-1,6-bi-phosphate is obtained. In particular, aldolase AldoB may be of human origin, and may have the amino acid sequence SEQ ID NO 1.

[0019] RhaD aldolase (or rhamnulose-1-phosphate aldolase) is capable of synthesizing many phosphorylated sugars from different aldehydes. Examples include L-fructose-1-phosphate, L-rhamnulose-1P, and L-xylulose-1P (Chen et al. (2020); ACS Synth Biol; 5(7): 607-618). Similarly, D-psicose-1-phosphate and D-sorbose-1-phosphate can be obtained (from DHAP and D-glyceraldehyde in both cases), L-fructose-1-phosphate or D-tagatose-1P (from DHAP and L-glyceraldehyde in both cases), L-rhamnulose-1-phosphate (from DHAP and lactaldehyde), or L-xylulose-1-phosphate (from DHAP with glycolaldehyde). A C4 sugar, such as erythrose-1-phosphate, can also be obtained with DHAP and formaldehyde.

[0020] YdjI aldolase can achieve a very wide range of sugars. Like AldoB and RhaD, it supports DHAP, but it offers a broad spectrum allowing longer carbon chains to be achieved.

[0021] Aldolases based on the use of DHA such as FSA (fructose-6-phosphate aldolase) or FbaB using GAP (glyceraldehyde-3-P aldolase), can be used in the same way.

[0022] According to another preferred characteristic of the present invention, the membrane anchoring peptide comprises a signal peptide of an Escherichia coli lipoprotein. A signal peptide is a peptide chain of a protein used to address it to a particular cellular compartment or in the periplasm in prokaryotes. In this case, the anchoring peptide will be chosen from those which have the ability to orient the polypeptide according to the invention towards the periplasmic space and to anchor itself there to one of the membranes, so that the enzymatic part of the polypeptide is maintained in an active configuration in the periplasmic space. Since the membrane is the lipid double layer delimiting the cell, the signal peptide is advantageously composed of a lipoprotein.

[0023] Periplasmic anchoring of a protein presents several problems relating to the addressing of the protein, its folding and the effect on the structure of the periplasm. According to an original embodiment, it was chosen to use a periplasmic anchoring system designated by the acronym APEx, described by Guo et al. (2014) in Biotechnol Lett; 36, n. 3, p. 609-616. This is formed from the leader peptide of the lipoprotein NlpA associated with the six amino acid peptide of the mature protein NlpA of sequence CDQSSS, the latter segment not being integrated into the thickness of the membrane, but protruding towards the periplasm. Thus, the six amino acids are a periplasmic part of NlpA and constitute the segment by which fusion with the aldolase sequence takes place. This system has the advantage of having efficient targeting to the cytoplasmic membrane and allowing satisfactory protein folding, so that the enzyme is fixed to the membrane in an active conformation.

[0024] Thus, according to a particular embodiment of the invention, the periplasmic anchoring peptide comprises the leader peptide of the lipoprotein NlpA and the six amino acid peptide of the mature protein NlpA of sequence CDQSSS. The anchoring peptide according to the invention may comprise the amino acid sequence SEQ ID NO 2.

[0025] Typically, aldolase and the periplasmic anchor peptide can be produced linked together as a fusion protein. The two segments of the fusion protein can be linked by a linker peptide, a role which is here played by the six amino acid sequence of the NlpA protein of sequence CDQSSS. The sequence of a fusion protein formed of AldoB and NlpA is shown in SEQ ID NO 3.

[0026] According to a second aspect, the present invention relates to a nucleotide sequence encoding a polypeptide comprising an aldolase and a bacterial periplasmic anchoring peptide, as described above. The nucleotide sequence may further encode the sequence of a suitable linker peptide for linking the aldolase and the anchoring peptide. In this case, it is the peptide of sequence CDQSSS. Preferably, according to the invention the nucleotide sequence may comprise the sequence SEQ ID NO 4.

[0027] For each of the amino acid and nucleotide sequences set out in the present application, there are homologous, equivalent or derived sequences. It is specified that the invention relates to all nucleic acid molecules having a sequence substantially identical to the sequences described, just as it includes polypeptides whose sequence is substantially identical to the amino acid sequences described. Furthermore, a person belonging to the technical field concerned knows that the nucleotide sequences defined here may have variants, as exist in nature due to differences between strains. The variations may be based on additions, substitutions and / or deletions. Variants may also result from the degenerate nature of the genetic code.

[0028] Preferably, the variants are substantially identical to the nucleic acid sequences described. More specifically, the variants in question have at least 80% of identity with the described sequences, preferably at least 90% identity, more preferably at least 95%, or at least 99% identity with the described sequences. It is desirable that the substantially identical sequences have a higher degree of identity with the described sequences than with any other sequence known in the prior art.

[0029] The nucleotide sequence according to the invention may be codon optimized for optimal expression in the host bacterial cell, for example in E. coli. The sequence may also comprise additional sequences necessary for the expression of the encoded polypeptide, such as promoter sequences, ribosome binding sites and nucleotide sequences for inducing expression of the protein. The person skilled in the art knows these sequences and knows how to implement them.

