Production of molecules by periplasmic enzymatic catalysis

Abstract

A method for producing a compound of interest from an organic substrate, in that it comprises the steps of: a) preparing a reaction mixture comprising, - at least two bacteria genetically modified to express in their periplasmic space at least one enzyme E1, E2, …, En, - an enzyme E1 being capable of catalyzing a first reaction R1 from said organic substrate to provide a first product P1, and optionally a first co-product CoP1, - each of the enzymes E2, …, En, being capable of catalyzing a reaction R2, …, Rn, from a product or a co-product obtained by a reaction Rn-1, to provide respectively a product P2, …, Pn, and optionally a co-product CoP2, …, CoPn, - said organic substrate, and - optionally, a co-factor of said organic substrate, a co-factor of said products obtained by a reaction Rn-1, called co-factors CoF1, CoF2, …, CoFn;a complementary substrate SC of a co-product obtained by a reaction Rn-1, b) - allowing the reaction mixture to react, and c) - separating the biomass from the supernatant and extracting from it said compound of interest consisting of one of the products P1, P2, …, Pn.;

Description

Title of the invention: Production of molecules by periplasmic enzymatic catalysis

[0001] The present invention belongs to the field of the production of molecules of interest by genetically modified microorganisms, and more particularly concerns the obtaining of compounds by enzymatic catalysis carried out in the bacterial periplasmic space.

[0002] It relates to a method for producing a compound of interest involving a mixture of bacteria, modified to express one or more enzymes in their periplasmic space, which together catalyze a reaction cascade for the production of a compound of interest. The invention also relates to a reaction mixture for the production of such a compound and a kit for preparing said medium.

[0003] The production of organic compounds by the petrochemical industry raises crucial problems for the future in terms of costs and environmental impact, particularly due to the large quantities of carbon released into the atmosphere and the use of non-renewable resources. The production of such compounds by biological means appears to be a non-polluting and sustainable alternative. It is still necessary to be able to carry out the appropriate reaction schemes and to obtain sufficient quantities with regard to the envisaged applications, according to economically acceptable methods. Indeed, if certain compounds can be obtained by a simple reaction, others require intermediate steps which complicate and increase the cost of the process. In addition, many reactions use cofactors which must be added to the reaction medium in quantities proportional to the substrate to be transformed, which increases costs accordingly.

[0004] Currently, various technologies implementing biological processes are used for the production of molecules. The most classic is the extraction of products of interest, for example aromas or therapeutic molecules, from organisms such as plants, by infusion maceration, solvent extraction, or other. However, plant extraction is limited in its applications, because it generally presents fairly low yields compared to the mass treated. It uses complex processes to isolate and purify the molecule of interest. Finally, the seasonal or geographical availability of the source organism strongly constrains extraction strategies.

[0005] Another technique, also well known, is based on the fermentative activity of microorganisms, genetically modified or not, whose cellular metabolism produces molecules of interest. The production of alcohol by yeasts is one of the oldest examples. Fermentation has certain advantages. It can be carried out all year round at a moderate cost, since the reactions are carried out by microorganisms. Theoretical yields are generally interesting, the contribution of synthetic biology and systems biology having greatly increased the efficiency of this type of system. However, fermentation has notable drawbacks arising from the living nature of the biocatalyst. Thus, in practice, theoretical yields are difficult to obtain, the cell always tending to redirect the flows used for the production of the molecule of interest towards its biomass.Furthermore, the molecule of interest is secreted into a fermentation medium necessarily containing a large number of other molecules, which complicates its separation and consumes a large quantity of water which must then be reprocessed. In addition, not all molecules can be produced by fermentation, either because of their toxicity to the cell or because the molecule is little or not excreted. Added to these difficulties is the development time of an industrial fermentative organism, which is on average around ten years.

[0006] More recently, enzymatic techniques have made it possible to synthesize high-value-added molecules without resorting to microorganisms. These techniques, in which purified enzymes catalyze reactions in vitro with great specificity, make it easy to control flows by controlling the reaction medium. The yields obtained can then be close to theoretical yields, or even reach them. The direct use of enzymes, immobilized or not, thus overcomes certain drawbacks of fermentation. However, these enzymes must be previously produced, purified and packaged, which makes the technique expensive.Even if the emergence of so-called "cell-free" systems has been able to partially resolve the problem of protein purification (enzymes) by synthesizing them concomitantly in the environment where the catalyzed reaction takes place, the cost of the cofactors often essential to the reaction remains too high for large-scale application of these techniques.

[0007] Thus, all these approaches have serious drawbacks. Moreover, they are limited to the production of certain compounds. An alternative technique possessing the advantages of fermentation and those of biocatalysis, but without their respective drawbacks, and which can be adapted at will to the synthesis of a wide range of molecules of interest, could meet current economic and environmental challenges.

[0008] For this purpose, the present invention provides a technology using the biological machinery of microbial cells to produce different enzymes capable of catalyzing the reactions of a reaction scheme leading from a substrate to a compound of interest. The principle is to use the periplasmic space provided by bacteria (essentially Gram-negative bacteria) as a controlled reaction space, in which a given enzyme transforms a substrate and excretes the product formed into the culture medium. The product and any co-products excreted by the first bacterium are then available to serve as a substrate for a bacterium expressing another enzyme, thus forming a second product, and so on.

[0009] Firstly, it has surprisingly appeared that bacteria modified to express different enzymes periplasmically could be used in the same reaction medium, to carry out in an orchestrated manner, each at least one of the different reactions of a reaction scheme comprising several steps. Indeed, according to the invention, the different transformed strains placed in the same reaction medium remain operational and carry out their function in harmony.

[0010] Secondly, it has been unexpectedly demonstrated that the periplasmic wall of bacteria does not hinder the circulation of products and reactants from one bacterium to another, so that not only are schemes comprising several reactions carried out in full, but also that the reaction kinetics are entirely satisfactory for obtaining the desired compound in just a few hours.

[0011] Sequential reaction schemes constituting complex biosynthetic pathways have been developed so as to be carried out by several bacteria, each expressing at least one enzyme in its periplasm. The periplasmic expression of enzymes, that is to say outside the cell itself, offers a controlled and simplified reaction medium, from which the compounds of interest can be extracted without difficulty. Indeed, according to the method which is the subject of the present invention, enzymes are produced and transferred into the periplasm of the bacteria, a compartment where they are confined and yet available and catalytically operational with respect to the compounds solubilized in the medium. Insofar as the periplasmic compartment is easily accessible to solutes, the reactions take place there in a protected environment which is much less complex than the cell cytoplasm.This externalization of reactions (outside the cell cytoplasm) avoids the problems of flow redirection, toxicity, and transport specific to the cell. The reaction medium is in fact simplified and can be controlled to improve enzymatic reactions, in particular by the addition of cofactors and activators contributing to optimal activity. The final extraction phase of the molecule of interest is also facilitated. Knowing that extraction can represent up to 70% of the production cost of a compound by fermentation, this aspect constitutes an essential asset.

[0012] A characteristic aspect of the proposed technique is that the process of producing the biomass is decoupled from that of producing the molecule of interest. The cell culture phase and the biochemical synthesis phase take place in separate reactors, with different media. As a result, the biomass in the reactor can be used for a new synthesis process, or be recovered at the end of the synthesis of a compound and reused for a subsequent synthesis cycle. It is then possible to carry out multiple reactions from the same biomass. The production costs of the biomass (cell multiplication of the different strains) are reduced accordingly, in particular due to the significantly lower volumes of water to be reprocessed. In doing so, the production costs remain substantially those of the microbial production costs.

[0013] Another notable advantage of the dissociation of the biomass production and compound synthesis processes is that competition in the use of carbon sources is avoided. In a conventional fermentation process, the aim is to maximize the use of carbon for the production of the compound of interest in order to achieve a high yield, but this optimization risks harming cell growth (biomass production), and also hindering the production of the compound of interest. The balance between cell growth and production of the compound of interest is therefore critical. On the contrary, in the inventive process, since biomass production is decoupled from molecule production, the carbon sources are distinct. It is therefore possible to optimize separately both the growth of the biomass and the production yield of the compound of interest.

[0014] The process that is the subject of the invention, which will be described in detail below, can be described as a modular system, insofar as different bacteria, each transformed to express at least one enzyme in its periplasm, can be cultivated and preserved, so as to provide at will the effectors of a wide range of reactions from which one can draw to construct a complete reaction scheme. The bacteria chosen to intervene in a defined scheme will be introduced into a single medium to act in concert in order to generate a product of interest. This approach, based on the combined implementation of cellular modules, has proven suitable for obtaining a wide variety of compounds, giving the inventive process an unprecedented universal character. In particular, molecules with high added value, rare or difficult to access (little excreted, toxic, racemic of isomers that are difficult to separate, ...), could be obtained with high yields and a particularly favorable economic and environmental balance sheet.

[0015] More specifically, the present invention relates to a method for producing a compound of interest from an organic substrate, which comprises the steps of: a) - prepare a reaction mixture comprising, in a suitable medium, - n bacteria, n being an integer at least equal to 2, each bacterium being generated tically modified to express in its periplasmic space at least one enzyme E1, E2, ..., En, - an enzyme El being capable of catalyzing a first reaction RI from said organic substrate to provide a first product PI, and optionally a first coproduct CoPl, - each of said enzymes E2, ..., En, being capable of catalyzing a reaction R2, ..., Rn, from a product or a co-product obtained by a reaction Rn-1, to respectively provide a product P2, ..., Pn, and optionally a co-product CoP2, ..., CoPn, - said organic substrate, and - optionally, a co-factor of said organic substrate, a co-factor of said products obtained by a reaction Rn-1, called co-factors CoFl, CoF2, ..., CoFn; a complementary substrate SC of a co-product obtained by a reaction Rn-1, b) - allowing the reaction mixture thus obtained to react, and c) - separating the biomass from the supernatant and extracting therefrom said compound of interest consisting of one of the products PI, P2, ..., Pn.

[0016] The inventive method uses at least two genetically modified bacteria, each capable of secreting at least one given enzyme. Depending on the reaction scheme developed to produce the desired compound, it may comprise several bacteria, for example more than ten, and even several dozen. This number results from the number of enzymes necessary to obtain a desired product from a given substrate (itself resulting from the number of reaction steps), which will decide the number of cellular modules to be used, without necessarily being equal. Indeed, the same bacterium can express more than one enzyme in which case, the number of enzymes is greater than the number of bacteria.

[0017] Each bacterium is responsible for the production of at least one particular enzyme, so that if n enzymes must intervene in the chosen reaction scheme, a maximum of n bacteria will be involved in the process. The expression "module" or "cellular module" may be used to designate a genetically transformed bacterium and one of the enzymes RI, R2, ..., Rn specifically expressed by it. It is specified that the integers from 1 to n can give an indication of the order of intervention of each enzyme in the chosen reaction scheme, without this being a systematic rule, some reaction schemes being linear, but others having ramifications. This is the case in particular when a module is used to regenerate a cofactor, which will be described in detail later.In any case, indices n and n-1, or n and n+1 apply to reactions directly consecutive to one another, downstream or upstream of the other, as well as to the enzymes and compounds involved in said reactions.

