Production of molecules catalyzed by periplasmic enzymes.

Abstract

1. A method for producing a compound of interest from an organic substrate, comprising: a) at least two bacteria genetically modified to express in their periplasmic space at least one enzyme E1, E2, ..., En, preferably a single enzyme, each of said n bacteria expressing an enzyme different from the other bacteria, an enzyme E1 capable of catalysing a first reaction R1 using said organic substrate to give a first product P1 and, optionally, a first co-product CoP1, each of enzymes E2, ...., En capable of catalysing a reaction R2, ..., Rn using a product or co-product obtained by reaction Rn-1 to give products P2, ..., Pn and, optionally, co-products CoP2, ..., CoPn, respectively, said organic substrate, and optionally a cofactor of said organic substrate, a cofactor of said product obtained by reaction Rn-1, called cofactors CoF1, CoF2, ..., CoFn; an additional substrate SC for the co-product obtained by reaction Rn-1. preparing a reaction mixture comprising: b) - allowing the reaction mixture to react, and c) - separating the biomass from the supernatant and extracting therefrom said compound of interest consisting of one of the products P1, P2, ..., Pn; The method includes:

Description

[Technical field]

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

[0002] The present invention relates to a method for the production of a compound of interest comprising a mixture (consortium) of bacteria modified to express in their periplasmic space one or more enzymes which together catalyze a reaction cascade for the production of the compound of interest. The present invention also relates to a kit for preparing the reaction mixture and said medium for the production of such a compound. [Background technology]

[0003] The production of organic compounds by the petrochemical industry raises significant future problems in terms of costs and environmental impact issues, especially due to the large amounts of carbon released into the atmosphere and the use of non-renewable resources. The production of such compounds by biological routes appears to be a non-polluting and sustainable alternative. It is still necessary to carry out a suitable reaction plan, following economically acceptable protocols, to be able to obtain sufficient quantities for the envisaged application. Indeed, while some compounds are accessible by simple reactions, others require intermediate steps that complicate the process and increase its costs. Furthermore, many reactions use cofactors that must be introduced into the reaction medium in amounts proportional to the substrate to be converted, which increases the costs accordingly.

[0004] Currently, various techniques using biological processes are used for the production of molecules. The most classical is the extraction of the product of interest, e.g., a fragrance or therapeutic molecule, from an organism such as a plant by hot or cold maceration, extraction with a solvent, or other means. However, the application of phytoextraction is limited because the yield is generally quite low compared to the amount processed. Phytoextraction uses complex methods to isolate and purify the molecule of interest. Finally, the seasonal or geographical availability of the source organism greatly limits the extraction strategy.

[0005] Another technique, also well known, is based on the fermentation activity of microorganisms, which may or may not be genetically modified, whose cellular metabolism produces a molecule of interest that is secreted into the culture medium. The production of alcohol by yeast is one of the oldest examples. Fermentation has certain advantages. It can be carried out all year round at reasonable costs, since the reactions are carried out by microorganisms. The theoretical yield is generally interesting, since the contributions of synthetic and systems biology have greatly increased the efficiency of this type of system. Nevertheless, fermentation has a significant disadvantage due to the living nature of the biocatalyst. Thus, in practice, it is difficult to obtain a theoretical yield, since the cells always tend to redirect the flows involved in the production of the molecule of interest towards their biomass. Moreover, the molecule of interest is secreted into the fermentation medium, which necessarily contains many other molecules, which complicates its separation and consumes a large amount of water that must be treated afterwards. Moreover, not all molecules can be produced by fermentation, either because of their toxicity to the cells or because they are almost never excreted at all. To these difficulties are added the average development time of about 10 years for industrial fermentation organisms. Thus, some limitations of production by fermentation can be identified: i) the coupling of growth and production limits the maximum yield of production, ii) the use of synthetic biology to engineer the microorganism and redirect carbon flux towards the product comes at the expense of growth, which affects the robustness of the method, iii) the operating conditions are limited to those compatible with microbial growth (pH, temperature, composition of the medium, oxygen load, etc.), iv) the substrates and products must not be cytotoxic; therefore, the maximum concentrations of substrates and products are established according to their toxicity, v) the substrates and products must be able to pass the cell membrane, and vi) the products are secreted into the culture medium full of organic acids and other by-products, complicating the purification of the products and increasing the overall cost of the method.

[0006] More recently, whole-cell bioproduction techniques have made it possible to overcome some of these disadvantages. Growth (enzyme production phase) and production are separated, and the substrate is dedicated only to the product synthesis. The operating conditions can be optimized to maximize production, and compatibility with growth is not required. Furthermore, purification of the product is simplified, since it is secreted into a simple medium free of growth co-products. Finally, if the transfer of the substrate / product through the cell membrane is rate limiting, one way to circumvent this constraint consists in permeabilizing the cell wall and membrane by chemical treatment (e.g., by adding detergents or solvents) or physical treatment (e.g., by using heat shock). Nevertheless, these procedures, in addition to causing cell damage, can interfere with downstream purification methods. Furthermore, the main limitation of such methods is the need for metabolic engineering to i) optimize the expression levels of the genes encoding the enzymes of the production pathway and ii) minimize intracellular side reactions. However, optimizing the amount of each enzyme in the enzyme cascade is essential for the overall performance of the method. In fact, each enzyme in a metabolic pathway is responsible for a specific biochemical reaction required for the synthesis of the final product. If the amount of enzyme present in the cascade is insufficient, the upstream substrate may accumulate, resulting in a decrease in the reaction rate and therefore in the production of the final product. Alternatively, an excessive amount of enzyme may also upset the balance of the enzyme cascade, resulting in a decrease in the efficiency of the entire process. Therefore, it is understood that it is worth optimizing the amount of each enzyme to maximize the overall reaction efficiency and the production of the final product. Although it is possible to easily adjust the concentration of purified enzymes in the reaction mixture, instead, when these enzymes are expressed in vivo, it is necessary to regulate the gene expression of each enzyme to achieve the optimal level of each enzyme.

[0007] Currently, metabolic engineering and synthetic biology are used to improve product yields and titers. Despite the constant development of new metabolic engineering tools, it is difficult to predict the level of enzyme production in cells. The variation in the expression level of a gene depends on several parameters, including the copy number of the gene determined by the origin of replication if the gene is carried on an expression plasmid, the level of transcription determined by the choice of promoter, and the level of translation by the ribosome binding site (RBS). However, the correlation between the efficiency of the expression system and the copy number, transcription or translation power is not linear. High expression represents a significant cellular metabolic burden and production and growth may be impaired. In general, optimization of gene expression is a combinatorial approach between these different elements to empirically determine the optimal combination.

[0008] Other enzymatic techniques have made it possible to synthesize molecules with high added value without resorting to microorganisms. Thanks to these techniques, where purified enzymes catalyze reactions in vitro with high specificity, it is possible to easily control the flow by controlling the reaction medium. The yields obtained are then close to or even reach the theoretical yield. The direct use of enzymes, immobilized or not, therefore overcomes some of the disadvantages of fermentation. However, these enzymes have to be produced, purified and packaged in advance, which makes this technique expensive. Although the advent of so-called cell-free systems has partially solved the problem of purifying proteins (enzymes) by synthesizing them simultaneously in the medium in which the catalytic reaction takes place, the cost of the cofactors, which are often essential for the reaction, remains too high for the large-scale use of these techniques. Thus, all these approaches have significant disadvantages. Moreover, they are limited to the production of certain compounds. It would be possible to meet current economic and environmental challenges if there were an alternative technique that has the advantages of fermentation and biocatalysis but without their respective disadvantages and can be adapted at will to the synthesis of a wide range of molecules of interest. Summary of the Invention [Means for solving the problem]

[0009] To this end, the present invention proposes a technique that uses 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 (mainly gram-negative bacteria) as a controlled reaction space, in which a given enzyme converts 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 substrates for a bacterium expressing another enzyme different from the enzyme produced by the first bacterium, thus forming a second product, and so on. This is called a cascade reaction, using a consortium of at least two bacteria, each expressing an enzyme different from the enzyme produced by the other bacterium. From a bioprocessing point of view, the increased accessibility of the substrates provided by the periplasm is a major advantage. The use of periplasmic secretion of enzymes improves the accessibility of the substrates compared to the so-called whole-cell bioprocesses with cytoplasmic enzymes mentioned above. Another advantage of expressing enzymes in the periplasm is the possibility to control the pH and saline composition of the reaction medium. The pH and its ionic composition of the periplasm are equivalent to the pH and its ionic composition of the surrounding medium, unlike the pH and ionic concentration of the cytoplasm, which are regulated by the microorganism to maintain homeostasis (Wilks JC and Slonczewski J, 2007). However, to the applicant's knowledge, no biological production method has been described so far that uses a consortium of at least two bacteria, each expressing an enzyme different from the enzyme expressed by the other bacteria in the periplasm, to carry out several catalytic steps in a cascade.

[0010] First, it surprisingly appeared that bacteria engineered to express different enzymes in the periplasm could be used in the same reaction medium, with each enzyme carrying out in an organized manner at least one of the different reactions of a reaction scheme comprising several steps. Indeed, according to the invention, different transformed strains exposed to the same reaction medium remain usable and perform their functions in unison.

[0011] Second, it was unexpectedly demonstrated that the periplasmic wall of the bacteria does not impede the circulation of products and reagents from one bacterium to another, so that not only can a scheme involving several reactions be carried out in its entirety, but the reaction kinetics are also entirely satisfactory to yield the desired compounds in just a few hours.

[0012] Furthermore, and despite the viscosity of the periplasm (gel-type state) coupled with the presence of sugars that may have the effect of slowing down the rate of enzymatic reactions (Brass et al., 1986), unlike the cytoplasm (liquid state) or aqueous solutions used in vitro, the Applicant has surprisingly found that the resistance of the medium does not prevent the execution of the reaction cascade according to the invention.

[0013] Finally, a major obstacle to the use of periplasm for bioproduction is known to be the instability of cofactors in this compartment, although cofactors are known to be essential in most metabolic pathways. For example, NAD (NAD+, whose reduced state is NADH) is widely distributed in metabolic reactions; it is a cofactor for more than 300 redox reactions (Zhou et al., 2011).

[0014] However, thanks to the method according to the invention using a consortium of microorganisms, it is possible to recycle the cofactors necessary for the enzymatic reaction, each of which is expressed in the periplasm of the different cells. Cofactors such as NAD(P)H cannot cross the cytoplasmic membrane of the bacterial cell, but can diffuse through the periplasmic space that separates the bacterial outer membrane from the cytoplasmic membrane. For this reason, the cofactors can move freely between the different cells of the consortium and can be efficiently recycled and reused for production.

[0015] It is therefore conceivable that sequential reaction schemes constituting complex biosynthetic pathways can be developed to be carried out by several bacteria, each of which expresses at least one enzyme in its periplasm. Expression of enzymes in the periplasm, i.e. outside the cell in the strict sense, provides a controlled and simplified reaction medium from which the compound of interest can be extracted without difficulty. In fact, according to the method that is the subject of the present invention, the enzymes are produced and transferred to the bacterial periplasm, a compartment in which they are confined but available and catalytically usable with respect to the compounds solubilized in the medium. In so far as the periplasmic compartment is easily accessible to solutes, the reactions take place therein in a protected environment that is much less complex than the cytoplasm. Such externalization of the reactions (outside the cytoplasm) avoids the problems of flux redirection, toxicity and transport specific to the cell. The reaction medium is in fact simplified and can be controlled to improve the enzymatic reactions, in particular by the addition of cofactors and activators that contribute to optimal activity. The final extraction step of the molecule of interest is also facilitated. This aspect is a necessary advantage, as extraction can account for up to 70% of the cost of producing a compound by fermentation.

