Generation of molecules by periplasmic enzyme catalysis

Abstract

A process for the production of a compound of interest from an organic substrate comprising the steps of: a) preparing a reaction mixture comprising-at least two bacteria, both genetically engineered to express in their periplasmic space at least one enzyme E1, E2,..., En, preferably a single enzyme, each of the n bacteria expresses an enzyme different from the other bacteria,-the enzyme E1 is capable of catalyzing a first reaction R1 to produce a first product P1 and optionally a first by-product CoP1 using the organic substrate,-each of the enzymes E2,..., En is capable of catalyzing a reaction R2,..., Rn to produce a product P2,..., Pn and optionally a by-product CoP2,..., CoPn, respectively, using the product or by-product obtained from the reaction Rn-1, the present invention relates to a method for preparing a target product, comprising the steps of:-a reaction of Rn-1,-said organic substrate, and-optionally, a cofactor of said organic substrate, a cofactor of said product obtained by reaction of Rn-1, called cofactors CoF1, CoF2,..., CoFn,-said organic substrate, and-optionally, a cofactor of said organic substrate, a cofactor of said product obtained by reaction of Rn-1; the invention relates to a method for producing a biomass, comprising the steps of: a) reacting the biomass with a substrate SC complementary to the by-product obtained by reacting Rn-1, b)-reacting the reaction mixture, and c)-separating the biomass from the supernatant and extracting therefrom the target compound consisting of one of the products P1, P2,..., Pn.

Description

Technical Field

[0001] The present invention is in the field of production of target molecules by genetically engineered microorganisms and, more particularly, relates to the production of compounds by enzyme catalysis performed in the bacterial periplasmic space.

[0002] It relates to a method for producing a target compound, the method involving a bacterial mixture (colony) modified to express one or more enzymes in its periplasmic space, the enzymes together catalyzing a reaction cascade to produce the target compound. The invention also relates to a reaction mixture for producing such a compound, and a kit for preparing the medium. Background Art

[0003] The production of organic compounds in the petrochemical industry raises key issues for the future in terms of cost and environmental impact, especially due to the large amount of carbon emissions into the atmosphere and the use of non-renewable resources. Producing such compounds by biological pathways seems to be a non-polluting and sustainable alternative. It is still necessary to be able to implement appropriate reaction schemes according to economically acceptable schemes and obtain sufficient quantities related to the intended application. In fact, although some compounds can be obtained by simple reactions, other compounds require intermediate steps, which can complicate the method and increase costs. In addition, many reactions use cofactors, which must be introduced into the reaction medium in an amount proportional to the substrate to be converted, which correspondingly increases costs.

[0004] Currently, various techniques that exploit biological processes are used to produce molecules. The most classic is to extract the target product, such as a fragrance or therapeutic molecule, from an organism such as a plant by soaking, maceration, solvent extraction or other means. However, the application of plant extraction is limited because the yield of plant extraction is usually quite low compared to the quality processed. It uses complex methods to isolate and purify the target molecule. Finally, the seasonal or geographical availability of the source organism greatly limits the extraction strategy.

[0005] Another well-known technology is based on the fermentative activity of microorganisms, which may or may not be genetically modified, whose cell metabolism produces the target molecule and secretes it 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 and at moderate costs, because the reaction is carried out by microorganisms. The theoretical yield is usually attractive, because the contributions of synthetic biology and systems biology have greatly improved the efficiency of such systems. However, fermentation has significant disadvantages, which originate from the activity of the biocatalyst. Therefore, in practice, it is difficult to obtain the theoretical yield, because the cell always tends to divert the production of the target molecule to the production of its biomass. In addition, the target molecule is secreted into the fermentation medium, which necessarily contains a large number of other molecules, which complicates its isolation and consumes a large amount of water, which then has to be treated. In addition, not all molecules can be produced by fermentation, either because they are toxic to the cells or because little or no molecule is excreted. In addition to these difficulties, the development time of industrial fermentation organisms is about ten years on average. Thus, several limitations of fermentation production can be identified: i) the coupling of growth and production limits the maximum yield, ii) the use of synthetic biology to engineer the microorganism and redirect the carbon flow towards the product comes at the expense of growth, which affects the robustness of the process, iii) the operating conditions are restricted to those compatible with microbial growth (pH, temperature, composition of the medium, oxygenation, etc.), iv) substrates and products must not be cytotoxic; therefore, the maximum concentrations of substrates and products are fixed based on their toxicity; v) substrates and products must be able to cross the cytoplasmic membrane; and vi) products are secreted into the culture medium containing organic acids and other by-products, complicating the purification of the product and increasing the overall cost of the process.

[0006] Recently, whole-cell bioproduction techniques have overcome many of these disadvantages. Growth (enzyme production phase) and production are separated; substrates are used only for product synthesis. Operating conditions can be optimized to maximize yield; they do not need to be compatible with growth. In addition, product purification is simplified because the product is secreted in a simple medium without growth byproducts. Finally, if the transfer of substrates / products across the cytoplasmic membrane is limited, one way to circumvent this limitation is to make the cell wall and membrane permeable by chemical treatment (e.g., by adding detergents or solvents) or physical treatment (e.g., by using heat shock). However, these procedures, in addition to causing cell damage, can interfere with downstream purification processes. In addition, the main limitation of this approach is the need for metabolic engineering to i) optimize the expression levels of genes encoding enzymes of the production pathway, and ii) minimize intracellular side reactions. However, optimizing the amount of each enzyme in the enzyme cascade is crucial to the overall performance of the method. In fact, each enzyme in the metabolic pathway is responsible for a specific biochemical reaction necessary to synthesize the final product. If the amount of enzyme in the cascade is insufficient, upstream substrates may accumulate, resulting in a decrease in the reaction rate and, therefore, a decrease in the yield of the final product. In turn, an excess of enzyme can also upset the balance of the enzyme cascade, reducing the efficiency of the entire process. Therefore, the value of optimizing the amount of each enzyme lies in maximizing the overall reaction efficiency and the yield of the final product.

[0007] While the concentration of purified enzymes in a reaction mixture can be easily adjusted, conversely, when these enzymes are expressed in vivo, their gene expression must be regulated to achieve optimal levels for each enzyme.

[0008] Currently, metabolic engineering and synthetic biology are used to improve the yield and titer of products. Although new metabolic engineering tools are constantly being developed, it is difficult to predict the level of enzyme production in cells. The variation of gene expression levels depends on multiple parameters, including the copy number of the gene (determined by the replication origin when it is carried by an expression plasmid), the transcription level (determined by the choice of the promoter), and the translation level (determined by the ribosome binding site (RBS)). However, the relationship between the efficiency of the expression system and the copy number, transcription or translation power is not linear. High expression may represent a significant cellular metabolic load, thereby impairing production and growth. Typically, the optimization of gene expression is a combination method between these different elements so that the best combination can be determined empirically.

[0009] Other enzymatic technologies allow the synthesis of high value-added molecules without the aid of microorganisms. In these technologies, purified enzymes catalyze reactions with high specificity in vitro, making it possible to easily control the production flow by controlling the reaction medium. The yields thus obtained can then be close to the theoretical yield or even reach the theoretical yield. Thus, the direct use of enzymes (whether or not they are immobilized) overcomes some of the disadvantages of fermentation. However, these enzymes must be produced, purified and packaged in advance, which makes the technology costly. Although the emergence of so-called cell-free systems has partially solved the problem of purifying proteins (enzymes) by simultaneously synthesizing them in the medium where the catalytic reaction takes place, the cost of the cofactors that are usually essential for the reaction is still too high for the large-scale application of these technologies. Therefore, all these methods have serious disadvantages. In addition, they are limited to the production of certain compounds. Alternative technologies that have the advantages of fermentation and biocatalysis, but do not have their respective disadvantages and can be freely adapted to the synthesis of a variety of target molecules can meet current economic and environmental challenges. Summary of the invention

[0010] For this reason, the present invention proposes a technology that uses the biological mechanism of microbial cells to produce different enzymes, and the enzyme can catalyze the reaction of the reaction scheme from substrate to target compound. The principle is to use the periplasmic space provided by bacteria (mainly Gram-negative bacteria) as a controlled reaction space, wherein a given enzyme transforms the substrate, and the formed product is excreted into the culture medium. Then, the product and any byproducts excreted by the first bacteria can be used as a substrate for bacteria expressing another enzyme (which is different from the enzyme produced by the first bacteria), thereby forming a second product, and so on. This is called a cascade reaction using a consortium of at least two bacteria, and the enzyme expressed by each bacterium is different from the enzyme produced by other bacteria. From the perspective of biological process, the increase in the accessibility of the substrate provided by the periplasm is a major advantage. Compared with the so-called whole-cell biological process using cytoplasmic enzymes mentioned above, the accessibility of the substrate can be improved using periplasmic secretory enzymes. Another advantage of expressing enzymes in the periplasm is that the pH and salt composition of the reaction medium can be controlled. The pH and ion composition of the periplasm are the same as those of the surrounding medium, while the pH and ion concentration of the cytoplasm are regulated by the microorganisms to maintain homeostasis (Wilks JC and Slonczewski J, 2007). However, to the best of the applicant's knowledge, no bioproduction method has ever been described that uses a consortium of at least two bacteria, each of which expresses an enzyme in the periplasm that is different from the enzyme expressed by the other bacteria, so that multiple catalytic steps are carried out in a cascade manner.

[0011] First, surprisingly, bacteria modified to express different enzymes in the periplasm can be used in the same reaction medium, each bacterium performing at least one different reaction in a reaction scheme comprising multiple steps in a coordinated manner. In fact, according to the present invention, different transformed strains contacted in the same reaction medium remain in normal operation and coordinate their functions.

[0012] Second, it was unexpectedly demonstrated that the bacterial periplasmic wall does not hinder the cycling of products and reagents from one bacterium to another, so that not only could protocols involving multiple reactions be carried out in their entirety, but the reaction kinetics were also very favorable, allowing the desired compounds to be obtained within hours.

[0013] Furthermore, despite the fact that the viscosity of the periplasm (gel state) is associated with the presence of sugars (Brass et al., 1986), which could slow down enzymatic reactions, unlike the cytoplasm (liquid state) or aqueous solutions used in vitro, the Applicant unexpectedly observed that the resistance of the medium does not hinder the performance of the reaction cascade according to the invention.

[0014] Finally, it is well known that a major obstacle to using the periplasm for bioproduction is the instability of cofactors in this region, which are essential in most metabolic pathways. For example, NAD (NAD+, in its reduced form NADH) is ubiquitous in metabolic reactions; it is a cofactor in more than 300 redox reactions (Zhou et al., 2011).

[0015] However, due to the method of the present invention using a consortium of microorganisms, it is possible to recycle the cofactors necessary for the enzymatic reaction, each enzyme being expressed in the periplasm of a different cell. Cofactors (e.g., NAD(P)H) cannot pass through the cytoplasmic membrane of bacterial cells, but they can diffuse through the periplasmic space that separates the bacterial outer membrane from the plasma membrane. This enables the cofactors to move freely between the different cells of the consortium, thereby enabling them to be efficiently recycled and reused for production.

[0016] Therefore, it is possible to develop a sequential reaction scheme comprising a complex biosynthetic pathway, whereby the scheme is carried out by a variety of bacteria, each of which expresses at least one enzyme in its periplasm. The periplasmic expression of the enzyme (i.e., outside the cell itself) provides a controlled and simplified reaction medium from which the target compound can be easily extracted. In fact, according to the method of the present invention, the enzyme is produced and transferred to the periplasm of the bacterium, where the enzyme is confined to the region, but is available and catalytic for the compound dissolved in the medium. As long as the periplasmic region is easy for solutes to enter, the reaction will occur in a protected environment, which is much simpler than the cytoplasm. This externalization of the reaction (outside the cytoplasm) avoids the redirection of the flow, toxicity and transportation problems that are unique to the cell. In fact, the reaction medium is simplified and can be controlled to improve the enzymatic reaction, particularly by adding cofactors and activators that help to achieve optimal activity. The final extraction stage of the target molecule is also facilitated. Since extraction can account for 70% of the cost of fermentation production compounds, this aspect is an important advantage.