[0030] In order to be able to express the polypeptide according to the invention, the nucleotide sequence must be inserted into a suitable host. This is why, according to a third aspect, the subject of the present invention is a vector comprising a nucleotide sequence encoding a polypeptide which comprises an aldolase and a bacterial periplasmic anchoring peptide, as described above. A vector is a vehicle which will allow the nucleotide sequence of interest to be transferred into the host cell. It can be chosen from the different types of vectors known to those skilled in the art, such as, for example, plasmids, viral vectors, artificial chromosomes of yeasts or bacteria. It should be noted that genomic integration of the nucleotide sequence according to the invention is also possible.

[0031] The vector comprising the nucleotide sequence of interest must be transformed into a suitable host. There are numerous transformation, transfection or transduction techniques that can be used to introduce the vector into the host cell, which those skilled in the art will be able to choose depending on the nature of the vector and the host used.

[0032] Thus, according to a fourth aspect, the present invention relates to a bacterium genetically modified to produce sugars of interest, phosphorylated or not, said bacterium being genetically modified to express a polypeptide which comprises an aldolase and a bacterial periplasmic anchoring peptide, as described above. Thanks to the anchoring peptide, the polypeptide produced by the genetically modified bacterium is addressed to the cytoplasmic membrane by the natural mechanisms of the cell.

[0033] A suitable host according to the present invention is a bacterium provided with an envelope, called periplasm, formed of a double membrane, one internal at the interface with the cytoplasm, and the other external at the interface with the external environment. This double membrane defines the periplasmic space in which the synthesis of the sugars of interest can take place. Such a host is typically a bacterial cell Gram negative.

[0034] The present invention can therefore be implemented in different Gram-negative bacteria as long as they have a periplasm. A large number of bacterial strains can be used, such as for example Pseudomonas, Klebsiella, Enterobacter, Helicobacter, Salmonella. Preferably, the bacterium is chosen from the species Escherichia coli.

[0035] According to a preferred feature of the invention, the bacterium is genetically modified to express a dihydroxyacetone-phosphate (DHAP) or dihydroxyacetone (DHA) dependent aldolase in the periplasmic space of said bacterium. Such an aldolase is capable of taking up DHAP or DHA and of catalyzing an electrophilic condensation reaction with an aldehyde to form a sugar of interest, phosphorylated or not depending on the donor chosen. This embodiment is advantageous because DHAP and DHA can be taken up by numerous aldolases.

[0036] With regard more particularly to the production of phosphorylated sugars, we have seen above that a major constraint is to be able to have extracellular DHAP in sufficient quantity whereas it is very expensive to produce by conventional techniques. This obstacle can now be overcome thanks to a technology developed by the Applicant which makes it possible to produce DHAP microbially and to excrete it from the cell into the culture medium. This technology is described in application WO2020 / 074582.

[0037] As explained above, depending on the aldehyde used, different sugars can be obtained thanks to a particular aldolase, and conversely a given aldehyde can result in several sugars or several isomers of a sugar depending on the aldolase catalyzing the reaction. According to a particular embodiment of the invention, said bacterium is modified to express an aldolase chosen from AldoB, RhaD, YdjI, FSA or FbaB. For example, when the aldolase AldoB is used, the bacterium expresses the amino acid sequence SEQ ID NO 1 (included in SEQ ID NO 3).

[0038] According to a fifth aspect, the present invention relates to a method for producing a sugar of interest which comprises a step consisting of cultivating a bacterium as described above in a suitable culture medium M1 comprising two compounds capable of forming said sugar by an aldol reaction.

[0039] The culture conditions may vary depending on the bacterial strain used and may be optimized by a person skilled in these techniques to ensure a maximum production rate of compounds of interest. The cultures may in particular take place at temperatures ranging from about 20°C to about 45°C, preferably between 30°C and 37°C and more preferably around 37°C. They may be subjected to agitation promoting oxygenation, to a speed of, for example, between 100 rpm and 250 rpm, and preferably between 180 rpm and 220 rpm. The culture protocol can be carried out in the usual manner, with production of isolated colonies of the genetically modified bacterium according to the invention on solid agar plates, followed by the production of a starter culture medium inoculated with a bacterial colony, the starter culture then being used to inoculate a larger volume of liquid medium. The cultures can be carried out at different scales ranging from volumes of 500 ml to 2000 liters depending on the production objectives (laboratory use or industrial production).