[0016] A distinctive aspect of the proposed technique is that the biomass production process is separated from the process of production of the molecule of interest. The cell culture and biochemical synthesis stages are carried out in separate reactors using different media. As a result, the biomass in the reactor can be used for a new synthesis process or recovered after the synthesis of the compound and reused for subsequent synthesis cycles. Multiple reactions can then be carried out from the same biomass. The cost of biomass production (cell growth of different strains) is correspondingly reduced, especially since the amount of water to be treated is significantly less. In doing so, the production cost remains substantially the microbial production cost. Another notable advantage of the separation of the biomass production and compound synthesis processes is that competition in the use of carbon sources is avoided. In conventional fermentation methods, the aim is to maximize the use of carbon for the production of the compound of interest to achieve high yields, but this optimization can have a negative impact on cell growth (biomass production) and therefore also hinder the production of the compound of interest. Therefore, the balance between cell growth and production of the compound of interest is critical. In contrast, in the method of the present invention, the carbon sources are different, since biomass production is separated from molecule production. Therefore, it is possible to separately optimize both biomass growth and production yield of the compound of interest. Moreover, the applicant has shown that it is possible to significantly increase the production of the product of interest by adjusting the ratio of each biocatalyst in the consortium (one bacterium expressing one enzyme). This is a significant advantage in conjunction with the modular system of the method of the present invention compared to previously mentioned methods that would require complex genetic modifications of the same bacteria or other tools to increase production. In particular, it is known that the efficiency of signal peptides depends on the target protein produced. For example, signal peptides that promote high levels of protein synthesis and secretion of one recombinant protein often result in low levels of protein synthesis and secretion of another recombinant protein (Karyolaimos et al., 2019). Since it is not possible to predict the performance of a signal peptide for a given recombinant protein, it is necessary to screen signal peptide libraries for signal peptides that promote high levels of protein synthesis and secretion.This approach is time-consuming and expensive. Thanks to the method according to the invention, it is possible to adjust the amount of each biocatalyst in the reaction mixture for producing the product in order to maximize the efficiency of the reaction (e.g. to increase the amount of biocatalyst to overcome the low efficiency of the signal peptide). It is therefore possible to use a single signal peptide (e.g. pelB) independently of the protein produced, to increase the proportion of biocatalysts in cases where the signal peptide does not produce the protein with the expected efficiency. This strategy facilitates the improvement of the production of the product, which is advantageous for industrial applications. The method that is the object of the invention, which will be described in detail below, can be described as a modular system in so far as different bacteria, each transformed to express in its periplasm at least one enzyme, preferably a single enzyme different from the enzymes expressed by the other bacteria of the system, can be cultivated and stored to provide effectors of a wide range of reactions as needed to approach the construction of a complete reaction scheme. We will speak of a library of biocatalysts, each of which contains bacteria expressing in its periplasm an enzyme different from the enzymes expressed by the other bacteria of the biocatalyst library. The bacteria selected to intervene in a defined scheme are introduced into a single medium and work in concert to produce the product of interest. This approach, based on the combinatorial execution of cellular modules, has proven to be suitable for obtaining a wide variety of compounds, endowing the method of the invention with a unique universal nature. In particular, molecules with high added value and rare or difficult to access (poorly excreted, toxic, difficult to separate racemic isomers, etc.) have been obtained with high yields and a particularly favorable economic and environmental balance.

[0017] Thus, the use of synthetic microbial consortia according to the method of the present invention has numerous advantages over prior art biological production methods: i) it overcomes the high metabolic load that a single strain traditionally has by splitting it and optimizing pathways in a modular manner; ii) it allows flexibility in balancing the metabolic flux between the individual modules by easily controlling the ratio of modified strains; iii) it prevents potential inhibition of metabolic intermediates on sensitive enzymes; iv) finally, thanks to its modular nature, it allows easy production of a variety of different chemical products by simply mixing different microbial strains. Thus, the method according to the present invention has the advantage of avoiding the problems associated with the simultaneous expression of several enzymes in a single cell and overcoming the complexities of regulating gene expression. Furthermore, the amount of each species of modified bacteria can be optimized for more efficient production of the product of interest.

[0018] More precisely, the present invention relates to a method for producing a compound of interest from an organic substrate, comprising the steps of: a)- In a suitable medium, - n bacteria, n being an integer at least equal to 2, each of which has been genetically modified to express in its periplasmic space at least one enzyme E1, E2, ... En, preferably a single enzyme, each of said n bacteria expressing an enzyme that is distinct from the other bacteria, - the enzyme E1 is capable of catalysing a first reaction R1 from said organic substrate to provide a first product P1 and, optionally, a first co-product CoP1; each of said enzymes E2, .... En is capable of catalysing a reaction R2, ..., Rn from a product or co-product obtained by reaction Rn-1 to provide a product P2, ..., Pn and, optionally, a co-product CoP2, ..., CoPn, respectively; - said organic substrate, and - optionally a cofactor of said organic substrate, a cofactor of said product obtained by reaction Rn-1, called cofactor CoF1, CoF2, ..., CoFn; a complementary substrate SC of the co-product obtained by reaction Rn-1 preparing a reaction mixture comprising: 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 P1, P2, ..., Pn; The present invention relates to a method comprising the steps of:

[0019] According to a particularly preferred embodiment, each bacterium expresses a single enzyme E1, E2, ..., En in its periplasmic space, and each of the n bacteria is genetically modified to express a different enzyme than the other bacteria.

[0020] The method of the invention uses at least two genetically modified bacteria, each of which is capable of secreting at least one given enzyme different from the enzyme expressed by the other bacteria. Depending on the reaction scheme developed to produce the desired compound, the reaction scheme may include several bacteria, for example more than 10 bacteria, and even several dozen bacteria. This number is derived from the number of enzymes necessary to obtain the desired product from a given substrate (which itself is derived from the number of reaction steps), which indicates the number of cell modules used, which are not necessarily equal. In fact, the same bacteria can express more than one enzyme, in which case the number of enzymes will be greater than the number of bacteria. However, according to a particular preferred embodiment, each bacterium expresses a single enzyme different from the enzyme expressed by the other bacteria. In particular, 2 to 20 bacteria, in particular 3 to 15, or even 4 to 12 may be used, each bacterium expressing an enzyme different from the enzyme expressed by the other bacteria.

[0021] Each bacterium is responsible for the production of at least one specific enzyme, preferably a single specific enzyme, so that if n enzymes can participate in the selected reaction scheme, then at most n bacteria will be involved in the process. The term "module" or "cell module" may be used to name the genetically transformed bacterium and one of the enzymes R1, R2, ..., Rn specifically expressed by this bacterium. Just to be sure, the integers 1 to n give an indication of the order of intervention of the respective enzymes in the selected reaction scheme, but this is not a systematic rule, since some reaction schemes are linear, while others are branched. This is especially true when modules are used to regenerate cofactors, which will be explained in detail below. In any case, the references n and n-1 or n and n+1 apply to the reactions that directly follow each other, as well as to the enzymes and compounds involved in said reactions, one downstream or upstream of the other.

[0022] Thus, reaction Rn-1 is defined as being immediately upstream of reaction Rn in the reaction scheme, so that product Pn-1 and, if any, co-product CoPn-1 resulting from reaction Rn-1 are present in the medium and available to serve as substrates or, if applicable, cofactors, respectively, for the immediately downstream reaction Rn. There is an exception to this general rule for the first reaction of the reaction scheme, insofar as the first reaction is necessarily carried out from a selected organic substrate that is converted by the action of a first enzyme R1 to give a first product P1 and, where appropriate, a first co-product CoP1. At each step, products P1, P2, ..., Pn and, optionally, co-products CoP1, CoP2, ..., CoPn are formed.

[0023] The reaction mixture prepared in the first step of the method according to the invention contains, in addition to the cellular modules resulting in a defined reaction scheme, a large amount of organic substrates constituting the starting material that is converted to finally obtain the desired target compound. This initial organic substrate undergoes a first reaction catalyzed by the enzymes of the first module. Then, during each subsequent reaction step, the product synthesized during the previous reaction can act as a substrate to give a new product, and so on. In this linear reaction scheme, the product finally obtained is the desired target compound.

[0024] Cofactors may be required for at least some of the reactions, which result in the corresponding coproducts. Here, the term "coproduct" specifically refers to chemical species formed from cofactors in the enzymatic reactions involved. It should be noted that these cofactors are generally capable of acting as cofactors in other reactions. It may therefore be necessary, where appropriate, to introduce into the reaction mixture a cofactor of the organic substrate of the first reaction, or of the product obtained by reaction Rn-1, which will act as a substrate in the subsequent reaction R.

[0025] In reaction Rn-1, when substrate Sn-1 and cofactor CoFn-1 react, coproduct CoPn-1 is formed alongside product Pn-1. The reaction scheme may provide that coproduct CoPn-1 is itself consumed as cofactor CoFn in the subsequent reaction Rn alongside complementary substrate. It is then particularly advantageous that reaction Rn can reorganize cofactor CoFn-1. In this way, it is possible to regenerate the first cofactor, which does not have to be added in large quantities to the reaction mixture. However, it is advisable to add said complementary substrate to the reaction mixture in sufficient amounts. In this description, the phrase "complementary substrate" refers to a compound that is different from the initial organic substrate and is used to react as a substrate alongside the cofactor. In this case, it becomes necessary to introduce into the reaction mixture a substrate complementary to the cofactor in question, in this case coproduct CoPn-1, which will react together in the subsequent reaction R.

[0026] In summary, it is understood that according to the defined reaction scheme, one or more cofactors can intervene, as can one or more complementary substrates. For this reason, in step a) of the method according to the invention, various cofactors and complementary substrates are added, where appropriate, to the reaction mixture.

[0027] The organic substrate can be any organic compound, the only limitation being that it is capable of constituting a substrate in an enzymatic reaction. The organic substrate can therefore be of very diverse nature, e.g., linear or branched hydrocarbons, possibly substituted, e.g., amines, acids, hydrocarbons, amino acids, or the like.

[0028] In step a) of the method of the invention, the cell modules and the organic substrate, optionally together with the cofactors and / or complementary substrates involved, are introduced into a suitable medium, i.e. a medium that the skilled person can easily prepare according to the methods of the techniques known to them. Such a medium is an aqueous medium, to which minerals and buffers are generally added, such as those given below as examples. This medium is to be distinguished from the reaction medium in the strict sense, which is defined as the aqueous phase in which the reactions of the reaction scheme take place. It includes the medium in which the bacteria are immersed and its periplasmic space, since solutes easily circulate from one to the other through the outer periplasmic wall. Simple buffers are used to facilitate the final purification of the compounds obtained.

[0029] Just to be clear, steps a) and b) do not have to be strictly consecutive. In fact, the method can be carried out in a continuous or semi-continuous mode, with continuous or sequential addition of substrates and reagents. Moreover, in certain cases, it is preferred not to add additional substrates and / or cofactors to the reaction mixture from the beginning, but within a certain period of time after the start of the reaction method. Likewise, one or more cell modules may be introduced in a delayed manner. In other words, according to this particular protocol, the action of step a) of the method takes place while step b) is beginning. This order of events is included in the context of the present invention.

[0030] It will be seen from the above that the process that is the object of the present invention offers multiple possibilities to design reaction schemes adapted to the synthesis of various compounds of interest, which can be decomposed in different ways that will be presented in detail shortly.

[0031] As already explained, the method according to the invention is based on the externalization of a catalytic reaction in the periplasm of bacteria. Gram-negative diderm bacteria are primarily concerned, but any other organism with a periplasm can be used. According to a preferred feature of the invention, the n genetically modified bacteria introduced into the reaction mixture are Gram-negative diderm bacteria, each selected from the families Enterobacteriaceae, Alcaligenaceae, Vibrionaceae and Pseudomonadaceae, for which the production of recombinant proteins has already been described (Stock et al., 1977; Eichmann et al., 2019; Karyolaimos et al., 2019). These bacteria may be enterobacteria, in particular belonging to the genera Salmonella, Yersinia or Escherichia. According to a more preferred embodiment, the bacterium is of the species Escherichia coli.

[0032] These bacteria are capable of expressing one of the enzymes E1, E2, ..., En in their periplasmic space, each of which expresses an enzyme different from that expressed by the other bacteria of the consortium. To do this, a genetic modification was induced to introduce into the cell a nucleic acid sequence encoding the enzyme of interest, accompanied by a nucleic acid sequence encoding a signal peptide. Each bacterium then produces a polypeptide in which two amino acid sequences are fused: one constituting the enzyme and the other constituting the signal peptide. The fused polypeptide is thus directed into the periplasmic space, where the signal peptide is removed. The enzyme is then free, but confined to the periplasmic compartment, and does not have the ability to pass through the outer wall. It has been confirmed that the enzymes remain in an active conformation and perform their catalytic functions perfectly, each of which is derived from the substrates available initially or which appear as the reaction progresses. Various signal peptides can be used in the context of the present invention, such as signal peptides selected from pelB, dsbA, EOX, lamB, MglB, MmAp, ompC, ompT, sufI, SfmC, STII, tolB, torA, torT, gIII, malE, ompA, phoA, or signal peptides known to those of skill in the art who know how to enable periplasmic expression using signal peptides (Karyolaimos et al., 2019).

