Bioreactor and microbe

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.

Description

[0001] BIOREACTOR AND MICROBE

[0002] FIELD OF THE INVENTION

[0003] 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.

[0004] BACKGROUND OF THE INVENTION

[0005] 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).

[0006] 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

[0007] 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.

[0008] 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.

[0009] 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.

[0010] Commercial bioprocesses currently exist for: a) Conversion of CO2and H2into methane, chemicals and protein using bacteria. b) Conversion of CO2, H2and O2into biopolymers, chemicals, carbohydrates, amino acids, and protein using bacteria. c) Conversion of CO, H2and 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.

[0011] With microbial bioprocesses converting CO2 and H2or CO2, H2and 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 pressurised CO2using spargers typically in an airlift or bubble column design. When photosynthetic microorganisms are grown in raceway ponds, carbon dioxide simply diffuses into the water during gentle mixing while light energy is provided by the sun. Because of the slow autotrophic growth rate of photosynthetic microorganisms, mass transfer of carbon dioxide into the medium is not the primary limitation on productivity. The primary limitation for bioprocesses which use photosynthetic microorganisms is typically related to light energy. Biochemically, this means the efficiency of using light energy for growth, whereas from a bioprocess perspective, this means the delivery and the penetration of light into the medium.

[0012] Photosynthetic microorganisms use light as the energy source to generate the ATP they require for carbon fixation and ultimately growth. However, photosynthetic microorganisms use inefficient carbon fixation pathways (Table 1) and therefore have a high ATP requirement per mole of carbon fixed. In contrast, chemolithotrophs are microorganisms which can use inorganic substrates like hydrogen as a source of energy. In particular, acetogens are able to grow autotrophically using H2as an energy source and CO2as the sole carbon source. Acetogens fix carbon through the extremely efficient Wood-Ljungdahl pathway (WLP) (Figure 1). The WLP is composed of two branches: the carbonyl branch and the methyl branch. In each branch, one molecule of CO2is reduced. In the methyl branch, a molecule of CO2is first reduced to formate and then bound to tetrahydrofolate (THF) generating formyl-THF, requiring one ATP molecule. Formyl-THF is dehydrated to methenyl-THF and sequentially reduced via methylene-THF to yield methyl-THF. In the carbonyl branch, CO2is reduced to enzyme-bound CO in a reaction catalysed by carbon monoxide dehydrogenase / acetyl-CoA synthase complex (CODH / ACS). In the final step of the WLP, the CODH / ACS complex catalyses the synthesis of acetyl-CoAfrom the methyl group generated from the methyl branch and the enzyme-bound CO generated from the carbonyl branch.

[0013] While acetogens use the efficient WLP for carbon fixation, the amount of ATP that they can generate from H2is low, thus limiting their growth rate. Increasingly productive systems (such as acetogenic microorganisms) capable of utilising both redox-mediated and light-mediated biochemical pathways for achieving sufficient ATP synthesis while utilising an energy efficient carbon fixation pathway are described in PCT / EP2022 / 086989. Therefore, using a recombinant acetogen that is able to grow autotrophically using the Wood-Ljungdahl pathway while also generating ATP through light will be able to grow more rapidly and generate organic compounds of interest more efficiently as demonstrated.

[0014] Table 1. Natural (inefficient) carbon fixation pathways.

[0015] In addition, light enables more dynamic level of control of engineered / modified microorganisms being housed within bioreactors. However, combining existing approaches faces a dual challenge of overcoming the requirement for efficient mass transfer of gases (in particular H2) and the requirement for uniform and efficient penetration of light into the bioreactor. Therefore, a bioprocess that uses a light-responsive acetogen requires suitable and carefully designed bioreactors.

[0016] In addition to the need for bioreactors suitable for such bioprocesses, the further fine tuning of these processes to capture and fix carbon sources offers an additional layer of control that is desirable. Microorganisms can sense and respond to their environments by detecting changes to temperature, pH, the presence of a substance (chemicals, sugars, antibiotics, etc.). These sensing systems within the microorganism can be re-engineered into artificial / synthetic control systems, termed biocircuitry. Generally, biocircuitry can be controlled via two forms of induction. Firstly, process induction involves the manipulation of process parameters such as pH and temperature. Secondly, chemical induction involves the presence (or absence) of an organic compound (such as a sugar) or chemical molecule (such as IPTG). Process induction has the advantage of providing more cost effective and enhanced controlled over the biocircuitry.