[0040] A large number of different media are suitable for culturing bacteria. As used herein, the term "medium" refers to a solid, liquid, or semi-solid support in which microorganisms can be cultured, for example, a gel. The media may be supplemented with additional components to enhance bacterial growth, for example, amino acids, sugars, lactose, glucose, mannose, glycerol, calcium, magnesium, iron, phosphate, sulfates, chlorides, acetates, citrates, etc. The media may also be supplemented with antibiotics or antimicrobials such as ampicillin, tetracycline, kanamycin, streptomycin, carbenicillin, erythromycin, and chloramphenicol. These antimicrobials may be used to selectively cultivate certain bacteria that contain a gene for resistance to a given antibiotic.Non-limiting examples of media that may be used to cultivate the bacteria of the invention include LB (lysogeny broth) medium, TB (terrify broth) medium, 2xYT medium, SOB (Super Optimal Broth) medium, SOC (Super Optimal Broth with Catabolite repression) medium, M9 (minimal salts media) media, NZCYM medium, or NZ amine medium.

[0041] According to the invention, the culture medium comprises the reagents involved in the aldolization leading to a sugar of interest, such as a rare sugar or a phosphorylated sugar. These reagents are the substrates of the membrane aldolase acting in the periplasm, typically an aldolase and a donor compound, which may comprise a phosphorus substituent. Different concentrations may be suitable, which a person skilled in the art will be able to relate to the cell population present. For example, the medium may comprise a donor compound at a content ranging from 0.1 mM to 12 mM, preferably between 1.2 mM and 9 mM. More particularly, when DHAP is used as a donor, it may have a concentration of approximately 1.2 mM to approximately 9 mM. The aldehyde is added to the medium so that the molar ratio with the donor compound may be between 3 / 1 and 20 / 1. Preferably, 10 equivalents of aldehyde are added.It is understood that these concentrations are those of the middle at the beginning of the culture, and that they are likely to evolve as the aldolization consumes the reagents and forms the targeted sugar.

[0042] For the reasons mentioned above, the donor compound present in the culture medium M1 may advantageously be DHAP or DHA. Thus, according to a preferred characteristic of the method which is the subject of the invention, the culture medium M1 comprises on the one hand an aldehyde and on the other hand DHAP or DHA. Many aldehydes may be used according to the invention, in particular those already mentioned above, without this being limiting.

[0043] The donor compound can be purchased from a distributor or prepared in a prior step to be added as such to the Ml medium. However, in an original manner, it can be provided in the form of a supernatant from a previously carried out microbial culture or a solution derived from said supernatant. This production method is particularly suitable for the production of phosphorylated sugars using DHAP. Indeed, a microbial strain transformed so as to synthesize DHAP and export it into its culture medium Mo can be cultivated under conditions as described in WO 2020 / 074582. After removal of the biomass, the culture supernatant So rich in DHAP is available. This microbial DHAP solution can then serve as a basis for the manufacture of a new culture medium, namely the Ml medium.

[0044] Thus, in a particularly interesting embodiment of the method which is the subject of the present invention, the culture medium M1 is prepared by the following protocol in which: - a bacterium capable of producing DHAP and secreting it is cultivated in a culture medium Mo, - at the end of this culture, the supernatant So containing the bacterial DHAP is separated from the biomass, and - the supernatant So is used to prepare the culture medium Ml.

[0045] According to one embodiment of the method which is the subject of the invention, a solution containing from 250 mg / l to 1500 mg / l of DHAP, preferably 300 mg / l to 1000 mg / l of DHAP, is prepared from the supernatant So, an aldehyde is added thereto in a ratio of 3 moles to 20 moles per mole of DHAP, preferably 8 to 12 equivalents of aldehyde, then a bacterium according to the invention is introduced into the medium M1 thus obtained.

[0046] Since the external periplasmic membrane of the bacterium is permeable to solutes, the phosphorylated compound and the aldehyde present in the culture medium are also found in the periplasmic space where they react under the catalytic action of aldolase. Thanks to the periplasmic anchoring of aldolase, the phosphorylated compound, for example DHAP, and the aldehyde form a phosphorylated sugar in the periplasm, which can then be exported to the culture medium. The phosphorylated sugar can then be recovered using one of the techniques available to those skilled in the art. The same process is at work for the production of other sugars by aldolization of an aldehyde in the presence of DHA, in particular rare sugars, which will thus be extracted without difficulty from the middle.

[0047] Thus, the method according to the invention advantageously further comprises a step in which the sugar obtained is extracted from the culture medium M1. The extraction can be carried out in various ways. A key step is the separation of the bacterial cells from the liquid medium which can be carried out by centrifugation, filtration or sedimentation. Once the liquid culture has been separated from the bacterial cells, the compound of interest can then be purified using standard techniques such as column chromatography, high performance liquid chromatography, ion exchange chromatography, thin layer chromatography and reversed phase chromatography.

[0048] Thanks to the present invention, it is possible to carry out bacterial production of phosphorylated sugars by the periplasmic expression of an enzyme synthesized from a nucleic acid sequence. The localization of the aldol reaction in the bacterial periplasm has proven to be an optimal production strategy, combining good accessibility to extracellular substrates to the catalyst and a high harvest of products of interest. These can be secreted into the culture medium and easily recovered, without the need for cell lysis. The quantities synthesized by the technology which is the subject of the present invention can be used for the sale of high value-added molecules.

[0049] Detailed description of embodiments of the invention

[0050] The present invention will be better understood, and relevant details will appear, in the light of the description which will be given of different variant embodiments.