[0033] Thus, according to the invention, each of the bacteria is genetically modified to express at least one polypeptide, preferably a single polypeptide, each comprising one of the enzymes E1, E2, ..., En linked to a signal peptide that directs the polypeptide into the periplasmic space of the bacterium.

[0034] According to another embodiment, the enzyme or a part thereof may be anchored in the periplasmic wall, in which case, according to the invention, one or more of said n bacteria are genetically modified to express a polypeptide comprising one of said enzymes E1, E2, ..., En linked to a membrane anchoring peptide comprising said signal peptide.

[0035] As already mentioned, a bacterium can be transformed to express in its periplasmic space more than one enzyme, i.e. two or more enzymes, responsible for catalyzing the same number of different reactions. These reactions may be sequential or non-sequential reactions of a reaction scheme. Thus, according to one embodiment of the present 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, such that said reactions RN-a and RN-b occur in the periplasmic space of said at least one bacterium. It is noted that, for the sake of clarity, this variant has not been developed for each of the embodiments described in the remainder of this description, but this variant is expressly included in the present invention.

[0036] The transformation of bacteria is carried out by the insertion of a suitable nucleic acid sequence using a suitable vector. One technique consists in introducing into the bacterial cytoplasm a plasmid containing a nucleic acid sequence capable of coding for the desired polypeptide. Another technique is based on the insertion of a nucleic acid sequence into the bacterial genome itself. These and other techniques are well known in the field of cell biotechnology and can be carried out without difficulty by the skilled artisan.

[0037] Once transformed, each variant is cultivated for biomass production and then used immediately or stored. It is convenient to create a series of bacteria capable of expressing different enzymes so as to have the same number of modules available to construct reaction schemes that can be carried out by the method according to the invention. This cell library can therefore be used to custom prepare the desired reaction mixture in step a) of the method according to the invention.

[0038] After the reaction mixture is prepared in a suitable medium, the reaction is allowed to proceed. This mode of proceeding corresponds to a batch production mode. It should be noted that production in a continuous or semi-continuous mode is also entirely possible, as will be explained below. The reaction chain is naturally interrupted when the initial organic substrate is exhausted. The compound of interest that is synthesized is essentially excreted in soluble form in the aqueous medium. According to the reaction scheme adopted, the compound consists of one of the products P1, P2, ..., Pn obtained in one of the reactions of the reaction scheme. After separation of the biomass, it can then be easily recovered in the supernatant, whose composition is simply compared with that resulting from other previously known techniques.

[0039] According to a particular embodiment of the invention, the method can use two cell modules, the first of which converts an 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 a desired compound. In this case, the reaction mixture comprises bacteria expressing a first enzyme E1 capable of catalyzing a first reaction R1 from said organic substrate to provide a first product P1, and bacteria 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.

[0040] This protocol may be the beginning of a larger scale reaction scheme, where the second product may 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 enzymes E3, ..., En capable of catalyzing reactions R3, ..., Rn from product Pn-1 obtained by reaction Rn-1 to provide product Pn, which is recovered in step c) as a compound of interest or consumed as a substrate for reaction Rn+1.

[0041] If a cofactor is required to carry out the first reaction, this cofactor is introduced into the reaction medium in step a). A first cell module is selected to convert the initial organic substrate and this cofactor (called the first cofactor CoF1), and a second module uses 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 may be separated from the medium to obtain the desired compound, or may participate as a substrate in a downstream reaction of a larger reaction scheme. According to a specific protocol, the coproduct CoP2 may participate as a substrate for another reaction.

[0042] Thus, according to an embodiment of the method according to the invention, the reaction mixture comprises a cofactor CoF1 of said organic substrate, a bacterium expressing an enzyme E1 capable of catalyzing a first reaction R1 from said organic substrate and the cofactor CoF1 to form a first product P1 and a first co-product 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 a second enzyme E2 capable of catalyzing a second reaction R2 from the first co-product CoP1 to form a second co-product CoP2, at least one of which is consumed in a third reaction or recovered in step c) as a compound of interest. The term "third reaction" here will be understood to refer to any one of the reactions that can occur downstream of the targeted reaction.

[0043] If the reaction scheme is larger, the above mechanism can be generalized. Indeed, each of the reactions Rn in the reaction scheme can use a cofactor CoFn that reacts with the product Pn-1 generated by the upstream reaction Rn-1 under the effect of the enzyme En. Then, the product Pn and the co-product CoPn are formed. The co-product CoPn can then be used as a cofactor CoFn+1 for the downstream reaction Rn+1. For that matter, the product Pn can be used as a substrate for the (other) downstream reaction Rn+1 if it is not the desired compound desired. This protocol can be generalized to one or more of the modules involved.

[0044] Thus, according to one embodiment of the method that is the object of the present invention, the reaction mixture comprises at least one cofactor CoFn, a product Pn-1 of reaction Rn-1 and at least one bacterium expressing an enzyme capable of catalyzing reaction Rn from said cofactor CoFn to form a product Pn and a co-product CoPn, at least one of the two being consumed in reaction Rn+1 or recovered in step c) as a compound of interest.

[0045] As seen, reaction Rn of the reaction scheme can use a cofactor as a substrate, which is generally a co-product generated by an upstream reaction. In this case, another compound, called a "complementary substrate", must be present alongside the cofactor, which gives a complementary product resulting from reaction Rn. This product may not be useful in the rest of the reaction scheme and may accumulate in the medium. Thus, according to one feature of the method that is the subject of the present invention, the reaction mixture can also contain at least one substrate SCn that is complementary to said at least one cofactor CoFn.

[0046] In the method according to the invention, said at least one cofactor CoFn can be introduced totally or partially into the reaction mixture in step a) or during step b). Indeed, it may be preferable to add a cofactor CoFn that is not involved in the first step of the reaction scheme only if a significant amount of product Pn is present in the medium, which will react with said cofactor CoFn. Thus, reaction kinetics are taken into account in order to optimize the production of the compound of interest.

[0047] Moreover, if a cofactor is required for the first reaction, it is particularly advantageous for the second (downstream) reaction to regenerate that cofactor. Indeed, cofactors are expensive compounds that directly impact production costs. Reaction schemes have been devised to meet this objective, where the second reaction consumes a co-product from the first reaction as a cofactor in the second reaction to reform the initial cofactor. In doing so, it is wise to provide a complementary substrate for the second reaction, which is chosen so that it is affordable. At the end of the second reaction, a second product, which may or may not be of interest, is formed, but above all, the cofactor of the first reaction is regenerated.

[0048] Thus, in a particularly advantageous embodiment of the method according to the invention, the at least one cofactor CoFn is at least partially formed in the reaction mixture by reaction Rn-1 catalyzed by enzyme En-1 capable of forming product Pn-1 and co-product CoPn-1, the co-product CoPn-1 being identical to the cofactor CoFn.

[0049] However, it is preferred to add a certain amount of this cofactor to the medium to start the process until the regeneration of the cofactor is sufficient to ensure a continuous supply of said cofactor. At this point, it turns out that a minimal amount is sufficient to start the reaction cycle. The amount of cofactor that needs to be introduced into the reaction mixture of the reaction concerned to proceed to completion is then significantly reduced with respect to the amount of the corresponding substrate with which it must react. The amount of cofactor can be 20 times less, but preferably 100 times less, more preferably 500 times less, even more preferably 1000 times less. It should be noted that certain cofactors that are synthesized in the cytoplasm, such as ATP, need only be present in small but sufficient amounts in the periplasmic space.

[0050] Thus, in this context, according to the invention, the amount of the first cofactor CoF1 introduced into the reaction mixture in step a) corresponds to a molar concentration up to 20 times lower than the initial molar concentration of said organic substrate.

[0051] As mentioned above, the initial organic substrate can be of various natures, including any kind of carbon-containing molecule. The method has in particular demonstrated its effectiveness in converting common carbohydrates (sugars), such as glucose, fructose, glycerol, or others, into compounds of interest, such as rare sugars, particularly phosphorylated sugars, specific isomers of rare sugars, dihydroxyacetone, and other molecules that are sought to exploit key synthetic pathways. Other organic compounds, including aldehydes, alcohols, organic acids, carbamates, hydrocarbons, and amino acids, can constitute the starting substrate. Examples include glyoxal, which provides glycolaldehyde, or L-phenylalanine, which is the starting point for the production of 2-phenylethanol. Moreover, the organic substrate can be a compound containing a single carbon atom, such as carbon dioxide.

[0052] Thus, according to one characteristic of the process that is the object of the present invention, the organic substrate of the first reaction is chosen from carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons, amino acids and carbon dioxide.

[0053] A large number of enzymes can be produced by the various transformed bacteria used in the method of the present invention. The skilled artisan knows how to identify suitable enzymes for the development of a reaction scheme leading from an organic substrate to a given compound of interest. The skilled artisan can select such enzymes from the enzymes listed and described or to be identified in the future.

[0054] Thus, according to a preferred feature of the method according to the invention, the enzymes E1, E2, ..., En are each selected from kinases, dehydrogenases, phosphatases, reductases, isomerases and transferases. The kinases may in particular be glycerol kinase, glucokinase, fructokinase, xylokinase, acetokinase or phosphofructokinase. The dehydrogenases are for example glycerol dehydrogenase or phosphite dehydrogenase. Reductases such as NADH reductase or glyoxal reductase can be used. The isomerases may be selected from xylose isomerase, xylulose isomerase, ribulose phosphate-3-epimerase, ribose-5-phosphate isomerase, and transketolase or transaldolase may be selected as transferase. Various phosphatases are also known.

[0055] Furthermore, multiple cofactors may be involved in one or another of the reactions of the reaction scheme. It should be noted that as cofactors, molecules involved belong to two categories, which form coproducts that can then play the role of cofactors in upstream or downstream reactions. Thus, preferably, in the method according to the invention, at least one of the enzymes E1, E2, ..., En is selected from the group consisting of ATP, ADP, AMP, UTP, UDP, UMP, NAD+, NADH, NADP+, NADPH, FAD, FADH 2 , Coenzyme A catalyzes a reaction that provides one of the co-products CoP1, CoP2, ..., CoPn selected from coenzyme A, or catalyzes a reaction that uses one of those co-products as a cofactor.

[0056] It has been seen that at the end of step c) of the method of the invention, the biomass is separated from the supernatant from which the compounds of interest are extracted. However, the work carried out has revealed an unexpected advantage of the method of the invention. In fact, it was found that this biomass, containing the n bacterial cells introduced in step a), can be reused as is in a new reaction cycle, with a yield at the same level as that observed during the first reaction cycle, and sometimes even greater. This leads to significant savings in terms of time, number of operations carried out, consumption of components and water, and finally from an economic point of view. This advantage arises from the principle of the invention, according to which the cell culture stage is separated from the biochemical synthesis stage: cell culture and biochemical synthesis are carried out in separate reactors, with different media.

[0057] The supernatant can be separated from the biomass by gravity flow, in which case it is possible to immediately reuse the biomass remaining in the reactor by adding only fresh substrate and, optionally, other required components.

[0058] Therefore, in a particularly advantageous manner, after step c) of the method object of the present invention, the biomass separated from the supernatant is recycled to prepare a new reaction mixture according to step a), with or without an intermediate storage step.

[0059] The method may be carried out in the context of a discontinuous batch culture as described above or with discontinuous flow (fed-batch type). The method may also be carried out in a semi-continuous manner or with continuous feeding, according to rules known in the art. In this case, steps a) and b) are not carried out sequentially, but more or less simultaneously.

[0060] For this reason, in a variant embodiment of the method according to the invention, steps a) and b) are carried out by continuously or at time intervals administering to the reaction mixture containing said n bacteria: - said organic substrate, and - optionally carried out by adding a cofactor of said organic substrate, a cofactor of said product obtained by reaction Rn-1, called cofactor CoF1, CoF2, ..., CoFn; a complementary substrate SC of the co-product obtained by reaction Rn-1, and by leaving the reaction mixture thus obtained to react continuously or semi-continuously.

