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EP-4735606-A2 - BIOREACTOR AND MICROBE

EP4735606A2EP 4735606 A2EP4735606 A2EP 4735606A2EP-4735606-A2

Abstract

The present invention relates to a bioreactor comprising, within a chamber, a genetically modified microorganism, wherein said microorganism comprises the components necessary for the biochemical conversion of an inorganic carbon source into acetyl-CoA, and a method for controlling said microorganism and thus the production of a microbially-produced biochemical product.

Inventors

  • ORTEGA, DAVID
  • WONG, Jin Xiang
  • SEMEIJN, Koen

Assignees

  • Phase Biolabs Ltd.

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A bioreactor comprising, within a chamber, a genetically modified microorganism, wherein said microorganism comprises the components necessary for the biochemical conversion of an inorganic carbon source into acetyl-CoA, wherein said components comprise: i. a source of reducing equivalents; ii. a light-dependent ion pump that generates an electrochemical ion gradient independently of the generation of reducing equivalents; and iii. a redox-dependent ion pump that generates an electrochemical ion gradient independently of a net change in the number of reducing equivalents.
  2. 2. The bioreactor according to claim 1 , wherein the electrochemical gradient generated by the ion pumps of (ii) and/or (iii) is utilised to generate ATP.
  3. 3. The bioreactor according to claim 1 or claim 2, wherein the inorganic carbon source is CO 2 .
  4. 4. The bioreactor according to any preceding claim, wherein the components comprise those of a linear carbon fixation pathway.
  5. 5. The bioreactor according to claim 4, wherein the linear carbon fixation pathway comprises components of the Wood-Ljungdahl pathway (WLP) required to generate acetyl-CoA.
  6. 6. The bioreactor according to claim 4 or 5, wherein said system comprises NADH- and NADPH-dependent reductases and an electron bifurcating reductase.
  7. 7. The bioreactor according to claim 6, wherein the NADH- and NADPH- dependent reductases are present together with a transhydrogenase that interchanges reducing equivalents from one species to another.
  8. 8. The bioreactor according to any preceding claim, wherein the components comprise one or more of: electron bifurcating hydrogenase, oxygen tolerant hydrogenase, formate dehydrogenase or carbon monoxide dehydrogenase.
  9. 9. The bioreactor according to any preceding claim, wherein the ion pumps of (ii) and (iii) are present as part of a membrane and require energy to pump ions across said membrane from areas of low electrochemical potential to areas of high electrochemical potential, thus generating an electrochemical ion gradient.
  10. 10. The bioreactor according to claim 9, wherein an ATP synthase uses said electrochemical ion gradient generated by the ion pumps of (ii) and/or (iii) to generate ATP.
  11. 11. The bioreactor according to claim 9 or 10, wherein the ion pumps of (ii) and (iii) and the ATP synthase depend preferentially on ions of the same species.
  12. 12. The bioreactor according to claim 9 or 10, wherein the ion pumps of (ii) and (iii) and the ATP synthase depend preferentially on ions of different species, and an antiporter ion pump is present to convert a chemical ion gradient of one species into a chemical ion gradient of a second species, to increase the electrochemical ion gradient that the ATP synthase is dependent upon.
  13. 13. The bioreactor according to any preceding claim, wherein the microorganism further comprises photosynthetic machinery of purple non-sulphur bacteria.
  14. 14. The bioreactor according to any preceding claim, wherein the lightdependant ion pump is a rhodopsin.
  15. 15. The bioreactor according to claim 14, wherein the rhodopsin is a bacteriorhodopsin, proteorhodopsin, deltarhodopsin, xanthorhodopsin, halorhodopsin, channelrhodopsin, archaerhodopsin, or bacterial sensory rhodopsin.
  16. 16. The bioreactor according to any preceding claim, wherein the redoxdependant ion pump is an Rnf or Ech protein complex.
  17. 17. The bioreactor according to any preceding claim, wherein the recombinant microorganism is an acetogen.
  18. 18. The bioreactor according to any of claims 1 to 16, wherein the recombinant microorganism is a purple non-sulphur bacterium.
  19. 19. The bioreactor according to any preceding claim, wherein the microorganism further comprises biochemical components necessary for converting the generated acetyl-CoA into a biochemical product.
  20. 20. The bioreactor according to claim 19, wherein the biochemical product is selected from one of the following compound classes: alcohols, sugars aldehydes, alkaloids, alkanes, alkenes, alkynes, amino acids, amines, aromatics, carboxylic acids, dicarboxylic acids, dienes, diols, esters, ethers, polymeric and monomeric chemicals, isoprenoids, polyketides, surfactants, terpenes, terpenoids, proteins, fats, and other secondary metabolites and/or a combination thereof.