[0018] Thus, a reaction Rn-1 is defined as being directly upstream of a reaction Rn in the reaction scheme, so that the product Pn-1 and the coproduct CoPn-1, if it exists, resulting from the reaction Rn-1, are present in the medium and available to serve respectively as a substrate, or where appropriate as a cofactor, for the reaction Rn directly downstream. This general principle suffers an exception concerning the first reaction of the reaction scheme, insofar as this is necessarily carried out from the chosen organic substrate which is transformed by the action of a first enzyme RI to give a first product PI, and where appropriate a first coproduct CoPl. At each step, a product PI, P2, ..., Pn is formed, as well as optionally a coproduct CoPl, CoP2, ..., CoPn.

[0019] The reaction mixture prepared in the first step of the process according to the invention comprises, in addition to the cellular modules materializing the defined reaction scheme, a quantity of the organic substrate constituting the raw material to be transformed to ultimately obtain the desired compound of interest. This initial organic substrate will undergo a first reaction catalyzed by the enzyme of the first module. Then, during each subsequent reaction step, the product synthesized during the immediately preceding reaction can act as a substrate to give a new product, and so on. In this linear reaction scheme, the product ultimately obtained will be the desired compound of interest.

[0020] Cofactors may be required at least for some of the reactions, which will give the corresponding coproducts. The term "coproduct" here specifically designates a chemical species formed from a cofactor in the enzymatic reaction concerned. It should be noted that these coproducts are generally able to act as cofactors in other reactions. Thus, it may be necessary to introduce into the reaction mixture, if necessary, a cofactor of the organic substrate for the first reaction, or a cofactor of the product obtained by a reaction Rn-1 which will act as a substrate in the following reaction R.

[0021] When in a reaction Rn-1, a substrate Sn-1 and a cofactor CoFn-1 react, a coproduct CoPn-1 is formed alongside a product Pn-1. A reaction scheme may provide for a coproduct CoPn-1 to itself be consumed as a cofactor CoFn in the following reaction Rn, alongside a complementary substrate. It is then particularly advantageous for the reaction Rn to be able to reform a cofactor CoFn-1. In this way, the first cofactor can be regenerated, which eliminates the need to add it massively to the reaction mixture. However, it is advisable to add said complementary substrate in sufficient quantity to the reaction mixture. In the present description, the expression "complementary substrate" designates a compound different from the initial organic substrate, which is used to react as a substrate alongside a cofactor.In this case, it will be necessary to introduce into the reaction mixture a substrate complementary to the cofactor concerned, in this case the coproduct CoPn-1, which will react together in the following reaction R.

[0022] In summary, it is understood that depending on the defined reaction scheme, one or more cofactors may be involved, as well as one or more complementary substrates. This is why, in step a) of the process according to the invention, where appropriate, the various cofactors and complementary substrates are added to the reaction mixture.

[0023] The organic substrate may be any organic compound, the only proviso being that it is capable of constituting a substrate in an enzymatic reaction. It may therefore be of a very diverse nature, for example a linear or branched and possibly substituted hydrocarbon, such as an amine, an acid, a carbohydrate, an amino acid, or other.

[0024] In step a) of the inventive process, the cellular modules and the organic substrate, with possibly the cofactors and / or complementary substrates involved, are introduced into a suitable medium, that is to say a medium that a person skilled in the art can prepare without difficulty according to the rules of the art that he masters. Such a medium is an aqueous medium, generally supplemented with minerals and a buffer, such as those given below as examples. It will be distinguished from the reaction medium strictly speaking which is defined as the aqueous phase in which the reactions of the reaction scheme take place: this includes the medium in which the bacteria are immersed as well as their periplasmic space, since the solutes circulate easily from one to the other through the external periplasmic wall. A simple buffer is used to facilitate the final purification of the compound obtained.

[0025] It is specified that the sequence of steps a) and b) may not be strictly successive. Indeed, the process can be carried out in continuous or semi-continuous mode, with permanent or sequential supply of substrate and reagents. Furthermore, in certain cases, it is preferable not to add a complementary substrate and / or a cofactor to the reaction mixture from the start, but to do so within a certain time after the initialization of the reaction process. Similarly, one or more cellular modules can be introduced in a delayed manner. In other words, according to this particular modality, an action of step a) of the process takes place while step b) has started. This chronology is included within the scope of the present invention.

[0026] It is understood from the above that the process which is the subject of the present invention offers multiple possibilities for designing reaction schemes suitable for the synthesis of various compounds of interest. It can be broken down in different ways which will now be presented in detail.

[0027] As already explained, the method according to the invention is based on an externalization of the catalytic reactions in the periplasm of bacteria. Gram-negative didermic bacteria are primarily concerned, but any other organism having a periplasm can also be used. According to a preferred characteristic of the invention, the n genetically modified bacteria introduced into the reaction mixture are Gram-negative didermic bacteria, each chosen from the families Enterobacteriaceae, Alcaligenaceae, Vibrionaceae, Pseudomonadaceae. They may in particular be enterobacteria belonging to the genera Salmonella, Yersinia or Escherichia. According to a further preferred embodiment, the bacteria are of the species Escherichia coli.

[0028] These bacteria are capable of expressing in their periplasmic space one of the enzymes E1, E2, ..., En. To do this, a genetic modification was carried out to introduce into the cells a nucleic sequence coding for the enzyme concerned, associated with a nucleic sequence coding for a signal peptide. Each bacterium then produces a polypeptide in which two amino acid sequences are fused: that constituting the enzyme and that of the signal peptide. The fusion polypeptide is thus sent to the periplasmic space, where the signal peptide is eliminated. The enzymes are then in the free state but confined in the periplasmic compartment, without having the capacity to cross the external wall. It was verified that they remained in an active conformation and that they perfectly ensured their catalytic function, each from the substrates available from the start or appearing as the reactions progressed.Different signal peptides may be used in the context of the present invention, such as pelB, dsbA, EOX, lamB, MglB, MmAp, ompC, ompT, sufl, SfmC, STII, tolB, torA, torT, gin, malE, ompA, phoA, or others chosen from those known to those skilled in the art, who know how to use them to enable periplasmic expression.

[0029] Thus according to the invention, each of the bacteria is genetically modified to express at least one polypeptide comprising respectively one of said enzymes El, E2, ..., En, linked to a signal peptide addressing said polypeptide in the periplasmic space of said bacterium.

[0030] According to another embodiment, the enzymes or some of them can be anchored to the periplasmic wall. In this case, according to the invention, one or more of said n bacteria are genetically modified to express a polypeptide comprising respectively one of said enzymes El, E2, ..., En, linked to a membrane anchoring peptide including said signal peptide.

[0031] As already mentioned, a bacterium can be transformed so as to express in its periplasmic space more than one enzyme, namely two or more, responsible for catalyzing as many different reactions. These reactions can be successive reactions of the reaction scheme or else distant reactions. Thus, according to one embodiment of the invention, at least one of said n bacteria is genetically modified to express in its periplasmic space at least two enzymes En-a and En-b capable of catalyzing at least two reactions Rn-a and Rn-b, so that said reactions Rn-a and Rn-b occur in the periplasmic space of said at least one bacterium. It is indicated that for the sake of clarity, this variant will not be developed for each embodiment set out in the remainder of the description, but that it is expressly included in the present invention.

[0032] The transformation of bacteria is carried out by insertion of the appropriate nucleic sequence using a suitable vector. One technique consists of introducing into the bacterial cytoplasm a plasmid comprising a nucleic sequence capable of encoding the desired polypeptide. Another technique is based on the insertion of a nucleic sequence into the bacterial genome itself. These techniques and others are known in the field of cellular biotechnology, and will be implemented without difficulty by a person skilled in the relevant field.

[0033] Once transformed, each variant is cultured for biomass production, then used immediately or stored. It is convenient to produce a series of bacteria capable of expressing different enzymes, so as to have as many modules for constructing reaction schemes that can be implemented by the method according to the invention. It will thus be possible to draw from this cell bank to custom-prepare the desired reaction mixture in step a) of the method according to the invention.

[0034] Once the reaction mixture is prepared in the appropriate medium, the reactions are allowed to proceed. This procedure corresponds to a discontinuous production mode (batch). Note that continuous or semi-continuous production is also entirely possible, as will be explained later. The reaction chain stops spontaneously when the initial organic substrate is exhausted. The synthesized compound of interest is essentially excreted in solubilized form in the aqueous medium. Depending on the reaction scheme adopted, the compound will consist of one of the products PI, P2, ..., Pn obtained in one or other of the reactions of the reaction scheme. After separation of the biomass, it can then be easily recovered in the supernatant, the composition of which is simple compared to that resulting from other techniques known to date.

[0035] According to a particular embodiment of the invention, the method can use two cellular modules, the first of which transforms the initial organic substrate, and the second of which uses the product of the first reaction to give a second product, which can be separated from the medium to obtain the desired compound. In this case, the reaction mixture comprises a bacterium expressing a first enzyme E1 capable of catalyzing the first reaction R1 from said organic substrate to provide a first product P1, and a bacterium expressing a second enzyme E2 capable of catalyzing a second reaction R2 from said first product P1, to form a second product P2, which is recovered in step c) as the compound of interest.

[0036] This modality may be the beginning of a more extended reaction scheme, in which case the second product can in turn be consumed as a substrate for a downstream reaction. In such an extended reaction scheme, the reaction mixture further comprises at least one bacterium expressing an enzyme E3, ..En, capable of catalyzing a reaction R3, ..., Rn, from a product Pn-1 obtained by a reaction Rn-1, to provide a product Pn, which is recovered in step c) as a compound of interest or is consumed as a substrate for a reaction Rn+1.

[0037] When a cofactor is required to carry out the first reaction, it is introduced in step a) into the reaction medium. The first cellular module is chosen to transform the initial organic substrate and this cofactor (called first cofactor CoFl), and the second module will use either the product P1 of the first reaction, to give a second product P2, or the coproduct CoP1 of the first reaction to give a second coproduct CoP2. The product P2 can be separated from the medium to obtain the desired compound, or else involved as a substrate in a reaction downstream of a more extensive reaction scheme. According to a particular modality, the coproduct CoP2 can also be involved as a substrate of another reaction.

[0038] Thus, in accordance with one embodiment of the method according to the invention, the reaction mixture comprises a cofactor CoFl of said organic substrate, a bacterium expressing an enzyme E1 capable of catalyzing a first reaction R1 from said organic substrate and the cofactor CoFl to form a first product P1 and a first coproduct CoP1; and a bacterium expressing a second enzyme E2 capable of catalyzing a second reaction R2 from the first product P1 to form a second product P2, or from the first coproduct CoP1 to form a second coproduct CoP2, at least one of the two being consumed in a third reaction, or recovered in step c) as a compound of interest. It is understood that the expression "third reaction" here designates any of the reactions that can take place downstream of the targeted reaction.

[0039] When the reaction scheme is more extensive, the above mechanism can be generalized. Indeed, each of the reactions Rn of a reaction scheme can implement a cofactor CoFn reacting with the product Pn-1 generated by the upstream reaction Rn-1 under the effect of an enzyme En. A product Pn and a coproduct CoPn are then formed. The coproduct CoPn can then be used as a cofactor CoFn+1 of a downstream reaction Rn+1. For its part, the product Pn can be used as a substrate of a (other) downstream reaction Rn+1, unless it is the compound of interest that one seeks to obtain. This modality can be generalized to one or more of the modules involved.