[0051] Example 1: Plasmids

[0052] Two commercial bacterial plasmids were used. - The plasmid pACT3 marketed by Addgene was chosen for in vivo expression under the control of the IPTG-inducible pTac promoter. It exhibits resistance to chloramphenicol. - The plasmid pET28b marketed by Novagen was used to express polyhistidine-tagged enzymes in the BL21(DE3) strain for purification. It exhibits resistance to kanamycin.

[0053] - Construction of plasmid pBC_P_001

[0054] Plasmid pBC_P_001 is constructed from plasmid pET28 and carries the Homo sapiens aldoB gene. The human aldoB gene was codon optimized for optimal expression in E. coli and synthesized by Eurofins. The aldoB fragment was amplified by PCR with primers aldoB_pET_F (GTTTAACTTTAAGAAGGAGATATACCATGGCACATCGCTTTCCGG), and aldoB_pET_R (ATCTCAGTGGTGGTGGTGGTGGTGCTCATACGTGTAACAGGCCGTAA ACA).

[0055] Then, it was assembled into the pET28b vector previously linearized by digestion with the NcoI / XhoI restriction enzymes with the NEBuilder HiFi assembly kit (Ref. E2621). The resulting plasmid pBC_P_001 was validated by sequencing by the company Eurofins genomics.

[0056] - Construction of plasmid BC_099

[0057] The plasmid is constructed from plasmid pACT3 and carries the Homo sapiens aldoB gene fused to the signal peptide sequence of the Escherichia coli lipoprotein NlpA.

[0058] The human aldoB gene was codon optimized for optimal expression in E. coli and synthesized by Eurofins. The plasmid pACT3 is used in this study for the in vivo expression of the aldolase AldoB fused at the N-terminus with the signal peptide of the E. coli lipoprotein NlpA for its targeting to the periplasm. The construction of the plasmid BC_099 was carried out with the NEBuilder HiFi assembly kit (Ref. E2621), from three fragments: - the plasmid pACT3 linearized by PCR with the primers pACT3_F (TAGAGTCG ACCTGCAGGCAT) and pACT3_R (ATTCTGTTTCCTGTGTGAAA); - the aldoB gene amplified by PCR with the primers aldoB_F (ATGGCACATCG CTTTCCGG) and aldoB_R (ATGCCTGCAGGTCGACTCTATTAATACGTGTAACAGGCCGTAAACA); - the nucleotide fragment coding for the 6 amino acid peptide of the E. coli NlpA protein amplified by PCR from genomic DNA with the primers NlpA_F (TTTCACACAGGAAACAGAATATGAAACTGACAACACATCATCTA) and NlpA_R (GCCGGAAAGCGATGTGCCATGCTGCTACTCTGGTCGCAAC).

[0059] The regions of homology between these three fragments, provided by the floating tails of the primers, allow homologous recombination. The resulting plasmid BC_099 was validated by sequencing by Eurofins genomics.

[0060] The amino acid sequence of the fusion protein of the aldolase AldoB with the anchor peptide NlpA is provided in SEQ ID NO 3 and the corresponding nucleotide sequence is provided in SEQ ID NO 4.

[0061] Example 2: Strains

[0062] Two parental strains of Escherichia coli supplied by the company NEB were used: - strain BL21 (DE3) (Ref. C2527H) whose genotype is fhuA2 [Ion] ompT gai (A DE3) [dcm] AhsdS ; - the NEB5-alpha strain (NEB®- Ref. C2987I) of genotype fhuA2 A(argF-lacZ)U169 phoA glnV44 ¢80 A(lacZ)M15 gyrA96 recAl relAl endAl thi-1 hsdR17.

[0063] The commercial strains BL21(DE3) and NEB5-alpha were transformed following the protocols provided by NEB, to obtain the following derived strains.

[0064] - Strain BC_ST_008, containing pBC_P_001;

[0065] The competent E. coli strain BL21(DE3) was used for the production of the aldolase AldoB-6*His with the plasmid pBC_P_001. This strain is suitable for very high protein production because it expresses T7 RNA polymerase compatible with the expression of the plasmid pET28. In addition, it is deficient in protease B which limits protein degradation during the purification procedure.

[0066] - Strain BC_092, containing pACT3

[0067] The competent E. coli strain NEB5-alpha derived from strain DH5a was used with plasmid pACT3 to provide an aldolase-free control.

[0068] - Strain BC_093, containing pBC_099

[0069] The competent E. coli strain NEB5-alpha derived from strain DH5a was used to express the aldolase AldoB periplasmically with plasmid pBC_099. This strain is resistant to T1 phages and does not possess the endA gene encoding the periplasmic endonuclease EndA.

[0070] Example 3: Culture media

[0071] The cells were cultured on rich LB (Luria-Bertani) medium during genetic manipulations, in solid or liquid culture, and during the first starter culture of the cells taken from the glycerol stock.