[0017] A characteristic aspect of the proposed technology is that the biomass production process is separated from the production process of the target molecule. The cell culture stage and the biochemical synthesis stage are carried out in separate reactors using different media. Therefore, the biomass in the reactor can be used for a new synthesis process, or can be recovered after the compound synthesis and reused in subsequent synthesis cycles. Then, multiple reactions can be carried out by the same biomass. The cost of biomass production (cell proliferation of different strains) is correspondingly reduced, especially because the amount of water to be treated is significantly reduced. In this way, the production cost is basically maintained at the level of microbial production costs. Another significant advantage of the separation of biomass production and compound synthesis processes is that competition for carbon source use is avoided. In conventional fermentation processes, the goal is to maximize the use of carbon to produce target compounds in order to achieve high yields, but such optimization may have an adverse effect on cell growth (biomass production), thereby also hindering the production of target compounds. Therefore, the balance between cell growth and target compound production is crucial. In contrast, in the method of the present invention, since biomass production is separated from molecule production, the carbon source is different. Therefore, the production yield of biomass growth and target compounds can be optimized separately. In addition, the applicant demonstrated that by adjusting the ratio of each biocatalyst (one bacterium expressing one enzyme) in the consortium, the production of the target product can be significantly enhanced. This is a considerable advantage compared to previously described methods that require complex genetic modification or other tools to enhance production of the same bacteria, which is related to the modular system of the method of the present invention. In particular, it is well known that the efficiency of signal peptides depends on the target protein to be produced. For example, a signal peptide that promotes high-level protein synthesis and secretion of one recombinant protein often leads to low-level protein synthesis and secretion of another recombinant protein (Karyolaimos et al., 2019). Since it is impossible to predict the performance of a signal peptide for a given recombinant protein, it is necessary to screen a signal peptide library to find a signal peptide that promotes high-level protein synthesis and secretion. This method is time-consuming and expensive. Thanks to the method of the present invention, the amount of each biocatalyst in the reaction mixture can be adjusted (e.g., increasing the amount of the biocatalyst to overcome the low efficiency of the signal peptide) to produce the product, thereby maximizing the efficiency of the reaction. Therefore, a single signal peptide (e.g., pelB) can be used regardless of the protein to be produced, and the ratio of the biocatalyst can be increased when the signal peptide does not have the expected efficiency for the production of the protein. This strategy can easily improve the production of the product, which is beneficial for industrial applications. This method, object of the present invention, which will be described in detail below, can be described as a modular system, since different bacteria can be cultured and stored, each of said bacteria being transformed to express in its periplasm at least one enzyme, preferably a single enzyme, different from the enzymes expressed by the other bacteria in the system, so as to provide effectors for various reactions as required to construct a complete reaction scheme.We will discuss biocatalyst libraries, each of which contains a bacterium expressing in its periplasm an enzyme that is different from the enzymes expressed by the other bacteria in the biocatalyst library. The selected bacteria participating in a particular protocol will be introduced into a single medium to act synergistically to produce the target product. This method based on the combined implementation of cell modules has been shown to be suitable for obtaining a variety of compounds, giving the method of the invention a unique versatility. In particular, molecules with high added value, rare or difficult to obtain (difficult to secrete, toxic, difficult to separate racemic isomers, etc.) have been obtained with high yields and a particularly favorable economic and environmental balance.

[0018] Therefore, the use of synthetic microbial consortia according to the method of the present invention has many advantages over prior art bioproduction methods: i) the high metabolic load usually borne by a single strain is overcome by dispersing the load and allowing the optimization of pathways in a modular manner; ii) the metabolic flux between the various modules can be flexibly balanced by easily controlling the proportion of the modified strains; iii) potential inhibition of sensitive enzymes by metabolic intermediates is prevented; iv) finally, due to its modular nature, a variety of different chemical products can be easily produced by simply mixing different microbial strains. Therefore, the method according to the present invention has the advantages of avoiding the problems associated with the co-expression of multiple enzymes in a single cell and overcoming the complexity of gene expression regulation. In addition, the amount of each modified bacterial species can be optimized to more efficiently produce the target product.

[0019] More specifically, the present invention relates to a method for producing a target compound from an organic substrate, comprising the following steps:

[0020] a) - preparing a reaction mixture in a suitable medium, said reaction mixture comprising,

[0021] - n bacteria, n being an integer at least equal to 2, each bacterium being genetically modified to express in its periplasmic space at least one enzyme E1, E2, ..., En, preferably a single enzyme, each of said n bacteria expressing an enzyme different from that of the other bacteria,

[0022] - an enzyme E1 capable of catalyzing a first reaction R1 to produce a first product P1 and optionally a first by-product CoP1 from said organic substrate,

[0023] - each of the enzymes E2, ..., En is capable of catalyzing the reactions R2, ..., Rn to produce the products P2, ..., Pn and optionally the by-products CoP2, ..., CoPn, respectively, as the product or by-product obtained from the reaction Rn-1,

[0024] - said organic substrate, and

[0025] - Optionally, the cofactor of the organic substrate, the cofactor of the product obtained by reaction Rn-1, is called cofactor CoF1, CoF2, ..., CoFn; the complementary substrate SC of the by-product obtained by reaction Rn-1,

[0026] b) reacting the reaction mixture thus obtained, and

[0027] c) separating the biomass from the supernatant and extracting therefrom said target compound consisting of one of the products P1, P2, ..., Pn.

[0028] According to a particular and 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 than the other bacteria.

[0029] The method of the present invention uses at least two genetically engineered bacteria, and each bacterium is capable of secreting at least one given enzyme, which is different from the enzyme expressed by other bacteria. According to the reaction scheme developed for producing the desired compound, it can include multiple bacteria, such as more than ten, or even dozens of species. This number is derived from the number of enzymes required for obtaining the desired product from a given substrate (itself derived from the number of reaction steps), which will indicate the number of cell modules to be used, and is not necessarily equal. In fact, the same bacterium can express more than one enzyme, in which case the number of enzymes is greater than the number of bacteria. However, according to a special and preferred embodiment, each bacterium will express a single enzyme, which is different from the enzyme expressed by other bacteria. In particular, 2 to 20 bacteria, especially 3 to 15 bacteria, or even 4 to 12 bacteria, can be used, and the enzyme expressed by each bacterium is different from the enzyme expressed by other bacteria.

[0030] Each bacterium is responsible for producing at least one specific enzyme, preferably a single specific enzyme, so if there are n enzymes that will participate in the selected reaction scheme, at most n bacteria will participate in the method. The expression "module" or "cell module" can be used to represent a genetically transformed bacterium and one of the enzymes R1, R2, ..., Rn specifically expressed by this bacterium. It should be clear that integers from 1 to n can indicate the order in which each enzyme participates in the selected reaction scheme, but this is not a systematic rule because some reaction schemes are linear and others have branches. This is especially true when modules are used to regenerate cofactors, which will be described in detail below. In any case, the numbers n and n-1 or n and n+1 apply to reactions that are directly continuous with other reactions downstream or upstream, as well as enzymes and compounds involved in the reactions.

[0031] Thus, reaction Rn-1 is defined as being directly upstream of reaction Rn in the reaction scheme, so that the product Pn-1 and the by-product CoPn-1 (if any) resulting from reaction Rn-1 are present in the medium and can serve as substrate or, if applicable, cofactor for the directly downstream reaction Rn, respectively. An exception to this general principle exists with respect to the first reaction of the reaction scheme, i.e., it must be carried out with a selected organic substrate which is converted by the action of the first enzyme R1 to obtain a first product P1 and, if appropriate, a first by-product CoP1. In each step, products P1, P2, ..., Pn and, optionally, by-products CoP1, CoP2, ..., CoPn are formed.

[0032] The reaction mixture prepared in the first step of the method of the present invention contains, in addition to the cell modules that produce the defined reaction scheme, a certain amount of organic substrate, which constitutes the starting material to be converted to finally obtain the desired target compound. This initial organic substrate undergoes a first reaction catalyzed by the enzyme of the first module. Then, in each subsequent reaction step, the product synthesized in the previous reaction can be used as a substrate to produce a new product, and so on. In this linear reaction scheme, the product finally obtained will be the desired target compound.

[0033] At least some reactions may require cofactors, which will produce corresponding byproducts. The term "byproduct" refers in particular to the chemical substance formed by the cofactor in the relevant enzymatic reaction. It should be noted that these byproducts are generally capable of acting as cofactors in other reactions. Therefore, in appropriate cases, it may be necessary to introduce the cofactor of the organic substrate of the first reaction or the cofactor of the product obtained by reaction Rn-1 (which will act as a substrate in the subsequent reaction R) into the reaction mixture.

[0034] When the substrate Sn-1 and the cofactor CoFn-1 react in the reaction Rn-1, the byproduct CoPn-1 and the product Pn-1 will be formed. The reaction scheme may provide that the byproduct CoPn-1 itself is consumed as a cofactor CoFn together with the complementary substrate in the subsequent reaction Rn. Then, it will be particularly advantageous for the reaction Rn to be able to reform the cofactor CoFn-1. In this way, the first cofactor can be regenerated without adding substances to the reaction mixture. However, it is recommended that the complementary substrate be added to the reaction mixture in sufficient amounts. In this specification, the expression "complementary substrate" means a compound different from the initial organic substrate, which is used to react as a substrate together with the cofactor. In this case, it is necessary to introduce a substrate complementary to the cofactor in question (in this case, the byproduct CoPn-1) into the reaction mixture, and they will react together in the subsequent reaction R.

[0035] In summary, it is understood that, according to the defined reaction scheme, one or more cofactors and also one or more complementary substrates may be involved. This is why, in step a) of the process of the invention, various cofactors and complementary substrates are added to the reaction mixture, where appropriate.

[0036] The organic substrate can be any organic compound, the only prerequisite being that it is capable of constituting a substrate for the enzymatic reaction. It can therefore be of a very diverse nature, such as straight-chain or branched hydrocarbons, and may be substituted, such as amines, acids, carbohydrates, amino acids, etc.

[0037] In step a) of the process of the invention, the cell module 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 can be prepared without difficulty by a person skilled in the art according to the technical rules known to him. This medium is an aqueous medium, to which minerals and buffers have generally been added, such as those illustrated below. It is to be distinguished from the reaction medium, which is defined as the aqueous phase in which the reactions in the reaction scheme take place: this includes the medium in which the bacteria are immersed and its periplasmic space, since solutes can easily circulate from one place to another through the periplasmic wall. Simple buffers are used to facilitate the final purification of the resulting compound.

[0038] It should be understood that 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 manner, wherein the substrate and reagent are added continuously or sequentially. Moreover, in some cases, it is better not to add the additional substrate and / or cofactor to the reaction mixture from the beginning, but to add it within a certain time after the start of the reaction method. Similarly, one or more cell modules can be introduced in a delayed manner. In other words, according to this particular scheme, the operation of step a) of the method is carried out at the beginning of step b). This time sequence is included in the context of the present invention.

[0039] As can be seen from the above, the method as the object of the present invention provides a variety of possibilities for designing reaction schemes to adapt to the synthesis of various target compounds. It can be decomposed in different ways, which will be described in detail below.

[0040] As mentioned above, the method according to the present invention is based on externalizing the catalytic reaction into the periplasm of bacteria. Mainly related to Gram-negative double-membrane bacteria, but any other organism with periplasm can also be used. According to a preferred feature of the present invention, the n kinds of genetically modified bacteria introduced into the reaction mixture are Gram-negative double-membrane bacteria, each selected from Enterobacteriaceae, Alcaligenaceae, Vibrionaceae and Pseudomonadaceae, wherein the production of recombinant proteins has been described (Stock et al., 1977; Eichmann et al., 2019; Karyolaimos et al., 2019). They can be intestinal bacteria belonging to Salmonella, Yersinia or Escherichia. According to a more preferred embodiment, the bacterium is an Escherichia coli species.

[0041] These bacteria are able to express one of the enzymes E1, E2, ..., En in their periplasmic space, each expressing a different enzyme from the enzymes expressed by the other bacteria in the consortium. To this end, genetic modification is induced to introduce into the cell a nucleic acid sequence encoding the relevant enzyme in combination with 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 fusion polypeptide is thus localized to the periplasmic space, where the signal peptide is removed. The enzyme is then free, but confined to the periplasmic region and cannot pass through the outer wall. It has been confirmed that they maintain an active conformation and that they perfectly perform their catalytic functions, each of which comes from a substrate available from the beginning or a substrate that appears as the reaction progresses. Various signal peptides can be used in the context of the present invention, such as pelB, dsbA, EOX, lamB, MglB, MmAp, ompC, ompT, sufI, SfmC, STII, tolB, torA, torT, gIII, malE, ompA, phoA, or other signal peptides selected from those known to those skilled in the art, who know how to use them to achieve periplasmic expression (Karyolaimos et al., 2019).

[0042] Therefore, according to the present invention, each bacterium is genetically modified to express at least one polypeptide, preferably a single polypeptide, comprising one of said enzymes E1, E2, ..., En, respectively, linked to a signal peptide localizing said polypeptide to the periplasmic space of said bacterium.

[0043] According to another embodiment, the enzyme or some of them may be anchored to the periplasmic wall. In this case, according to the invention, one or more of the n bacteria are genetically modified to express a polypeptide comprising one of the enzymes E1, E2, ..., En, respectively, linked to a membrane anchoring peptide comprising the signal peptide.