[0040] This is why, according to one embodiment of the method which is the subject of the invention, the reaction mixture comprises at least one cofactor CoFn, at least one bacterium expressing an enzyme En capable of catalyzing a reaction Rn from the product Pn-1 of a reaction Rn-1 and said cofactor CoFn to form a product Pn and a coproduct CoPn, at least one of the two being consumed in a reaction Rn+1, or recovered in step c) as a compound of interest.

[0041] As has been seen, a reaction Rn of the reaction scheme can use as a substrate a cofactor, which is generally a coproduct generated by an upstream reaction. In this case, it is appropriate for another compound, called a "complementary substrate", to be present alongside it, which will give a complementary product at the end of the reaction Rn. The latter may not be useful in the rest of the reaction scheme and may accumulate in the medium. This is why, according to a characteristic of the process which is the subject of the invention, the reaction mixture may further comprise at least one complementary substrate SCn of said at least one cofactor CoFn.

[0042] In the process according to the invention, said at least one CoFn cofactor may be introduced in whole or in part into the reaction mixture in step a), or during step b). It is indeed sometimes preferable to add a CoFn cofactor not involved in the first steps of the reaction scheme, only when the product Pn which is to react with said CoFn cofactor is present in the medium in a significant quantity. The reaction kinetics is thus taken into account to optimize the production of the compound of interest.

[0043] Furthermore, when a cofactor is necessary for a first reaction, it is particularly advantageous for a second reaction (downstream reaction) to allow its regeneration. Indeed, cofactors are expensive compounds which directly impact production costs. A reaction scheme has been devised to meet this objective, in which the second reaction consumes the coproduct from the first reaction as a cofactor in the second reaction, to reform the initial cofactor. In doing so, it is appropriate to provide a complementary substrate for the second reaction, which will be chosen so that it has a moderate cost price. At the end of the second reaction, a second product is formed which may or may not be of interest, but above all the cofactor from the first reaction is regenerated.

[0044] Thus, in a particularly interesting embodiment of the method according to the present invention, said at least one cofactor CoFn is formed at least in part in the reaction mixture by a reaction Rn-1 catalyzed by an enzyme En-1 capable of forming a product Pn-1 and a coproduct CoPn-1, said coproduct CoPn-1 being identical to the cofactor CoFn.

[0045] It is however preferable to bring a certain quantity of this cofactor into the medium, in order to initiate the process until the regeneration of the cofactor is sufficient to ensure a continuous supply of said cofactor. However, it has appeared that a minimal quantity is sufficient to initiate the cycle of reactions. The quantity of the cofactor that it is necessary to introduce into the reaction mixture for a progress complete of the reaction concerned is then significantly reduced compared to the quantity of the corresponding substrate with which it must react. It may be at least 20 times lower, but preferably at least 100 times lower, even more preferably at least 500 times lower, and even better at least 1000 times lower. Note that certain cofactors synthesized in the cell cytoplasm, such as ATP for example, may be present in the periplasmic space in a small but sufficient quantity.

[0046] Thus, in this context, according to the invention, the quantity of the first cofactor CoFl introduced into the reaction mixture in step a) corresponds to a molar concentration at least 20 times lower than the initial molar concentration of said organic substrate.

[0047] As indicated above, the initial organic substrate can be of diverse nature, including all kinds of carbon molecules. The process has in particular demonstrated its effectiveness in transforming common carbohydrates (sugars) such as glucose, fructose, glycerol, or others, into compounds of interest such as rare sugars, in particular phosphorylated sugars, particular isomers of rare sugars, dihydroxyacetone and other molecules sought to provide access to important synthesis routes. Other organic compounds can constitute the starting substrate, among which aldehydes, alcohols, organic acids, carbamates, hydrocarbons, amino acids. Examples that can be cited are glyoxal providing glycolaldehyde, or L-phenylalanine, the starting point for the production of 2-phenylethanol.The organic substrate can also be, by extension, a compound comprising a single carbon atom, such as carbon dioxide.

[0048] Thus, according to a characteristic of the process which is the subject of the present invention, the organic substrate of the first reaction is chosen from carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons, amino acids, carbon dioxide.

[0049] Many enzymes can be produced by the various transformed bacteria used in the inventive method. A person skilled in the art knows how to identify those suitable for developing a reaction scheme leading from an organic substrate to a given compound of interest. He can select them from those which are listed and described or which will be identified in the future.

[0050] Thus, in accordance with a preferred characteristic of the method according to the invention, the enzymes E1, E2, ..., En, are each chosen from kinases, dehydrogenases, phosphatases, reductases, isomerases, transferases. The kinases may be in particular a glycerol kinase, a glucokinase, a fructokinase, a xylokinase, an acetate kinase, or a phosphofructokinase. The dehydrogenases are for example glycerol dehydrogenase, or phosphite dehydrogenase. Re- Ductases such as NADH reductase, glyoxal reductase can be used. Isomerases can be chosen from xylose isomerase, xylulose isomerase, ribulose-phosphate-3-epimerase, ribose-5-phosphate isomerase, while transketolase, or transaldolase can be chosen as transferase. Various phosphatases are also known.

[0051] Furthermore, multiple cofactors may be involved in one or other of the reactions of the reaction scheme. It should be noted that since cofactors form coproducts which can in turn act as cofactors in an upstream or downstream reaction, the molecules concerned belong to both categories. Thus, preferably, in the method according to the invention, at least one of the enzymes E1, E2, ..., En, catalyzes a reaction providing one of the coproducts CoP1, CoP2, ..., CoPn, chosen from ATP, ADP, AMP, UTP, UDP, UMP, NAD+, NADH, NADP+, NADPH, FAD, FADH2, coenzyme A, or catalyzes a reaction using one of them as a cofactor.

[0052] It has been seen that at the end of step c) of the inventive process, the biomass is separated from the supernatant from which the compound of interest is extracted. However, the work carried out has highlighted an unexpected advantage of the inventive process. It has in fact appeared that this biomass, containing the cells of the n bacteria brought in step a), can be reused as is in a new cycle of reactions, with a yield of the same level and sometimes even higher than that observed during the first reaction cycle. This results in a significant saving in terms of time, number of manipulations to be carried out, consumption of ingredients and water and finally, in economic terms. This advantage stems from the principle of the invention according to which the cell culture phase is dissociated from the biochemical synthesis phase: they take place in separate reactors, with different media.

[0053] The supernatant can be separated from the biomass by gravity flow, and in this case, it is possible to directly reuse the biomass remaining in the reactor, by adding only fresh substrate and optionally the other necessary ingredients.

[0054] Thus, in a particularly advantageous manner, after step c) of the process which is the subject of the present invention, the biomass separated from the supernatant is reused to prepare a new reaction mixture according to step a), with or without an intermediate preservation step.

[0055] The method may be implemented in the context of discontinuous batch cultures (so-called batch) as described above, or with discontinuous flow (fed batch type). It may also be carried out in a semi-continuous regime or with continuous feeding, according to known rules of the art. In this case, steps a) and b) do not take place sequentially, but more or less simultaneously.

[0056] This is why, in an alternative embodiment of the method according to the invention, steps a) and b) are carried out by adding to the reaction mixture containing said n bacteria, permanently or at time intervals, - said organic substrate, and - optionally, a cofactor of said organic substrate, a cofactor of said products obtained by an Rn-1 reaction, called cofactors CoFl, CoF2, ..., CoFn; a complementary substrate SC of a coproduct obtained by an Rn-1 reaction, and allowing the reaction mixture thus obtained to react in continuous or semi-continuous mode.

[0057] It is clear from the above that the approach adopted by the method for producing compounds of interest as just described has numerous advantages that overcome the difficulties encountered with conventional techniques and is very general in scope. The externalization of the reactions makes it possible to use the biochemical machinery of the cell to provide the enzymatic effectors of reactions taking place in the delimited space of the periplasm, while allowing the reagents and products involved in the reactions to circulate. The design by combinable modules makes it possible to develop complete original reaction schemes and to implement them quickly. The synthesis of a given product of interest is thus facilitated, but also its final purification, since the aqueous bioproduction medium is very simple.

[0058] It should be added that the process, based on the production of reusable biomass, is much less expensive than known enzymatic processes (when they exist). It is therefore possible to industrially produce molecules of all kinds, including complex or difficult to access ones.

[0059] It is thus understood that the process which is the subject of the invention represents a novel alternative to current production methods, and offers a powerful tool for accelerating the development of new products.

[0060] According to a second aspect, the present invention relates to a reaction mixture for the production of a compound of interest from an organic substrate. Such a reaction mixture is essentially identical to that prepared in step a) of the production process described above.

[0061] Thus, the subject of the present invention is a reaction mixture for the production of a compound of interest from an organic substrate, containing, in a suitable medium: - n bacteria, n being an integer at least equal to 2, each bacterium being genetically modified to express in its periplasmic space at least one enzyme E1, E2, ..., En, - an enzyme E1 being capable of catalyzing an RI reaction from said substrate organic to provide a product PI, and optionally a co-product CoPl, - each of said at least one enzyme E2, ..En, being capable of catalyzing a reaction R2, ..., Rn, from a product or a co-product obtained by a reaction Rn-1, to respectively provide a product P2, ..., Pn, and optionally a co-product CoP2, . ..,CoPn, - said organic substrate, and - optionally, a cofactor of said organic substrate, a cofactor of said products obtained by an Rn-1 reaction, called cofactors CoFl, CoF2, ..., CoFn; a complementary substrate SC of a coproduct obtained by an Rn-1 reaction.

[0062] The reaction mixture is therefore a mixed composition comprising the suspended cells of at least two bacteria, and various soluble reagents, including the organic substrate to be transformed to obtain the compound of interest. Reference may be made to the preceding description to define the various methods of producing this mixture.

[0063] Finally, according to a third aspect of the present invention, the use of a reaction mixture as described above is claimed, for producing a compound of interest chosen from carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons, amino acids. For example, a reaction mixture comprising a carbohydrate can be used to produce a phosphorylated sugar or DHA (dihydroxyacetone), or a reaction mixture comprising an amino acid can be used to produce a nitrogenous organic compound.

[0064] The present invention will be better understood and relevant details will appear in the light of the description which will be given of different embodiments, in relation to the appended figures.

[0065] [Fig.l]: Production of glycerol-3-phosphate over time from glycerol by two cellular modules (BL21-Glpk and BL21-AckA).

[0066] [Fig.2a]: Effect of the concentration of cellular modules (BL21-Glpk and BL21-AckA) on the production of glycerol-3-phosphate.

[0067] [Fig.2b]: Effect of reaction volume - production of glycerol-3-phosphate in volumes of 2 ml, 20 ml, 200 ml and 2000 ml.

[0068] [Fig.3a]: Production of D-glucose-6-phosphate from D-glucose by two cellular modules BL21-Glk and BL21-AckA.

[0069] [Fig.3b]: Production of D-glucose-6-phosphate from D-glucose by two cellular modules BL21-nlpA-Glk and BL21-nlpA-ackA.

