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US-20260125713-A1 - PRODUCTION OF MOLECULES BY PERIPLASMIC ENZYMATIC CATALYSIS

US20260125713A1US 20260125713 A1US20260125713 A1US 20260125713A1US-20260125713-A1

Abstract

A process for producing a compound of interest from an organic substrate, including the steps of: a) preparing a reaction mixture including at least two bacteria genetically modified to express, in their periplasmic space, at least one enzyme, each of said bacteria expressing an enzyme different from the other bacteria, an enzyme being capable of catalyzing a first reaction using said organic substrate so as to provide a first product, each of the enzymes being capable of catalyzing a reaction using a product or a coproduct obtained by a reaction, so as to provide respectively a product, b) leaving the reaction mixture to react, and c) separating the biomass from the supernatant and extracting therefrom said compound of interest consisting of one of the products.

Inventors

  • CLÉA LACHAUX
  • Eric Grondin
  • Yvan Cam

Assignees

  • BIOC3

Dates

Publication Date
20260507
Application Date
20230504
Priority Date
20220506

Claims (20)

  1. 1 . A process for producing a compound of interest from an organic substrate, comprising the steps of: a) preparing a reaction mixture comprising, in a suitable medium, n bacteria, n being an integer at least equal to 2, each bacterium being genetically modified to express in its periplasmic space at least one enzyme E1, E2, . . . En, each of said n bacteria expressing an enzyme different from the other bacteria, an enzyme E1 being able to catalyze a first reaction R1 from said organic substrate to provide a first product P1, each of said enzymes E2, . . . , En, being capable of catalyzing a reaction R2, . . . , Rn, from a product or a coproduct obtained by a reaction Rn−1, to provide, respectively, a product P2, . . . , Pn, said organic substrate, b) allowing the reaction mixture thus obtained to react, and c) separating the biomass from the supernatant and extracting therefrom said compound of interest consisting of one of the products P1, P2, . . . , Pn.
  2. 2 . The process according to claim 1 , wherein said n genetically modified bacteria are Gram-negative diderm bacteria, each chosen from the Enterobacteriaceae, Alcaligenaceae, Vibrionaceae, or Pseudomonadaceae family.
  3. 3 . The process according to claim 1 , wherein each of the n bacteria is genetically modified to express at least one polypeptide comprising, respectively, one of said enzymes E1, E2, . . . , En, linked to a signal peptide addressing said polypeptide into the periplasmic space of said bacterium.
  4. 4 . The process according to claim 3 , wherein one or more of said n bacteria are genetically modified to express a polypeptide comprising, respectively, one of said enzymes E1, E2, . . . , En, linked to a membrane anchoring peptide including said signal peptide.
  5. 5 . The process according to claim 1 , wherein at least one of said n bacteria is genetically modified to express in its periplasmic space at least two enzymes En-a and En-b capable of catalyzing at least two reactions Rn-a and Rn-b, so that said reactions RN-a and RN-b occur in the periplasmic space of said at least one bacterium.
  6. 6 . The process according to claim 1 , wherein the reaction mixture comprises a bacterium expressing a first enzyme E1 capable of catalyzing the first reaction R1 from said organic substrate to provide a first product P1, and a bacterium expressing a second enzyme E2 capable of catalyzing a second reaction R2 from said first product P1, to form a second product P2, which is recovered in step c) as a compound of interest, or is consumed as a substrate of a third reaction.
  7. 7 . The process according to claim 6 , wherein the reaction mixture further comprises at least one bacterium expressing an enzyme E3, . . . , En, capable of catalyzing a reaction R3, . . . , Rn, from a product Pn−1 obtained by a reaction Rn−1, to provide a product Pn, which is recovered in step c) as a compound of interest or is consumed as a substrate of a reaction Rn+1.
  8. 8 . The process according to claim 1 , wherein the reaction mixture comprises a cofactor CoF1 of said organic substrate, a bacterium expressing an enzyme E1 capable of catalyzing a first reaction R1 from said organic substrate and the cofactor CoF1 to form a first product P1 and a first coproduct CoP1; and a bacterium expressing a second enzyme E2 capable of catalyzing a second reaction R2 from the first product P1 to form a second product P2, or from the first coproduct CoP1 to form a second coproduct CoP2, at least one of the two being consumed in a third reaction, or recovered in step c) as a compound of interest.
  9. 9 . The process according to claim 8 , wherein the reaction mixture comprises at least one cofactor CoFn, at least one bacterium expressing an enzyme capable of catalyzing a reaction Rn from the product Pn−1 of a reaction Rn−1 and said cofactor CoFn to form a product Pn and a coproduct CoPn, at least one of the two being consumed in a reaction Rn+1, or recovered in step c) as a compound of interest.
  10. 10 . The process according to claim 9 , wherein the reaction mixture can also comprise at least one substrate SCn complementary to said at least one cofactor CoFn.
  11. 11 . The process according to claim 1 , wherein said at least one cofactor CoFn can be introduced entirely or partially into the reaction mixture in step a), or during step b).
  12. 12 . The process according to claim 1 , wherein at least one cofactor CoFn is formed at least in part in the reaction mixture by a reaction Rn−1 catalyzed by an enzyme En−1 capable of forming a product Pn−1 and a coproduct CoPn−1, said coproduct CoPn−1 being identical to the cofactor CoFn.
  13. 13 . The process according to claim 12 , wherein the quantity of the first cofactor CoF1 introduced into the reaction mixture in step a) corresponds to a molar concentration at least 20 times lower than the initial molar concentration of said organic substrate.
  14. 14 . The process according to claim 1 , wherein the organic substrate of the first reaction is chosen from carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons, amino acids, and carbon dioxide.
  15. 15 . The process according to claim 1 , wherein the enzymes E1, E2, . . . , En are each chosen from kinases, dehydrogenases, phosphatases, reductases, isomerases, and transferases.
  16. 16 . The process according to claim 1 , wherein at least one of the enzymes E1, E2, . . . , En catalyzes a reaction providing one of the coproducts CoP1, CoP2, . . . , CoPn, chosen from ATP, ADP, AMP, UTP, UDP, UMP, NAD+, NADH, NADP+, NADPH, FAD, FADH 2 , coenzyme A, or catalyzes a reaction using one of them as cofactor.
  17. 17 . The process according to claim 1 , wherein after step c) of the process that is the object of the present invention, the biomass separated from the supernatant is reused to prepare a new reaction mixture according to step a), with or without an intermediate preservation step.
  18. 18 . The process according to claim 1 , wherein steps a) and b) are carried out by adding to the reaction mixture containing said n bacteria, continuously or at intervals of time, a cofactor of said organic substrate, a cofactor of said products obtained by a reaction Rn−1, called cofactors CoF1, CoF2, . . . , CoFn; a complementary substrate SC of a coproduct obtained by a reaction Rn−1, and by allowing the reaction mixture thus obtained to react continuously or semi-continuously.
  19. 19 . A reaction mixture for the production of a compound of interest from an organic substrate, wherein it contains, in a suitable medium: n bacteria, n being an integer at least equal to 2, each bacterium being genetically modified to express in its periplasmic space at least one enzyme E1, E2, . . . En, each of said n bacteria expressing an enzyme different from the other bacteria, an enzyme E1 being able to catalyze a first reaction R1 from said organic substrate to provide a first product P1, each of said enzymes E2, . . . , En, being capable of catalyzing a reaction R2, . . . , Rn, from a product or a coproduct obtained by a reaction Rn−1, to provide, respectively, a product P2, . . . , Pn, and optionally a coproduct CoP2, . . . , CoPn, and said organic substrate.
  20. 20 . A method for producing a compound of interest chosen from carbohydrates, aldehydes, alcohols, organic acids, carbamates, hydrocarbons, and amino acids, which comprises a step using the reaction mixture according to claim 19 .