[0044] As previously mentioned, bacteria can be modified to express more than one enzyme in its periplasmic space, that is, two or more enzymes responsible for catalyzing many different reactions. These reactions can be continuous reactions or discontinuous reactions of the reaction scheme. Therefore, according to one embodiment of the present invention, 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, and the enzymes are capable of catalyzing at least two reactions Rn-a and Rn-b, so that the reactions RN-a and RN-b are carried out in the periplasmic space of the at least one bacterium. It should be noted that, for the sake of clarity, this variant will not be developed for each embodiment described in the rest of the specification, but it is explicitly included in the present invention.

[0045] The transformation of bacteria is carried out by inserting the appropriate nucleic acid sequence using a suitable vector. One technique is to insert a plasmid containing a nucleic acid sequence capable of encoding the desired polypeptide into the bacterial cytoplasm. Another technique is based on inserting the nucleic acid sequence into the bacterial genome itself. These techniques and others are known in the field of cell biotechnology and will be readily implemented by a qualified person in the relevant field.

[0046] After conversion, each variant is cultured for biomass production and then used or stored immediately. It is convenient to produce a series of bacteria capable of expressing different enzymes, thereby obtaining as many modules as possible for constructing a reaction scheme that can be implemented by the method according to the invention. Therefore, the cell bank can be used to customize the preparation of the desired reaction mixture in step a) of the method according to the invention.

[0047] Once the reaction mixture has been prepared in a suitable medium, the reaction can be carried out. This mode of treatment corresponds to a batch production mode. It should be noted that production in a continuous or semi-continuous mode is also entirely possible, as explained below. When the initial organic substrate is exhausted, the reaction chain will spontaneously interrupt. The synthesized target compound is essentially secreted in the aqueous medium in a dissolved form. Depending on the reaction scheme adopted, the compound will consist of one of the products P1, P2, ..., Pn obtained in one of the reactions in the reaction scheme. After separation of the biomass, the compound can be easily recovered in the supernatant, and its composition is simpler than that obtained by other previously known technologies.

[0048] According to a particular embodiment of the present invention, the method can use two cell modules, wherein the first cell module converts the initial organic substrate, and the second cell module uses the product of the first reaction to produce a second product, which can be separated from the medium to obtain the desired compound. In this case, the reaction mixture comprises bacteria expressing a first enzyme E1 and bacteria expressing a second enzyme E2, wherein the first enzyme E1 is capable of catalyzing a first reaction R1 to obtain a first product P1 from the organic substrate, and the second enzyme E2 is capable of catalyzing a second reaction R2 to form a second product P2 from the first product P1, and the second product P2 is recovered as a target compound in step c).

[0049] This scheme can be the beginning of a more extensive reaction scheme, in which case the second product can in turn be consumed as a substrate for a downstream reaction. In such an extended reaction scheme, the reaction mixture further comprises at least one bacterium expressing an enzyme E3, ..., En, which is capable of catalyzing reactions R3, ..., Rn to obtain product Pn from product Pn-1 obtained by reaction Rn-1, which product Pn is recovered in step c) as a target compound or consumed as a substrate for reaction Rn+1.

[0050] When a cofactor is needed to carry out the first reaction, the cofactor is introduced into the reaction medium in step a). A first cell module is selected to convert the initial organic substrate and the cofactor (called the first cofactor CoF1), and the second module will use the product P1 of the first reaction to produce the second product P2, or use the byproduct CoP1 of the first reaction to produce the second byproduct CoP2. The product P2 can be separated from the medium to obtain the desired compound, or the product P2 can participate as a substrate in a downstream reaction of a larger reaction scheme. According to a particular scheme, the byproduct CoP2 can also participate as a substrate in another reaction.

[0051] Therefore, according to one embodiment of the method of the present invention, the reaction mixture comprises the cofactor CoF1 of the organic substrate, bacteria expressing enzyme E1 and bacteria expressing second enzyme E2, wherein the enzyme E1 is capable of catalyzing a first reaction R1 to form a first product P1 and a first byproduct CoP1 from the organic substrate and cofactor CoF1, and the second enzyme E2 is capable of catalyzing a second reaction R2 to form a second product P2 from the first product P1, or to form a second byproduct CoP2 from the first byproduct CoP1, wherein at least one of the two is consumed in a third reaction, or is recovered as a target compound in step c). It should be understood that the expression "third reaction" herein refers to any reaction that can occur downstream of the target reaction.

[0052] The above mechanism can be generalized when the reaction scheme is more extensive. In fact, each reaction Rn in the reaction scheme can use the cofactor CoFn to react with the product Pn-1 produced by the upstream reaction Rn-1 under the action of the enzyme En. Then, the product Pn and the byproduct CoPn are formed. The byproduct CoPn can then be used as a cofactor CoFn+1 for the downstream reaction Rn+1. As such, the product Pn can be used as a substrate for (other) downstream reactions Rn+1, unless it is the target compound sought. The scheme can be generalized to one or more modules involved.

[0053] This is why, according to one embodiment of the method that is the object of the present invention, the reaction mixture comprises at least one cofactor CoFn, at least one bacterium expressing an enzyme capable of catalyzing the reaction Rn to form the product Pn and the by-product CoPn from the product Pn-1 of the reaction Rn-1 and the cofactor CoFn, at least one of which is consumed in the reaction Rn+1 or is recovered as the target compound in step c).

[0054] As can be seen, the reaction Rn in the reaction scheme can use as substrate a cofactor, which is usually a by-product produced by an upstream reaction. In this case, there should be another compound in addition thereto, called a "complementary substrate", which will produce a complementary product produced by the reaction Rn. This product may be useless in the rest of the reaction scheme and may accumulate in the medium. This is why, according to a feature of the method that is the object of the present invention, the reaction mixture may also contain at least one substrate SCn complementary to the at least one cofactor CoFn.

[0055] In the process according to the invention, the at least one cofactor CoFn can be introduced in whole or in part into the reaction mixture in step a) or in step b). In fact, it is sometimes preferred to add the cofactor CoFn that does not participate in the first step of the reaction scheme only when the product Pn of the reaction with the cofactor CoFn is present in large quantities in the medium. Therefore, the reaction kinetics are taken into account to optimize the production of the target compound.

[0056] In addition, when a cofactor is necessary for a first reaction, it would be particularly advantageous for the second reaction (downstream reaction) to allow its regeneration. In fact, cofactors are expensive compounds that directly affect the production costs. In order to achieve this goal, a reaction scheme has been designed in which the second reaction consumes the by-product of the first reaction as a cofactor for the second reaction to reform the initial cofactor. In doing so, it is recommended to provide a complementary substrate for the second reaction, which will be selected so that it has a moderate cost. At the end of the second reaction, a second product will be formed, which may or may not be the target product, but most importantly, the cofactor of the first reaction has been regenerated.

[0057] Therefore, in a particularly advantageous embodiment of the process according to the invention, the at least one cofactor CoFn is at least partially formed in the reaction mixture by reaction Rn-1, which is catalyzed by the enzyme En-1 and is capable of forming the product Pn-1 and the by-product CoPn-1, which is identical to the cofactor CoFn.

[0058] However, it is preferred to add a certain amount of cofactor to the medium to start the process until the regeneration of the cofactor is sufficient to ensure a continuous supply of said cofactor. Now, it is obvious that very small amounts are sufficient to start the reaction cycle. Then, the amount of cofactor that must be introduced into the reaction mixture to allow the relevant reaction to proceed completely is significantly reduced compared to the amount of the corresponding substrate that must be reacted with it. The amount may be at least 20 times lower, but preferably at least 100 times lower, more preferably at least 500 times lower, even more preferably at least 1000 times lower. It should be noted that some cofactors synthesized in the cytoplasm, such as ATP, may be present in the periplasmic space in small but sufficient amounts.

[0059] In this case, therefore, according to the invention, the amount of first cofactor CoF1 introduced into the reaction mixture in step a) corresponds to a molar concentration that is at least 20 times lower than the initial molar concentration of the organic substrate.

[0060] As mentioned above, the initial organic substrate can have various properties, including any type of carbon-containing molecules. The method has particularly demonstrated its effectiveness in converting common carbohydrates (sugars) (e.g., glucose, fructose, glycerol, etc.) into target compounds (e.g., rare sugars, especially phosphorylated sugars, specific isomers of rare sugars, dihydroxyacetone and other molecules seeking to obtain important synthetic pathways). Other organic compounds can constitute the starting substrate, including aldehydes, alcohols, organic acids, carbamates, hydrocarbons and amino acids. Examples include glyoxal providing glycolaldehyde, or L-phenylalanine as the starting point for producing 2-phenylethanol. Further, the organic substrate can also be a compound comprising a single carbon atom, such as carbon dioxide.

[0061] Thus, according to one characteristic of the process object of the present invention, the first reacted organic substrate is chosen from carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons, amino acids and carbon dioxide.

[0062] The various transformed bacteria used in the inventive method can produce a variety of enzymes. Those skilled in the art know how to identify those enzymes suitable for developing reaction schemes for obtaining a given target compound from an organic substrate. They can select these enzymes from the enzymes listed and described or the enzymes that will be identified in the future.

[0063] Therefore, according to a preferred feature of the inventive method, enzymes E1, E2, ..., En are each selected from kinases, dehydrogenases, phosphatases, reductases, isomerases and transferases. Kinases can be glycerol kinase, glucokinase, fructokinase, xylokinase, acetate kinase or phosphofructokinase in particular. Dehydrogenases are, for example, glycerol dehydrogenases or phosphite dehydrogenases. Reductases can be used, such as NADH reductases or glyoxal reductases. Isomerases can be selected from xylose isomerases, xylulose isomerases, ribulose phosphate-3-epimerases, ribose-5-phosphate isomerases, and transketolases or transaldolases can be selected from transferases. A variety of phosphatases are also known.

[0064] In addition, multiple cofactors may be involved in one or more reactions in the reaction scheme. It should be noted that as cofactors forming byproducts, these byproducts can also play the role of cofactors in upstream or downstream reactions, so related molecules belong to these two categories. Therefore, preferably, in the method according to the present invention, at least one of enzymes E1, E2, ..., En catalyzes a reaction providing a byproduct CoP1, CoP2, ..., CoPn, wherein the byproduct CoP1, CoP2, ..., CoPn is selected from ATP, ADP, AMP, UTP, UDP, UMP, NAD+, NADH, NADP+, NADPH, FAD, FADH2, coenzyme A, or catalyzes a reaction using one of them as a cofactor.

[0065] It can be seen that at the end of step c) of the inventive method, the biomass is separated from the supernatant and the target compound is extracted from the supernatant. However, the work performed has revealed the unexpected advantages of the inventive method. In fact, it seems that the biomass containing the n kinds of bacterial cells introduced in step a) can be reused as is in a new reaction cycle, with the same or even higher yield level observed in the first reaction cycle. This has significant savings in terms of time, the number of operations to be performed, the consumption of ingredients and water, and the final economy. This advantage derives from the principle of the present invention, according to which the cell culture stage is separated from the biochemical synthesis stage: they are carried out in separate reactors, using different media.

[0066] The supernatant can be separated from the biomass by gravity flow, in which case the remaining biomass in the reactor can be directly reused by simply adding fresh substrate and optionally other necessary ingredients.

[0067] Thus, in a particularly advantageous manner, after step c) of the process object of the invention, the biomass separated from the supernatant is reused for preparing a new reaction mixture according to step a), with or without an intermediate storage step.

[0068] The method can be implemented in the case of discontinuous batch cultivation as described above, or by discontinuous flow (fed batch type). According to rules known in the art, it can also be carried out in a semi-continuous mode or in a continuous supply mode. In this case, steps a) and b) are not carried out in sequence, but more or less simultaneously.

[0069] That is why, in a variant embodiment of the method according to the invention, steps a) and b) are carried out by adding continuously or intermittently to the reaction mixture containing the n bacteria

[0070] - said organic substrate, and

[0071] - Optionally, the cofactor of the organic substrate, the cofactor of the product obtained by reaction Rn-1, is called cofactor CoF1, CoF2, ..., CoFn; the complementary substrate SC of the by-product obtained by reaction Rn-1, and the reaction mixture obtained thereby is reacted continuously or semi-continuously.

[0072] As can be seen from the above, the method for producing the target compound just described has many advantages, can cope with the difficulties encountered by conventional technologies, and has a very wide range of applications. The externalization of the reaction actually makes it possible to use the biochemical machinery of the cell to provide enzymatic effectors of the reaction that occurs in the space defined by the periplasm, while allowing the reagents and products involved in the reaction to circulate. The combinable modular design allows the rapid development and implementation of complete original reaction schemes. Therefore, the synthesis of a given target product and its final purification are facilitated because the aqueous biological production medium is very simple.

[0073] It should be added that, since the method relies on the production of reusable biomass, it is much cheaper than the known enzymatic methods (if they exist at all), thus enabling the industrial production of a wide range of molecules, including those that are complex or difficult to obtain.