[0070] [Fig.4]: Production of fructose-1-phosphate from D-fructose by two modules BL21-KhkC and BL21-AckA cells.

[0071] [Fig.5]: Production of glucose-6-phosphate with reuse of the BL21-G1K and BL21-AckA cellular modules.

[0072] [Fig.6]: Production of dihydroxyacetone from glycerol by the BL21-GldA and BL21-Nox modules.

[0073] [Fig.7]: Production of glycolaldehyde from glyoxal by the BL21-YvgN module, with or without the BL21-PtxD regeneration module.

[0074] [Fig.8]: Consumption of D-xylose by the isomerization reaction alone (BL21-XylA) and by the total reaction (BL21-XylA, BL21-XylB, BL21-AckA, BL21-AphA).

[0075] [Fig.9]: Production of D-xylulose from D-xylose by the isomerization reaction alone (BL21-XylA) and by the total reaction (BL21-XylA, BL21-XylB, BL21-AckA, BL21-AphA).

[0076] [Fig. 10]: Migration on SDS-PAGE gel of the periplasmic fractions of the modules XylA, XylB, AcKA, Rpe, RpiA, TktA, Tal, PfkA and the BL21 WT control.

[0077] [Fig. 11]: Production of D-fructose-l,6-bisphosphate from D-xylose using eight cell modules.

[0078] [Fig. 12]: Production of the intermediate D-sedoheptulose-7-phosphate during the production of D-fructose-l,6-bisphosphate with eight modules.

[0079] MATERIALS AND METHODS

[0080] 1- Plasmids

[0081] - The plasmid pET22b (ori pBR322, ampicillin, T7 promoter, pelB) and the plasmid pET26b (ori pBR322, kanamycin, T7 promoter, pelB) are commercial plasmids carrying the pelB signal sequence at the N-terminus and a His-tag sequence at the C-terminus. They were used for the expression of a gene of interest coding for an enzyme that will be transported thanks to the PelB fusion peptide into the periplasm of the cell.

[0082] - The plasmid pACT3 (ori pl5A, chloramphenicol, Ptac promoter) was used to anchoring of proteins in the periplasm. The plasmid was modified to have the leader sequence of the lipoprotein NlpA at the N-terminus.

[0083] - The plasmid pETDuet-1 (ori pBR322, ampicillin, T7 promoter) is designed for the co-expression of two genes of interest. The vector carries two cloning sites, each preceded by a T7 promoter, a lac operator, and a Shine-Dalgarno sequence. The plasmid pETDuet-1 was modified here by integrating the PelB signal sequence after the two ribosome binding sites. The genes integrated into this modified plasmid allow the co-expression of two free enzymes in the periplasm under the control of two IPTG-inducible T7 promoters.

[0084] The genes are amplified from the genomic DNA of Escherichia coli MG1655 by a technique known per se, for example by PCR (Polymerase Chain Reaction) using the appropriate primers, then inserted into the plasmids pET22b, pET26b, pACT3 or pETDuet-1 previously linearized with the NEBuilder® kit by recom homologous combination. If specified, synthetic genes were directly ordered from Genscript® and cloned by them directly into the plasmid pET22b or pET26b.

[0085] The primers used for gene amplification are of a generally known type. Those skilled in the art know how to choose and use them in the context of PCR techniques. The following primers are cited as examples relating to the above-mentioned plasmids: GLPK_pET22b; Khk-C_pET22b; ackA_pET26b; gldA_pET22b; pET22B_yhbO; cadA_pET22b; XylA_pET22b; XylB_pET22b; GLK_pET22b; GLK_pET22b; pET22B_rpe; pET22B_rpiA; pET22b_tktA; pET22b_tal; pET22b_pfkA; nlpA_glk; pACT3_glk; nlpA_ackA; pACT3_ackA; pETDuet_xylA ; pETDuet_xylB ; pET22b_ARO8; pET22b_ARO10; pET22b_ADH5.

[0086] 2 - Construction of the strains

[0087] The BL21(DE3) or BL21(DE3) gold strain was chosen for the transformation of plasmids pET22b or pET26b (depending on the resistance cassette) in order to be able to use T7 polymerase. The transformations were carried out following the TSS protocol described by Chung and Miller (Chung et al., 1993). The last two strains were constructed so that the enzyme was anchored in the periplasm, all the others so that it was free. The strains used are given in Table 1.

[0088] [Table 1] Escherichia coli strains used Overexpressed gene (Organism) Module name Plasmid Resistance Strain: Genotype xylA (E.coli) BL21-XylA pET22b - xylA Amp BL21 (DE3) : E. coli str. B F-ompT gai dcm Ion hsdSB(rB-mB-) X(DE3 [lad lacUV5-T7pO7 indl sam7 nin5]) xylB (E.coli) BL21-XylB pET22b - xylB ackA (E.coli) BL21-AckA pET26 ackA r-Ecoli (Ecoli) BL21-Rpe pET22b - rpe Amp rpiA (E.coli) BL21-RpiA pET22b - rpiA tktA (E.coli) BL21-TktA pET22b - tktA talA (E.coli) BL21-TalA pET22b - tala pfkA (E.coli-BlA-BlA) pfkA Nox (Lactobacillus sanfranciscensis) BL21-Nox pET22b - nox cadA (E.coli) BL21-CadA pET22b - cadA gldA (E.coli) BL21-GldA pET22b - gldA yvgN (E.coli) gBY-v1-YbN ybnh pET22b. (E.coli) BL21-YhbO pET22b - yhbo glk (E.coli) BL21-Glk pET22b - glk glpK (E.coli) BL21-GlpK pET22b - glpk khkC (H.sapiens) BL21-KhkC pET2b -monas pcDxD stutzeri) BL21-PtxD pET22b - ptxD aphA (E.coli) BL21-AphA pET22b-apha / BL21 pET22b-MSC xylA (E.coli) xylB (E.coli) BL21-XylA-XylB pETDuet 1-BAR-BAR-80-xylB (S.cerevisiae) BL21-ARO8 pET22b-ARO8 AR01O (S.cerevisiae).cerevisiae) BL21-ARO1 0 pET22b-ARO10 ADH5 (S.cerevisiae) BL21-ADH5 pET22b-ADH5 glK (E.coli) BL21-nlpA-Glk pACT3-nlpA-glk Chm . ackA (E.coli) BL21-nlpA-AckA p ACT 3 -nlp A- ackA

[0089] 3 - Culture media and conditions

[0090] Induced whole cell production

[0091] The strains are taken from a glycerol stock stored at -80°C, in 5 ml of TB medium buffered to pH 7 with the appropriate antibiotic, and placed at 37°C overnight. The composition of the TB medium is given in Table 2.

[0092] [Table 2] Composition of TB medium buffered at pH 7 Compound Quantity Unit Yeast extract 24 g / 1 Tryptone 20 g / 1 Glycerol 5 g / 1 Kh2PO4 0.017 M Na2HPO4 0.072 M

[0093] The next day, the preculture is diluted to 1 / 100 in 50 ml of fresh TB medium, the culture is shaken at 200 rpm at 37°C. When the optical density at 600 nm (DO600nm) reaches a value between 0.4 and 0.6, the cells are induced with 0.1 mM IPTG. They are then placed at 20°C with shaking at 200 rpm, for approximately 18 hours.

[0094] Outsourced production of target molecules

[0095] The induced whole cells expressing one or more enzymes in their periplasm (or modules) are centrifuged at 4500 rpm for 10 minutes and resuspended in sterile water to reach a defined quantity of cells expressed in uDO. The modules carrying the enzymes, chosen according to the needs of a predefined reaction scheme, are introduced together into the reaction mixture. This includes a buffer to stabilize the pH, if necessary activators and cofactors, the substrate and a known quantity of each cell module. The composition is deliberately as simple as possible to ensure optimal enzymatic activity and minimized production cost. The substrates and cofactors were purchased from Sigma® or Carbosynth®, except for the acetyl phosphate used for ATP regeneration which was synthesized according to the method described in (Crans et al., 1983), from phosphoric acid (85%, 2 mol) and ethyl acetate (2 mol), two low-cost compounds.

[0096] 4 - Quantification and analysis of extracellular products

[0097] Monitoring of substrate and product concentrations is carried out by chroma- high pressure ion chromatography (HPIC) or high performance liquid chromatography (HPLC). Different detection methods have been used depending on the target molecules.

[0098] HPIC analyses

[0099] The samples are centrifuged at 14800 rpm for 2 minutes, then filtered at 0.2 µm. The samples to be analyzed are diluted to 100th in milli-Q water in vials equipped with a pre-pierced stopper and placed at 15°C in an automatic sample changer.

[0100] • Amperometric detector (HPIC - PAD)

[0101] The analysis of certain substrates and products is carried out by ion chromatography (Dionex™ ICS-6000 ion chromatography System) coupled with pulsed amperometric detection or PAD (Dionex™ ICS-6000 Electrochemical Detector). The molecules are separated on a Dionex™ Carbopac™ PA1 column (2x250mm) preceded by a pre-column of the same type (2x50mm) at 25°C.

[0102] Two elution methods were used. The method used for the determination of sugars (xylose, glucose, fructose), organophosphorus compounds (glucose-6P, fructose-IP, fructose-1,6bP, glycerol-3P), methylglyoxal and aldehydes such as glycolaldehyde is as follows: the NaOH concentration is maintained at 100 mM from 0 to 30 min while the sodium acetate (NaOAc) concentration is gradually varied. From 0 to 2 min, 0 mM NaOAc; from 2 min to 15 min, increase from 0 to 500 mM NaOAc; from 15 min to 23 min, concentration maintained at 500 mM NaOAc; from 23 min to 23.1 min, decrease from 500 mM to 0 mM NaOAc; from 23.1 min to 30 min maintenance at 0 mM NaOAc.

[0103] • Conductometric detector (HPIC - CD)

[0104] The analysis of anions (organophosphorus compounds, phosphate, chloride, sulfate, organic acids, etc.) is carried out by ion chromatography (Dionex™ ICS-6000 ion chromatography System) equipped with a conductivity meter (Dionex™ ICS-6000 CD Conductivity Detector). The molecules are separated on a Dionex™ lonPac™ AS11-HC column (2x250mm) preceded by a pre-column of the same type (2x50mm) at 25°C.

[0105] The concentration gradients are carried out with the EGC 500 KOH eluent generator at a flow rate of 0.35 ml / min. The following gradient was used to best separate the intermediate compounds of the reaction scheme presented in Example 7. Before each injection, a 7-min conditioning at 2 mM NaOH is carried out. The first step of the gradient is an isocratic elution at a NaOH concentration of 10 mM for 3 min followed by an increase in the NaOH concentration in several steps: 1) increase from 10 mM to 50 mM in 9 min; 2) increase in 50 mM to 100 mM in 7 min; 3) hold at 50 mM for 3 min; 4) decrease the NaOH concentration to 10 mM; 5) hold at 10 mM for 4 min. At the column outlet, an ADRS_2mm suppressor (87mA) is used to improve detection. For the other examples, the NaOH concentration gradient presented below was used for the determination of anionic metabolites. The first step is an isocratic elution at a NaOH concentration of 2 mM for 4 min followed by a multi-step increase in the NaOH concentration: 1) increase from 2 mM to 100 mM in 11 min; 2) hold at 100 mM for 10 min; 3) decrease to 2 mM and hold for 1 min.