Description

BIOREACTOR AND MICROBE FIELD OF THE INVENTION The present invention relates to a bioreactor for the controlled growth of a genetically modified microorganism, particularly those intended to produce and further utilise acetyl-CoA, and methods for controlling the production of a biochemical product via microorganisms, said microorganisms comprising both a light-dependent ion pump and a redox-dependent ion pump. In particular, the present invention relates to a means for modulating said microorganism using light. BACKGROUND OF THE INVENTION With rising concern about climate change, the conversion of C1 carbon compounds produced as a result of burning fossil fuels is of great commercial and environmental interest due to the large volumes of these compounds generated as an industrial waste. This includes carbon monoxide (CO), methane (CH4) and carbon dioxide (CO2), all of which are gases at ambient temperature. Other C1 compounds include formate (CH2O2) and methanol (CH3OH). The use of microorganisms to reduce industrial waste, for example, by removing C1 carbon compounds such as CO2 (including CO2 captured from the atmosphere, and CO2 produced directly from a process), is known in the art, and has the advantage of reducing industrial waste while simultaneously producing products of commercial interest (Peplow M. The race to upcycle CO2 into fuels, concrete and more. Nature 603, 780-783 (2022); Liew F et al. Gas Fermentation- A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks. Front Microbiol 7, 694 (2016)). Bioprocesses, such as gas fermentation or carbon fixation, can utilise microorganisms to convert a given input into an output/product of higher value. The efficiency, productivity and cost of a bioprocess depends not only on the SUBSTITUTE SHEET (RULE 26) microorganisms utilised to catalyse the bioprocess, but also on the system surrounding and supporting the operation of microorganisms. As such, a bioreactor is a key component in a bioprocess housing any microorganisms. Bioreactors in the art typically involve passing inputs (or ‘feedstocks’) such as liquid (e.g., sugars, methanol) or gas (e.g., carbon dioxide (CO2), hydrogen (H2)) feedstocks through a medium containing a microorganism. Bioreactors which use liquids as the primary input typically require a simple design as liquid inputs can easily be dissolved in aqueous solvents (water) with agitation. In contrast, where gases are the primary input for the bioprocess, this complicates both the design of the bioreactor and the operation of the bioprocess, as efficient mixing of the liquid and gas inputs is required. Further, if the primary inputs are gases, the productivity of the bioprocess can be severely limited by the “mass transfer” achieved by the bioreactor. Mass transfer refers to the ability to dissolve gases in liquid. Bioreactors must be carefully designed according to the type of input, and to match the requirements of the microorganisms housed within them, in order to enhance the efficiency of the bioprocess. The design and functionality of a bioreactor is of upmost importance due to the challenge of achieving efficient mass transfer (i.e. , dissolving gases). The efficiency of mass transfer increases with the use of increasing levels of energy when mixing gases. Therefore, efficient mass transfer is usually achieved using high levels of mixing energy, which results in a process with poor economics. Currently, bioreactors have been developed for gas fermentation processes, and photobioreactors have been developed separately. Bioprocesses involving light will typically utilise/cultivate photosynthetic microorganisms, such as algae or cyanobacteria. Commercial bioprocesses currently exist for: a) Conversion of CO2 and H2 into methane, chemicals and protein using bacteria. b) Conversion of CO2, H2 and O2 into biopolymers, chemicals, carbohydrates, amino acids, and protein using bacteria. c) Conversion of CO, H2 and CO2 into chemicals and protein using bacteria. d) Conversion of CO2 and light into fatty acids, short chain fatty acids, pigments, among other organic compounds using cyanobacteria or algae. With microbial bioprocesses converting CO2 and H2 or CO2, H2 and O2 into a desired product, the microorganisms are able to take up the gases very quickly, and thus the primary limitation is mass transfer of the gases. This is due to the low solubility of H2 (O2 and CO2 are much easier to dissolve in water). While the commercial bioprocesses shown above in items a), b) and c) all face the challenge of mass transfer, the bioreactors used in these processes are materially different to bioreactors using algae. In the former, airlift and loop reactors are commonplace, while in the latter, tubular and raceway ponds (with sunlight) or photobioreactors are used. Bioprocesses that grow or use photosynthetic microorganisms require photobioreactors to provide light while introducing pr