[0061] From the above, it becomes clear that the approach adopted by the method for producing a compound of interest just described has numerous advantages that answer the challenges encountered by conventional techniques and is very general in scope. The externalization of the reaction actually makes it possible to use the biochemical machinery of the cell to supply the enzymatic effectors of the reaction taking place in the space bounded by the periplasm, while leaving the reagents and products involved in the reaction in circulation. Thanks to the combinable modular design, completely original reaction schemes can be developed and rapidly implemented. Thus, the synthesis of a given product of interest is promoted, as is its final purification, because the aqueous bioproduction medium is very simple.

[0062] It should be added that the method is much cheaper than known enzymatic methods (if they exist), since it relies on the production of reusable biomass. It is therefore possible to industrially produce any kind of molecule, including those that are complex or difficult to reach.

[0063] It is therefore understood that the method that is the object of the present invention represents an unprecedented alternative to current production methods and provides a powerful tool for accelerating the development of new products.

[0064] 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 the reaction mixture prepared in step a) of the production method described above.

[0065] Thus, the present invention provides a method for the preparation of a compound comprising the steps of: - n bacteria, n being an integer at least equal to 2, each of which has been genetically modified to express in its periplasmic space at least one enzyme E1, E2, ..., En, preferably a single enzyme, each of said n bacteria expressing an enzyme that is distinct from the other bacteria; - an enzyme E1 is capable of catalysing a first reaction R1 from said organic substrate to provide a first product P1 and, optionally, a first co-product CoP1; each of said enzymes E2, ..., E n is capable of catalysing a reaction R2, ..., R n from a product or a co-product obtained by reaction R n-1 to provide a product P2, ..., P n and, optionally, a co-product CoP2, ..., CoP n , respectively; - said organic substrate, and - optionally a cofactor of said organic substrate, a cofactor of said product obtained by reaction Rn-1, called cofactor CoF1, CoF2, ..., CoFn; a complementary substrate SC of the co-product obtained by reaction Rn-1 The present invention relates to a reaction mixture for the production of a compound of interest from an organic substrate, comprising:

[0066] According to a particularly preferred embodiment, each bacterium is genetically modified to express a single enzyme E1, E2, ..., En in its periplasmic space, each of said n bacteria expressing a different enzyme from the other bacteria.

[0067] The reaction mixture is therefore a mixed composition comprising at least two bacteria in suspension, cells and various soluble reagents, including the organic substrate to be converted, to obtain the compound of interest. Reference may be made to the above description to define the various embodiments of this mixture. According to a particular embodiment, the reaction mixture comprises at least three bacteria, in particular at least four bacteria, or even at least five bacteria, and in particular at least six bacteria, each of which is genetically modified to express an enzyme different from the enzymes expressed by the other bacteria.

[0068] Finally, according to a third aspect of the present invention, the use of the above reaction mixture is claimed to produce a compound of interest selected from carbohydrates, aldehydes, alcohols, organic acids, carbamic acids, hydrocarbons, and amino acids. For example, a reaction mixture containing carbohydrates can be used to produce phosphorylated sugars or dihydroxyacetone (DHA), or a reaction mixture containing amino acids can be used to produce nitrogenous organic compounds.

[0069] The invention will be better understood and its details will emerge in the light of the description given of various embodiments, with reference to the attached drawings, in which: [Brief description of the drawings]

[0070] [Figure 1] Sequential production of glycerol-3-phosphate in glycerol form by two cellular modules (BL21-Glpk and BL21-AckA) [Figure 2a] Effect of the concentration of cell modules (BL21-Glpk and BL21-AckA) on the production of glycerol-3-phosphate [Figure 2b] Effect of reaction volume - production of glycerol-3-phosphate at volumes of 2 ml, 20 ml, 200 ml and 2000 ml [Figure 3a] Production of D-glucose-6-phosphate from D-glucose by two cellular modules, BL21-Glpk and BL21-AckA [Figure 3b] Production of D-glucose-6-phosphate from D-glucose by two cellular modules, BL21-nlpA-Glk and BL21-nlpA-AckA. [Figure 4] Production of fructose-1-phosphate from D-fructose by two cellular modules, BL21-KhkC and BL21-AckA [Diagram 5] Production of glucose-6-phosphate by reusing BL21-Glk and BL21-AckA cell modules [Figure 6]Production of dihydroxyacetone from glycerol by the BL21-Gldk and BL21-Nox modules [Figure 7] Production of glycolaldehyde from glyoxal by the BL21-YvgN module with or without the BL21-PtxD regeneration module [Figure 8] Consumption of D-xylose by the isomerization reaction alone (BL21-XylA) and by the entire reaction (BL21-XylA, BL21-XylB, BL21-AckA, BL21-AphA) [Figure 9] Production of D-xylulose from D-xylose by the isomerization reaction alone (BL21-XylA) and by the entire reaction (BL21-XylA, BL21-XylB, BL21-AckA, BL21-AphA) [Figure 10] SDS-PAGE gel electrophoresis of periplasmic fractions of XylA, XylB, AckA, Rpe, RpiA, TktA, Tal, PfkA modules and control BL21 WT. [Figure 11] Production of D-fructose-1,6-phosphate from D-xylose using an eight-cell module [Figure 12] Production of D-sedoheptulose-7-phosphate, an intermediate in the production of D-fructose-1,6-diphosphate, using eight modules [Figure 13] Monitoring the concentration of 2-phenylethanol by HPLC in a synthetic consortium consisting of strains BL21-ARO8, BL21-ARO10, BL21-ADH5 and BL21-PtxD in the presence of 10 mM L-phenylalanine and in the control BL21-WT. [Figure 14]SDS-PAGE gel of periplasmic fractions of BL21-AckA, BL21-GlpK, BL21-AckA-GlpK biocatalysts and negative control BL21-W. M: Precision Plus Protein™ All Blue Marker, BIO-RAD®; 1: BL21-AckA (2 OD600nm / ml); 2: BL21-GlpK (2 OD600nm / ml); 3: BL21-GlpK-AckA (2 OD600nm / ml), 4: BL21-GlpK (1 OD600nm / ml), BL21-GlpK (1 OD600nm / ml), 5: BL21-WT (2 OD600nm / ml); Black dashed box: 56 kDa GlpK protein; White dashed box: 43 kDa AckA protein. [Figure 15] Production of glycerol-3-phosphate from glycerol by strain BL21-GlpK-AckA, a synthetic consortium composed of two modules BL21-AckA and BL21-GlpK supplied in varying ratios, and the control BL21-WT. [Figure 16] Glucose-6-phosphate production in a synthetic consortium consisting of two BL21-Glk, BL21-AckA modules and in a BL21-WT control in the presence of glucose and without added ATP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] Materials and Methods 1-Plasmid - Plasmid pET22b (ori pBR322, ampicillin, T7 promoter, pelB) and plasmid pET26b (ori pBR322, kanamycin, T7 promoter, pelB) are commercially available plasmids carrying the pelB signal sequence at the N-terminus and a His-tag sequence at the C-terminus. These plasmids were used for the expression of genes of interest encoding enzymes that are transported into the periplasm of the cells by a PelB fusion peptide. - The plasmid pACT3 (ori p15A, chloramphenicol, Ptac promoter) was used to anchor the protein in the periplasm. This plasmid was modified to carry the N-terminal leader sequence of the lipoprotein NlpA. - Plasmid pETDuet-1 (ori pBR322, ampicillin, T7 promoter) is designed for the simultaneous expression of two genes of interest. The vector carries two cloning sites, one after the T7 promoter, one after the lac operator and one after the Shine-Dalgarno sequence. Plasmid pETDuet-1 was modified here by incorporating the PelB signal sequence after two ribosome binding sites. The expression cassette (T7-RBS-pelB) is identical to that of pET22b. The genes incorporated in this modified plasmid allow the simultaneous expression of two free enzymes in the periplasm under the control of two T7 promoters inducible with IPTG.

[0072] The genes are amplified by techniques known per se, for example by polymerase chain reaction (PCR) using suitable primers, from the genomic DNA of E. coli MG1655 and then inserted by homologous recombination into the plasmids pET22b, pET26b, pACT3 or pETDuet-1 previously linearized with the NEBuilder® kit. Where indicated, the synthetic genes were ordered directly from the company GenScript® and cloned directly by them into the plasmids pET22b or pET26b.

[0073] The primers used for gene amplification are of generally known type. The skilled artisan knows how to select and use primers in the context of PCR techniques. According to a particular embodiment, the primers for the amplification of each of the genes are prepared using the kit and the instructions of the manufacturer NEB "NEBuilder HiFi DNA Assembly Master Mix & NEBuilder HiFi DNA Assembly Cloning Kit E2621 ​​E5520" (https: / / www.neb.com / - / media / nebus / files / manuals / manuale2621_e5520.pdf?rev=67d09a762b2c47ed8df3a0bd1cb59a1b&hash=02CF464149634283F5633CA1DE4D2FDF). In particular and according to the manufacturer's recommendations, the PCR primers used in the construction of HiFi DNA must have two sequence components: i) an overlapping sequence necessary for the construction of the adjacent fragment, ii) a gene-specific sequence necessary for the priming of the template during PCR. To obtain efficient assembly of PCR fragments into vectors, it is suggested to use overlaps of 15-30 nucleotides with a Tm equal to or higher than 48°C (assuming AT pairs = 2°C and GG pairs = 4°C). Examples of the above plasmids include the following primers: GLPK_pET22b; Khk-C_pET22b; ackA_pET26b; gldA_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_glpK_ack; pET22b_ARO8; pET22b_ARO10; pET22b_ADH5.

[0074] 2- Strain Construction Strains BL21(DE3) or BL21(DE3)Gold were selected for transformation with plasmid pET22b or pET26b (depending on the resistance cassette) to allow the use of T7 polymerase. Transformations were carried out according to the TSS protocol described by Chung and Miller (Chung et al., 1993). The last two strains were constructed such that the enzyme was anchored in the periplasm, all others were constructed such that the enzyme was free. The strains used are given in Table 1.

[0075] [Table 1A]

[0076] [Table 1B]

[0077] 3- Culture media and conditions Induced whole-cell production Strains are drawn up from glycerol stocks stored at -80°C in 5 ml of TB medium buffered to pH 7 with the appropriate antibiotics and placed overnight at 37°C. The composition of TB medium is given in Table 2.

[0078] [Table 2]

[0079] The following day, the preculture is diluted 1:100 into 50 ml of fresh TB medium and the culture is stirred at 200 rpm at 37°C. When the optical density at 600 nm (OD600nm) reaches a value between 0.4 and 0.6, the cells are induced with 0.1 mM IPTG. The cells are then placed at 20°C for approximately 18 hours with stirring at 200 rpm.

[0080] Exogenous production of target molecules Induced whole cells (or modules) expressing one or more enzymes in their periplasm are centrifuged at 4500 rpm for 10 min and resuspended in sterile water to reach a defined amount of cells expressed at ODU. Modules carrying enzymes selected according to the requirements of a predefined reaction scheme are introduced together in a reaction mixture. This reaction mixture contains a buffer to stabilize the pH, activators and cofactors if necessary, substrates and a known amount of each cell module. The composition is intentionally as simple as possible to ensure optimal enzyme activity and minimized production costs. Substrates and cofactors were purchased from Sigma® or Carbosynth®, with the exception of acetyl phosphate used for ATP regeneration, which was synthesized from two low-cost compounds, phosphoric acid (85%, 2 mol) and ethyl acetate (2 mol), according to the method described by Crans et al., 1983.

[0081] 4- Quantification and analysis of extracellular products Substrate and product concentrations are monitored by high pressure ion chromatography (HPIC) or by high performance liquid chromatography (HPLC). Depending on the target molecule, different detection methods were used.

[0082] HPIC analysis The samples were centrifuged at 14,800 rpm for 2 min and then 0.2 μm filtered. The samples to be analyzed were diluted 1 in 100 in Milli-Q water in flasks with pre-pierced stoppers and placed in the autosampler at 15°C.