[0017] Currently, biocircuitry is typically controlled in microorganisms which respond to redox-based or light-based changes to their environment (Shimizu-Sato S et al. A light-switchable gene promoter system. Nat Biotechnol 20, 1041-1044 (2002); Zhang J et al. Engineering an NADPH / NADP+ Redox Biosensor in Yeast. ACS Synth Biol, 5, 12, 1546-1556 (2016)). At present, there is a need to layer an additional, more dynamic level control of microorganisms which will allow for increasingly efficient and easily modulated production of biochemical products.

[0018] Accordingly, it can be seen that developing improved approaches to the use and control of microorganisms in bioreactors for increasingly efficient conversion of C1 carbon compounds, and a method which allows for fine-tuned regulation of these microorganisms and thus the production of a microbially-produced biochemical product is needed in the art. Therefore, it is clear that the provision of new and increasingly efficient bioreactors capable of delivering a variety of inputs to both support and fine-tune the operation of the microorganisms housed within it would provide a contribution to the art. Such bioreactors and the provision of a greater level of control of such microorganisms would prove useful in the reduction of industrial waste gas levels, via the renewable generation of desirable and commercially important products.

[0019] SUMMARY OF THE INVENTION

[0020] The present invention is based on the surprising discovery that a series of designs of bioreactors which are able to achieve efficient (low energy input) mass transfer of CO2 and H2while also providing illumination at the required intensity and wavelength and pattern would be able to provide the inputs necessary for a modified microorganism to generate additional energy (ATP) while simultaneously exerting control over a genetically modified microorganism via a novel mechanism. The design of an efficient bioreactor that can achieve effective delivery of CO2, H2and light depends on three parameters: the geometry of the bioreactor and how liquid is circulated, the introduction and mixing of gases (for mass transfer) and the delivery of light into the vessel.

[0021] Thus, in accordance with a first aspect of the invention, there is provided 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.

[0022] The present invention is also based on the surprising finding that that the production of desired biochemical products via microorganisms genetically engineered to utilise both redox-dependent ion pumps and light-dependent ion pumps can be modulated at an increasingly efficient level using synthetic biocircuitry which responds to light as an inducer. Using light as an inducer allows for the concentration and presence of light-responsive proteins of interest to be modified according to the wavelength of light used and the function of the light- responsive protein. Accordingly, different wavelengths of light, or different durations of exposure to light, can be utilised in combination with unique biocircuitries within microorganisms to increase or decrease a concentration of a protein. Increased protein concentrations may thus be achieved for example by inducing or increasing protein expression, reducing protein degradation, and / or increasing protein stability. Similarly, decreased protein concentrations may be achieved for example by abolishing or decreasing protein expression, increasing protein degradation, and / or decreasing protein stability. Using light as a means for modulating the production of desired biochemical products via microorganisms is beneficial in that it is direct, non-toxic to the cell, and easily programmable.

[0023] Therefore, in a second aspect of the invention, there is provided a method for controlling the production of a microbially-produced biochemical product, wherein the product is derived from the conversion of acetyl-CoA, comprising culturing a genetically engineered microorganism under suitable conditions, said microorganism comprising: (i) a light-dependent ion pump that generates an electrochemical ion gradient independently of the generation of reducing equivalents; and

[0024] (ii) a redox-dependent ion pump that generates an electrochemical ion gradient independently of a net change in the number of reducing equivalents; and wherein said culturing comprises modulating the exposure of the microorganism to light to drive the light-dependent ion pump to control the generation of the electrochemical ion gradient.

[0025] In a third aspect of the invention, there is provided a genetically modified acetogen comprising a recombinant rhodopsin and a recombinant nucleic acid molecule comprising a sequence encoding a light-activated protein, preferably wherein the light-activated protein is partitioned and then reconstituted through a light- activated partner, more preferably wherein the light-activated protein is a light- activated DNA or RNA binding protein.

[0026] DESCRIPTION OF FIGURES

[0027] The invention is described with reference to the accompanying drawings.

[0028] Figure 1 shows the Wood-Ljungdahl pathway (WLP) for the synthesis of acetyl- CoAfrom gaseous carbon sources.

[0029] Figures 2A-2D show a number of example bioreactor geometries and modes of liquid circulation.