[0074] It will therefore be appreciated that the process which is the object of the present invention represents an unprecedented alternative to current production methods and provides a powerful tool to accelerate the development of new products.

[0075] According to a second aspect, the present invention relates to a reaction mixture for producing a target compound from an organic substrate. This reaction mixture is substantially the same as the reaction mixture prepared in step a) of the above production method.

[0076] Therefore, the present invention relates to a reaction mixture for producing a target compound from an organic substrate, comprising in a suitable medium:

[0077] - n bacteria, n being an integer at least equal to 2, each bacterium being genetically modified to express in its periplasmic space at least one enzyme E1, E2, ..., En, preferably a single enzyme, each of said n bacteria expressing an enzyme different from that of the other bacteria,

[0078] - an enzyme E1 capable of catalyzing a first reaction R1 to produce a first product P1 and optionally a first by-product CoP1 from said organic substrate,

[0079] - each of the enzymes E2, ..., En is capable of catalyzing the reactions R2, ..., Rn to produce the products P2, ..., Pn and optionally the by-products CoP2, ..., CoPn, respectively, as the product or by-product obtained from the reaction Rn-1,

[0080] - said organic substrate, and

[0081] - Optionally, the cofactor of the organic substrate, the cofactor of the product obtained by reaction Rn-1, is referred to as cofactor CoF1, CoF2, ..., CoFn; the complementary substrate SC of the by-product obtained by reaction Rn-1.

[0082] According to a particular and 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 than the other bacteria.

[0083] Thus, the reaction mixture is a mixed composition comprising suspended cells of at least two bacteria and various soluble reagents, including an organic substrate to be transformed to obtain the target compound. Various embodiments of the mixture can be defined with reference to the previous description. According to a particular embodiment, the reaction mixture comprises at least 3 bacteria, in particular at least 4 bacteria, or even at least 5 bacteria, in particular at least 6 bacteria, each of which is genetically modified to express an enzyme different from the enzymes expressed by the other bacteria.

[0084] Finally, according to a third aspect of the invention, the use of a reaction mixture as described above for producing a target compound selected from carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons and amino acids is claimed. For example, a reaction mixture comprising carbohydrates can be used to produce phosphorylated sugars or dihydroxyacetone (DHA), or a reaction mixture comprising amino acids can be used to produce nitrogen-containing organic compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The present invention will be better understood through the description of various embodiments with reference to the accompanying drawings, in which its details will become apparent.

[0086] Figure 1 : Production of glycerol-3-phosphate from glycerol over time by two cell modules (BL21-Glpk and BL21-AckA).

[0087] Figure 2a : Effect of the concentration of cell modules (BL21-Glpk and BL21-AckA) on glycerol-3-phosphate production.

[0088] Figure 2b : Effect of reaction volume - glycerol-3-phosphate production in 2 ml, 20 ml, 200 ml and 2000 ml volumes.

[0089] Figure 3a : Production of D-glucose-6-phosphate from D-glucose by two cell modules BL21-Glk and BL21-AckA.

[0090] Figure 3b : Production of D-glucose-6-phosphate from D-glucose by two cell modules BL21-nlpA-Glk and BL21-nlpA-AckA.

[0091] Figure 4 : Production of fructose-1-phosphate from D-fructose by two cell modules BL21-KhkC and BL21-AckA.

[0092] Figure 5 : Production of glucose-6-phosphate using duplicated BL21-Glk and BL21-AckA cell modules.

[0093] Figure 6 : Production of dihydroxyacetone from glycerol via BL21-GldA and BL21-Nox modules.

[0094] Figure 7 : Production of glycolaldehyde from glyoxal by the BL21-YvgN module with or without the BL21-PtxD regeneration module.

[0095] Figure 8 : Consumption of D-xylose by individual isomerization reactions (BL21-XylA) and by the overall reaction (BL21-XylA, BL21-XylB, BL21-AckA, BL21-AphA).

[0096] Fig. 9 : Production of D-xylulose from D-xylose by a single isomerization reaction (BL21-XylA) and by an overall reaction (BL21-XylA, BL21-XylB, BL21-AckA, BL21-AphA).

[0097] Fig.10 : SDS-PAGE gel migration of the periplasmic fraction of the XylA, XylB, AckA, Rpe, RpiA, TktA, Tal, PfkA modules and the control BL21WT.

[0098] Fig.11 : Production of D-fructose-1,6-bisphosphate from D-xylose using eight cell modules.

[0099] Fig.12 : Production of the intermediate D-sedoheptulose-7-phosphate in the production of D-fructose-1,6-bisphosphate using eight modules.

[0100] Fig.13 .2-Phenylethanol concentrations in a synthetic consortium consisting of strains BL21-ARO8, BL21-ARO10, BL21-ADH5, and BL21-PtxD and the control BL21-WT were monitored by HPLC in the presence of 10 mM L-phenylalanine.

[0101] Fig.14 . SDS-PAGE gel of periplasmic fractions of BL21-AckA, BL21-GlpK, BL21-AckA-GlpK biocatalyst and negative control BL21-W.

[0102] M:Precision Plus protein TM All Blue Marker,BIO- 1:BL21-AckA (2OD 600nm / ml); 2:BL21-GlpK (2OD 600nm / ml); 3:BL21-GlpK-AckA (2OD 600nm / ml), 4:BL21-GlpK (1OD 600nm / ml), BL21-GlpK (1OD 600nm / ml), 5:BL21-WT (2OD 600nm / ml); black dotted line box: 56 kDa GlpK protein; white dotted line box: 43 kDa AckA protein.

[0103] Fig.15 Production of glycerol-3-phosphate from glycerol by the strain BL21-GlpK-AckA, a synthetic consortium consisting of the two modules BL21-AckA and BL21-GlpK (supplied in variable ratios), and the control BL21-WT.

[0104] Fig.16 Glucose-6-phosphate production in the presence of glucose and without added ATP in a synthetic consortium consisting of 2BL21-Glk, BL21-AckA modules and a BL21-WT control. DETAILED DESCRIPTION

[0105] Materials and methods

[0106] 1-Plasmid

[0107] - Plasmid pET22b (ori pBR322, ampicillin, T7 promoter, pelB) and plasmid pET26b (oripBR322, kanamycin, T7 promoter, pelB) are commercial plasmids carrying the pelB signal sequence at the N-terminus and the His tag sequence at the C-terminus. They are used to express target genes encoding enzymes that will be transported into the periplasm of cells via the PeIB fusion peptide.

[0108] - Plasmid pACT3 (ori p15A, chloramphenicol, Ptac promoter) is used to anchor the protein in the periplasm. This plasmid was modified to have the N-terminal leader sequence of lipoprotein NlpA.

[0109] - Plasmid pETDuet-1 (ori pBR322, ampicillin, T7 promoter) is designed for co-expression of two target genes. The vector carries two cloning sites, each preceded by a T7 promoter, a lac operator and a Shine-Dalgarno sequence. Plasmid pETDuet-1 is modified here by integrating the PelB signal sequence after the two ribosome binding sites. The expression cassette (T7-RBS-pelB) is identical to that of pET22b. The genes integrated into this modified plasmid enable co-expression of the two free enzymes in the periplasm under the control of two IPTG-inducible T7 promoters.

[0110] These genes are amplified from the genomic DNA of E. coli MG1655 by known techniques, such as polymerase chain reaction (PCR) using appropriate primers, and then inserted into the pre-used The kit linearizes the plasmids pET22b, pET26b, pACT3, or pETDuet-1 by homologous recombination. If specified, synthetic genes are directly obtained from Order and clone directly from it into plasmid pET22b or pET26b.

[0111] Primers used for gene amplification are of generally known types. Those skilled in the art know how to select and use them in the context of PCR techniques. According to a particular embodiment, primers for amplifying each gene are prepared using a kit and the instructions of manufacturer NEB "NEBuilder HiFi DNA Assembly Master Mix & NEBuilder HiFi DNA Assembly CloningKit E2621 ​​E5520" (https: / / www.neb.com / - / media / nebus / files / manuals / manuale2621_e5520.pdf?rev=67d09a762b2c47ed8df3a0bd1cb59a1b&hash=02CF464149634283F5633CA1DE4D2FDF). In particular, according to the manufacturer's recommendations, PCR primers for assembling HiFi DNA must have two sequence components: i) overlapping sequences, which are required for the assembly of adjacent fragments; ii) sequences specific to genes, which are required for the initiation of templates during PCR. For efficient assembly of PCR fragments in a vector, an overlap of 15 to 30 nucleotides with a Tm equal to or greater than 48°C (assuming AT pairs = 2°C and GC pairs = 4°C) is recommended. 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.

[0112] 2- Construction of strains

[0113] Strain BL21 (DE3) or BL21 (DE3) gold was selected for transformation plasmid pET22b or pET26b (depending on the resistance cassette) so that T7 polymerase could be used. Transformation was performed according to the TSS protocol described by Chung and Miller (Chung et al., 1993). The last two strains were constructed to anchor the enzyme in the periplasm, and all other strains were constructed to free the enzyme. The strains used are listed in Table 1.

[0114] [Table 1] Escherichia coli strains used

[0115]

[0116]

[0117] 3- Culture media and conditions

[0118] Induced whole cell production

[0119] The strains were removed from glycerol stocks stored at -80°C and placed in 5 ml of TB medium (buffered to pH 7 with appropriate antibiotics) at 37°C overnight. The composition of TB medium is shown in Table 2.

[0120] [Table 2] Composition of TB medium buffered to pH 7

[0121] Compound quantity unit Yeast Extract 24 g / l Tryptone 20 g / l glycerin 5 g / l <![CDATA[Kh2PO4]]> 0.017 M <![CDATA[Na2HPO4]]> 0.072 M

[0122] The next day, the preculture was diluted to 1 / 100 in 50 ml of fresh TB medium and the culture was stirred at 200 rpm at 37° C. When the optical density at 600 nm (OD 600 nm) reached a value between 0.4 and 0.6, the cells were induced with 0.1 mM IPTG. They were then placed at 20° C. and stirred at 200 rpm for about 18 hours.

[0123] External production of target molecules

[0124] The induced whole cells (or modules) expressing one or more enzymes in their periplasm are centrifuged at 4500 rpm for 10 minutes and resuspended in sterile water to reach a defined amount of cells expressed as ODU. The modules carrying the enzymes selected according to the requirements of the predefined reaction scheme are introduced together into the reaction mixture. This contains a buffer for stabilizing the pH, activators and cofactors if necessary, substrates and a known amount of each cell module. The composition is deliberately kept as simple as possible to ensure optimal enzyme activity and minimum production costs. Substrates and cofactors are all obtained from or All the ingredients were purchased except acetyl phosphate for ATP regeneration, which was synthesized from phosphoric acid (85%, 2 mol) and ethyl acetate (2 mol), two low-cost compounds, according to the method described by Crans et al. (1983).

[0125] 4- Quantification and analysis of extracellular products

[0126] Substrate and product concentrations are monitored by high pressure ion chromatography (HPIC) or high performance liquid chromatography (HPLC). Different detection methods are used depending on the target molecule.

[0127] HPIC analysis

[0128] The samples were centrifuged at 14,800 rpm for 2 minutes and then filtered at 0.2 μm. The samples to be analyzed were diluted 1 / 100 with milli-Q water in flasks equipped with pre-perforated stoppers and placed in an autosampler at 15°C.

[0129] Ampereometric detector (HPIC-PAD)

[0130] Ion chromatography (Dionex TM ICS-6000 ion chromatography system) with pulsed amperometric detection or PAD (Dionex TM ICS-6000 electrochemical detector) for specific substrate and product analysis. The molecules were heated at 25°C on a Dionex TM Carbopac TM The separation was carried out on a PA1 column (2 x 250 mm) preceded by a guard column of the same type (2 x 50 mm).

[0131] 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), methylglyoxal, and aldehydes (e.g., glycolaldehyde) was as follows: the NaOH concentration was kept at 100 mM from 0 to 30 minutes, and the sodium acetate (NaOAc) concentration was gradually changed. From 0 to 2 minutes, 0 mM NaOAc; from 2 minutes to 15 minutes, it was increased from 0 to 500 mM NaOAc; from 15 minutes to 23 minutes, the concentration was kept at 500 mM NaOAc; from 23 minutes to 23.1 minutes, it was decreased from 500 mM to 0 mM NaOAc; from 23.1 minutes to 30 minutes, it was kept at 0 mM NaOAc.

[0132] Conductivity detector (HPIC-CD)

[0133] The conductivity was measured by a conductivity meter (Dionex TMIon chromatography (Dionex ICS-6000CD conductivity detector) TM ICS-6000 ion chromatography system) to analyze anions (organophosphorus compounds, phosphates, chlorides, sulfates, organic acids, etc.). The molecules were heated at 25°C on a Dionex TM IonPac TM The separation was performed on an AS11-HC column (2 x 250 mm) preceded by a guard column of the same type (2 x 50 mm).