[0072] LB environment

[0073] The rich LB medium is composed of 10 g / L of tryptone, 5 g / L of yeast extracts and 5 g / L of NaCl. Agar at a level of 15 g / L is added to obtain a solid medium. The LB medium, with or without agar, is sterilized by autoclaving for 20 minutes at 110°C before use.

[0074] Middle M9 The cultures for the production of DHAP are carried out in M9 mineral medium containing 10 g / l of glucose. Its composition is as follows: 18 g / L Na2HPO4 * 12 H2 O; 3 g / L KH2PO4; 0.5 g / L NaCl; 2 g / L NH4C1; 0.5 g / L MgSO4 * 7 H2O; 0.015 g / L CaCl2 * 2 H2O; 0.01 g / L FeCl3; 6.0.10 3 g / L Thiamine HCl; 0.49.10 3 g / L NaEDTA; 1.8.103 g / L CoCl2 * 6 H2O; 1.8.103 g / L ZnC12SO4 * 7 H2O; 0.4.103 g / L Na2MO4 * 2 H2O; 0.1.10 3 g / L H3BO3; 1.2.10 3 g / L MnSO4 * H2O; 1.2.10 3 g / L CuCl2 * 2 H2O.

[0076] Example 4: Process for the production of D-fructose-l-phosphate and D-xylulose-l-phosphate from commercial DHAP

[0077] The BC_093 strain is taken up on LB agar plates and left at 37°C overnight. from glycerol stocks where it is stored at -80°C. A clone isolated from a dish is taken and cultured in liquid LB medium at 200 rpm, at 37°C, overnight. The next day, a dilution is made in fresh liquid LB medium at 37°C at 200 rpm. After 6 hours, 0.1 mM IPTG is added to the medium to induce the expression of the NlpA-aldoB fusion protein in the periplasm for strain BC_093. The culture is maintained overnight at 200 rpm, 30°C.

[0078] The BC_092 strain is taken up on LB agar plates and left at 37°C overnight from glycerol stocks where it is stored at -80°C. A clone isolated on the plate is taken and cultured in liquid LB medium at 200 rpm, at 37°C overnight. The next day, a dilution is carried out in fresh liquid LB medium at 37°C at 200 rpm. After 6 hours, 0.1 mM IPTG is added to the medium and then the culture is maintained overnight at 200 rpm, 30°C.

[0079] The culture of the bacterial strain BC_093 expressing the aldolase AldoB in its periplasm is centrifuged at 4000 rpm for 10 min. The cell pellet is resuspended in a solution of 50 Mm HEPES buffered to PH 7.5 containing 500 mg / L of DHAP (SIGMA-Ref 51269) and 10 eq of aldehyde. DL-glyceraldehyde (SIGMA G5001) is used for the production of D-fructose-1-phosphate while glycolaldehyde (SIGMA G6805) is used for the production of D-xylulose-1-phosphate. The volume of the reaction mixture is calculated so as to concentrate the cells by a factor of 2, for an initial bacterial culture volume of 10 ml the reaction volume will be 5 ml. This mixture is placed at 30°C, 200 rpm minimum for 4 hours. Samples are taken during the reaction, they are filtered (0.22 µm) and stored at -20°C.

[0080] Strain BC_092 was used to provide an aldolase-free control. For the other controls, the reaction mixture was adapted, DHAP or aldehyde was replaced by water.

[0081] Example 5: Process for the production of D-fructose-l-phosphate and D-xylulose-l-phosphate from microbial DHAP

[0082] Production of microbial DHAP

[0083] A bacterial culture allowing the production of DHAP is carried out according to the method presented in WO 2020 / 074582. The transformed E. coli strain YC20 is cultivated in the M9 medium under the conditions described in WO 2020 / 074582. The culture is centrifuged at 10,000 rpm and sterilized with a 0.22 pm filter. The biomass is removed to keep only the culture supernatant So containing the DHAP. The solution (supernatant So) contains from 300 mg / L to 1000 mg / L of DHAP. It is buffered at pH 7.5 and stored at -20°C.

[0084] The BC_093 strain is on an LB agar plate and left at 37°C overnight from glycerol stocks where it is stored at -80°C. A clone isolated on a plate is taken and put into Culture in liquid LB medium at 200 rpm, 37°C overnight. The next day, dilution is made in fresh liquid LB medium at 37°C at 200 rpm. After 6 hours, 0.1 mM IPTG is added to the medium to induce the expression of the NlpA-aldoB fusion protein in the periplasm for strain BC_093. The culture is maintained overnight at 200 rpm, 30°C.

[0085] The culture of the bacterial strain BC_093 expressing the aldolase AldoB in its periplasm is centrifuged at 4000 rpm for 10 min. The cell pellet is resuspended in the supernatant So containing 650 mg / L of microbial DHAP buffered at pH 7.5 to which 10 eq of aldehyde has been added. D-glyceraldehyde (SIGMA G5001) is used for the production of D-fructose-l-phosphate, glycolaldehyde (SIGMA G6805) is used for the production of D-xylulose-1-phosphate. This mixture is then placed at 30°C, 200 rpm at least for 4 hours. Samples are taken during the reaction, they are filtered (0.22 pm) and stored at - 20°C.