[0083] Amperometric detector (HPIC-PAD) Analysis of certain substrates and products is performed by ion chromatography (Dionex™ ICS-6000 Ion Chromatography Instrument) coupled with a pulsed amperometric detector or PAD (Dionex™ ICS-6000 Electrochemical Detector). Molecules are separated on a Dionex™ Carbopac™ PA1 column (2 x 250 mm) preceded by a guard column (2 x 50 mm) of the same type at 25°C.

[0084] Two elution methods were used. The method used for the determination of sugars (xylose, glucose, fructose), organophosphorus compounds (glucose-6P, fructose-1P, fructose-1,6bP, glycerol-3P), aldehydes such as methylglyoxal and glycolaldehyde is as follows: NaOH concentration is maintained at 100 mM from 0 to 30 min and sodium acetate concentration (NaOAc) is gradually changed: 0 to 2 min, 0 mM NaOAc; 2 to 15 min, increase from 0 to 500 mM NaOAc; 15 to 23 min, concentration is maintained at 500 mM NaOAc; 23 to 23.1 min, decrease from 500 mM to 0 mM NaOAc; 23.1 to 30 min, maintain at 0 mM NaOAc.

[0085] Conductivity detector (HPIC-CD) Anions (organophosphorus compounds, phosphate, chloride, sulfate, organic acids, etc.) are analyzed by ion chromatography (Dionex™ ICS-6000 Ion Chromatography Instrument) equipped with a conductivity meter (Dionex™ ICS-6000 CD Conductivity Detector). Molecules are separated on a Dionex™ IonPac™ AS11-HC column (2 x 250 mm) preceded by a guard column of the same type (2 x 50 mm) at 25°C.

[0086] The concentration gradient is generated with an eluent generator EGC 500 KOH at a flow rate of 0.35 ml / min. The following gradient was used to best separate the intermediate compounds from the reaction scheme presented in Example 7. Before each injection, a 7-minute conditioning with 2 mM NaOH is performed. The first step of the gradient is an isocratic elution with a NaOH concentration of 10 mM for 3 minutes, followed by an increase in the NaOH concentration in several steps: 1) increase from 10 mM to 50 mM in 9 minutes; 2) increase from 50 mM to 100 mM in 7 minutes; 3) hold at 50 mM for 3 minutes; 4) decrease in NaOH concentration to 10 mM; 5) hold at 10 mM for 4 minutes. At the column outlet, an ADRS 2 mm suppressor (87 mA) is used to improve detection. In another example, the NaOH concentration gradient presented below was used to determine anionic metabolites. The first step was an isocratic elution at a NaOH concentration of 2 mM for 4 min, followed by a multi-step increase in NaOH concentration: 1) increasing from 2 mM to 100 mM in 11 min; 2) holding at 100 mM for 10 min; 3) decreasing to 2 mM and holding for 1 min.

[0087] UHPLC-UV / RI analysis Sugars and organic acids are analyzed by UHPLC (Dionex Ultimate™ 3000) equipped with a refractometer (Shodex™ RI-101) and a UV detector (Dionex UltiMate™ 3000 Diode Array Detector 3000 (RS)). Molecules are separated on a Phenomenex™ ROA-Organic Acids H+ column (8%) (300 x 7.8 mm) equipped with a guard column of the same type (50 x 7.8 mm). Elution is performed isocratically for 35 min at a flow rate of 0.5 ml / min. The samples to be analyzed are 0.2 μm filtered before injection.

[0088] HPLC-Mass spectrometry The presence of fructose-1-phosphate is detected by UHPLC (Dionex UltimateTM 3000) equipped with a mass spectrometer (Thermo Fisher Scientific™). The molecules are separated on an ACQUITY UPLC BEH Amide column (130 Å, 1.7 μm, 2.1 x 30 mm) preceded by a guard column (ACQUITY BEH Shield RP18 1.7 μm VANGUARD). Mobile phase A consists of ultrapure and methylphosphonic acid (1.8 mM), organic mobile phase B consists of acetonitrile and formic acid (1.3 mM). The concentration gradient used is as follows, with a flow rate of 0.35 ml / min: 1 min isocratic with 90% organic mobile phase, followed by several steps of reduction in the organic mobile phase fraction: 82% in 3 min, 78% in 6.5 min and 50% in 8 min. This composition is held constant for 10 min (50% organic phase), followed by an equilibration period of 7 min.

[0089] Glycerol-3-phosphate C13 (G3P-C13) is used as an internal standard. An elution gradient is created. An injection mixture is prepared with an insert containing 100 μl of 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 when processing results using Chromeleon™ software.

[0090] 5- Protein extraction by osmotic shock Cells are grown overnight at 37°C in 2 ml of LB medium, agitated at 200 rpm. The cell suspension is transferred to an Eppendorf tube and centrifuged (2 ml; 5 min; 14,000g; 4°C). The pellet is resuspended in 500 μl of buffer 1 and incubated on ice for 20 min. The tube is inverted periodically to avoid sedimentation. The cell mixture is centrifuged (15 min; 14,000g; 4°C). The supernatant is discarded and the cell pellet is resuspended in 125 μl of buffer 2. After a further incubation on ice for 10-20 min with periodic inversion, the cells are centrifuged (15 min; 14,000g; 4°C). The supernatant containing the periplasmic fraction was collected, then denatured at 95°C for 10 min and deposited on an SDS-PAGE gel (45 min, 180V, 400mA) to confirm the presence of the enzyme and its molecular weight. The size marker used is Precision Plus Protein™ All Blue Standards #1610373EDU. The composition of the buffer is given in Table 3.

[0091] [Table 3] EXAMPLES

[0092] Example 1 Production of phosphorylated compounds Phosphorylation is a reaction that allows cells to sequester essential metabolites in the cytoplasm and prevent their loss by diffusion to the external environment. Negatively charged phosphorylated compounds cannot diffuse through the phospholipid bilayer. Thus, sugar metabolism always involves a phosphorylation step. For example, sugars such as D-glucose, D-fructose, D-threalose, L-rhamnulose, D-xylose and L-arabinose are trapped in the cytoplasm as 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, respectively. The same applies to other carbon sources that can be assimilated by microorganisms, such as polyols: mannitol, sorbitol and glycerol, which are phosphorylated to mannitol-1-phosphate, sorbitol-6-phosphate and glycerol-3-phosphate, respectively. Externalizing phosphorylation according to the present invention solves this export problem and makes microbial production of these compounds feasible.

[0093] To do this, a strain of E. coli was transformed to express a kinase in its periplasm that catalyzes the phosphorylation of sugars (first module). This reaction consumes a cofactor, ATP, and forms ADP as a coproduct. The second module was made by transforming a strain of E. coli to express acetate kinase (AckA) in its periplasm. This enzyme catalyzes the reaction using acetyl phosphate and ADP to form acetate and ATP.

[0094] The reaction scheme is as follows:

[0095] [ka]

[0096] [Text in image] Acetic acid Acetyl phosphate Acetate kinase

[0097] The examples below illustrate the production of phosphorylated compounds of interest from three organic substrates (glycerol, glucose and fructose) with the intervention of the cofactors ATP and ADP, corresponding to this reaction scheme. Due to the fact that the BL21-AckA module is involved, which performs the regeneration of ATP, the amount of ATP introduced into the initial reaction mixture is significantly less than the amount of substrate (in this case 32 times less in one of the examples and 60 times less in the other two).

[0098] Thus, the use of the BL21-AckA module allows considerable savings, since the cost of ATP is roughly 300 euros / kg. However, another important reason motivates the regeneration of the ATP cofactor. Indeed, some kinases are inhibited by ADP. For example, glycerol kinase is inhibited by small amounts of ADP (Kic=500 μM). However, in the first reaction, one ADP molecule is generated per molecule of product formed. In the case of inhibition by ADP, the production of the compound of interest is therefore not possible unless the ADP formed is continuously removed, which is done here by a reaction that regenerates ATP. Using the regeneration module allows the production of phosphorylated molecules.

[0099] Example 1.1 Production of glycerol-3-phosphate from glycerol Glycerol-3-phosphate is the entry point for the phospholipid pathway. This molecule of interest has been produced by microorganisms in fed-batch bioreactors at amounts of 325 mg / l, but its export proved to be a major problem (Popp et al., 2008). Furthermore, the phenomenon of glycerol dephosphorylation occurs inside the cell.

[0100] Two modules are used: the first expresses glycerol kinase from E. coli to produce glycerol-3-phosphate from glycerol and ATP, and the second functions to regenerate ATP from E. coli AckA via acetate kinase. Both the BL21-Glpk and BL21-AckA modules are derived from the BL21(DE3) chassis.

[0101] Both modules are cultivated in TB medium and induced with IPTG as described (Materials and Methods, point 3) before being harvested to carry out the production of glycerol-3-phosphate in simple medium. The reaction mixture is: Module 1: 2 ODU / ml BL21-Glk cells Module 2: 2 ODU / ml BL21-AckA cells Substrate: Glycerol (160 mM) Cofactor: ATP (5mM) Complementary substrate: acetyl phosphate (220 mM) Buffer: HEPES (50 mM) and MgSO 4 (15mM) Includes.

[0102] The glycerol-3-phosphate production reaction is carried out in a volume of 2 ml of reaction mixture by resuspension of the pellet containing an equivalent amount of 2 ODU of Glpk and AckA cells in a volume of 2 ml. The tube is then placed at 25° C. with stirring. Samples are taken at 0 h after addition of the enzyme and after 1 h, 3 h, 5 h and 22 h in the reaction mixture. The results are presented in FIG. 1. After 22 h of reaction, a production of 1.4 g / l of glycerol-3-phosphate was obtained.

[0103] Effect of cell concentration on yield The same protocol was repeated using twice the module concentration. A yield of 81.5% or a production of 12 g / l was obtained in 23 h, which corresponds to 37 times the maximum production obtainable by the microbial pathway (Popp et al., 2008). Thus, the cell concentration has an important effect and can be optimized.

[0104] Effect of cell concentration on reaction rate To evaluate the effect of the amount of cells on the production rate, different concentrations were used under the same experimental conditions. The glycerol-3-phosphate concentrations obtained in 1 h show that the reaction rate is proportional to the cell concentration (Figure 2a). Productivities ranging from 1 g / l / h to 4 g / l / h can therefore be obtained. These yields show that the scale-up of the method according to the invention is possible.

[0105] This example demonstrates the possibility of producing phosphorylated polyols by the method object of the present invention, which helped to overcome the obstacles related to their export and dephosphorylation by intracellular phosphatases.

[0106] Effect of reaction volume The method according to the invention is intended to produce molecules at different scales, from laboratory to industrial level. To evaluate the effect of increasing the volume of the reaction mixture on glycerol-3-phosphate production, reactions were carried out in volumes of 2 ml, 20 ml, 200 ml and 2000 ml. The modules used were, as before, BL21-Glpk and BL21-AckA. The reaction mixture was: Module 1: 2 ODU / ml BL21-Glk cells Module 2: 2 ODU / ml BL21-AckA cells Substrate: Glycerol (25 mM) Cofactor: ATP (5mM) Complementary substrate: acetyl phosphate (25 mM) Buffer: HEPES (50 mM) and MgSO 4 (15mM) It is composed of:

[0107] Samples were taken after addition of the cell biocatalyst (module) to the mixture at 0 h and then at 1, 3, 4 and 5 h of reaction. Under the conditions described, with 25 mM acetyl phosphate, the theoretical maximum glycerol phosphate production 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%. A 100% yield is reached after 3 h when the reaction volume is 200 ml or 2000 ml. These results highlight the interchangeable character of the method, an essential prerequisite for scale-up.

[0108] Moreover, the product is stable at 2000 ml. According to these results, a scale-up of the process by a factor of 1000 is quite feasible and even advantageous for production.

[0109] (Example 1.2) Production of glycerol-6-phosphate from glucose Glucose-6-phosphate is the entry point for glycolysis and the pentose phosphate pathway, and two protocols for its production were examined, one with the enzyme free in the periplasm and one with the same enzyme anchored to the inner membrane.