[0030] Figures 3A-3E show the process of gas mixing via spargers or ejectors and their placement.

[0031] Figures 4A and 4B show the process of introducing light into a bioreactor. Arrows from light sources, guides and LED strips indicate direction of light. Figures 5A and 5B show preferred embodiments of the bioreactor of the present invention.

[0032] Figures 6A-6C show the activity of a modified acetogen cultured in an external loop bioreactor illuminated with light.

[0033] Figure 7 demonstrates the functionality of light-dependent bacteriorhodopsins when expressed in wild-type C. autoethanogenum.

[0034] Figure 8 shows an inducible promoter known in the art.

[0035] Figure 9 shows inducible light-based promoter known in the art.

[0036] Figures 10A and 10B show novel Boolean logic gates created by combining inducible promoters with other light responsive biochemical components or promoters.

[0037] Figure 11 shows in vivo data demonstrating increased growth of recombinant acetogenic microorganisms when a light-dependent bacteriorhodopsin is present in the system. (A) Comparison of growth on gases in the absence of a light source in wild type Acetobacterium woodii (A. woodii), and A. woodii harbouring one of two empty expression plasmids, one of which was used to express the bacteriorhodopsin tested. (B) All-trans retinal is essential for the functionality of bacteriorhodopsin. A comparison was made of growth on gases in the presence of a light source with wild type A. woodii expressing the bacteriorhodopsin from plasmid pMTL-84151 in the absence of all-trans retinal, wild type A. woodii expressing the bacteriorhodopsin from plasmid pMTL-84151 in the presence of all-trans retinal, and A. woodii harbouring the empty expression plasmid pMTL- 84151. Figure 12 demonstrates that the functionality of light-dependent bacteriorhodopsins BR2 (A) and BR1 (B) when expressed in wild type A. woodii, and that the activity of bacteriorhodopsin is dependent on the presence of all-trans retinal. Functionality was demonstrated by measuring the change in pH of the medium with cell suspensions of A. woodii expressing bacteriorhodopsin in the presence or the absence of all-trans retinal when illuminated by a light source.

[0038] Figures 13A and 13B show that the expression of different proteins can be controlled in response to illumination via specific wavelengths of light in synthetic microorganisms.

[0039] Figure 14 shows that the concentration of different target proteins can be controlled through tunable degradation of the proteins via the expression of an adaptor protein in synthetic microorganisms.

[0040] DETAILED DESCRIPTION

[0041] The inventors of the present invention have surprisingly found that a bioreactor which is able to achieve efficient (low energy input) mass transfer of CO2and H2while also providing illumination at the required intensity and wavelength and pattern, is able to simultaneously provide the inputs necessary for a genetically modified microorganism to generate additional energy while exerting control over the genetically modified microorganism. The bioreactor of the present invention is therefore specifically configured to efficiently deliver CO2, H2and light to genetically modified microorganisms capable of catalysing both redox-mediated and light-mediated bioprocesses in order to drive a more efficient gas fermentation / carbon fixation. Such use of multiple biochemical pathways within a genetically modified microorganism allows for fine-tuned, dynamic modulation of the microorganism. Modulation or control can be exerted by manipulating parameters such as light, gas feed rate and composition, temperature, pH, macronutrients (e.g., phosphate, nitrogen, or ammonia), key micronutrients (vitamins) or inorganics (trace metals) as the microorganisms will respond to variations, excessiveness, or limitations in one or more of these parameters. Accordingly, by controlling the bioprocess mediated by the genetically modified microorganisms, the present invention allows for enhanced control and therefore increased efficiency of the production of commercially desirable products by the microorganisms.

[0042] Even further, the inventors of the present invention have surprisingly found that unique geometries of said design and how liquid is moved, the mixing of gases (mass transfer) and the delivery of light into the vessel can enhance the efficiency of bioreactors used in the fermentation of gases.

[0043] In accordance with a first aspect of invention, there is provided 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.

[0044] The bioreactor of the present invention will comprise at least: a chamber, one or more means of input, one or more means of output, and a genetically modified microorganism. The genetically modified microorganisms will be comprised within the chamber of the bioreactor. As disclosed hereafter, the microorganisms are genetically modified such that they can make use of both redox-mediated and light-mediated biochemical pathways. A such, these microorganisms are amenable to modulation via both their redox-mediated and light-mediated biochemical pathways for production of ATP via generation of electrochemical gradients. In the context of the bioreactor, this modification allows for fine-tuned and dynamic modulation of gas fermentation / carbon fixation at both the redox and light-based level in microorganisms. Accordingly, a bioreactor has been designed to allow this dual-level control i.e., to control the microorganisms using light as well as CO2 and H2.