[0086] Example 6: Production of D-fructose-l-phosphate from microbial DHAP

[0087] The recombinant bacteria BC_093 obtained in Example 2 were cultured according to the method described in Example 5 above. At the end of the culture, 621 mg / L of fructose-1-P were produced, as shown by the results presented in Table 1: Measurement of the production of D-fructose-l-phosphate (mg / L) from microbial DHAP by the strain BC_093, in the presence and absence of glyceraldehyde.

[0088] [Tableauxl] Time (h) with glyceraldehyde without glyceraldehyde 0 0 0 2 173 0 4 559 0 6 606 0 23 704 0 28 621 0

[0089] These results indicate that aldolase is indeed active in the periplasm. The ability of the strain to produce fructose-1-P in the presence of DHAP and aldehyde is demonstrated.

[0090] Example 7: Production of D-fructose-l-phosphate from commercial DHAP

[0091] The same protocol as above was implemented to produce D-fructose-1-phosphate from commercial DHAP. Control tests were carried out under the same conditions, with DHAP alone and with glyceraldehyde alone. Another test was conducted by replacing the bacterial strain BC-093 with the strain BC_092 not expressing AldoB in the presence of glyceraldehyde. The results are presented in: - Table 2: Measurement of D-fructose-1-phosphate production (mg / L) from 500 mg / L commercial DHAP by strain BC_093 in the presence of glyceraldehyde. Controls: DHAP alone, glyceraldehyde alone) and - Table 3: Control of D-fructose-1-phosphate production (mg / L) from 500 mg / L commercial DHAP by the BC_092 strain not expressing AldoB in the presence of glyceraldehyde.

[0092] [Tables2] Time (h) DHAP alone Glyceraldehyde alone Glyceraldehyde and DHAP 0 0 0 0 3 0 0 4.67 4 0 0 6.8 22 0 0 14 26 0 0 16.9

[0093] [Tables3] Time (h) Glyceraldehyde and DHAP 0 0 3 0 4 0 22 0 26 0

[0094] These results show that the production of D-fructose-1-phosphate is well correlated with the presence of the aldolase AldoB, DHAP and glyceraldehyde. They also make it possible to affirm that the production of phosphorylated sugar observed in the presence of glyceraldehyde and microbial DHAP (example 6) is indeed linked to the aldolization between these two substrates, and is not due to an unidentified compound in the culture medium for the production of microbial DHAP.

[0095] Example 8: Production of D-xylulose-l-phosphate from commercial DHAP

[0096] The recombinant bacteria BC_093 obtained in Example 2 were cultured in accordance with the method described in Example 4 above, taking as substrate glycolaldehyde instead of glyceraldehyde. At the end of the culture, more than 80 mg / L of D-xylulose-1P were produced. Control tests were carried out under the same conditions, with DHAP alone, and with glycolaldehyde alone. Another test was conducted by replacing the bacterial strain BC-093 with the strain BC_092 not expressing AldoB in the presence of glycolaldehyde. The results are presented in - Table 4: Measurement of D-xylulose-1-phosphate production (mg / L) from 500 mg / L of commercial DHAP by strain BC_093 in the presence of glycolaldehyde. Controls: DHAP alone, glycolaldehyde alone and - Table 5: Control of D-xylulose-1-phosphate production (mg / L) from 500 mg / L commercial DHAP by the BC_092 strain not expressing AldoB in the presence of glycolaldehyde.

[0097] [Tables4] Time (h) DHAP alone Glycolaldehyde alone Glycolaldehyde and DHAP 0 0 0 0 3 0 0 26 4 0 0 41 22 0 0 75 26 0 0 80.2

[0098] [Tableauxô] Time (h) Glycolaldehyde and DHAP 0 0 3 0 4 0 22 0 26 0

[0099] The production of D-xylulose-1-P from DHAP and glycolaldehyde shows that AldoB is capable of utilizing DHAP with different aldehydes. This shows that by using a different aldehyde, a different phosphorylated sugar can be synthesized. The developed technology can thus make it possible to synthesize a whole range of phosphorylated sugars, including sugars that are rare and / or difficult to produce in large quantities, which are of interest in medical or industrial applications.

[0100] Example 9: Quantitative analysis of metabolites

[0101] HPLC grade acetonitrile and standards (Dihydroxyacetone, DHAP, D- fructose-1-phosphate, D-xylulose-5-phosphate) were purchased from Sigma except for C13 Glycerol-3-Phosphate which was purchased from Omicron Biochemicals, Inc.