[0106] UHPLC-UV / RI analyses

[0107] The analysis of sugars and organic acids is done by UHPLC (Dionex Ultimate™ 3000) equipped with a refractometer (Shodex™ RL 101) and a UV detector (Dionex UltiMate™ 3000 Diode Array Detectors 3000(RS)). The molecules are separated on a Phenomenex™ ROA-Organic Acid H+ (8%) column (300x7.8mm) with a precolumn of the same type (50x7.8mm). Elution is done isocratic (H2SO4 5mM) at a flow rate of 0.5 ml / min for 35 minutes. The samples to be analyzed are filtered at 0.2 pm before injection.

[0108] HPLC analyses - mass spectrometry

[0109] The presence of fructose-1-phosphate is detected by UHPLC (Dionex UlthnateTM 3000) equipped with a mass spectrometer (Thermo Scientific™). The molecules are separated on an ACQUITY UPLC BEH Amide Column (130Å, 1.7 pm, 2.1x30mm) preceded by a precolumn (ACQUITY BEH Shield RP 18 1.7pM VANGUARD). Mobile phase A is composed of ultrapure water and methylphosphonic acid (1.8 mM), and organic mobile phase B of acetonitrile and formic acid (1.3 mM). The concentration gradient used is as follows, at a flow rate of 0.35 ml / min: 1 min isocratic to 90% of the organic mobile phase, followed by decreases in the fraction of the organic mobile phase in several steps: 82% at 3 min; 78% at 6.5 min; 50% at 8 min. This composition is kept constant for 10 min (50% organic phase) followed by an equilibration time of 7 min.

[0110] Glycerol-3-phosphate C13 (G3P-C13) is used as an internal standard. An elution gradient is performed. The injected mixture is prepared in inserts containing 100 μl of the undiluted filtered sample, 100 μl of eluent (acetonitrile + 1.3 mM formic acid) and 10 μl of internal standard (G3P-C13). The final dilution of the sample is 2.1. This dilution factor is taken into account in the processing of the results with the ChromeleonTM software.

[0111] 5 - Extraction of proteins by osmotic shock

[0112] The cells are cultured in 2 ml of LB medium overnight, at 37°C and with shaking at 200 rpm. The cell suspension is transferred into an Eppendorf tube and centrifuged (2 ml; 5 min; 14000 g; 4°C). The pellet is resuspended in 500 μl of buffer No. 1 and incubated for 20 min on ice. The tubes are inverted regularly to avoid sedimentation. The cell mixture is centrifuged (15 min; 14000 g; 4°C). The supernatant is discarded and the cell pellet is resuspended in 125 μl of buffer No. 2. After a further incubation of 10 to 20 minutes on ice, with regular inversion, the cells are centrifuged (15 min; 14000 g; 4°C). The supernatant containing the periplasmic fraction is recovered, then denatured for 10 min at 95°C and loaded onto an SDS-PAGE gel (45 min, 180 V, 400 mA) to verify the presence of the enzyme and its molecular weight. The size marker used is Precision Plus Protein™ Garlic Blue Standards #1610373EDU. The composition of the buffers is given in Table 3.

[0113] [Table 3]: Composition of buffers No. 1 and No. 2 Buffer N°1 Buffer N°2 0.2 M Tris - HCl (pH 8.0) 0.01 M Tris HCl (pH 8.0) 200 g / 1 sucrose 0.005 g / 1 MgSO4 0.1 M EDTA 0.2% SDS 1% Triton X-100

[0114] EXEMPLE 1 : Production of phosphorylated compounds

[0115] Phosphorylation is a reaction that allows cells to sequester essential metabolites in the cytoplasm and prevent their loss by diffusion into the external environment. Indeed, negatively charged phosphorylated compounds cannot diffuse through the phospholipid bilayer. Thus, the catabolism of sugars always includes a phosphorylation step. For example, sugars such as D-glucose, D-fructose, D-threalose, L-rhamnulose, D-xylose and L-arabinose are respectively trapped in the cytoplasm in the form of D-glucose-6-phosphate, D-fructose-1-phosphate, D-threalose-6-phosphate, L-rhamnulose-6-phosphate, D-sorbitol-6-phosphate, D-xylulose-5-phosphate and D-ribulose-5-phosphate. The same applies to other sources of carbon that can be assimilated by microorganisms, such as polyols: mannitol, sorbitol and glycerol respectively phosphorylated into mannitol-1-phosphate, sorbitol-6-phosphate and glycerol-3-phosphate.Outsourcing the phosphorylation reaction makes it possible to solve this export problem and to achieve microbial production of these compounds, in accordance with the present invention.

[0116] To do this, a strain of Escherichia coli was transformed to express in its periplasm a kinase catalyzing the phosphorylation of a sugar (first module). This reaction consumes a cofactor, in this case ATP, and forms ADP in as a co-product. A second module was constructed by transforming an Escherichia coli strain to express acetate kinase (AckA) in its periplasm. This enzyme catalyzes a reaction using acetyl phosphate and ADP to form acetate and ATP.

[0117] The reaction scheme is as follows:

[0118] [Chem.l] kinase (BL21-kinase) Substrate ---------> Substrate-phosphate ATP ADP Acetate Acetyl Phosphate Acetate kinase (BL21-AckA)

[0119] The examples below illustrate the production of phosphorylated compounds of interest from three organic substrates (glycerol, glucose, fructose), with the intervention of the cofactors ATP and ADP, corresponding to this reaction scheme. Because the BL21-AckA module intervenes, carrying out the regeneration of ATP, the quantity of ATP introduced into the initial reaction mixture is significantly lower than that of the substrate (in this case 32 times lower for one of the examples and 60 times lower for the other two).

[0120] The use of the BL21-AckA module thus allows substantial savings to be made because the cost of ATP is around €300 / kg. However, another important reason motivates the regeneration of the ATP cofactor. Indeed, certain kinases are inhibited by ADP. Glycerol kinase, for example, is inhibited by small quantities of ADP (Kic = 500 pM). However, in the first reaction, one molecule of ADP is generated per molecule of product formed. In the case of inhibition by ADP, the production of the compound of interest is therefore impossible unless the ADP formed is permanently eliminated, which is done here thanks to the ATP regenerating reaction. The use of a regeneration module thus allows the production of phosphorylated molecules.

[0121] EXAMPLE 1.1: Production of glycerol-3-phosphate from glycerol

[0122] Glycerol-3-phosphate is the entry point for the phospholipid pathway. This molecule of interest has been produced at a rate of 325 mg / l by microbial means in a fed-batch bioreactor, but its export has proven to be a major problem (Popp et al. 2008). In addition, a phenomenon of dephosphorylation of glycerol occurs inside the cell.

[0123] Two modules are used: the first module expresses the E. coli glycerol kinase which produces glycerol-3-phosphate from glycerol and ATP, the second module has the function of regenerating ATP via the E. coli acetate kinase AckA. These two BL21-Glpk and BL21-AckA modules are obtained from a BL21(DE3) chassis.

[0124] Both modules are cultured and induced with IPTG in TB medium as described (Materials and Methods, point 3) before being recovered to carry out the production of glycerol-3-phosphate in a simple medium. The reaction mixture comprises: Module 1: 2 uDO / ml of BL21-Glk cells Module 2: 2 uDO / ml of BL21-AckA cells Substrate: glycerol (160 mM) Cofactor: ATP (5 mM) Complementary substrate: ethyl phosphate (220 mM) Buffered solution: HEPES (50 mM) and MgSO4 (15 mM)

[0125] The glycerol-3-phosphate production reactions are carried out in 2 ml volumes of reaction mixture by resuspension of the pellet containing an equivalent amount of Glpk and AckA cells of 2uDO in a 2 ml volume. The tubes are then placed at 25°C with stirring. Samples are taken from the reaction mixture after the addition of enzyme at 0 hours, and after 1 hour, 3 hours, 5 hours and 22 hours. The results are shown in [Fig.l]. After 22 h of reaction, a production of 1.4 g / l glycerol-3-phosphate was obtained.

[0126] Influence of cell concentration on yield

[0127] The same protocol was repeated with a doubled module concentration. A production of 12 g / l was obtained in 23 h, corresponding to a yield of 81.5%, i.e. 37 times more than the maximum production obtained by microbial means (Popp et al. 2008). Thus, the cell concentration has a significant influence and can be optimized.

[0128] Influence of cell concentration on reaction rate

[0129] In order to evaluate the effect of the quantity of cells on the production rate, different concentrations were used, under the same experimental conditions. The glycerol-3-phosphate concentrations obtained in one hour show that the reaction rate is proportional to the cell concentration ([Fig.2a]). Productivities between 1 g / l / h and 4 g / l / h can therefore be obtained. These yields indicate that a scale-up of the process according to the invention is possible.

[0130] This example demonstrates the possibility of producing phosphorylated polyols by the process which is the subject of the invention. This made it possible to overcome the obstacles linked to export and dephosphorylation by intracellular phosphatases.

[0131] Influence of reaction volume

[0132] The process according to the invention is intended to produce molecules at different scales, from the laboratory to the industrial level. In order to evaluate the effect of increasing the volume of the reaction mixture on the production of glycerol- 3-phosphate, the reaction was carried out in volumes of 2 ml, 20 ml, 200 ml and 2000 ml. The modules used are as previously BL21-Glk and BL21-AckA. The reaction mixture is composed of: Module 1: 2 uDO / ml of BL21-Glk cells Module 2: 2 uDO / ml of BL21-AckA cells Substrate: Glycerol (25mM) Cofactor: ATP (5mM) Complementary substrate: Ethyl phosphate (25mM) Buffered solution: HEPES (50mM) and MgSO4 (15mM)

[0133] Samples were taken after the addition of the cellular biocatalysts (modules) to the mixture at 0 h, then at 1 h, 3 h, 4 h and 5 h of reaction. Under the conditions described, with 25 mM ethyl phosphate, the theoretical maximum production of glycerol-phosphate is 4.3 g / l. The production kinetic profiles observed at 2 ml and 20 ml are similar. The maximum production is 3.3 g / l after 3 h with a yield of 76%. When the reaction volume is 200 ml or 2000 ml, 100% of the yield is reached after 3 h.

[0134] Furthermore, a degradation of the product is visible for productions from 2 ml to 200 ml, while at 2000 ml the product is stable. According to these results, scaling up the process by a factor of 1000 is entirely possible, and it is even favorable to production.

[0135] EXAMPLE 1.2: Production of glucose-6-phosphate from glucose

[0136] Glucose-6-phosphate is an entry point for the glycolysis pathway and the pentose phosphate pathway. For this production, two modalities have been tested, one with free enzymes in the periplasm, the other with the same enzymes anchored to the inner membrane.