[0045] As used herein, the term “bioreactor” refers to a device that contains live microorganisms and converts one or more inputs into one or more outputs. For the avoidance of doubt, a “bioreactor” contains biochemically active, viable microorganisms at a level sufficient to convert input into output. Preferably, the one or more inputs are light, carbon dioxide (CO2) and hydrogen (H2). The ‘inputs’ may also be referred to herein as ‘feedstocks’ in that they comprise a material which is to be supplied to or fuel the bioprocesses occurring within the bioreactor. More preferably, the one or more outputs are a desirable product which may be of commercial interest. Examples of a desirable product include, but are not limited to alcohols, sugars, proteins, 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, other secondary metabolites, or any combination thereof.

[0046] The “bioreactor apparatus” is the apparatus of the bioreactor without the presence of microorganisms i.e., it is the components of the bioreactor before the addition of a microorganism. The bioreactor apparatus must contain at least a chamber, one or more inlets, and one or more outlets.

[0047] It is envisaged that the microorganisms contained within a chamber of the bioreactor will be recombinant. As used herein in any aspect of the invention, the terms “modified”, “genetically engineered”, “recombinant microorganism” and “genetically modified” in the context of the present invention, refer to a microorganism (for instance, a strain of bacteria) that has undergone genetic engineering such that its DNA has been altered by the introduction of new DNA, excision of native DNA, or other modifications to its sequence or structure. Recombinant microorganisms have been genetically modified or “engineered” such that they are altered with respect to the naturally occurring cell. Recombinant DNA methods commonly involve the introduction of new DNA via a vector, for example, a plasmid. Such methods are well known to those skilled in the art. Use of recombinant strains of microorganisms (e.g., bacteria) may confer advantageous properties, for example, recombinant bacterial strains may be given the ability to express heterologous proteins which are not naturally expressed within the bacteria, or reprogramming of the host’s genetic instructions.

[0048] The chamber is a receptacle that is water-tight and gas-tight and can therefore retain a fluid medium containing a microorganism, as well as any gases which are delivered into the chamber. The chamber may be any shape, such as a cube, cuboid, or cylinder. The bioreactor is preferably constructed from materials which are smooth. The material may be polished to achieve a smooth finish. Further, the materials may be chemically non-reactive (i.e. , inert), and corrosion-resistant. Preferably, the bioreactor is constructed from metals, such as aluminium, or metal alloys, such as stainless steel or carbon steel. Examples of other materials from which the bioreactor may be constructed include, but are not limited to plastics, such as acrylic plastic, glass, such as plexiglass or borosilicate glass, or rubbers, such as silicone rubber or butyl rubber. The bioreactor may be constructed from any of the materials disclosed herein, or a combination thereof. In one embodiment, the bioreactor is fully opaque or partially opaque. In another embodiment, the bioreactor may comprise a window or transparent sections to allow light to enter the chamber from an external light source. In an alternative embodiment, the walls of the bioreactor are fully opaque, and illumination will occur from an internal light source.

[0049] The bioreactor of the present invention may be of any capacity, but preferably the bioreactor will be of an industrial relevant scale. For example, the bioreactor capacity may be at least 5000 litres, at least 6000 litres, at least 7000 litres, at least 8000 litres, at least 9000 litres, at least 10000 litres, at least 11000 litres, at least 12000 litres, at least 13000 litres, at least 14000 litres, at least 15000 litres, at least 16000 litres, at least 17000 litres, at least 18000 litres, at least 19000 litres, at least 20000 litres, at least 25000 litres, at least 30000 litres, at least 35000 litres, at least 40000 litres, at least 45000 litres, at least 50000 litres, at least 55000 litres, at least 60000 litres, at least 65000 litres, at least 70000 litres, at least 75000 litres, at least 80000 litres, at least 85000 litres, at least 90000 litres, at least 100000 litres, at least 125000 litres, at least 150000 litres, at least 200000 litres, at least 225000 litres, at least 250000 litres, at least 300000 litres, at least 325000 litres, at least 350000 litres, at least 375000 litres, at least 400000 litres, at least 425000 litres, at least 450000 litres, at least 475000 litres, at least 500000 litres, or more than 500000 litres. Such a capacity will be capable of suitably containing the fluid medium which is used to cultivate the genetically modified microorganisms. The bioreactor may not be 100% filled with fluid medium, but may be filled anywhere between 60-90% with fluid medium, preferably between 60-85% with fluid medium. The fluid medium will be any suitable growth medium for the microorganisms to grow. The skilled person will be aware of suitable media which may be available and commonly used in the art.