[0134] The concentration gradient was generated using the eluent generator EGC 500KOH at a flow rate of 0.35 ml / min. The following gradient was used to best separate the intermediate compounds from the reaction scheme shown in Example 7. Before each injection, a 7-minute conditioning with 2 mM NaOH was performed. The first step of the gradient was an isocratic elution for 3 minutes at a NaOH concentration of 10 mM, followed by increasing the NaOH concentration in multiple steps: 1) from 10 mM to 50 mM in 9 minutes; 2) from 50 mM to 100 mM in 7 minutes; 3) maintained at 50 mM for 3 minutes; 4) reducing the NaOH concentration to 10 mM; 5) maintained at 10 mM for 4 minutes. At the column outlet, an ADRS 2 mm suppressor (87 mA) was used to improve detection. For other examples, the NaOH concentration gradient described below was used to determine anionic metabolites. The first step was an isocratic elution at 2 mM NaOH for 4 min, and then the NaOH concentration was increased in multiple steps: 1) from 2 mM to 100 mM in 11 min; 2) maintained at 100 mM for 10 min; 3) decreased to 2 mM and maintained for 1 min.

[0135] UHPLC-UV / RI analysis

[0136] By using a refractometer (Shodex TM RI-101) and UV detector (Dionex UltiMate TM 3000 Diode Array Detector 3000 (RS)) UHPLC (Dionex Ultimate TM 3000) to analyze sugars and organic acids. TM Separation on ROA-Organic Acid H+ column (8%) (300 x 7.8 mm) equipped with a guard column of the same type (50 x 7.8 mm). Elution was performed isocratically (5 mM H2SO4) at a flow rate of 0.5 ml / min for 35 minutes. The samples to be analyzed were filtered at 0.2 μm before injection.

[0137] HPLC-mass spectrometry

[0138] Thermo Fisher Scientific TM The presence of fructose-1-phosphate was detected by UHPLC (Dionex UltimateTM3000). The molecules were purified by ACQUITY UPLC BEH amide column ( 1.7μm, 2.1x30mm) with a guard column (ACQUITY BEH Shield RP18 1.7μm VANGUARD) in front of the column. Mobile phase A consisted of ultrapure water and methylphosphonic acid (1.8mM), and organic mobile phase B consisted of acetonitrile and formic acid (1.3mM). The concentration gradient used was as follows, with a flow rate of 0.35ml / min: isocratic elution with 90% organic mobile phase in 1 minute, then the organic mobile phase fraction was reduced in multiple steps: 82% at 3 minutes, 78% at 6.5 minutes, and 50% at 8 minutes. This composition was kept constant for 10 minutes (50% organic phase), followed by an equilibrium time of 7 minutes.

[0139] Glycerol-3-phosphate C13 (G3P-C13) was used as an internal standard. An elution gradient was created. The injection mixture was prepared in 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 was 2.1. This dilution factor will be taken into account when processing the results using the ChromeleonTM software.

[0140] 5- Extraction of proteins by osmotic shock

[0141] The cells were cultured overnight in 2 ml of LB medium at 37°C with stirring at 200 rpm. The cell suspension was transferred to an Eppendorf tube and centrifuged (2 ml; 5 minutes; 14,000 g; 4°C). The pellet was resuspended in 500 μl of buffer 1 and incubated on ice for 20 minutes. The test tubes were inverted periodically to avoid precipitation. The cell mixture was centrifuged (15 minutes; 14,000 g; 4°C). The supernatant was discarded and the cell pellet was resuspended in 125 μl of buffer 2. After further incubation on ice (periodic inversion) for 10 to 20 minutes, the cells were centrifuged (15 minutes; 14,000 g; 4°C). The supernatant containing the periplasmic fraction was recovered, then denatured at 95°C for 10 minutes and deposited on an SDS-PAGE gel (45 minutes, 180 V, 400 mA) to verify the presence of the enzyme and its molecular weight. The size marker used was Precision Plus Protein TM All Blue Standards #1610373EDU. The composition of the buffer is shown in Table 3.

[0142] [Table 3]: Composition of buffer 1 and 2

[0143]

[0144]

[0145] Example 1: Production of phosphorylated compounds

[0146] Phosphorylation is a reaction that allows cells to isolate essential metabolites in the cytoplasm and prevents them from losing by diffusing into the external environment. Negatively charged phosphorylated compounds cannot diffuse through the phospholipid bilayer. Therefore, the catabolism of sugar always includes a phosphorylation step. For example, sugars such as D-glucose, D-fructose, D-trehalose, L-rhamnose, D-xylose and L-arabinose are restricted in the cytoplasm in the form of D-glucose-6-phosphate, D-fructose-1-phosphate, D-trehalose-6-phosphate, L-rhamnose-6-phosphate, D-sorbitol 6-phosphate, D-xylulose-5-phosphate and D-ribulose-5-phosphate respectively. It is also applicable to other carbon sources that can be absorbed by microorganisms, such as polyols: mannitol, sorbitol and glycerol, which are phosphorylated to mannitol-1-phosphate, sorbitol-6-phosphate and glycerol-3-phosphate respectively. According to the present invention, the phosphorylation reaction is externalized to solve this output problem, and the microbial production of these compounds is carried out.

[0147] For this reason, the E. coli strain is transformed to express a kinase that catalyzes sugar phosphorylation in its periplasm (the first module). This reaction consumes the cofactor ATP and forms ADP as a byproduct. The second module is constructed by transforming the E. coli strain to express acetate kinase (AckA) in its periplasm. This enzyme catalyzes the reaction of acetyl phosphate and ADP to form acetate and ATP.

[0148] The reaction scheme is as follows:

[0149] [Chemical formula 1]

[0150]

[0151] The following examples illustrate that, according to this reaction scheme, the target phosphorylated compound is produced from three organic substrates (glycerol, glucose and fructose) by the participation of cofactors ATP and ADP. Due to the BL21-AckA module for ATP regeneration, the amount of ATP introduced into the initial reaction mixture is significantly less than the amount of substrate (in this case, one of the examples is 32 times less and the other two examples are 60 times less).

[0152] Therefore, using the BL21-AckA module enables substantial savings, since the cost of ATP is about 300 € / kg. However, another important reason motivates the regeneration of the ATP cofactor. In fact, 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 produced for each product molecule formed. Therefore, in the case of inhibition by ADP, it is impossible to produce the target compound unless the formed ADP is constantly removed, which is done here by a reaction that regenerates ATP. Using the regeneration module enables the production of phosphorylated molecules.

[0153] Example 1.1: Production of glycerol-3-phosphate from glycerol

[0154] Glycerol-3-phosphate is an entry point to the phospholipid pathway. This target molecule was produced microbially in a fed-batch bioreactor with a yield of 325 mg / l, but its export proved to be a major problem (Popp et al., 2008). In addition, glycerol dephosphorylation occurs intracellularly.

[0155] Two modules were used: the first module expressed glycerol kinase from E. coli, which produced glycerol-3-phosphate from glycerol and ATP, and the second module had the function of regenerating ATP via acetate kinase from E. coli AckA. Both the BL21-Glpk and BL21-AckA modules were from the BL21(DE3) chassis.

[0156] Both modules were grown in TB medium and induced with IPTG as described (Materials and methods, point 3), and then recovered for glycerol-3-phosphate production in simple medium. The reaction mixture contained:

[0157] Module 1: 2 ODU / ml BL21-Glk cells

[0158] Module 2: 2 ODU / ml BL21-AckA cells

[0159] Substrate: Glycerol (160mM)

[0160] Cofactor: ATP (5mM)

[0161] Complementary substrate: acetyl phosphate (220 mM)

[0162] Buffer solution: HEPES (50 mM) and MgSO4 (15 mM)

[0163] The glycerol-3-phosphate production reaction was performed in a 2 ml reaction mixture by resuspending the pellet of Glpk and AckA cells containing an equal amount of 2 ODU in a volume of 2 ml. The tube was then placed at 25°C and stirred. Samples were taken from the reaction mixture at 0 hour and 1 hour, 3 hours, 5 hours, and 22 hours after the addition of the enzyme. The results are shown in Table 1. Figure 1 After 22 hours of reaction, a yield of 1.4 g / l glycerol-3-phosphate was obtained.

[0164] Effect of cell concentration on yield

[0165] The same protocol was repeated, but the module concentration was doubled. The yield reached 12 g / l in 23 h, corresponding to a yield of 81.5%, which is 37 times the maximum yield obtained by the microbial pathway (Popp et al., 2008). Therefore, the cell concentration has an important effect and can be optimized.

[0166] Effect of cell concentration on reaction rate

[0167] To evaluate the effect of cell number on production rate, different concentrations were used under the same experimental conditions. The glycerol-3-phosphate concentrations obtained within one hour showed that the reaction rate was proportional to the cell concentration ( Figure 2a ). Thus, productivity of 1 g / l / h to 4 g / l / h can be obtained. These productivity rates show that the process of the present invention can be scaled up.

[0168] This example demonstrates the possibility of producing phosphorylated polyols by the method that is the object of the present invention. This helps to overcome the obstacles associated with the export and dephosphorylation of phosphatases within cells.

[0169] Effect of reaction volume

[0170] The method according to the invention is intended to produce molecules at different scales, from laboratory level to industrial level. In order to evaluate the effect of increasing the reaction mixture volume on the production of glycerol-3-phosphate, the reaction was carried out at a volume of 2 ml, 20 ml, 200 ml and 2000 ml. As before, the modules used were BL21-Glk and BL21-AckA. The reaction mixture consisted of:

[0171] Module 1: 2 ODU / ml BL21-Glk cells

[0172] Module 2: 2 ODU / ml BL21-AckA cells

[0173] Substrate: Glycerol (25 mM)

[0174] Cofactor: ATP (5mM)

[0175] Complementary substrate: acetyl phosphate (25 mM)

[0176] Buffer solution: HEPES (50 mM) and MgSO4 (15 mM)

[0177] Samples were taken at 0, 1, 3, 4 and 5 hours of reaction after adding the cell biocatalyst (module) to the mixture. Under the conditions described, the theoretical maximum glycerophosphate yield was 4.3 g / l using 25 mM acetyl phosphate. The production kinetics observed at 2 ml and 20 ml were similar. The maximum yield after 3 hours was 3.3 g / l, with a yield of 76%. When the reaction volume was 200 ml or 2000 ml, the yield reached 100% after 3 hours. These results highlight the convertible nature of the method, which is an essential prerequisite for scale-up.

[0178] Furthermore, the product was stable at 2000 ml. Based on these results, it is entirely possible to scale up the process 1000-fold and even facilitate production.

[0179] Example 1.2: Production of glycerol-6-phosphate from glucose

[0180] Glucose-6-phosphate is the entry point for the glycolytic pathway and the pentose phosphate pathway. For this production, two protocols were tested, one with free enzyme in the periplasm and the other with the same enzyme anchored on the inner membrane.

[0181] Protocols using free enzymes

[0182] The two modules used are the BL21-Glk module expressing the E. coli Glk kinase, and the BL21-AckA ATP regeneration module expressing the E. coli acetate kinase. These enzymes are present in the periplasm in a free state. The two modules are cultured in TB medium and induced with IPTG, and then introduced into the reaction mixture medium in an amount of 2 ODU in 2 ml. A control sample containing strain BL21-WT is prepared. The reaction mixture contains:

[0183] Module 1: 2 ODU / ml BL21-Glk cells

[0184] Module 2: 2 ODU / ml BL21-AckA cells

[0185] Substrate: 300 mM glucose

[0186] Cofactor: 5mM ATP

[0187] Complementary substrate: 125 mM acetyl phosphate

[0188] Buffer solution: HEPES (50 mM) and MgSO4 (15 mM)

[0189] Figure 3a It was shown that glucose-6-phosphate was indeed synthesized, and a concentration of 30.8 g / L was obtained in 5 hours of reaction, i.e., a productivity of 96%, which was limited by the amount of acetyl phosphate (125 mM). Therefore, a plateau was reached very quickly. After 22 hours, glucose-6-phosphate was observed to decrease due to molecular degradation. No production was observed in the control strain.

[0190] Protocol using anchoring enzymes

[0191] The modules used here (BL21-nlpA-Glk and BL21-nlpA-AckA) express the same two enzymes (glucokinase Glk and acetate kinase AckA), but they are fused to the anchor peptide nlpA, so that both enzymes are anchored to the inner membrane of the periplasm of the respective modules. The modules were cultured and tested according to the same protocol as above.

[0192] The reaction scheme is as follows:

[0193] [Chemical formula 2]

[0194]

[0195] Figure 3b The production of glucose-6-phosphate was shown, with a concentration of 24.8 g / l obtained after 3 hours of reaction, i.e., a yield of 76%, limited by the amount of acetyl phosphate (125 mM). After 22 hours, a decrease in glucose-6-phosphate was observed due to molecular degradation. No production was observed with the control strain.