Description

The present invention belongs to the field of production of molecules of interest by genetically modified microorganisms, and more particularly concerns the production of compounds by enzymatic catalysis conducted in the bacterial periplasmic space. It relates to a process for the production of a compound of interest involving a mixture of bacteria (consortium), modified to express one or more enzymes in their periplasmic space, which together catalyze a reaction cascade for the production of a compound of interest. The invention also relates to a reaction mixture for the production of such a compound and a kit for preparing said medium. The production of organic compounds by the petrochemical industry raises crucial future problems in terms of cost and environmental impact issues, especially due to the large amounts of carbon released into the atmosphere and the use of non-renewable resources. The production of such compounds by biological pathways appears as a non-polluting and sustainable alternative. It is still necessary to be able to carry out the appropriate reaction schemes and to obtain sufficient quantities in relation to the applications envisaged, according to economically acceptable protocols. Indeed, while some compounds can be obtained by a simple reaction, others require intermediate steps that complicate and increase the cost of the process. In addition, many reactions use cofactors which must be introduced into the reaction medium in a quantity proportional to the substrate to be transformed, which increases the costs accordingly. Currently, various technologies using biological processes are used for the production of molecules. The most classic is the extraction of products of interest, for example flavors or therapeutic molecules, from organisms such as plants, by infusion maceration, extraction by solvent, or other means. However, plant extraction is limited in its applications, because it generally has fairly low yields compared to the mass processed. It uses complex processes to isolate and purify the molecule of interest. Finally, the seasonal or geographical availability of the source organism greatly constrains extraction strategies. Another technique, also well known, is based on the fermentative activity of microorganisms that are may or may not be genetically modified, whose cellular metabolism produces molecules of interest, secreted in a culture medium. The production of alcohol by yeasts is one of the oldest examples. Fermentation has certain advantages. It can be carried out all year round at a moderate cost, since the reactions are carried out by microorganisms. Theoretical yields are generally interesting, as the contribution of synthetic biology and systems biology has greatly increased the efficiency of this type of system. Nevertheless, fermentation has notable disadvantages arising from the living nature of the biocatalyst. Thus, in practice, theoretical yields are difficult to obtain, since the cell always tends to redirect the flows engaged in the production of the molecule of interest toward its biomass. Moreover, the molecule of interest is secreted into a fermentation medium necessarily containing a large number of other molecules, which complicates its separation and consumes a large amount of water that must then be treated. Furthermore, not all molecules can be produced by fermentation, either because of their toxicity to the cell or because little to none of the molecule is excreted. Added to these difficulties is the development time of an industrial fermentative organism, of about ten years on average. Thus, several limitations of production by fermentation can be identified: i) the coupling of growth and production limits the maximum yield of production, ii) the use of synthetic biology to engineer a microorganism and redirect the carbon flow to the product is done at the expense of growth, which impacts the robustness of the process, iii) the operating conditions are limited to conditions compatible with microbial growth (pH, temperature, composition of the medium, oxygenation, etc.), iv) the substrates and products must not be cytotoxic; the maximum concentration of the substrate and the product are therefore fixed according to their toxicity, v) the substrate and product must be able to cross the cytoplasmic membrane, and vi) the product is secreted into a culture medium loaded with organic acids and other by-products, complicating the purification of the product and increasing the overall cost of the process. More recently, whole cell bioproduction techniques have made it possible to overcome several of these disadvantages. Growth (enzyme production phase) and production are decoupled; the substrate is only dedicated to product synthesis. The operating conditions can be optimized to maximize production; compatibility with growth is not required. In addition, the purification of the product is simplified because the product is secreted in a simple mediu