[0050] Within the bioreactor (i.e., within the chamber) there may also be means for creating turbulence in the bioreactor e.g., stirring, mixing, or agitating the fluid medium contained within the chamber. One or more means for creating turbulence may be present, which may be attached to an internal surface or wall of the chamber. The means can be of any shape or configuration, as will be apparent to the skilled man. The means is preferably impermeable to water and is made of a resilient material such as rubber, more preferably a rigid material such as metal, glass, or acrylic plastic. The means may be flat or substantially flat, i.e., it has a planar configuration or is sheet-like or designed to be a shape to induce patterns of turbulence (curved or spiralled turbulence).

[0051] The means for creating turbulence is advantageous as it forces the fluid containing the microorganism and the gases to mix, thereby improving the efficiency of the conversion of the inputs into desirable products / output. In one embodiment, means for creating turbulence is a fixed means for creating turbulence. As such, the fixed means for creating turbulence may be a baffle. As used herein, the term “baffle” is to be given its usual meaning in the art, namely a deflector plate that affects fluid flow. The fixed means for agitation, such as a baffle, may be present within an external loop of the bioreactor and / or within the chamber of the bioreactor. One or more baffles may be present in order to optimize mixing of the contents within the bioreactor. For example, at least one baffle, at least two baffles, at least three baffles, at least four baffles, at least five baffles, at least six baffles, at least seven baffles, at least eight baffles, at least nine baffles, at least ten baffles, at least eleven baffles, at least twelve baffles, or more than twelve baffles may be present. The baffles may be arranged such that they are positioned on opposite sides of the chamber or external loops of the bioreactor. The baffles may be parallel to one another, and / or in a staggered arrangement.

[0052] Alternative fixed means for creating turbulence may be an impeller. However, impellers rely on the use of moving parts. Moving parts can be detrimental to the microorganisms present in the bioreactor. Therefore, in another embodiment, the means for creating turbulence does not rely on moving parts. Turbulence is ideally created using static mixers such as a baffle which also requires less energy than mechanical mixers which rely on moving parts due to low shear and low energy. In combination with turbulence, there may also be circulation of fluids achieved using liquid pumps, nozzles etc, and gas flow achieved by ejectors, nozzles, etc (Figures 2 and 3). The advantage of using circulation of liquids and flow of gases is that the microorganisms within the chamber are gently mixed, and not damaged or killed during agitation. It is envisaged that a preferred bioreactor of the present invention will achieve efficient mixing of contents using a combination of baffles and liquid / gas circulation.

[0053] The bioreactor will contain at least one inlet (inlet means) and at least one outlet (outlet means) through which the inputs can be added, and also through which microorganisms and suitable media (such as water and nutrients) and gases can be added (inlet) and removed (outlet). Desirable products may be removed via the outlet. Where there are inlets and outlets (i.e., openings within the bioreactor), there may be a filter present at said opening to prevent biomass (i.e., microorganisms contained within the bioreactor) from passing through the opening. Such a filter will be intended to prevent contamination of the external environment or other parts external to the bioreactor which are connected to the bioreactor via an inlet or outlet means (e.g., any inputs / feedstocks, feeding vessels, outputs, etc). The filter may be a microfilter (i.e., with a pore size of 0.1 - 10 pm), an ultrafilter (i.e., with a pore size of 0.01 - 0.1 pm), or a nanofilter (i.e., with a pore size 1-10 nm) of which is capable of preventing microorganisms from passing through the filter.

[0054] The inlet is preferably controlled by a valve that allows regulation of the flow through the inlet. The inputs necessary to maintain microorganisms and gases may be introduced into the bioreactor via the inlet. The inlet device used for introducing gases into a bioreactor is a sparger.