[0196] Conclusion: when enzyme is free or anchored, production reaction is possible.In the present embodiment, it is faster to produce glucose-6-phosphate by anchoring enzyme than to produce by free enzyme, but productive rate is lower.Can implement the method according to the present invention with transformed bacteria, the transformed bacteria produces anchoring or free periplasmic enzyme.

[0197] Example 1.3: Production of glycerol-1-phosphate from fructose

[0198] Fructose-1-phosphate is a sugar phosphorylated at position 1 from which the target synthon glyceraldehyde can be produced. Its biosynthesis has never been described.

[0199] As mentioned above, two modules are required. One module carries the sequence encoding the human (Homo sapiens) KhkC fructokinase, which is able to phosphorylate sugars at position 1 (BL21-KhkC). The other module expresses the E. coli acetate kinase responsible for regenerating the ATP cofactor (BL21-AckA, already described).

[0200] For this test, 4 ODU / ml of each module was added to the reaction mixture, i.e., 8 ODU in total in 2 ml. A control sample containing strain BL21-WT was prepared. The reaction mixture contained:

[0201] Module 1: 2 ODU / ml BL21-KhkC cells

[0202] Module 2: 2 ODU / ml BL21-AckA cells

[0203] Substrate: 300 mM fructose

[0204] Cofactor: 5mM ATP

[0205] Complementary substrate: 125 mM acetyl phosphate

[0206] Buffer solution: HEPES (50 mM) and MgSO4 (5 mM)

[0207] Prepare a BL21-WT (2 ODU / ml, also called OD 600nm =2) of the control sample.

[0208] Figure 4 The results shown in show that the reaction was almost complete after 5 hours. It reached a plateau after 22 hours with a fructose-1-phosphate concentration of 1.3 g / l. The reaction was limited by acetyl phosphate (125 mM). The control strain did not produce any product.

[0209] Conclusion: Therefore, the method according to the present invention is suitable for the production of phosphorylated sugars. First, it was determined that it is possible and effective to produce phosphorylated sugars by externalizing the phosphorylation reaction in the periplasm. Secondly, the use of the same strain (BL21-AckA) in multiple reaction schemes illustrates the flexibility of the method and its economic benefits, because the biomolecular engineering work is limited to obtain the required modules. Finally, the advantage of the module designed for regeneration of cofactors is that it can use low concentrations of ATP to produce phosphorylated molecules. Therefore, it is possible to design reaction schemes using small amounts of ATP naturally present in the periplasmic region.

[0210] Example 2: Storage and reuse of cells

[0211] In the method according to the invention, the use of a module implemented in a separate manner from the preceding cell cultivation phase makes it possible to carry out a plurality of reaction cycles with the same biomass and to preserve this biomass over time.

[0212] Glucose-6-phosphate production was performed by reusing cells of the BL21-GLK and BL21-AckA modules that had been used in previous production. For this purpose, a first production was performed in which the cells were suspended in a 2 ml reaction mixture (8 ODU per cell type). Then, the cells used for the first production were separated from the supernatant and passed through the centrifugal separation module. The precipitate was recovered and stored at 4°C. Four days later, the cells were resuspended in water and introduced into a new 2 ml volume of reaction mixture in an amount of 8 ODU per cell type.

[0213] The reaction mixture used in the two cycles consisted of:

[0214] Module 1: 8 ODU / ml BL21-GlK cells

[0215] Module 2: 8 ODU / ml BL21-AckA cells

[0216] Substrate: 300 mM glucose

[0217] Cofactor: 5mM ATP

[0218] Complementary substrate: 220 mM acetyl phosphate

[0219] Buffer solution: HEPES (50 mM) and MgSO4 (5 mM)

[0220] Figure 5 The results in show that the output of glucose-6-phosphate is 50g / l in 6 hours. If the final concentration of the resulting is 65g / l, the reaction is complete. Therefore, the reaction yield is 75%. By reusing the cells, the glucose-6-phosphate produced is 1.6 times more than when the cells are used for the first time (producing 30g / L). Therefore, the method according to the present invention can preserve and use the same biocatalyst for many times without losing efficiency, thereby reducing the cost associated with biomass production.

[0221] Example 3: Production of dihydroxyacetone from glycerol

[0222] Dihydroxyacetone (DHA) is a tanning agent widely used in the cosmetics industry. DHA is produced using the method according to the invention. It includes recycling the redox cofactors involved in the reaction.

[0223] For this reason, two modules were constructed. The E. coli strain was transformed to express the E. coli glycerol dehydrogenase in its periplasm, and the enzyme catalyzes the conversion of glycerol into DHA (the first BL21-GldA module). The reaction consumes the cofactor NAD+, and forms NADH as a byproduct. The second module (BL21-Nox module) was constructed by transforming the E. coli strain to express the Lactobacillus sanfrancisco NADH reductase (NOX) in its periplasm. The enzyme catalyzes the reaction of oxygen (O2) and NADH, generates water (H2O) and NAD+, and NAD+ participates in the first reaction after regeneration. The plasmid pET26b carrying the nox gene (which encodes NADH oxidase NOX) of Lactobacillus sanfrancisco was ordered by GenScript and transformed into the bacterial strain BL21-WT.

[0224] The reaction scheme is as follows:

[0225] [Chemical formula 3]

[0226]

[0227] Both modules were grown in TB medium and induced with IPTG and then recovered for DHA production. The reaction mixture contained:

[0228] Module 1: 4 ODU / ml BL21-GldA cells

[0229] Module 2: 4 ODU / ml BL21-Nox cells

[0230] Substrate: 20 mM glycerol

[0231] Cofactor: 5mM NAD+

[0232] Buffer solution: 100 mM (Tris pH 8 or Gly pH 9).

[0233] Negative controls without substrate were prepared. In addition, negative controls were prepared in which each strain was replaced by BL21(DE3) cells that do not express GldA or NOX enzymes. Since the optimal pH for GldA is 9 and the optimal pH for NOX is 7.5, tests were performed at pH 8 and 9 to find the best compromise for reaction function. Measurements were performed after 5 hours, as Figure 6 shown.

[0234] At pH 8 and pH 9, both modules produced DHA, whereas no DHA was produced in the wild-type control. The amount of DHA obtained was 260 mg / l at pH 8 and 330 mg / l at pH 9, demonstrating the feasibility of the proposed reaction scheme.

[0235] Example 4: Production of glycolaldehyde from glyoxal

[0236] Glycolaldehyde is a highly reactive molecule present in cellular metabolism and can be used as a platform molecule like most aldehydes. However, it is too expensive for industrial use. Aldehydes are rarely produced by microorganisms due to their reactivity, which makes them toxic to cells and unstable. Their ability to react with membrane proteins and lipids makes their removal more complicated. Glycolaldehyde can be obtained by reducing glyoxal, which is inexpensive but significantly toxic.

[0237] To this end, two modules were constructed. Escherichia coli strains were transformed to express Bacillus subtilis glyoxal reductase YvgN in its periplasm, which catalyzes the conversion of glyoxal into glycolaldehyde (the first BL21-YvgN module). The reaction consumes the cofactor NADPH and forms NADP as a byproduct (Dudek et al., 2013). The second module (BL21-PtxD) was constructed by transforming Escherichia coli strains to express phosphite dehydrogenase PtxD (a NADP reductase) in its periplasm. The ptxD gene of Pseudomonas stutzeri encoding the enzyme contains 18 mutations to ensure improved catalytic efficiency, stability at 37°C, and management of 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 codon-optimized for expression in E. coli. Plasmids pET22b and pET26b have the signal sequence pelB, which is responsible for protein export to the periplasm; pET22b is resistant to ampicillin, and pET26b is resistant to kanamycin. These plasmids were then transformed into BL21-WT to obtain the BL21-YvgN and BL21-PtxD modules.

[0238] The reaction scheme is as follows:

[0239] [Chemical formula 4]

[0240]

[0241] The reaction mixture comprises:

[0242] Module 1: 1 ODU / ml BL21-YvgN cells.

[0243] Module 2: 1 ODU / ml BL21-PtxD cells (except for pre-test)

[0244] Substrate: 10 mM glyoxal

[0245] Cofactor: 0.4 mM NADPH

[0246] Complementary substrate: 10 mM phosphite

[0247] Buffer solution: 50 mM HEPES pH 7.5 and 10 mM KCl.

[0248] In preliminary tests, the production of aldehydes by periplasmic glyoxal reductase was performed alone, without a regeneration system. In a second step, the reaction was started by adding BL21-PtxD inducing cells at a concentration of 1 ODU and 10 mM of phosphite to the reaction mixture in order to regenerate the produced NADP into NADPH. The reaction was placed at a temperature of 37°C and stirred at 200 rpm. Measurements were performed at 0 h, 1 h, 3 h and 5 h.

[0249] Figure 7 The results in the study showed that glycolaldehyde was detected as early as 1 hour after the reaction, both with and without the regeneration system, but the yield had already reached a plateau in the absence of PtxD due to the consumption of NADPH. In contrast, in the presence of the BL21-PtxD module, the reaction continued due to the regeneration of NADPH. After 5 hours, glycolaldehyde production was 25 times higher, with a yield of 37%.

[0250] Therefore, the BL21-YvgN and BL21-PtxD modules efficiently produce glycolaldehyde from glyoxal in the periplasmic region without adversely affecting the bacterial cells. This indicates that aldehyde compounds can be produced from toxic precursors by the method according to the present invention.

[0251] Example 5: Production of alcohol from amino acids

[0252] A reaction scheme has been developed for the production of 2-phenylethanol from L-phenylalanine. The reaction scheme is based on the use of four separate modules. Three modules were constructed to express the Saccharomyces cerevisiae ARO8, ARO10 and ADH5 genes, respectively, the first module encoding L-phenylalanine ketoglutarate aminotransferase, the second module encoding phenylpyruvate carboxylase, and the third module encoding 2-phenylethanol dehydrogenase. A fourth module expressing phosphite dehydrogenase PtxD was designed for NADH regeneration.

[0253] The reaction scheme is as follows:

[0254] [Chemical formula 5]

[0255]

[0256] The third reaction generates the target compound, which is coupled to the regeneration reaction of the cofactor NADH from the byproduct NAD+.

[0257] The reaction mixture is as follows:

[0258] Module 1: 5OD / ml BL21-ARO8 cells

[0259] Module 2: 5OD 600nm / ml BL21-ARO10 cells

[0260] Module 3: 5OD 600nm / ml BL21-ADH5 cells

[0261] Module 4: 5OD 600nm / ml BL21-PtxD cells

[0262] Substrate: L-phenylalanine (10 mM)

[0263] Cofactors: 2-ketoglutarate (10mM), MgCl2 (5mM), ZnCl2 (5mM), thiamine (0.2mM), pyridoxal (0.1mM), NADH (1mM)

[0264] Complementary substrate: phosphite (100 mM)

[0265] Buffer solution: HEPES pH 7 (100 mM).

[0266] 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.

[0267] 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 the consortium was tested and validated by measuring the appearance of 2-phenylethanol by HPLC. Fig.13 ]The results showed that 65 mg / l of 2-phenylethanol was synthesized in 5 hours of reaction.

[0268] Example 6: Production of rare sugar isomers

[0269] Rare sugars are mainly produced by isomerization of common sugars. The difficulty is that isomerization is a reversible reaction. The goal here is to limit the reverse reaction by converting the rare sugar formed into a new compound such as a phosphorylated sugar, which is then dephosphorylated to obtain the target isomer.

[0270] In this embodiment, 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 as a cofactor. The reaction scheme continues with two downstream reactions, i.e., a reaction that consumes the formed ADP and acetyl phosphate to regenerate ATP, and a dephosphorylation reaction of D-xylulose-5-phosphate to return D-xylulose.

[0271] Four different modules are used.

[0272] - The first module (BL21-XylA) is an E. coli strain transformed to express the xylose isomerase XylA present in E. coli in its periplasm.

[0273] - The second module (BL21-XylB) is an E. coli strain transformed to express the E. coli kinase XylB, which phosphorylates xylulose to xylulose-5P by consuming ATP molecules.

[0274] - Use the BL21-AckA module presented above to regenerate ATP.

[0275] - The fourth module (BL21-AphA) is an E. coli strain transformed to express the E. coli broad-spectrum phosphatase AphA in its periplasm (Passariello et al., 2006).

[0276] The complete reaction scheme is shown below.