[0137] Modality with free enzymes

[0138] The two modules used are the BL21-Glk module expressing the Escherichia coli Glk kinase, and the BL21-AckA ATP regeneration module where the Escherichia coli acetate kinase is expressed. The enzymes are found in the free state in the periplasm. Both modules are cultured and induced with IPTG in TB medium, then introduced into the reaction mixture medium at a level of 2 uDO in 2 ml. A control sample comprising the BL21-WT strain is prepared. The reaction mixture comprises: Module 1: 2 uDO / ml of BL21-Glk cells Module 2: 2 uDO / ml of BL21-AckA cells Substrate: 300 mM glucose Cofactor: 5 mM ATP Complementary substrate: 125 mM acetyl phosphate Buffered solution: HEPES (50 mM) and MgSO4 (15 mM)

[0139] [Fig.3a] shows that glucose-6-phosphate is well synthesized, with a concentration of 30.8 g / l obtained after 5 h of reaction, i.e. a yield of 96%, limited by the quantity of acetyl phosphate (125 mM). A plateau is therefore reached quickly. After 22 h, a decrease in glucose-6-phosphate is observed, due to the degradation of the molecule. No production is observed with the control strain.

[0140] Modality with anchored enzymes

[0141] The modules used here (BL21-nlpA-Glk and BL21-nlpA-ackA) express the same two enzymes (glucokinase Glk and acetate kinase AckA), but these are fused to the nlpA anchor peptide, so that both enzymes are anchored to the inner membrane of the periplasm in their respective module. The modules are cultured and tested according to the same protocol as above.

[0142] The reaction scheme is as follows:

[0143] [Chem.2] Glucokinase (BL21-nlpA-Glk) Glucose ---------------► Glucose-6-phosphate ATP ADP Acetate ------- Acetyl Phosphate Acetate kinase (BL21-nlpA-AckA)

[0144] [Fig.3b] shows a production of glucose-6-phosphate at a concentration of 24.8 g / l obtained after 3 hours of reaction, i.e. a yield of 76%, limited by the quantity of acetyl phosphate (125 mM). After 22 hours, a decrease in glucose-6-phosphate is observed, due to the degradation of the molecule. No production is observed with the control strain.

[0145] Conclusion: Production reactions are possible when the enzymes are free or anchored, in this example the production of glucose-6-phosphate from anchored enzymes is faster than from free enzymes but the yield is lower. The method according to the invention can be implemented with transformed bacteria generating periplasmic enzymes, whether anchored or free.

[0146] EXAMPLE 1.3: Production of fructose-1-phosphate from fructose

[0147] Fructose-1-phosphate is a sugar phosphorylated in position 1 from which one can generate glyceraldehyde, a synthon of interest. Its synthesis by biological means has never been described.

[0148] As before, two modules are required. One carries the sequence coding for the Homo sapiens KhkC fructokinase which is capable of phosphorylating sugars at position 1 (BL21-KhkC). The other expresses the Escherichia coli acetate kinase responsible for regenerating the ATP cofactor (BL21-AckA already described).

[0149] For this test, 4 uDO / ml of each module were added to the reaction mixture, for a total of 8 uDO in 2 ml. A control sample comprising the BL21-WT strain is prepared. The reaction mixture comprises: Module 1: 2 uDO / ml of BL21-KhkC cells Module 2: 2 uDO / ml of BL21-AckA cells Substrate: 300 mM fructose Cofactor: 5 mM ATP Complementary substrate: 125 mM acetyl phosphate Buffered solution: HEPES (50 mM) and MgSO4 (5 mM)

[0150] The results presented in [Fig.4] show that the reaction is almost complete after 5 hours. It reaches a plateau after 22 hours with a concentration of 1.3 g / L in fructose-1-phosphate. The reaction is limited by acetyl phosphate (125 mM). No production is obtained with the control strain.

[0151] Conclusion: The process that is the subject of the invention is thus suitable for the production of phosphorylated sugars. Firstly, it is established that the production of phosphorylated sugars by externalization of phosphorylation reactions in the periplasm is possible and efficient. Secondly, the use of the same strain (BL21-AckA) in several reaction schemes illustrates the flexibility of the process and its economic interest, by limiting the biomolecular engineering work to obtain the required modules. Finally, the interest of a module designed for the regeneration of the cofactor is to be able to use low concentrations of ATP for the production of phosphorylated molecules. It is therefore possible to design reaction schemes using ATP naturally present in small quantities in the periplasmic compartment.

[0152] EXAMPLE 2: Conservation and reuse of cells

[0153] In the method according to the invention, the use of modules implemented in a manner decoupled from the prior cell culture phase makes it possible to carry out several reaction cycles with the same biomass and to conserve this biomass over time.

[0154] Glucose-6-phosphate production was carried out by reusing cells from the BL21-GLK and BL21-AckA modules that had already been used in a previous production. For this purpose, a first production was carried out in which the cells were suspended in 2 ml of reaction mixture (8 uDO for each cell type). Then the cells used for this first production were separated from the supernatant, and the modules separated by centrifugation. The pellets were recovered and stored at 4°C. After four days, the cells were resuspended in water and introduced into a new volume of the reaction mixture of 2 ml at a level of 8 uDO for each cell type.

[0155] The reaction mixture used for both cycles comprises: Module 1: 8 uDO / ml of BL21-G1K cells Module 2: 8 uDO / ml of BL21-AckA cells Substrate: 300 mM glucose Cofactor: 5 mM ATP Complementary substrate: 220 mM acetyl phosphate Buffered solution: HEPES (50 mM) and MgSO4 (5 mM)

[0156] The results presented in [Fig.5] show a production of 50 g / l of glucose-6-phosphate in 6 hours. The reaction would be complete if the final concentration obtained was 65 g / l. The yield of the reaction is therefore 75%. By reusing the cells, 1.6 times more glucose-6-phosphate was produced than during their first use where 30 g / l had been produced. The process according to the invention therefore makes it possible to preserve and use the same biocatalyst several times without loss of efficiency, and thus to reduce the cost linked to the production of biomass.

[0157] EXAMPLE 3: Production of dihydroxyacetone from glycerol

[0158] Dihydroxyacetone (DHA) is a tanning agent widely used in the cosmetics industry. The process according to the invention was implemented to produce DHA. It includes the recycling of the redox cofactors involved in the reaction.

[0159] To do this, two modules were constructed. An Escherichia coli strain was transformed to express in its periplasm an Escherichia coli glycerol dehydrogenase catalyzing the conversion of glycerol to DHA (first module BL21-GldA). This reaction consumes a cofactor, namely NAD+, and forms NADH as a coproduct. A second module (module BL21-Nox) was constructed by transforming an Escherichia coli strain to express in its periplasm the NADH reductase (NOX) from Lactobacillus sanfranciscensis. This enzyme catalyzes a reaction using acetyl phosphate and NADH to form acetate and NAD+, which is then regenerated to take part in the first reaction (Hartley et al., 2017). Plasmid pET26b carrying the nox gene from L. sanfransiscensis which encodes the NADH oxidase NOX was ordered from Genecript and transformed into strain BL21-WT.

[0160] The reaction scheme is as follows:

[0161] [Chem.3] Glycerol dehydrogenase (BL21-GldA) Glycerol -----------------► Dihydroxyacetone NAD+ NADH,H+ H2O ◄--------------- O2 NADH reductase (BL21-Nox)

[0162] Both modules are cultured and induced with IPTG in TB medium before being recovered to carry out DHA production. The reaction mixture comprises: Module 1: 4 uDO / ml of BL21-GMA cells Module 2: 4 uDO / ml of BL21-Nox cells Substrate: 20 mM glycerol Cofactor: 5 mM NAD+ Buffered solution: 100 mM (Tris pH 8 or Gly pH 9).

[0163] Negative controls without substrate were carried out. In addition, negative controls where each strain is replaced by BL21(DE3) cells not expressing the GldA or NOX enzymes were carried out. The optimal pH of GldA being 9 while that of NOX is 7.5, tests were carried out at pH 8 and 9 in order to find the best compromise for the functioning of the reaction. The measurements were carried out after 5 hours and are presented in [Fig.6].

[0164] DHA production is obtained in the presence of both modules, at pH 8 and pH 9, whereas no production appears in the wild-type control. A DHA quantity of 260 mg / l at pH 8 and 330 mg / l at pH 9 was obtained, demonstrating the feasibility of the proposed reaction scheme.

[0165] EXAMPLE 4: Production of glycolaldehyde from glyoxal

[0166] Glycolaldehyde is a highly reactive molecule present in cellular metabolism and can be used, like most aldehydes, as a platform molecule. However, its price is too high for industrial use. Due to their very reactivity, which makes them toxic to cells and unstable, aldehydes are rarely produced microbially. Their export is further complicated by their ability to react with membrane proteins and lipids. Glycolaldehyde could be obtained by reduction of glyoxal, which is inexpensive but clearly toxic.

[0167] To do this, two modules were constructed. A strain of Escherichia coli was transformed to express in its periplasm the glyoxal reductase YvgN from Bacillus subtilis, catalyzing the conversion of glyoxal into glycolaldehyde (first module BL21-YvgN). This reaction consumes a cofactor, namely NADPH, and forms NADP as a coproduct (Dudek et al., 2013). A second module (BL21-PtxD) was constructed by transforming an Escherichia coli strain to express the phosphite dehydrogenase PtxD, a NADP reductase, in its periplasm. The Pseudomonas stutzeri ptxd gene that encodes this enzyme includes 18 mutations ensuring increased catalytic efficiency, stability at 37°C, and support for both NAD and NADP substrates. The Bacillus subtilis yvgn gene encodes glyoxal reductase (GR). The wild-type version was used. The expression plasmids pET22b-PtxD mutant and pET26b-YvgN were ordered from Genescript. The ptxd gene was codon optimized for expression in E. coli. Plasmids pET22b and pET26b possess the pelB signal sequence responsible for protein export into the periplasm; pET22b is resistant to ampicillin and pET26b to kanamycin.These plasmids were then transformed into BL21-WT to yield the BL21-YvgN and BL21-PtxD modules.

[0168] The reaction scheme is as follows:

[0169] [Chem.4] Glyoxal reductase (BL21-YvgN) Glyoxal > Glycolaldehyde NADPH,H+ NADP+ Phosphate Phosphite Phosphite dehydrogenase (BL21-PtxD)

[0170] The reaction mixture comprises: Module 1: 1 uDO / ml of BL21-YvgN cells. Module 2: 1 uDO / ml of BL21-PtxD cells (except for pre-test) Substrate: 10 mM glyoxal Cofactor: 0.4 mM NADPH Complementary substrate: 10 mM phosphite Buffered solution: 50 mM HEPES pH 7.5 and 10 mM KCl.

[0171] In a preliminary test, aldehyde production by periplasmic glyoxal reductase was carried out alone, without a regeneration system. In a second step, the reactions are initiated by adding induced BL21-PtxD cells to the reaction mixture at a concentration of 1 uDO, and 10 mM phosphite in order to regenerate the produced NADP into NADPH. The reactions are placed at a temperature of 37 °C with stirring at 200 rpm. Measurements are carried out at 0 hours, 1 hour, 3 and 5 hours.

[0172] The results shown in [Fig.7] show that glycolaldehyde is detected from 1 h of reaction with and without regeneration system, but production already reaches a plateau in the absence of PtxD due to NADPH depletion. On the contrary, in the presence of the BL21-PtxD module, the reaction continues thanks to NADPH regeneration. After 5 h, glycolaldehyde production is 25 times higher, for a yield of 37%.