[0055] The outlet is preferably controlled by a valve that allows regulation of the flow through the outlet. Desired products or outputs may be removed or collected via the outlet.

[0056] A single opening that acts as both inlet and outlet may be present, or there may be separate inlet and outlet means. Multiple inlets and outlets are also possible. Inputs may be continually added and removed via the inlet and outlet such that the level of fluid within the bioreactor (chamber) is constant or substantially constant. The inlet and outlet may be situated on opposite surfaces of the chamber, or next to or substantially next to each other, or distributed evenly throughout the surface. The bioreactor may also contain one or more ports which can be used when connecting two or more bioreactors together. The ports allow the media etc within the bioreactor to flow from one bioreactor to the next. Alternatively, the ports may be "closed" so that media cannot flow from one bioreactor to the other.

[0057] Figure 2 shows a number of example bioreactor geometries. The section of a bioreactor where gas rises is called a riser. In loop reactor designs, the section of a bioreactor where liquid moves downwards is called a downcomer. Bioreactors are typically only filled up to 60-85% of the total volume, referred to as the working volume. The horizontal dashed lines in Figure 2 demonstrate an approximate liquid level. The fluid medium in a bioreactor needs to be continuously mixed to ensure nutrients, salts, etc, are evenly spread throughout the fluid medium. In sugar-based processes, mixing (or agitation) is typically achieved using an impeller that spins. This is acceptable for smaller bioreactors (with capacities of less than 50,000 litres) and when producing extremely valuable products (e.g., biopharmaceuticals), but at very large volume this approach is extremely energy intensive and expensive, so alternative methods are used. The simplest method is to use the geometry of the bioreactor and the introduced liquid to cause currents that achieve mixing via different flows / circulation. Accordingly, in a preferred embodiment, the bioreactor of the present invention utilises the flow or circulation of liquids to promote mixing of fluids or create turbulence. Shown in Figure 2 are some standard bioreactor designs which can be used.

[0058] Within Figure 2A, five bioreactor designs (labelled A-E) are shown. Bioreactor design A shows a bubble column bioreactor. Bioreactor designs B and C show internal loop bioreactors with slightly different designs. Bioreactor design D shows a plug-flow bioreactor (horizontal tube). Bioreactor design E shows an external loop bioreactor.

[0059] Figure 2 primarily focuses on illustrating how liquid moves through a bioreactor. Liquid can flow bottom to top as shown, or introduced at the top and removed from bottom, and this applies to all models. Two-dimensional models are shown, but as would be appreciated by the skilled person, bioreactors are three- dimensional, meaning that for example, an internal riser is a tube (rather than simply a line), thus in effect this creates a tube within a tube.

[0060] The flow or circulation of the liquid caused by the introduction of liquids is sufficient to mix the bioreactor contents. The inlet flow of liquids can be adjusted to achieve a sufficient level of mixing, as will be apparent to one skilled in the art. Liquid may be introduced into the bioreactor using a nozzle. As would be appreciated by the skilled person, the shape and design of the nozzle can impact the velocity of the liquid and therefore the mixing and circulation of liquid and thus is an important design consideration. As used herein, the term “nozzle” will be given its usual meaning in the art and refers to a device designed to control the direction and velocity of a fluid flow as it enters (or indeed exits) an enclosed region such as a tube or pipe. Types of nozzles which may be used include, but are not limited to, flat fan, hollow cone, full cone, or solid stream.

[0061] A liquid outlet is required as liquid must be constantly removed to maintain a constant liquid level (or working volume) in the bioreactor. Liquid may be introduced at the top or the bottom of the bioreactor and similarly, removed from the top of bottom of the bioreactor. Accordingly, in one embodiment, a means for liquid input is positioned at the top of the bioreactor. Further, a means for liquid output may be positioned at the bottom of the bioreactor. In an alternative embodiment, the liquid input means may be positioned at any side of the bioreactor. Liquid can flow up one or more risers and flow down one or more downflows within the bioreactor.

[0062] Figure 2B and Figure 2C illustrate variations of the external loop reactor design (designs E1-E5). The external loop can reconnect on the lower lateral side of the riser (Figure 2C, bioreactor design E4) or can reconnect with the bottom of the riser (Figure 2C, bioreactor design E5).