[0277] [Chemical formula 6]

[0278]

[0279] The modules were cultured in TB medium and induced with IPTG, then recovered for D-xylose production. The reaction mixture contained:

[0280] Module 1: 8 ODU / ml BL21-XylA cells

[0281] Module 2: 8 ODU / ml BL21-XylB cells

[0282] Module 3: 8 ODU / ml BL21-AckA cells

[0283] Module 4: 8 ODU / ml BL21-AphA cells

[0284] Substrate: D-xylose (300 mM)

[0285] Cofactor: ATP (5mM)

[0286] Complementary substrate: acetyl phosphate (125 mM)

[0287] Buffer solution: HEPES pH 7.5 (50 mM), MgSO4 (5 mM), MnSO4 (3 mM)

[0288] The method was started with modules 1, 2 and 3, each expressing an enzyme in the reaction mixture. The test tube was placed at 37°C and stirred at 200 rpm. Module 4 expressing AphA phosphatase was added 4 hours after the start of the reaction, and the pH was adjusted to 6. The reaction was monitored at 7 hours and 22 hours. At the same time, the reaction was carried out using the BL21-Xyla module alone to evaluate the isomerization reaction alone. The D-xylose concentration during the reaction was monitored by HPIC ( Figure 8 ) and D-xylulose concentration ( Fig. 9 ) changes.

[0289] Figure 8 The results showed that the consumption of D-xylose substrate was similar in both protocols. However, after 4 h, the D-xylulose yield of the complete reaction was two times lower than that of the isomerization reaction alone ( Fig. 9 ). The reason for this difference is that the production of D-xylulose-5P in the full protocol leads to the consumption of D-xylulose. Its presence could not be detected by the D-xylulose assay but was confirmed by mass spectrometry.

[0290] Module 4 carrying the phosphatase AphA was added to the reaction mixture at T = 4 hours, catalyzing the dephosphorylation of D-xylulose-5P to D-xylulose. Thus, an increase of 2 g / ml and 8 g / l of D-xylulose was observed 3 and 18 hours after the introduction of the phosphatase, respectively. The amount of D-xylulose in the complete reaction (12.8 g / l) exceeded that in the isomerization reaction alone (10.2 g / l), a significant increase of 2.6 g / l.

[0291] The reason is that during the isomerization reaction, a balance is created between the two substances, D-xylose and D-xylulose, so D-xylose cannot be completely converted. The presence of the BL21-XylB kinase module in the reaction mixture can limit D-xylulose to the phosphorylated form, which can no longer be converted into D-xylose. D-xylulose-5P accumulates in the reaction mixture, and the addition of the BL21-AphA phosphatase can improve the recovery of D-xylulose compared to the separate isomerization. By pulling the isomerization to the phosphorylated form of the rare sugar, a higher conversion rate will be obtained.

[0292] These results first show that isomerization, phosphorylation and dephosphorylation by BL21-XylA, BL21-XylB and BL21-AphA modules are feasible. They also prove that four different modules work together to achieve complex reaction schemes, including ATP regeneration mechanisms. Therefore, in the case of the method as the object of the present invention, the coordinated and sequential use of all types of modules has been verified.

[0293] Example 7: Production of D-fructose-1,6-diphosphate from D-xylose

[0294] The conversion of D-xylose to D-fructose-1,6-bisphosphate is part of the pentose phosphate pathway, which is the natural assimilation pathway of D-xylose into the cytoplasm. The conversion is performed by a reaction scheme implementing nine reactions carried out by eight different modules.

[0295] The eight modules used were composed of E. coli strains, each transformed to express in its periplasm an enzyme present in E. coli:

[0296] 1-Xylose isomerase XylA, which isomerizes D-xylose to D-xylulose

[0297] 2-Xylulose kinase XylB, which phosphorylates D-xylulose to D-xylulose-5P

[0298] 3-Acetate kinase AckA, which regenerates ATP from ATP and acetyl phosphate

[0299] 4-Ribulose-phosphate-3-epimerase Rpe, which converts D-xylulose-5P to D-ribulose-5P

[0300] 5-Ribose-5-phosphate isomerase RpiA, which produces D-ribose-5P from D-ribulose-5P

[0301] 6-Transketolase Tkt, which converts D-xylulose-5P and D-ribose-5P to D-glyceraldehyde-3P and D-sedoheptulose-7P

[0302] 7-Transaldolase Tal, which converts D-glyceraldehyde-3P and D-sedoheptulose-7P to D-fructose-5P and D-erythrose-4P

[0303] 8-6-phosphofructokinase 1PfkA, which converts D-fructose-5P to D-fructose-1,6bP

[0304] The complete reaction scheme is shown below.

[0305] [Chemical formula 7]

[0306]

[0307] The presence of enzymes in the periplasm of the eight modules was verified by recovering the periplasmic fraction of each strain by osmotic shock. Table 4 gives the size of the enzymes and the amount of cells used for osmotic shock (OD 600nm multiplied by the culture volume).

[0308] [Table 4]: Molecular weight (kilodaltons) of proteins XylA, XylB, AckA, Rpe, RpiA, TktA, TalA and PfkA, and optical density of cultures used for osmotic shock

[0309] protein XY Xb AHr Rpe R TKMv TalA PcqI Size (kDa) 44-49 52 43.3 24 22-27 73 35 35 OD 600nm 2.84 2.04 9.2 2.38 1.86 4.46 1.34 8.74

[0310] Fig.10 Results of SPS-PAGE gel analysis are presented. They show that expression of the enzymes expected from the eight modules was verified in the periplasm.

[0311] The modules were cultured in TB medium and induced with IPTG and then recovered to produce D-fructose-1,6bP in a reaction mixture comprising:

[0312] Module 1: 8 ODU / ml BL21-XylA cells

[0313] Module 2: 8 ODU / ml BL21 XylB cells

[0314] Module 3: 8 ODU / ml BL21 AckA cells

[0315] Module 4: 8 ODU / ml BL21-Rpe cells

[0316] Module 5: 8 ODU / ml BL21-RpiA cells

[0317] Module 6: 8 ODU / ml BL21-Tkt cells

[0318] Module 7: 8 ODU / ml BL21-Tal cells

[0319] Module 8: 8 ODU / ml BL21-PfkA cells

[0320] Substrate: 100 mM D-xylose

[0321] Cofactor: 3mM ATP

[0322] Complementary substrate: 100 mM acetyl phosphate

[0323] Buffer solution: HEPES pH 7.5 (50 mM), MgSO4 (20 mM), MnSO4 (1 mM) and thiamine pyrophosphate (1 mM) CoCl2 (0.1 mM)

[0324] D-fructose-1,6bP was measured immediately, 1 hour, 3 hours and 4 hours after the modules were added to the reaction mixture. Two controls were performed, one without substrate and the other without modules (the eight modules were replaced with BL21 WT cells). Fig.11 After 1 hour of reaction, the production of D-fructose-1,6-bisphosphate was 1.3 g / l, reaching a maximum of 2.8 g / l within 3 hours. No production of D-fructose-1,6-bisphosphate was measured in either control reaction.

[0325] D-Sedoheptulose-7-phosphate ( Fig.12 ). In fact, the XylB and PfkA carrier modules catalyze the only irreversible reaction in the reaction scheme, D-sedoheptulose-7-phosphate, which is located in the middle of a reaction chain consisting of reversible reactions, the products of which are in equilibrium with each other. D-sedoheptulose-7-phosphate was determined immediately, 1 hour, 3 hours and 4 hours after the modules were added to the reaction mixture. It was 130 mg / l after 4 hours in the complete reaction, but was absent in the control sample. Other intermediate compounds were difficult to detect with the analytical method used, but the level of D-sedoheptulose-7P was representative of their concentrations and was therefore similar. Thus, D-fructose-1,6-bisphosphate was successfully produced according to a complex reaction scheme.

[0326] These results show that the method of the invention based on reaction externalization works efficiently even for long and branched reaction chains involving many modules. It is also particularly advantageous to be able to reconstruct the native complex metabolic pathways of the cytoplasm in this way.

[0327] Example 8: Effect of a combination of two bacteria each expressing one enzyme on product (G3P) production compared to one bacterium expressing both enzymes

[0328] The production of glycerol-3-phosphate is based on the catalytic activity of glycerol kinase and acetate kinase. In order to demonstrate the value of using a synthetic consortium (two bacteria each expressing a different enzyme) compared to bacteria expressing two 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 this end, the plasmid pETDuet-pelB-glpK-pelB-ackA was constructed and transformed into E. coli, and the plasmid contains two cloning sites and is controlled by two T7 promoters. This biocatalyst is referred to as BL21-GLK-AckA in this article. The design of the vector pETDuet makes the expression cassette of pETDuet identical to that of pET22b. Therefore, the purpose of the comparison of strains BL21-AckA, BL21-GlpK and BL21-GlpK-AckA is to achieve the effect of co-expression without being affected by promoters, RBS or signal peptides.

[0329] After osmotic shock, the concentration of each strain was 2OD 600nm The periplasmic fractions of strains BL21-AckA and BL21-AckA-GlpK were recovered from 2 ml cell suspensions of 100 μg / mL. We observed that the 43 kDa AckA band in BL21-AckA-GlpK was at least two times lower than that in BL21-AckA. This result demonstrates that the co-expression of the two enzymes in the same bacterium has a negative impact on the production of the enzyme AckA in the periplasm of the biocatalyst BL21-AckA-GlpK (a bacterium expressing both enzymes). By comparing the 56 kDa GlpK bands of strains BL21-GlpK and BL21-GlpK-AckA, we also observed that the co-expression of the two enzymes in the same bacterium has a negative impact on the production of the periplasmic GlpK.

[0330] Finally, we extracted the periplasmic fraction from the mixture of BL21-AckA and BL21-GlpK (the bacterial consortium of the present invention), where the concentration of each strain was 1 OD 600nm , conditions close to standard co-culture conditions. AckA and GlpK bands were more pronounced in the co-culture (combination according to the invention) than in the periplasmic fraction extracted from the biocatalyst co-expressing AckA and GlpK (one bacterium expressing both enzymes). [ Fig.14 ] These results indicate that better expression of each enzyme can be obtained using a bacterial consortium compared to using the same bacteria co-expressing both enzymes.

[0331] We also observed that the signal peptide pelB made the production of enzyme AckA better than that of enzyme GlpK in the periplasm of strains BL21-AckA and BL21-GlpK, respectively. This observation is not surprising, as 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 reaction efficiency by increasing the amount of BL21-GlpK to compensate for the low efficiency of PelB. This strategy can easily increase the production of glycerol-3P, which is beneficial for industrial use. In contrast, the biocatalyst BL21-GlpK-AckA, which co-expresses the enzymes GlpK and AckA, does not provide this modularity. Each expression cassette (promoter and / or RBS and / or signal peptide) must be rebuilt to regulate the expression of GlpK and AckA; this is more complicated and time-consuming.

[0332] We also compared the production of G3P by the biocatalyst BL21-GlpK-AckA (a bacterium expressing both enzymes) and the synthetic consortium BL21-GlpK and BL21-AckA (according to the present invention). The reaction mixture consisted of:

[0333] Module: BL21-GlpK and / or BL21-AckA and / or BL21-GlpK-AckA cells (OD 600nm总计 =2)

[0334] Substrate: 160 mM glycerol

[0335] Cofactors: 5mM ATP, 15mM MgSO4

[0336] Complementary substrate: 220 mM acetyl phosphate

[0337] Buffer solution: 50 mM HEPES buffer, pH 7.5

[0338] The 5 ml reaction mixture was placed at 37°C and stirred at 200 rpm for 24 hours. 200 μL samples were taken at T0h, T0.5h, T1h, T2h, T3h, T5h and T24h.

[0339] The BL21-GlpK-AckA biocatalyst was able to produce 2.1 grams of G3P per liter in 24 hours. However, by using a mixture of the two biocatalysts, BL21-GlpK and BL21-AckA, in equal proportions (50% each), it was possible to produce 5 grams of G3P per liter in 24 hours, or more than 2 times the yield compared to a system that co-expressed both enzymes in one bacterium. Furthermore, by adjusting the ratio of the two biocatalysts, it was possible to further optimize G3P production. In fact, using a mixture of 92.5% BL21-GlpK and 7.5% BL21-AckA, G3P production reached 10 grams per liter in 5 hours. [ Fig.15 ]. These results show that by adjusting the ratio of BL21-GlpK and BL21-AckA biocatalysts, the bioproduction of G3P can be significantly increased. A significant advantage of using the consortium according to the invention is that the ratio of each biocatalyst can be easily adjusted in an easier and more reliable manner than using complex genetic engineering in the same bacterium co-expressing multiple enzymes.

[0340] Example 9: Production of Extracellular ATP

[0341] The purine adenosine 5'-triphosphate (ATP) is not only a universal intracellular energy transporter, but also plays an important role as an extracellular signaling molecule. It is well known that ATP is 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 bacterial ATP secretion and its extracellular effects remain to be elucidated (Spari D and Beldi G, 2020).