[0102] To confirm the nature of the metabolites produced by the bacteria, analyses were performed according to Mathon et al., 2017 with modifications. DHAP and G3P were analyzed using the Thermo Scientific UltiMate 3000 BioRS Systems Dionex liquid chromatography apparatus coupled to the Thermo Scientific™ MSQ Plus™ Single Quadrupole Mass Detector mass spectrometer. Chromatography was performed using an Acquity UPLC BEH amide column (Waters, 130Â, 1.7 pm, 2.1 mm x 150 mm) and an Acquity UPLC BEH VanGuard Pre-column amide column (Waters, 130Â, 1.7 pm, 2.1 mm x 5 mm) using a binary gradient. Solvent A is composed of 5 mM methylphosphonic acid (Sigma-Aldrich, 289868-1g) in aqueous phase and solvent B is acetonitrile with 3.5 mM triethylamine (Fluka-Sigma-Aldrich, 65897-50 ml-F) and 1.3 mM formic acid (Fisher Chemical, Al 17-50-50 ml).The injection volume of the pre-filtered samples (with a 0.2 pms filter) is 2 pl and a flow rate of 0.35 ml / min is applied. A temperature of 15°C is used for the autosampler. Instrument control, data acquisition, design and analysis report generation is performed via Chromeleon™ Chromatography Data System (CDS) Software.

[0103] The mass spectrometer is equipped with an Electron Spray Ionization (ESI) source used in negative ionization mode. The source temperature is 550°C, the needle voltage is -3.5 kV and the cone voltage is -45 V. DHAP, D-fructose-lP, D-xylulose-lP were analyzed using single ion tracking (SIM) with a mass to charge ratio (m / z) of 169, 259, 229, respectively and for the internal standard 13C Glycerol-3-Phosphate, the m / z ratio is 174. The samples were quantified using the response factor of DHAP and glycerol-3-P with the internal standard. All standard solution stocks for each individual analyte were prepared by deionized water (DI water, 18.2 MΩ-cm *, Millipore) in acetonitrile solution. Samples were diluted in acetonitrile before analysis (1:1, v:v).

[0104] SEQID NO 1: Amino acid sequence of fructose-bisphosphate aldolase (AldoB) from (YHomo sapiens MAHRFPALTQ EQKKELSEIA QSIVANGKGI LAADESVGTM GNRLQRIKVE NTEENRRQFR EILFSVDSSI NQSIGGVILF HETLYQKDSQ GKLFRNILKE KGIVVGIKLD QGGAPLAGTN KETTIQGLDG LSERCAQYKK DGVDFGKWRA VLRIADQCPS SLAIQENANA LARYASICQQ NGLVPIVEPE VIPDGDHDLE HCQYVTEKVL AAVYKALNDH HVYLEGTLLK PNMVTAGHAC TK-KYTPEQVA MATVTALHRT VPAAVPGICF LSGGMSEEDA TLNLNAINLC PLPKPWKLSF SYGRALQASA LAAWGGKAAN KEATQEAFMK RAMANCQAAK GQYVHTGSSG AASTQSLFTA CYTY

[0105] SEQ ID NO 2: sequence of lipoprotein acids NlpA & E.COH MKLTTHHLRT GAALLLAGIL LAGCDQSSSD AKHIKVGVIN GAEQDVAEVA KKVAKEKYGL DVELVGFSGS LLPNDATNHG ELDANVFQHR PFLEQDNQAH GYKLVAVGNT FVFPMAGYSK KIKTVAQIKE GATVAIPNDP TNLGRALLLL QKEKLITLKE GKGLLPTALD ITDNPRHLQI MELEGAQLPR VLDDPKVDVA IISTTYIQQT GLSPVHDSVF IEDKNSPYVN ILVAREDNKN AENVKEFLQS YQSPEVAKAA ETIFNGGAVP GW

[0106] SEQ ID NO 3: Sequence of amino acids from the protein fusion of AldoB and NlpA

[0107] Bold characters: NlpA signal peptide comprising the six amino acid linker peptide, marked by the double underline) - Underlined characters: AldoB enzyme codon optimized MKLTTHHLRTGAALLLAGILLAGCDQSSSMAHRFPALTQEQKKELSEIAQ SIVANGKGILAADESVGTMGNRLORIKVENTEENRROFREILFSVDSSINOSIGG VILFHETLYOKDSOGKLFRNILKEKGIVVGIKLDOGGAPLAGTNKETTIOGLDG LSERCAOYKKDGVDFGKWRAVLRIADOCPSSLAIOENANALARYASICOONG LVPIVEPEVIPDGDHDLEHCOYVTEKVLAAVYKALNDHHVYLEGTLLKPNMV TAGHACTKKYTPEOVAMATVTALHRTVPAAVPGICFLSGGMSEEDATLNLNA INLCPLPKPWKLSFSYGRALOASALAAWGGKAANKEATOEAFMKRAMANCO AAKGOYVHTGSSGAASTOSLFTACYTY

[0108] SEQ ID NO 4: Codon-optimized nucleotide sequence of E. coli, encoding the fusion protein of AldoB and NlpA