[0110] Free enzyme protocol The two modules used are the BL21-Glk module expressing the E. coli Glk kinase and the BL21-AckA ATP regeneration module in which the E. coli acetate kinase is expressed. The enzyme is found free in the periplasm. The two modules are cultivated in TB medium and induced with IPTG, then introduced into the reaction mixture at a volume of 2 ODU in 2 ml. A control sample is prepared containing the strain BL21-WT. The reaction mixture is: Module 1: 2 ODU / ml BL21-Glk cells Module 2: 2 ODU / ml BL21-AckA cells Substrate: 300 mM glucose Cofactor: 5mM ATP Complementary substrate: 125 mM acetyl phosphate Buffer: HEPES (50 mM) and MgSO 4 (15mM) Includes.

[0111] Figure 3a shows that glucose-6-phosphate is indeed synthesized at a concentration of 30.8 g / L, i.e. a yield of 96%, obtained from a 5-hour reaction, limited by the amount of acetyl phosphate (125 mM). A plateau is therefore reached quickly. After 22 hours, a decrease in glucose-6-phosphate is observed, due to the degradation of the molecule. No production is observed in the control strain.

[0112] Immobilized enzyme protocols The modules used here (BL21-nlpA-Glk and BL21-nlpA-AckA) express the same two enzymes (glucokinase Glk and acetate kinase AckA) but fused to the anchor peptide nlpA such that both enzymes are anchored to the inner membrane of the periplasm in their respective modules. The modules are cultivated and tested following the same protocol as above.

[0113] The reaction scheme is as follows:

[0114] [ka]

[0115] [Text in image] Acetic acid Acetyl phosphate Acetate kinase

[0116] Figure 3b shows the production of glucose-6-phosphate, limited by the amount of acetyl phosphate (125 mM), at a concentration of 24.8 g / L, i.e. a yield of 76%, obtained after 3 hours of reaction. After 22 hours, a decrease in glucose-6-phosphate is observed, due to degradation of the molecule. No production is observed in the control strain.

[0117] Conclusion: The productive reaction is possible if the enzyme is free or immobilized. In this example, the production of glucose-6-phosphate from the immobilized enzyme is faster than from the free enzyme, but the yield is lower. The method according to the invention can be carried out with transformed bacteria producing the periplasmic enzyme, whether immobilized or free.

[0118] (Example 1.3) Production of glycerol-1-phosphate from fructose Fructose-1-phosphate is a sugar phosphorylated at the 1-position from which the desired synthon glyceraldehyde can be generated, the biological synthesis of which has never been described.

[0119] As before, two modules are required: one harboring a sequence encoding the Homo sapiens KhkC fructokinase capable of phosphorylating sugars at position 1 (BL21-KhkC), and the other expressing the E. coli acetate kinase responsible for regenerating the ATP cofactor (BL21-AckA, already described).

[0120] In this test, 4 ODU / ml of each module was added to the reaction mixture, i.e. a total of 8 ODU in 2 ml. A control sample was prepared containing strain BL21-WT. The reaction mixture was: Module 1: 2 ODU / ml BL21-KhkC cells Module 2: 2 ODU / ml BL21-AckA cells Substrate: 300mM fructose Cofactor: 5mM ATP Complementary substrate: 125 mM acetyl phosphate Buffer: HEPES (50 mM) and MgSO 4 (5mM) Includes.

[0121] Strain BL21-WT (OD 2 ODU / ml) 600nm A control sample containing 10 mM NaCl (referred to as 10 mM NaCl = 2) is prepared.

[0122] The results presented in Figure 4 show that the reaction is nearly complete after 5 hours. The reaction reaches a plateau after 22 hours, with fructose-1-phosphate at a concentration of 1.3 g / l. The reaction is limited by acetyl phosphate (125 mM). No product is obtained in the control strain.

[0123] Conclusion: The method according to the invention is thus suitable for the production of phosphorylated sugars. Firstly, it is established that the production of phosphorylated sugars by externalizing the phosphorylation reaction to the periplasm is possible and efficient. Secondly, the use of the same strain (BL21-AckA) in several reaction schemes illustrates the flexibility of the method and its economic benefits, by limiting the biomolecular engineering work to obtain the required modules. Finally, the advantage of the modules designed for the regeneration of cofactors is that they can use low concentrations of ATP for the production of phosphorylated molecules. It is therefore possible to design reaction schemes using ATP, which is naturally present in small amounts in the periplasmic compartment.

[0124] Example 2 Cell preservation and reuse In the method according to the invention, the use of a module carried out in a manner separated from the previous cell culture stage makes it possible to carry out several reaction cycles with the same biomass and to preserve this biomass for a long time.

[0125] Glucose-6-phosphate production was carried out by reusing cells from the BL21-GLK and BL21-AckA modules already used in previous productions. For this purpose, a first production was carried out in which the cells were resuspended in 2 ml of reaction mixture (8 ODU for each cell type). The cells used for this first production were then separated from the supernatant and the modules were separated by centrifugation. The pellet was collected and stored at 4° C. After 4 days, the cells were resuspended in water and introduced into a new volume of 2 ml of reaction mixture in an amount of 8 ODU for each cell type.

[0126] The reaction mixture used for both cycles was: Module 1: 8 ODU / ml BL21-GlK cells Module 2: 8 ODU / ml BL21-AckA cells Substrate: 300 mM glucose Cofactor: 5mM ATP Complementary substrate: 220 mM acetyl phosphate Buffer: HEPES (50 mM) and MgSO 4 (5mM) It consists of:

[0127] The results presented in Figure 5 show a production of 50 g / l of glucose-6-phosphate in 6 hours. The reaction was considered to be complete when the final concentration obtained was 65 g / l. Hence, the reaction yield is 75%. By reusing the cells, 1.6 times more glucose-6-phosphate was produced than when the cells were used for the first time and 30 g / L was produced. Thus, the method according to the invention makes it possible to store the same biocatalyst and use it several times without losing efficiency, thus reducing the costs associated with biomass production.

[0128] Example 3 Production of dihydroxyacetone from glycerol. Dihydroxyacetone (DHA) is a tanning agent that is widely used in the cosmetics industry. The method according to the present invention was used to produce DHA. The method involves recycling of the redox cofactors involved in the reaction.

[0129] To do this, two modules were constructed. A strain of E. coli was transformed to express in its periplasm E. coli glycerol dehydrogenase, which catalyzes the conversion of glycerol to DHA (the first, BL21-GldA module). This reaction consumes the cofactor NAD+ and forms NADH as a coproduct. The second module (BL21-Nox module) was constructed by transforming a strain of E. coli to express Lactobacillus sanfranciscensis NADH reductase (NOX) in its periplasm. This enzyme converts oxygen (O 2 ) and catalyzes the reaction using NADH to produce water (H 2 O) and NAD+, which is regenerated and participates in the first reaction. Plasmid pET26b carrying the nox gene of L. sanfranciscensis encoding the NADH oxidase NOX was ordered from GenScript and transformed into strain BL21-WT.

[0130] The reaction scheme is as follows:

[0131] [ka]

[0132] [Text in image] Glycerol dehydrogenase Glycerol Dihydroxyacetone NADH reductase

[0133] The two modules were harvested and grown in TB medium and induced with IPTG before carrying out the production of DHA. The reaction mixture was: Module 1: 4 ODU / ml BL21-GldA cells Module 2: 4 ODU / ml BL21-Nox cells Substrate: 20 mM glycerol Cofactor: 5mM NAD+ Buffer: 100 mM (Tris pH 8 or Gly pH 9) Includes.

[0134] Negative controls without substrate were prepared. Additionally, negative controls were prepared in which each strain was replaced by BL21(DE3) cells expressing neither the GldA nor the NOX enzymes. Since the pH optimum of GldA is 9 and that of NOX is 7.5, tests were performed at pH 8 and 9 to find the best compromise for the functioning of the reactions. Measurements were performed after 5 hours and are shown in Figure 6.

[0135] DHA production was obtained at pH 8 and pH 9 in the presence of the two modules, with no production observed in the wild-type control. Amounts of DHA of 260 mg / l at pH 8 and 330 mg / l at pH 9 were obtained, demonstrating the feasibility of the proposed reaction scheme.

[0136] Example 4 Production of glycolaldehyde from glyoxal. Glycolaldehyde is a highly reactive molecule present in cellular metabolism and, like most aldehydes, can be used as a platform molecule. Nevertheless, glycolaldehyde is too expensive for industrial use. Due to its reactivity, which makes it toxic and unstable for cells, aldehydes are rarely produced using microorganisms. Aldehydes can react with membrane proteins and lipids, further complicating their export. It is thought that glycolaldehyde can be obtained by reducing the inexpensive but obviously toxic glyoxal.

[0137] To do this, two modules were constructed. A strain of E. coli was transformed to express in its periplasm the Bacillus subtilis glyoxal reductase YvgN, which catalyzes the conversion of glyoxal to glycolaldehyde (the first BL21-YvgN module). This reaction consumes the cofactor NADPH and forms NADP as a coproduct (Dudek et al., 2013). The second module (BL21-PtxD) was constructed by transforming a strain of E. coli to express in its periplasm the phosphate dehydrogenase PtxD, an NADP reductase. The Pseudomonas stutzeri ptxD that encodes this enzyme contains 18 mutations that ensure increased catalytic efficiency, stability at 37 °C, and handling of the two substrates NAD and NADP. The Bacillus subtilis yvgn gene encodes glyoxal reductase (GR). The wild-type version was used. Expression plasmids pET22b-PtxD mutant and pET26b-YvgN were ordered from Genescript. The ptxD gene was ordered codon optimized for expression in E. coli. Plasmids pET22b and pET26b carry the signal sequence pelB, responsible for protein export into the periplasm; pET22b is resistant to ampicillin and pET26b is resistant to kanamycin. These plasmids were then transformed into BL21-WT to give the BL21-YvgN and BL21-PtxD modules.

[0138] The reaction scheme is as follows:

[0139] [ka]

[0140] [Text in image] Glyoxal reductase Glycolaldehyde Phosphite dehydrogenase

[0141] The reaction mixture was: Module 1:1 ODU / ml BL21-YvgN cells Module 2: 1 ODU / ml BL21-PtxD cells (excluding pre-test) Substrate: 10 mM glyoxal Cofactor: 0.4 mM NADPH Complementary substrate: 10 mM phosphite Buffer: 50 mM HEPES pH 7.5 and 10 mM KCl Includes.

[0142] In preliminary tests, the production of aldehydes by periplasmic glyoxal reductase was carried out alone without a regeneration system. In the second step, the reaction was started by the addition of the reaction mixture of BL21-PtxD-induced cells at a concentration of 1 ODU and 10 mM phosphite to regenerate the produced NADP to NADPH. The reaction was placed at a temperature of 37°C with stirring at 200 rpm. Measurements are taken at 0, 1, 3 and 5 hours.

[0143] The results presented in Figure 7 show that glycolaldehyde is detected as early as 1 hour of reaction with and without the regeneration system, but production already reaches a plateau in the absence of PtxD due to the depletion of NADPH. In contrast, in the presence of the BL21-PtxD module, the reaction continues thanks to the regeneration of NADPH. After 5 hours, glycolaldehyde production is increased 25-fold with a yield of 37%.

[0144] Therefore, the BL21-YvgN and BL21-PtxD modules efficiently produce glycolaldehyde from glyoxal in the periplasmic compartment without any detrimental effects on the bacterial cell, demonstrating that the method according to the present invention allows the production of aldehyde compounds from toxic precursors.

[0145] Example 5 Production of alcohol from amino acids A reaction scheme for the production of 2-phenylethanol from L-phenylalanine has been developed. The reaction scheme is based on the use of four separate modules. Three modules were constructed expressing the S. cerevisiae ARO8, ARO10 and ADH5 genes, respectively; the first gene encodes L-phenylalanine oxoglutarate aminotransferase, the second gene encodes phenylpyruvate carboxylase, and the third gene encodes 2-phenylethanol dehydrogenase. The fourth module expressing the phosphite dehydrogenase PtxD is designed for NADH regeneration.

[0146] The reaction scheme is as follows:

[0147] [ka]

[0148] [Text in image] Decarboxylase Dehydrogenase Phenylalanine Phenylpyruvic acid Phenylacetaldehyde 2-Phenylethanol Phosphite dehydrogenase

[0149] It is this third reaction that results in the coupling with the regeneration of the cofactor NADH from the coproduct NAD+ to produce the compound of interest.