[0173] The BL21-YvgN and BL21-PtxD modules therefore efficiently produce glycolaldehyde from glyoxal in the periplasmic compartment, without any harmful effect on the bacterial cells. This demonstrates that it is possible, using the method according to the invention, to produce aldehyde compounds from a toxic precursor.

[0174] EXAMPLE 5: Production of an alcohol from an amino acid

[0175] A reaction scheme has been developed that allows the production of 2-phenylethanol from L-phenylalanine. This reaction scheme is based on the use of four separate modules. Three modules were constructed that respectively express the ARO8, ARO10, ADH5 genes of S. cerevisiae, one coding for L-phenylalanine oxo-glutarate aminotransferase, the second for phenylpyruvate carboxylase and the third for 2-phenylethanol dehydrogenase. A fourth module expressing the phosphite dehydrogenase PtxD is designed for the regeneration of NADH.

[0176] The reaction scheme is as follows:

[0177] [Chem.5] TmnsamÜMtse Oécaitxnylase I8U1-ARO8) (8L21*ÂRO18J (ÎOl-A&W ----------■ / \ j ( \ ieui-Ptx»)

[0178] This is the third reaction producing the compound of interest which gives rise to a coupling with a regeneration of the cofactor NADH from the coproduct NAD+.

[0179] EXAMPLE 6: Production of rare sugar isomers

[0180] The production of rare sugars is mainly done by isomerization of a common sugar. The difficulty arises from the fact that isomerization is a reversible reaction. The objective here is to limit the reverse reaction by transforming the rare sugar formed into a new compound, for example into a phosphorylated sugar, which is then dephosphorylated to obtain the isomer of interest.

[0181] In this example, a first reaction isomerizes a simple sugar, D-xylose, into a rare sugar, D-xylulose. A second reaction phosphorylates D-xylulose into D-xylulose-5-phosphate, in the presence of ATP acting as a cofactor. The reaction scheme continues with two downstream reactions, namely on the one hand a reaction consuming the formed ADP and acetyl phosphate to regenerate ATP, and on the other part of a dephosphorylation reaction of D-xylulose-5-phosphate, giving back D-xylulose.

[0182] Four different modules are used.

[0183] - The first module (BL21-XylA) is a transformed Escherichia coli strain to express in its periplasm the xylose isomerase XylA present in E. coli.

[0184] - The second module (BL21-XylB) is a transformed Escherichia coli strain to express a kinase, XylB from E. coli, which allows xylulose to be phosphorylated into xylulose-5P by consuming one molecule of ATP.

[0185] - The BL21-AckA module already seen above is used to regenerate ATP.

[0186] - The fourth module (BL21-AphA) is a transformed Escherichia coli strain to express in its periplasm the broad-spectrum phosphatase AphA of E. coli (Passariello et al., 2006).

[0187] The complete reaction scheme is presented below.

[0188] [Chem.6] [ ' XyloSe îsonwase ' Phosphatase SP&é to {BL2VXyiA} (BUI-XyB) JBUl-AphA) 1 D-xytase ...............* 0-xyluKw .................> ----------► ' / * ' îo: ' ~ 1 T F2; < ATP ADP! ] WL / LJ Acetate ........... Phosphate of acetic acid Acetate kinase ■ 7 (BUEAckA)

[0189] The modules are cultured and induced with IPTG in TB medium before being recovered to carry out the production of D-xylulose. The reaction mixture comprises: Module 1: 8 uDO / ml of BL21-XylA cells Module 2: 8 uDO / ml of BL21-XylB cells Module 3: 8 uDO / ml of BL21-AckA cells Module 4: 8 uDO / ml of BL21-AphA cells Substrate: D-xylose (300 mM) Cofactor: ATP (5 mM) Complementary substrate: acetyl phosphate (125 mM) Buffered solution: HEPES pH 7.5 (50 mM), MgSO4 (5 mM), MnSO4 (3 mM)

[0190] The process is initiated with modules 1, 2 and 3 each expressing an enzyme in the reaction mixture. The tubes are placed at a temperature of 37 °C with stirring at 200 rpm. The addition of module 4 expressing the phosphatase AphA is done 4 hours after the start of the reaction and the pH is adjusted to 6. The reaction is monitored at 7 a.m. and then at 10 p.m. In parallel, a reaction is carried out with the BL21-XylA module alone, in order to evaluate the simple isomerization reaction. The evolution of the concentrations of D-xylose ([Fig.8]) and D-xylulose ([Fig.9]) during the reaction was followed by HPIC.

[0191] [Fig.8] shows that the substrate D-xylose is consumed in similar quantities in both modalities. On the other hand, after 4 hours, the production of D-xylulose is half as much for the complete reaction as for the isomerization reaction alone ([Fig.9]). This difference is explained by the consumption of D-xylulose due to the production of D-xylulose-5P in the complete scheme. Its presence is not detectable by the D-xylulose assay method, but has been confirmed by mass spectrometry.

[0192] Module 4 carrying the phosphatase AphA, added to the reaction mixture at T=4 h, catalyzes the dephosphorylation of D-xylulose-5P to D-xylulose. Thus, 3 hours and 18 hours after the introduction of the phosphatase, an increase of 2 g / m and 8 g / l of D-xylulose is observed, respectively. The amount of D-xylulose in the complete reaction (12.8 g / l) exceeds that of the isomerization reaction alone (10.2 g / l), a significant gain of 2.6 g / l.

[0193] This is explained by the fact that during the isomerization reaction, an equilibrium is created between the two species D-xylose and D-xylulose, so that there can be no total conversion of D-xylose. The presence of the kinase module BL21-XylB in the reaction mixture makes it possible to trap D-xylulose in phosphorylated form, which can then no longer be converted into D-xylose. D-xylulose-5P accumulates in the reaction mixture, and the addition of the phosphatase BL21-Apha makes it possible to recover D-xylulose in increased quantity compared to a simple isomerization. By thus pulling the isomerization towards a phosphorylated form of the rare sugar, a higher conversion rate is obtained.

[0194] These results show firstly that isomerization, phosphorylation and dephosphorylation by the BL21-XylA, BL21-XylB and BL21-AphA modules are possible. They also attest that four different modules worked together to achieve a complex reaction scheme, including an ATP regeneration mechanism. The synergistic and sequential use of all kinds of modules in the process of the invention is thus validated.

[0195] EXAMPLE 7: Production of D-fructose-1,6-bisphosphate from D-xylose

[0196] The conversion of D-xylose to D-fructose-1,6-bisphosphate is part of the pentose phosphate pathway, which is the natural assimilation pathway of D-xylose into the cytoplasm. This conversion was carried out by a reaction scheme involving nine reactions carried out by eight different modules.

[0197] The eight modules used are constructed from a strain of Escherichia coli, each transformed to express in its periplasm an enzyme present in E. coli: 1 - xylose isomerase XylA, which isomerizes D-xylose into D-xylulose

[0198]

[0199] 2 - xylulose kinase XylB, which phosphorylates D-xylulose to D-xylulose-5P 3 - acetate kinase AckA, which regenerates ATP from ATP and acetyl phosphate 4 - ribulose-phosphate-3-epimerase Rpe, which converts D-xylulose-5P into D-ribulose-5P 5 - ribose-5-phosphate isomerase RpiA, which produces D-ribose-5P from D-ribulose-5P 6 - Tkt transketolase which transforms D-xylulose-5P and D-ribose-5P into D-glyceraldehyde-3P and D-sedoheptulose-7P 7 - transaldolase Tal which transforms D-glyceraldehyde-3P and D-se-doheptulose-7P into D-fructose-5P and D-erythrose-4P 8 - 6-phosphofructokinase 1 PfkA which transforms D-fructose-5P into D-fructose-1,6bP The complete reaction scheme is shown below. [Chem. 7] D-xyiose 1 -BL21-XylA D-xylulose acetate D-ribulose-5P t _->ATP —. J 2-BL21- _ .. 3-BL21-AckA f 5-BL21 - RpiA J olzi | ADP ; XylB X 4-BL21- D-xylulose-5P ethyl phosphate P® D-ribose-5P । ..................f........................."I 6 - BL21 -TktA D-glyceraldehyde-3P D-sedoheptulose-7P acetate D-fructose-6P 7-BL21-TalA D-erythrose-4P 3-BL21-AckA " " 8-BL21-PfkA "—.ADP ....." acetyl-P D-fructose-1,6bP

[0200]

[0201] The presence of enzymes in the periplasm of the eight modules was verified by recovering the periplasmic fraction by osmotic shock for each of the strains. The size of the enzymes and the quantity of cells used for osmotic shock (DO600nm multiplied by the culture volume) are given in Table 4. [Table 4]: Molecular weight in kilodaltons of the proteins XylA, XylB, AckA, Rpe, RpiA, TktA, TalA and PfkA and optical density of the culture used for shock osmotic Protein XylA

[0202] The results of the SPS-PAGE gel analysis are given in [Fig. 10]. They show that the expression in the periplasm of the enzymes expected for the 8 modules is validated.

[0203] The modules were cultured and induced with IPTG in TB medium before being recovered to carry out the production of D-fructose-1,6bP in the reaction mixture comprising: Module 1: 8 uDO / ml of BL21-XylA cells Module 2: 8 uDO / ml of BL21-XylB cells Module 3: 8 uDO / ml of BL21-AckA cells Module 4: 8 uDO / ml of BL21- Rpe cells Module 5: 8 uDO / ml of BL21-RpiA cells Module 6: 8 uDO / ml of BL21-Tkt cells Module 7: 8 uDO / ml of BL21-Tal cells Module 8: 8 uDO / ml of BL21-PfkA cells Substrate: 100 mM D-xylose Cofactor: 3 mM ATP Complementary substrate: 100 mM acetyl phosphate Buffered solution: HEPES pH 7.5 (50 mM), MgSO4 (20 mM), MnSO4 (1 mM) and thiamine pyrophosphate (1 mM) CoC12 (0.1 mM)

[0204] The D-fructose-1,6bP assay was performed immediately after adding the modules to the reaction mixture, after 1 hour, 3 hours and 4 hours. Two controls were performed, one without substrate and the other without module (the eight modules are replaced by BL21 WT cells). The results are shown in [Fig.11]. After 1 h of reaction, the production of D-fructose-1,6-bisphosphate is 1.3 g / l and reaches a maximum of 2.8 g / l in 3 h. No production of D-fructose-1,6-bisphosphate is measured in the two control reactions.