[0342] Alvarez et al. demonstrated that in E. coli, DH5αATP is rapidly hydrolyzed in the periplasmic space. The steady-state extracellular ATP concentration in the periplasmic space was determined to be 24 ± 3 μM / 10 10 Bacteria. (Alvarez et al., 2017).

[0343] In the case of the production of phosphorylated molecules (e.g. glucose-6-phosphate), ATP is an essential cofactor for phosphorylation. However, ATP is an expensive molecule to produce and generally needs to be regenerated from cheaper molecules (e.g. ADP).

[0344] The use of an ATP regeneration module (e.g. BL21-AckA) enables the reuse of trace amounts of ATP present in the periplasm for the production of phosphorylated molecules. This reduces production costs and makes efficient use of the cell's natural resources.

[0345] Therefore, the method according to the present invention enables the production of G6P without the addition of commercially available ATP.

[0346] The first reaction mixture comprises:

[0347] Module 1: 2 ODU / ml BL21-Glk cells

[0348] Module 2: 2 ODU / ml BL21-AckA cells

[0349] Substrate: 300 mM glucose

[0350] Complementary substrate: 250 mM acetyl phosphate

[0351] Buffer solution: HEPES (50 mM) and MgSO4 (15 mM). When the cell concentration of each biocatalyst is 2 OD600nm / ml, it will be able to produce 4 g / l of glucose-6-phosphate without adding ATP. This shows that ATP production is sufficient to support glucose-6P production at this cell density.

[0352] The number of E. coli cells per 1 OD 600nm depends on many factors, including growth conditions, culture medium, and bacterial strain used. However, there is an empirical correlation between the optical density at 600nm and the number of bacterial cells. In general, for E. coli, an optical density of 1 at 600nm corresponds to approximately 8 x 10 8 The cell concentration of cells / ml (https: / / www.agilent.com / store / biocalculators / calcODBacterial.jsp). Fig.16 Under the conditions of , the optical density of each biocatalyst at 600 nm was 2 / ml, and the cell concentration was 3.2 x 10 9 Considering the 24 ± 3 μm / 10 provided by Alvarez et al. 10 The ATP concentration of each bacterium, the ATP concentration in the test would be 7.68 μM. This amount of ATP is capable of synthesizing 4 g / L glucose-6P or 15,000 μM; the BL21-AckA module is able to exceed the possible production of naturally occurring extracellular ATP by 2000 times. These results show that the method of the present invention is able to optimize trace amounts of cofactors for bioproduction, which is particularly interesting from an economic point of view.

[0353] References

[0354] Alvarez, C.L., G. Corradi, N. Lauri, I. Marginedas-Freixa, M.F. Leal Denis, N. Enrique, S.M. Mate, V. Milesi, M.A. Ostuni, V. Herlax, and P.J. Schwarzbaum. 2017. "Dynamic regulation of extracellular ATP in Escherichia coli." Biochem J 474(8): 1395-1416. doi: 10.1042 / bcj20160879.

[0355] Brass, J.M, C.F. Higgins, M. Folley, P.A. Rugman, J. Birmingham, and P.B. Garland. 1986. Lateral diffusion of proteins in the periplasm of Escherichia coli. J. Bacteriol. 165: 787–794.

[0356] Chung, C.T., and R.H. Miller. 1993. "Preparation and storage of competent Escherichia coli cells." Methods Enzymol 218: 621-7. doi: 10.1016 / 0076-6879(93)18045-e.

[0357] Crans, Debbie C., and George M. Whitesides. 1983. "A convenient synthesis of disodium acetyl phosphate for use in situ ATP cofactor regeneration." The Journal of Organic Chemistry 48(18): 3130-3132. doi: 10.1021 / jo00166a048.

[0358] Dudek, Hanna M., Petra Popken, Edwin van Bloois, Wouter A. Duetz, and Marco W. Fraaije. 2013. "A Generic, Whole-Cell–Based Screening Method for Baeyer-Villiger Monooxygenases." Journal of Biomolecular Screening 18(6): 678-687. doi:10.1177 / 1087057113480390.

[0359] Eichmann J, Oberpaul M, Weidner T, Gerlach D, Czermak P. Selection of High Producers From Combinatorial Libraries for the Production of Recombinant Proteins in Escherichia coli and Vibrio natriegens. Front Bioeng Biotechnol. 2019 Oct 4; 7: 254. doi:10.3389 / fbioe.2019.00254. PMID: 31637238; PMCID: PMC6788121.

[0360] Karyolaimos A, Ampah-Korsah H, Hillenaar T, Mestre Borras A, Dolata K M, Sievers S, Riedel K, Daniels R, de Gier JW. Enhancing recombinant protein yields in the E. coli periplasm by combining signal peptide and production rate screening. Front Microbiol. 2019; 10: 1511.

[0361] Mergulhao FJ, Monteiro GA. 2007. Periplasmic targeting of recombinant proteins in Escherichia coli. Methods in Molecular Biology 390: 47-61.

[0362] Passariello, C., C. Forleo, V. Micheli, S. Schippa, R. Leone, S. Mangani, M. C. Thaller, and G. M. Rossolini. 2006. "Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655." Biochim Biophys Acta 1764(1): 13 - 9. doi:10.1016 / j.bbapap.2005.08.028.

[0363] Popp, A., H. T. Nguyen, K. Boulahya, C. Bideaux, S. Alfenore, S. E. Guillouet, and E. Nevoigt. 2008. "Fermentative production of L - glycerol 3 - phosphate utilizing a Saccharomyces cerevisiae strain with an engineered glycerol biosynthetic pathway." Biotechnol Bioeng 100(3): 497 - 505. doi:10.1002 / bit.21777.

[0364] Spari D, Beldi G. Extracellular ATP as an Inter - Kingdom Signaling Molecule: Release Mechanisms by Bacteria and Its Implication on the Host. Int J Mol Sci. 2020 Aug 4; 21(15): 5590. doi:10.3390 / ijms21155590. PMID: 32759857; PMCID: PMC7432876.

[0365] Stock, J. B., B. Rauch, and S. Roseman. 1977. Periplasmic space in Salmonella typhimurium and Escherichia coli. J. Biol. Chem. 252: 7850–7861.

[0366] Wilks JC,Slonczewski JL.pH of the cytoplasm and periplasm ofEscherichia coli:rapid measurement by green fluorescent protein fluorimetry.JBacteriol.2007Aug;189(15):5601-7.doi:10.1128 / JB.00615-07.Epub 2007Jun 1.PMID:17545292;PMCID:PMC1951819.

[0367] Zhou Y,Wang L,Yang F,Lin X,Zhang S,Zhao ZK.Determining the extremesof the cellular NAD(H)level by using an Escherichia coli NAD(+)-auxotrophicmutant.Appl Environ Microbiol.2011Sep;77(17):6133-40.doi:10.1128 / AEM.00630-11.Epub2011Jul 8.PMID:21742902;PMCID:PMC3165392。

Claims

1. A method for producing a target compound from an organic substrate, characterized in that It includes the following steps: a) - preparing a reaction mixture in a suitable medium, said reaction mixture comprising, - n bacteria, n being an integer at least equal to 2, each bacterium being genetically modified to express in its periplasmic space at least one enzyme E1, E2, ..., En, preferably a single enzyme, each of said n bacteria expressing an enzyme different from that of the other bacteria, - an enzyme E1 capable of catalyzing a first reaction R1 to produce a first product P1 and optionally a first by-product CoP1 from said organic substrate, - each of the enzymes E2, ..., En is capable of catalyzing the reactions R2, ..., Rn to produce the products P2, ..., Pn and optionally the by-products CoP2, ..., CoPn, respectively, as the product or by-product obtained from the reaction Rn-1, - said organic substrate, and - Optionally, the cofactor of the organic substrate, the cofactor of the product obtained by reaction Rn-1, is called cofactor CoF1, CoF2, ..., CoFn; the complementary substrate SC of the by-product obtained by reaction Rn-1, b) reacting the reaction mixture thus obtained, and c) separating the biomass from the supernatant and extracting therefrom said target compound consisting of one of the products P1, P2, ..., Pn.

2. The method according to claim 1, characterized in that The n genetically engineered bacteria are Gram-negative double-membrane bacteria, each selected from the family Enterobacteriaceae, Alcaligenaceae, Vibrionaceae or Pseudomonadaceae.

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, comprising one of the enzymes E1, E2, ..., En, respectively, linked to a signal peptide that localizes the polypeptide to the periplasmic space of the bacterium.

4. The method according to the preceding claim, characterized in that One or more of the n bacteria are genetically modified to express polypeptides, each of which comprises one of the enzymes E1, E2, ..., En, which are linked to a membrane anchor peptide comprising the signal peptide.

5. A method according to any one of the preceding claims, characterised in that 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, and the enzymes are capable of catalyzing at least two reactions Rn-a and Rn-b, so that the reactions RN-a and RN-b are carried out in the periplasmic space of the at least one bacterium.

6. A method according to any one of the preceding claims, characterised in that The reaction mixture comprises bacteria expressing a first enzyme E1 and bacteria expressing a second enzyme E2, wherein the first enzyme E1 is capable of catalyzing a first reaction R1 to obtain a first product P1 from the organic substrate, and the second enzyme E2 is capable of catalyzing a second reaction R2 to form a second product P2 from the first product P1, and the second product P2 is recovered as a target compound in step c) or consumed as a substrate for a third reaction.

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

8. A method according to any one of the preceding claims, characterised in that The reaction mixture comprises the cofactor CoF1 of the organic substrate, bacteria expressing enzyme E1 and bacteria expressing a second enzyme E2, wherein the enzyme E1 is capable of catalyzing a first reaction R1 to form a first product P1 and a first byproduct CoP1 from the organic substrate and the cofactor CoF1, and the second enzyme E2 is capable of catalyzing a second reaction R2 to form a second product P2 from the first product P1, or to form a second byproduct CoP2 from the first byproduct CoP1, wherein at least one of the two is consumed in the third reaction, or is recovered as a target compound in step c).

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

10. The method according to the preceding claim, characterized in that The reaction mixture may further comprise at least one substrate SCn complementary to the at least one cofactor CoFn.

11. The method according to any one of the preceding claims, characterized in that The at least one cofactor CoFn can be introduced fully or partly into the reaction mixture in step a) or in step b).

12. A method according to any one of the preceding claims, characterised in that Said 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 byproduct CoPn-1, said byproduct CoPn-1 being identical to the cofactor CoFn.

13. The method according to the preceding claim, 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 at least 20 times lower than the initial molar concentration of the organic substrate.

14. A method according to any one of the preceding claims, characterised in that The first reacted organic substrate is selected from the group consisting of carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons, amino acids and carbon dioxide.

15. A method according to any one of the preceding claims, characterised in that The enzymes E1, E2, . . . , En are each selected from the group consisting of kinases, dehydrogenases, phosphatases, reductases, isomerases and transferases.

16. A method according to any one of the preceding claims, characterised in that At least one of the enzymes E1, E2, ..., En catalyzes a reaction that provides one of the byproducts CoP1, CoP2, ..., CoPn, wherein the byproduct CoP1, CoP2, ..., CoPn is selected from ATP, ADP, AMP, UTP, UDP, UMP, NAD+, NADH, NADP+, NADPH, FAD, FADH2, coenzyme A, or catalyzes a reaction that uses one of them as a cofactor.

17. A method according to any one of the preceding claims, characterised in that After step c) of the process object of the present invention, the biomass separated from the supernatant is reused for the preparation of a new reaction mixture according to step a), with or without an intermediate storage step.

18. A method according to any one of the preceding claims, characterised in that Steps a) and b) are performed by continuously or intermittently adding to the reaction mixture containing the n bacteria - Optionally, the cofactor of the organic substrate, the cofactor of the product obtained by reaction Rn-1, is called cofactor CoF1, CoF2, ..., CoFn; the complementary substrate SC of the by-product obtained by reaction Rn-1, and the reaction mixture obtained thereby is reacted continuously or semi-continuously.

19. A reaction mixture for producing a target compound from an organic substrate, characterized in that It comprises in a suitable medium: - n bacteria, n being an integer at least equal to 2, each bacterium being genetically modified to express in its periplasmic space at least one enzyme E1, E2, ..., En, preferably a single enzyme, each of said n bacteria expressing an enzyme different from that of the other bacteria, - an enzyme E1 capable of catalyzing a first reaction R1 to produce a first product P1 and optionally a first by-product CoP1 from said organic substrate, - each of the enzymes E2, ..., En is capable of catalyzing the reactions R2, ..., Rn to produce the products P2, ..., Pn and optionally the by-products CoP2, ..., CoPn, respectively, as the product or by-product obtained from the reaction Rn-1, - said organic substrate, and - Optionally, the cofactor of the organic substrate, the cofactor of the product obtained by reaction Rn-1, is referred to as cofactor CoF1, CoF2, ..., CoFn; the complementary substrate SC of the by-product obtained by reaction Rn-1.

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

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