[0150] The reaction mixture is as follows: Module 1: 5 OD / ml BL21-ARO8 cells Module 2: 5 OD 600nm / ml BL21-ARO10 cells Module 3: 5 OD 600nm / ml BL21-ADH5 cells Module 4: 5 OD 600nm / ml BL21-PtxD cells Substrate: L-phenylalanine (10 mM) Cofactors: 2-ketoglutarate (10 mM), MgCl 2 (5 mM), ZnCl 2 (5mM), thiamine (0.2mM), pyridoxal (0.1mM), NADH (1mM) Complementary substrate: phosphite (100 mM) Buffer: HEPES pH7 (100mM)

[0151] The 2 ml reaction was placed at 33° C. and stirred at 200 rpm for 5 hours. Samples were taken at 0, 1, 3 and 5 hours.

[0152] A synthetic consortium of four bacterial biocatalysts BL21-ARO8, BL21-ARO10, BL21-ADH5 and BL21-PtxD was used to produce 2-phenylethanol from L-phenylalanine. The effectiveness of this consortium was tested and confirmed by measuring the appearance of 2-phenylethanol by HPLC [Figure 13]. The results showed that the synthesis of 65 mg / l of 2-phenylethanol was obtained in a 5-hour reaction.

[0153] Example 6 Production of rare sugar isomers Rare sugars are mainly produced by isomerization of common sugars. The difficulty arises from the fact that isomerization is a reversible reaction. The aim here is to limit the reversibility of the reaction by converting the rare sugars formed into new compounds, e.g. phosphorylated sugars, which are then dephosphorylated to obtain the desired isomer.

[0154] In this example, the first reaction isomerizes the monosaccharide D-xylose to the rare sugar D-xylulose. The second reaction phosphorylates D-xylulose to D-xylulose-5-phosphate in the presence of ATP, which acts as a cofactor. The reaction scheme continues with two downstream reactions, a reaction that consumes the formed ADP and acetyl phosphate to regenerate ATP, and a reaction that dephosphorylates D-xylulose-5-phosphate back to D-xylulose.

[0155] Four different modules are used. - The first module (BL21-XylA) is a strain of E. coli transformed to express in its periplasm the xylose isomerase XylA present in E. coli. - The second module (BL21-XylB) is a strain of E. coli transformed to express the E. coli kinase, XylB, which phosphorylates xylulose to xylulose-5P by consuming an ATP molecule. - Regenerate ATP using the BL21-AckA module already seen above. - The fourth module (BL21-AphA) is a strain of E. coli transformed to express the E. coli broad-spectrum phosphatase AphA in its periplasm (Passariello et al., 2006).

[0156] The complete reaction scheme is presented below.

[0157] [ka]

[0158] [Text in image] Xylose isomerase Addition in 4 hours Acetyl phosphate Acetate kinase

[0159] Both modules are grown in TB medium and induced with IPTG before being harvested and run to produce D-xylose. The reaction mixture is: Module 1: 8 OD / ml BL21-XylA cells Module 2: 8 OD / ml BL21-XylB cells Module 3: 8 OD / ml BL21-AckA cells Module 4: 8 OD / ml BL21-AphA cells Substrate: D-xylose (300 mM) Cofactor: ATP (5mM) Complementary substrate: acetyl phosphate (125 mM) Buffer: HEPES pH7.5 (50mM), MgSO 4 (5 mM), MnSO 4 (3mM) Includes.

[0160] The method is started 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. Module 4 expressing AphA phosphatase is added 4 hours after the start of the reaction and the pH is adjusted to 6. The reaction is monitored at 7 hours and then at 22 hours. In parallel, a reaction is run with the BL21-Xyla module alone to evaluate a simple isomerization reaction. The evolution of the concentrations of D-xylose (FIG. 8) and D-xylulose (FIG. 9) during the reaction was monitored by HPIC.

[0161] Figure 8 shows that D-xylulose substrate is consumed in similar amounts in both protocols. However, after 4 hours, the production of D-xylulose was half that of the isomerization reaction alone in the complete reaction (Figure 9). This difference is explained by the consumption of D-xylulose due to the production of D-xylulose-5P in the complete scheme, the presence of which was not detectable by the D-xylulose assay method but was confirmed by mass spectrometry.

[0162] 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 h and 18 h after introduction of the phosphatase, an increase of 2 g / m and 8 g / l of D-xylulose, respectively, is observed. The amount of D-xylulose in the complete reaction (12.8 g / l) exceeds that of the isomerization reaction alone (10.2 g / l), giving a significant increase of 2.6 g / l.

[0163] 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 BL21-XylB kinase module in the reaction mixture allows to capture D-xylulose in a phosphorylated form, which can then no longer be converted to D-xylose. D-xylulose-5P accumulates in the reaction mixture, and the addition of the BL21-AphA phosphatase allows an increased recovery of D-xylulol compared to simple isomerization. Higher conversion rates are obtained by directing the isomerization towards the phosphorylated form of the rare sugar.

[0164] These results show for the first time that isomerization, phosphorylation and dephosphorylation are possible with the BL21-XylA, BL21-XylB and BL21-AphA modules. These results prove that four different modules cooperate to achieve a complex reaction scheme, including a mechanism for ATP regeneration. The synergistic and sequential use of all kinds of modules in the context of the method object of the present invention is thus confirmed.

[0165] Example 7 Production of D-fructose-1,6-bisphosphate from D-xylose The conversion of D-xylose to D-fructose-1,6-bisphosphate is part of the pentose phosphate pathway, the natural route for assimilation of D-xylose into the cytoplasm. This conversion was carried out by a reaction scheme involving nine reactions implemented by eight different modules.

[0166] The eight modules used were constructed from strains of E. coli, each transformed to express enzymes present in E. coli in its periplasm: 1-Xylose isomerase XylA, which isomerizes D-xylose to D-xylulose 2-Xylulose kinase XylB, which phosphorylates D-xylulose to D-xylulose-5P 3-Acetate kinase AckA, which regenerates ATP from ADP and acetyl phosphate 4-Ribulose-phosphate-3-epimerase Rpe, which converts D-xylulose-5P to D-ribulose-5P 5-Ribulose-5-phosphate isomerase RpiA, which produces D-ribose-5P from D-ribulose-5P 6 -Transketolase Tkt, which converts D-xylulose-5P and D-ribose-5P to D-glyceraldehyde-3P and D-sedoheptulose-7P 7-Transaldolase Tal, which converts D-glyceraldehyde-3P and D-sedoheptulose-7P to D-fructose-5P and D-erythrose-4P 8- 6-Phosphofructokinase 1 PfkA, which converts D-fructose-5P to D-fructose-1,6bP

[0167] The complete reaction scheme is presented below.

[0168] [ka]

[0169] [Text in image] Acetic acid Ethyl Phosphate D-Glyceraldehyde-3P Acetyl-P

[0170] The presence of the enzymes in the periplasm of the eight modules was confirmed by harvesting the periplasmic fraction by osmotic shock for each of the strains. The enzyme sizes and the amount of cells (OD 600 nm times medium volume) used for the osmotic shock are given in Table 4.

[0171] [Table 4]

[0172] The results of the SPS-PAGE gel analysis are given in Figure 10. The results show that the predicted periplasmic expression of the enzymes for the eight modules is confirmed.

[0173] The module has been retrieved. Module 1: 8 ODU / ml BL21-XylA cells Module 2: 8 ODU / ml BL21 XylB cells Module 3: 8 ODU / ml BL21 AckA cells Module 4: 8 ODU / ml BL21-Rpe cells Module 5: 8 ODU / ml BL21- RpiA cells Module 6: 8 ODU / ml BL21-Tkt cells Module 7: 8 ODU / ml BL21-Tal cells Module 8: 8 ODU / ml BL21- PfkA cells Substrate: 100mM D-xylose Cofactor: 3mM ATP Complementary substrate: 100mM acetyl phosphate Buffer: HEPES pH 7.5 (50mM), MgSO 4 (20 mM), MnSO 4 (1 mM) and thiamine pyrophosphate (1 mM), CoCl 2 (0.1mM) The reaction mixture was grown in TB medium and induced with IPTG before producing D-fructose-1,6bP.

[0174] Determination of D-fructose-1,6bP was carried out immediately after addition of the modules to the reaction mixture, and after 1 h, 3 h and 4 h. Two controls were carried out, one without substrate and one without modules (8 modules replaced by BL21 WT cells). The results are presented in FIG. 11. After 1 h of reaction, the production of D-fructose-1,6-diphosphate is 1.3 g / l, reaching a maximum of 2.8 g / l at 3 h. In the two control reactions, the production of D-fructose-1,6-diphosphate is not measured.

[0175] D-sedoheptulose-7-phosphate was also assayed (FIG. 12). In fact, the XylB and PfkA carrier modules catalyze the only irreversible reaction in the reaction scheme, while D-sedoheptulose-7-phosphate is in the middle of the reaction chain composed of reversible reactions whose products are in equilibrium with each other. D-sedoheptulose-7-phosphate was assayed immediately after the addition of the modules to the reaction mixture, as well as after 1, 3 and 4 hours. D-sedoheptulose-7-phosphate is 130 mg / l after 4 hours in the complete reaction and is absent in the control sample. Although the other intermediate compounds are difficult to detect using the analytical method used, the level of D-sedoheptulose-7P represents their concentration, and therefore the concentrations are similar. Thus, the production of D-fructose-1,6-bisphosphate according to the complex reaction scheme was successfully carried out.

[0176] These results demonstrate that the method according to the invention, based on the externalization of reactions, works efficiently even with long, branched chains containing many molecules. It is also particularly advantageous that native complex metabolic pathways in the cytoplasm could be reconstituted in this way.

[0177] Example 8 Effect of a consortium of two bacteria, each expressing one enzyme, compared to one bacteria expressing two enzymes on the production of product (G3P). Glycerol-3-phosphate production is based on the catalytic activity of glycerol kinase and acetate kinase. To demonstrate the value of using a synthetic consortium (two bacteria each expressing a different enzyme) compared to a bacterium expressing both enzymes, a whole-cell biocatalyst was created that co-expresses E. coli glycerol kinase GlpK and E. coli acetate kinase AckA in its periplasm. To do this, a plasmid pETDuet-pelB-glpK-pelB-ackA containing two cloning sites under the control of two T7 promoters was constructed and transformed into E. coli. This biocatalyst is referred to herein as BL21-GLK-AckA. The design of the vector pETDuet was performed such that the expression cassette of pETDuet is identical to that of pET22b. Thus, a comparison of strains BL21-AckA, BL21-GlpK and BL21-GlpK-AckA has for its object the effect of co-expression without the influence of the promoter, RBS or signal peptide.

[0178] The periplasmic fractions of strains BL21-AckA and BL21-AckA-GlpK were cultured at 2 OD for each of the strains. 600nm The 56 kDa GlpK band was recovered after osmotic shock from 2 ml of cell suspension concentrated at 100°C. We observe that the 43 kDa AckA band is less than two times lower in BL21-AckA-GlpK compared to BL21-AckA. This result demonstrates the negative effect of the co-expression of the two enzymes in the same bacterium on the production of the enzyme AckA in the periplasm of the biocatalyst BL21-AckA-GlpK (one bacterium expressing the two enzymes). By comparing the 56 kDa GlpK band in strains BL21-GlpK and BL21-GlpK-AckA, we also observe the negative effect of the co-expression of the two enzymes in the same bacterium on the periplasmic GlpK production.

[0179] Finally, we extracted periplasmic fractions from a mixture of BL21-AckA and BL21-GlpK (a bacterial consortium according to the invention), where each of the strains was cultured at 1 OD, conditions that approximate standard co-cultivation conditions. 600nmThe AckA and GlpK bands are more prominent in the co-culture (consortium according to the invention) compared to the periplasmic fraction extracted from a biocatalyst co-expressing AckA and GlpK (one bacterium expressing the two enzymes) [Figure 14]. These results show that the use of a consortium of bacteria makes it possible to obtain a better expression of each enzyme compared to the use of the same bacterium co-expressing both enzymes.