[0109] The sequence coding the NlpA signal peptide is in bold, the optimized ALDOB codon sequence is underlined. ATGAAACTGACAACACATCATCTACGGACAGGGGCCGCATTATTGCT GGCCGGAATTCTGCTGGCAGGTTGCGACCAGAGTAGCAGCATGGCACA TCGCTTTCCGGCTCTGACGCAAGAACAGAAGAAAGAATTGTCAGAGATTGC GCAGTCCATCGTGGCGAATGGCAAAGGTATTCTGGCCGCAGATGAGAGCG TGGGAACCATGGGTAATCGGCTTCAACGCATCAAAGTGGAGAACACCGAA GAAAATCGGCGTCAGTTTCGCGAAATCCTCTTTAGCGTCGACAGTAGCATC AATCAGTCCATTGGCGGGGTGATTCTGTTTCACGAAACGCTGTACCAGAAA GACTCGCAAGGGAAACTGTTCCGCAACATTCTGAAAGAGAAAGGCATTGT GGTGGGCATCAAACTGGATCAGGGTGGCGCACCCTTAGCTGGCACTAATA AGGAGACAACGATTCAAGGCCTCGATGGGCTTTCTGAACGTTGTGCCCAGT ATAAGAAGGATGGTGTTGACTTTGGCAAATGGCGCGGTACTGCGTATCG CTGATCAGTGCCTAGCAGTCTGGCCATTCAGGAAAAACGCGAATGCGTTAG CACGCTATGCGAGCATTTGCCAACAGAACGGCCTGTTCCCATTGTCGAGC CGGAAGTAATCCCGGATGCGCGGTGTATAAAGCCCTGAAATGCACATCACGGTGTACTGAAGACCCGAACATGGTAACTGCCGGACATGCCT GCACCAAGAAATACACCCCTGAACAAGTCGCCATGGCAACAGTTACGGCT TTACACCGCACTGTTCCAGGGCGGGTTCGGTTTCCTTCAGGGTG GCATGTCGGAAGAAGATGCCACCCTCAACCTGAATGCGATCAACTTGTGTC CACTGCCGAAGCCGTGGAAACTGTCATTCTCTATGGACGTGCCTTCAGG CCAGTGCGTTAGCGGCATGGGGTGGGAAAGCGGCAAACAAAGAAGCTACC CAGGAAGCCTTCATGAAACGTGCAATGGCGAACTGTCAAGCTGCGAAAGG CCAGTACGTCCATACCGGTTCTAGCGGTGCTGCATCGACACAATCTCTGTT ACGGCCTGTTACACGTATTAA

Claims

Claims

1. Polypeptide comprising an aldolase capable of catalyzing an aldolization reaction, in the periplasm of a bacterium, between two compounds, and a bacterial membrane anchoring peptide.

2. Polypeptide according to claim 1, characterized in that it comprises a dihydroxyacetone-phosphate (DHAP)-dependent aldolase capable of catalyzing a condensation reaction between an aldehyde and DHAP, or a dihydroxyacetone (DHA)-dependent aldolase capable of catalyzing a condensation reaction between an aldehyde and DHA.

3. Polypeptide according to claim 1 or 2, characterized in that the aldolase is chosen from AldoB, RhaD, YdjI, FSA or FbaB.

4. Polypeptide according to one of the preceding claims, characterized in that the membrane anchoring peptide comprises the signal peptide of an Escherichia coli lipoprotein.

5. Polypeptide according to the preceding claim, characterized in that the membrane anchoring peptide comprises the leader peptide of the lipoprotein NlpA and the six amino acid peptide of the mature protein NlpA of sequence CDQSSS.

6. Nucleotide sequence encoding a polypeptide according to one of the preceding claims.

7. Vector comprising a nucleotide sequence according to the preceding claim.

8. Bacteria genetically modified to produce phosphorylated sugars, characterized in that said bacterium is genetically modified to express a polypeptide according to one of claims 1 to 5

9. d J. Genetically modified bacteria according to the preceding claim, characterized in that said bacterium is Escherichia coli.

10. Bacterium according to one of claims 8 or 9, characterized in that it is genetically modified to express a dihydroxyacetone-phosphate (DHAP) or dihydroxyacetone (DHA) dependent aldolase in the periplasmic space of said bacterium

11. Genetically modified bacteria according to one of claims 8 to 10, characterized in that said bacterium is modified to express an aldolase chosen from AldoB, RhaD, YdjI, FSA or FbaB.

12. A method of producing a sugar, characterized in that it comprises a step of cultivating a bacterium according to one of claims 8 to 11 in a suitable culture medium Ml comprising two compounds capable of forming said sugar by an aldol reaction.

13. Method according to the preceding claim, characterized in that the culture medium M1 comprises on the one hand an aldehyde and on the other hand DHAP or DHA.

14. Method according to the preceding claim, characterized in that the culture medium Ml is prepared by the following protocol in which: - a bacterium capable of producing DHAP and secreting it is cultivated in a culture medium Mo, - at the end of this culture, the supernatant So containing the bacterial DHAP is separated from the biomass, and - the supernatant So is used to prepare the culture medium Ml.

15. Method according to the preceding claim, characterized in that it further comprises a step in which the sugar obtained is extracted from the culture medium M1.