[0205] D-sedoheptulose-7-phosphate was also measured ([Fig. 12]). Indeed, the modules carrying XylB and PfkA catalyze the only irreversible reactions in the reaction scheme, and D-sedoheptulose-7-phosphate is in the middle of the reaction chain composed of reversible reactions whose products balance each other. D-sedoheptulose-7-phosphate was measured immediately after adding the modules to the reaction mixture, after 1 hour, 3 hours and 4 hours. It is 130 mg / l after 4

[0206]

[0207]

[0208]

[0209]

[0210] hours in the complete reaction and is absent from the control samples. The other intermediate compounds are difficult to detect with the analytical methods used, but the level of D-sedoheptulose-7P is representative of their concentration, which is therefore similar. The production of D-fructose-1,6-bisphosphate according to a complex reaction scheme was therefore successfully achieved. These results demonstrate that the process of the invention, based on the externalization of reactions, works efficiently, including for a long and branched chain, involving numerous modules. It is also particularly interesting to have been able to reconstitute a complex, naturally cytoplasmic metabolic pathway in this way. EXAMPLE 8: Production of D-fructose-1,6-bisphosphate from D-xylose The conversion of D-xylose to D-fructose-1,6-bisphosphate is carried out here by a reaction scheme involving nine reactions carried out by seven different modules. The reaction scheme of the previous example is modified by replacing the first two modules BL21-XylA and BL21-XylB with a single module expressing both enzymes XylA and XylB in its periplasm. The complete reaction scheme is shown below. [Chem. 8] D-xylose 1 - BL21-XylA-xylB T acetate t_______>ATP 2-BL21-AckA L. ADP ethyl phosphate D-xylulose ------3 - BL21- i Rpe / » A" D-xylulose-5P D-ribulose-5P 4-BL21-RpiA r D-ribose-5P 5 - BL21-TktA D-glyceraldehyde-3P D-sedoheptulose-7P acetate --------------6-BL21-TalA D-fructose-6P D-erythrose-4P T ► A TP! 2-BL21-AckA k ' S 7-BL21-PfkA ADP A- -" acetyl-P D-fructose-1,6bP

[0211] The presence of enzymes in the periplasm of the seven modules was verified by recovering the periplasmic fraction by osmotic shock for each of the strains and migration on gel, as before.

[0212] The modules were cultured and induced with IPTG in TB medium before being recovered to carry out the production of D-fructose-1,6bP in the reaction mixture comprising: Module 1: 8 uDO / ml of BL21-XylA-XylB cells Module 2: 8 uDO / ml of BL21-AckA cells Module 3: 8 uDO / ml of BL21- Rpe cells Module 4: 8 uDO / ml of BL21-Rpi cells Module 5: 8 uDO / ml of BL21-Tkt cells Module 6: 8 uDO / ml of BL21-Tal cells Module 7: 8 uDO / ml of BL21-PfkA cells Substrate: 100 mM D-xylose Cofactor: 3 mM ATP Complementary substrate: 100 mM acetyle phosphate Buffered solution: HEPES pH 7.5 (50 mM), MgSO4 (20 mM), MnSO4 (1 mM) and thiamine pyrophosphate (1 mM) CoC12 (0.1 mM) REFERENCES

[0213] Alvarez, CL, G. Corradi, N. Lauri, I. Marginedas-Freixa, MF Leal Denis, N. Enrique, SM Mate, V. Milesi, MA Ostuni, V. Herlax, and PJ Schwarzbaum. 2017. “Dynamic regulation of extracellular ATP in Escherichia coli.” Biochem J 474 (8):1395–1416. doi: 10.1042 / bcj20160879.

[0214] Chung, C. T., and R. H. Miller. 1993. “Preparation and storage of competent Escherichia coli cells.” Methods Enzymol 218:621-7. doi: 10.1016 / 0076-6879(93)18045-e.

[0215] Crans, Debbie C., and George M. Whitesides. 1983. "A convenient synthesis of disodium acetyl phosphate for use in in situ ATP cofactor régénération." The Journal of Organic Chemistry 48 (18):3130-3132. doi: 10.1021 / jo00166a048.

[0216] Dudek, Hanna M., Petra Popken, Edwin van Bloois, Wouter A. Duetz, and Marco W. Fraaije. 2013. "A Generic, Whole-Cell-Based Screening Method for Baeyer-Villiger Monooxygenases." Journal of Biomolecular Screening 18 (6):678-687. doi: 10.1177 / 1087057113480390.

[0217] Hartley, C. J., N. G. French, J. A. Scoble, C. C. Williams, Q. I. Churches, A. R. Frazer, M. C. Taylor, G. Coia, G. Simpson, N. J. Turner, and C. Scott. 2017. "Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme compléments for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis." PLoS One 12 (ll):e0184183. doi: 10.1371 / joumal.pone.0184183.

[0218] Passariello, C., C. Forleo, V. Micheli, S. Schippa, R. Leone, S. Mangani, M. C. Thaller, and G. M. Rossolini. 2006. "Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655." Biochim Biophys Acta 1764 (1):13-9. doi: 10.1016 / j.bbapap.2005.08.028.

[0219] Popp, A., H. T. Nguyen, K. Boulahya, C. Bideaux, S. Alfenore, S. E. Guillouet, and E. Nevoigt. 2008. "Fermentative production of L-glycerol 3-phosphate utilizing a Sac-charomyces cerevisiae strain with an engineered glycerol biosynthetic pathway." Biotechnol Bioeng 100 (3):497-505. doi: 10.1002 / bit.21777.

Claims

Claims

1. A method for producing a compound of interest from an organic substrate, characterized in that it comprises the steps of: a) - preparing a reaction mixture comprising, in a suitable medium, - n bacteria, n being an integer at least equal to 2, each bacterium being genetically modified to express in its periplasmic space at least one enzyme E1, E2, ..., En, each of said n bacteria expressing an enzyme different from the other bacteria, - an enzyme E1 being capable of catalyzing a first reaction RI from said organic substrate to provide a first product PI, and optionally a first co-product CoP1, - each of said enzymes E2, ..., En, being capable of catalyzing a reaction R2, ..., Rn, from a product or a co-product obtained by a reaction Rn-1, to provide respectively a product P2, ..., Pn, and optionally a co-product CoP2, ..., CoPn, - said organic substrate, and - optionally, a cofactor of said organic substrate, a cofactor of said products obtained by an Rn-1 reaction, called cofactors CoFl, CoF2, ..., CoFn; a complementary substrate SC of a coproduct obtained by an Rn-1 reaction, b) - allowing the reaction mixture thus obtained to react, and c) - separating the biomass from the supernatant and extracting therefrom said compound of interest consisting of one of the products PI, P2, ..., Pn, characterized in that said n genetically modified bacteria are Gram-negative diderm bacteria, each chosen from the Enterobacteriaceae family and. in that each of said n bacteria is genetically modified to express at least one polypeptide comprising respectively one of said enzymes El, E2, ..., En, linked to a signal peptide addressing said polypeptide in the periplasmic space of said bacterium or to a membrane anchoring peptide including said signal peptide.

2. Method according to claim 1, characterized in that the reaction mixture comprises a bacterium expressing a first enzyme E1 capable of catalyzing the first reaction RI from said organic substrate to provide a first product PI, and a bacterium expressing a second enzyme E2 capable of catalyzing a second reaction R2 from of said first product PI, to form a second product P2, which is recovered in step c) as the compound of interest, or is consumed as a substrate of a third reaction.

3. Method according to the preceding claim, characterized in that the reaction mixture further comprises at least one bacterium expressing an enzyme E3, ..., En, capable of catalyzing a reaction R3, ..., Rn, from a product Pn-1 obtained by a reaction Rn-1, to provide a product Pn, which is recovered in step c) as a compound of interest or is consumed as a substrate of a reaction Rn+1.

4. Method according to any one of the preceding claims, characterized in that the reaction mixture comprises a cofactor CoFl of said organic substrate, a bacterium expressing an enzyme E1 capable of catalyzing a first reaction R1 from said organic substrate and the cofactor CoFl to form a first product P1 and a first coproduct CoP1; and a bacterium expressing a second enzyme E2 capable of catalyzing a second reaction R2 from the first product P1 to form a second product P2, or from the first coproduct CoP1 to form a second coproduct CoP2, at least one of the two being consumed in a third reaction, or recovered in step c) as a compound of interest.

5. Method according to the preceding claim, characterized in that the reaction mixture comprises at least one cofactor CoFn, at least one bacterium expressing an enzyme En capable of catalyzing an Rn reaction from the product Pn-1 of an Rn-1 reaction and said cofactor CoFn to form a product Pn and a coproduct CoPn, at least one of the two being consumed in an Rn+1 reaction, or recovered in step c) as a compound of interest.

6. Method according to the preceding claim, characterized in that the reaction mixture further comprises at least one complementary substrate SCn of said at least one cofactor CoFn.

7. Process according to any one of the preceding claims, characterized in that said at least one cofactor CoFn is introduced in whole or in part into the reaction mixture in step a), or during step b).

8. Method according to one of the preceding claims, characterized in that said at least one cofactor CoFn is formed at least in part in the reaction mixture by a reaction Rn-1 catalyzed by an enzyme En-1 capable of forming a product Pn-1 and a coproduct CoPn-1, said coproduct CoPn-1 being identical to the cofactor CoFn.

9. Process according to the preceding claim, characterized in that the quantity of the first cofactor CoFl introduced into the reaction mixture in step a) corresponds to a molar concentration at least 20 times lower than the initial molar concentration of said organic substrate.

10. Process according to any one of the preceding claims, characterized in that the organic substrate of the first reaction is chosen from carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons, amino acids, carbon dioxide.

11. Method according to one of the preceding claims, characterized in that the enzymes El, E2, ..., En, are each chosen from kinases, dehydrogenases, phosphatases, reductases, isomerases, transferases.

12. Method according to one of the preceding claims, characterized in that at least one of the enzymes El, E2, ..., En, catalyzes a reaction providing one of the coproducts CoPl, CoP2, ..., CoPn, chosen from ATP, ADP, AMP, UTP, UDP, UMP, NAD+, NADH, NADP+, NADPH, FAD, FADH2, coenzyme A, or catalyzes a reaction using one of them as a cofactor.

13. Method according to one of the preceding claims, characterized in that after step c), the biomass separated from the supernatant is reused to prepare a new reaction mixture according to step a), with or without an intermediate preservation step.

14. Method according to one of the preceding claims, characterized in that steps a) and b) are carried out by adding to the reaction mixture containing said n bacteria, continuously or at time intervals, - optionally, a cofactor of said organic substrate, a cofactor of said products obtained by an Rn-1 reaction, called cofactors CoFl, CoF2, ..., CoFn; a complementary substrate SC of a coproduct obtained by an Rn-1 reaction, and by allowing the reaction mixture thus obtained to react in continuous or semi-continuous mode.

15. Reaction mixture for the production of a compound of interest from an organic substrate, characterized in that it contains, in a suitable medium: - n Gram-negative diderm bacteria each chosen from the Enterobacteriaceae family, n being an integer at least equal to 2, each bacterium being genetically modified to express in its periplasmic space at least one enzyme El, E2, En, each of said n bacteria expressing an enzyme different from the other bacteria, - an enzyme E1 being capable of catalyzing a reaction RI from said organic substrate to provide a product PI, and optionally a co-product CoP1, - each of said at least one enzyme E2, ..., En, being capable of catalyzing a reaction R2, ..., Rn, from a product or a co-product obtained by a reaction Rn-1, to respectively provide a product P2, ..., Pn, and optionally a co-product CoP2, ..., CoPn, - said organic substrate, and - optionally, a cofactor of said organic substrate, a cofactor of said products obtained by an Rn-1 reaction, called cofactors CoFl, CoF2, ..., CoFn; a complementary substrate SC of a coproduct obtained by an Rn-1 reaction.

16. Use of a reaction mixture according to claim 15 for producing a compound of interest chosen from carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons, amino acids.

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