[0180] We also observe that the signal peptide pelB allows a better production of the enzyme AckA than the enzyme GlpK in the periplasm of strains BL21-AckA and BL21- GlpK, respectively. This finding is not surprising since the effectiveness of the signal peptide depends on the target protein. The amount of BL21-GlpK and BL21- AckA in the glycerol-3P production reaction mixture can be adjusted to maximize the efficiency of the reaction by increasing the amount of BL21-GlpK, thus compensating for the low efficiency of PelB. This strategy allows to easily improve the production of glycerol-3P, which is advantageous for industrial use. In comparison, the biocatalyst BL21-GlpK-AckA, which co-expresses the enzymes GlpK and AckA, does not provide this modularity. Each of the expression cassettes (promoter and / or RBS and / or signal peptide) would have to be reconstructed to regulate the expression of GlpK and AckA; this is more complicated and time consuming.

[0181] We also compared the production of G3P by the biocatalyst BL21-GlpK-AckA (one bacterium expressing the two enzymes) and the synthetic consortium BL21-GlpK and BL21-AckA (according to the present invention). Module: BL21-GlpK and / or BL21-AckA and / or BL21-GlpK-AckA cells (OD 600nm 全体 = 2) Substrate: 160 mM glycerol Cofactors: 5 mM ATP, 15 mM MgSO4 Complementary substrate: 220 mM acetyl phosphate Buffer: 50mM HEPES buffer pH 7.5 It is composed of:

[0182] The 5 ml reactions are placed at a temperature of 37° C. with stirring at 200 rpm and last for 24 hours. Samples of 200 μL are taken at times T0, T0.5, T1, T2, T3, T5 and T24.

[0183] The BL21-GlpK-AckA biocatalyst can produce 2.1 grams of G3P per liter in 24 hours. However, by using a mixture of the two types of biocatalysts, BL21-GlpK and BL21-AckA, in the same ratio (50% each), it is possible to produce 5 grams of G3P per liter in 24 hours, or more than twice the production of one bacterial system co-expressing the two enzymes. Furthermore, the production of G3P can be further optimized by adjusting the ratio of the two types of biocatalysts. In fact, by using a mixture of 92.5% BL21-GlpK and 7.5% BL21-AckA, the production of G3P can reach 10 grams per liter in only 5 hours [Figure 15]. These results indicate that the G3P bioproduction can be significantly improved by adjusting the ratio of BL21-GlpK and BL21-AckA biocatalysts. A considerable advantage of the use of a consortium according to the invention is that the ratio of each biocatalyst can be easily adjusted in a much easier and more reliable manner than through the use of complex genetic engineering within the same bacterium to co-express several enzymes.

[0184] Example 9 Production of ATP from extracellular sources Purine adenosine 5'-triphosphate (ATP) is not only a universal intracellular energy transporter, but also plays an important role as an extracellular signaling molecule. ATP is well known as a messenger for intercellular communication in multicellular organisms, but older phylogenetic unicellular organisms such as yeast or bacteria also use ATP as an extracellular signaling molecule. However, the mechanism of ATP secretion by bacteria and its extracellular involvement still need to be elucidated (Spari D and Beldi G, 2020).

[0185] Alvarez et al. showed that E. coli DH5α ATP is rapidly hydrolyzed in the periplasmic space. The steady-state extracellular ATP concentration in the periplasmic space is 24 ± 3 μM / 10 10 It was determined to be a bacterium (Alvarez et al., 2017).

[0186] In the context of the production of phosphorylated molecules such as glucose-6-phosphate, ATP is an essential cofactor for phosphorylation, but ATP is an expensive molecule to produce and must often be regenerated from cheaper molecules such as ADP.

[0187] By using an ATP regeneration module such as BL21-AckA, the minute amounts of ATP present in the periplasm can be reused for the production of phosphorylated molecules, thereby reducing production costs and efficiently utilizing the natural resources of the cell.

[0188] Thus, G6P production has been achieved according to the methods of the present invention without the addition of commercially available ATP.

[0189] The first reaction mixture comprises: Module 1: 2 ODU / ml BL21-Glk cells Module 2: 2 ODU / ml BL21-AckA cells Substrate: 300 mM glucose Complementary substrate: 250 mM acetyl phosphate Buffer: HEPES (50 mM) and MgSO 4 (15mM) At a cell concentration of 2 OD600nm / ml for each biocatalyst, 4 g / l of glucose-6-phosphate can be produced without the addition of ATP, suggesting that ATP production is sufficient to support glucose-6P production at this cell density.

[0190] The number of E. coli cells per 1 OD 600 nm depends on many factors, including the growth conditions, culture medium, and bacterial strain used. However, there is an empirical correlation between the optical density at 600 nm and the number of bacterial cells. In general, for E. coli, an optical density at 600 nm of 1 is approximately 8 × 10 8 This corresponds to a cell concentration of 3.2 × 10 9 ATP 24±3μm / 10 cells / ml provided by Alvarez et al. 10 Taking into account the concentration of bacteria, the concentration of ATP in the test is 7.68 μM. This amount of ATP would allow the synthesis of 4 g / L glucose-6P or 15,000 μM; the BL12-AckA module would allow more than 2000 times the production possible from naturally occurring extracellular ATP. These results suggest that the method of the invention makes it possible to optimize trace cofactors for bioproduction, which is particularly interesting from an economic point of view. (References) TIFF2025514570000013.tif223170TIFF2025514570000014.tif132170

Claims

1. 1. A method for producing a compound of interest from an organic substrate, comprising: Here's how: a)- In a suitable medium, - n bacteria, n being an integer at least equal to 2, each of which has been genetically modified to express in its periplasmic space at least one enzyme E1, E2, ..., En, preferably a single enzyme, each of said n bacteria expressing an enzyme that is distinct from the other bacteria; - the enzyme E1 is capable of catalysing a first reaction R1 from said organic substrate to provide a first product P1 and, optionally, a first co-product CoP1; each of said enzymes E2, ..., E n is capable of catalysing a reaction R2, ..., R n from a product or co-product obtained by reaction R n-1 to provide a product P2, ..., P n and, optionally, a co-product CoP2, ..., CoP n , respectively; - said organic substrate, and - optionally a cofactor of said organic substrate, a cofactor of said product obtained by reaction Rn-1, called cofactor CoF1, CoF2, ..., CoFn; a complementary substrate SC of the co-product obtained by reaction Rn-1 preparing a reaction mixture comprising: 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 P1, P2, ..., Pn; A method comprising the steps of:

2. The method of claim 1, wherein the n genetically modified bacteria are Gram-negative Diderm bacteria, each selected from the family Enterobacteriaceae, Alcaligenesaceae, Vibrionaceae, or Pseudomonadaceae.

3. 3. The method according to claim 1 or 2, characterized in that each of the n bacteria is genetically modified to express at least one polypeptide, preferably a single polypeptide, each comprising one of the enzymes E1, E2, ..., En linked to a signal peptide that directs the polypeptide into the periplasmic space of the bacterium.

4. 4. The method of claim 3, wherein one or more of the n bacteria are genetically modified to express a polypeptide, each comprising one of the enzymes E1, E2, ..., En linked to a membrane-anchoring peptide that includes the signal peptide.

5. 5. The method according to claim 1, wherein at least one of the n bacteria is genetically modified to express at least two enzymes En-a and En-b in its periplasmic space capable of catalyzing at least two reactions RN-a and RN-b, such that the reactions RN-a and RN-b occur in the periplasmic space of the at least one bacterium.

6. 6. The method according to claim 1, characterized in that the reaction mixture comprises bacteria expressing a first enzyme E1 capable of catalysing a first reaction R1 from the organic substrate to provide a first product P1, and bacteria expressing a second enzyme E2 capable of catalysing a second reaction R2 from the first product P1 to form a second product P2, which P2 is recovered in step c) as a compound of interest or consumed as a substrate for a third reaction.

7. 7. The method according to claim 6, characterized in that the reaction mixture further comprises at least one bacterium expressing enzymes E3, ..., En capable of catalysing reactions R3, ..., Rn from a product Pn-1 obtained by reaction Rn-1 to provide a product Pn, which is recovered in step c) as a compound of interest or consumed as a substrate for reaction Rn+1.

8. 8. The method according to claim 1 , characterized in that the reaction mixture comprises a cofactor CoF1 for the organic substrate, a bacterium expressing an enzyme E1 capable of catalysing a first reaction R1 from the organic substrate and the cofactor CoF1 to form a first product P1 and a first co-product CoP1, and a bacterium expressing a second enzyme E2 capable of catalysing a second reaction R2 from the first product P1 to form a second product P2 or a second enzyme E2 capable of catalysing a second reaction R2 from the first co-product CoP1 to form a second co-product CoP2, at least one of which is consumed in a third reaction or recovered in step c) as a compound of interest.

9. 9. The method according to claim 8, characterized in that the reaction mixture comprises at least one cofactor CoFn, a product Pn-1 of reaction Rn-1 and at least one bacterium expressing an enzyme capable of catalyzing reaction Rn from said cofactor CoFn to form a product Pn and a co-product CoPn, at least one of the two being consumed in reaction Rn+1 or recovered in step c) as a compound of interest.

10. 10. The method according to claim 9, characterized in that the reaction mixture can also contain at least one substrate SCn complementary to said at least one cofactor CoFn.

11. 11. The method according to claim 1, characterized in that the at least one cofactor CoFn can be totally or partially introduced into the reaction mixture in step a) or during step b).

12. 12. The method according to claim 1, wherein the at least one cofactor CoFn is at least partially formed in the reaction mixture by a reaction Rn-1 catalyzed by an enzyme En-1 capable of forming a product Pn-1 and a co-product CoPn-1, and the co-product CoPn-1 is identical to the cofactor CoFn.

13. 13. The method according to claim 12, characterized in that the amount of the first cofactor CoF1 introduced into the reaction mixture in step a) corresponds to a molar concentration that is 20 times lower or less than the initial molar concentration of the organic substrate.

14. 14. The process according to any one of claims 1 to 13, characterized in that the organic substrate of the first reaction is selected from carbohydrates, aldehydes, alcohols, organic acids, carbamic acids, hydrocarbons, amino acids and carbon dioxide.

15. 15. The method according to any one of claims 1 to 14, characterized in that the enzymes E1, E2, ..., En are each selected from kinases, dehydrogenases, phosphatases, reductases, isomerases and transferases.

16. At least one of the enzymes E1, E2, …, En is ATP, ADP, AMP, UTP, UDP, UMP, NAD+, NADH, NADP+, NADPH, FAD, FADH 2 16. The method according to claim 1 , characterized in that the method catalyzes a reaction providing one of the co-products CoP1, CoP2, ..., CoPn selected from coenzyme A, or catalyzes a reaction using one of these co-products as a cofactor.

17. 17. The method according to any one of claims 1 to 16, characterized in that, after step c) of the method that is the object of the present invention, the biomass separated from the supernatant is recycled to prepare a new reaction mixture according to step a), with or without an intermediate storage step.

18. Steps a) and b) comprise continuously or at time intervals adding to a reaction mixture containing said n bacteria: - said organic substrate, and 18. The method according to claim 1 , characterized in that it is carried out by optionally adding a cofactor of the organic substrate, a cofactor of the product obtained by reaction Rn-1, called cofactors CoF1, CoF2, ..., CoFn; a complementary substrate SC of the co-product obtained by reaction Rn-1, and by leaving the reaction mixture thus obtained to react continuously or semi-continuously.

19. The reaction mixture is added in a suitable medium. - n bacteria, n being an integer at least equal to 2, each of which has been genetically modified to express in its periplasmic space at least one enzyme E1, E2, ..., En, preferably a single enzyme, each of said n bacteria expressing an enzyme that is distinct from the other bacteria; - an enzyme E1 is capable of catalysing a first reaction R1 from said organic substrate to provide a first product P1 and, optionally, a first co-product CoP1; each of said enzymes E2, ..., E n is capable of catalysing a reaction R2, ..., R n from a product or a co-product obtained by reaction R n-1 to provide a product P2, ..., P n and, optionally, a co-product CoP2, ..., CoP n , respectively; - said organic substrate, and - optionally a cofactor of said organic substrate, a cofactor of said product obtained by reaction Rn-1, called cofactor CoF1, CoF2, ..., CoFn; a complementary substrate SC of the co-product obtained by reaction Rn-1 A reaction mixture for producing a compound of interest from an organic substrate, comprising:

20. 20. Use of the reaction mixture according to claim 19 for producing a compound of interest selected from carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons and amino acids.

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