[0063] In another configuration, the riser (main body of the bioreactor) can be positioned horizontally, and the external loop can extend out from the main body of the bioreactor (Figure 2D). Again, the external loop can reconnect to the bottom side of the main body of the bioreactor (Figure 2D, bioreactor design E6) or to the lateral side of the bioreactor (Figure 2D, bioreactor design E7). By changing the positioning of the inlet nozzle for liquid the circulation and flow of liquid can be changed or reversed (Figure 2B, bioreactor design E2; Figure 2B, bioreactor design E3; Figure 2C, bioreactor design E5; and Figure 2D, bioreactor design E8).

[0064] In accordance with the invention, the bioreactor is configured to provide an input of CO2, H2and light. As regards the provision of gases such as CO2 and H2, the gases are pressurised to be injected into a bioreactor via an inlet. The inlet means for gases may be the same or different inlet means as used for liquids. Accordingly, in one embodiment, the bioreactor comprises a gas compressor to introduce gases into the bioreactor.

[0065] In a preferred embodiment, gases may be introduced via an inlet such as a nozzle or sparger (Figure 3). As such, the bioreactor apparatus may contain a sparge unit functionally connected to the (gas) inlet, such that the gas passes from the (gas) inlet, through the sparge unit and subsequently into the chamber. One or more spargers may be present in the bioreactor. The gas is therefore preferably “sparged” into the chamber. As used herein, the terms “sparging”, “sparge” and “sparged” are to be given their usual meaning in the art, i.e., relating to the introduction of a gas into a fluid such that it may be dissolved in the liquid, in a process known as mass transfer. As would be appreciated by the skilled person, gases cannot flow downwards as they are lighter than liquid and thus naturally flow towards the top of the bioreactor. Therefore, pressurised gases are injected via a sparger or nozzle. As the gases dissolve into the liquid (mass transfer), liquid-rich gas can flow downwards towards the bottom of the bioreactor via circulation of the liquid.

[0066] As used herein, the term “mass transfer” will be known to those skilled in the art and refers to the net transference or movement of a component in a mixture from an area in which its concentration is high to an area in which its concentration is lower (i.e., dissolving of gas in a liquid). As gases dissolve in the liquid, their distribution becomes more uniform in space. Injection of gases into the bioreactor may be mediated by an inlet such as a nozzle or sparger. Particularly effective mixing can be achieved by injecting a gas through an inlet such as a nozzle. An example of a suitable pressure for injecting gas through a nozzle or sparger is between 1 and 25 bar, more preferably between 2 and 3 bar. Nozzles and spargers are of great importance in mass transfer since the shape and design of the nozzle or sparger determines the size of the bubbles of gas introduced into the bioreactor. In particular, smaller bubbles (i.e., created by nozzles or spargers with smaller perforations / holes) have a larger surface area to volume ratio, thus resulting in improved mass transfer. As such, the shape, number and location / distribution of nozzles can be used to improve mass transfer and thus mixing of gases. Improved mass transfer is also achieved via improved means for creating turbulence.

[0067] In this embodiment, the gas inlet is preferably separate to the inlet for other inputs and microorganisms. A tube, such as a dispersion tube, may be used to introduce gases into the bioreactor. In a further embodiment, some or all of the gas input can be sparged into a separate vessel, referred to as a feeding vessel, containing a liquid such as water (i.e. , to introduce ‘pre-mixed’ gases into the bioreactor). A preferred feeding vessel has at least a 500-litre capacity. The gas that is sparged into the feeding vessel will dissolve into the liquid, which can then be introduced into the bioreactor chamber through an inlet. Sparging the gases into a feeding vessel containing a liquid thus allows mixing of gases to be carried out externally of the chamber i.e., prior to, such that gases are ‘pre-mixed’ before entering the chamber. Sparging the gases directly into the chamber allows for mixing of gases to occur within the chamber.

[0068] Figure 3 shows the process of gas mixing, and the effect on liquid circulation with different bioreactor geometries. To introduce gases into a bioreactor they must first be pressurized using a compressor (Figures 3A-D), and then injected via a nozzle or sparger. In the case of external loop bioreactors, the direction of flow of the liquid in the loop can be concurrent or counter current with the flow of gases. The former is achieved simply due to the mixing effects of the gases; however, the latter requires the use of an additional pump. Further, the input of liquid does not necessarily need to be at the bottom, it can be along the sides, near the top or from the top, different positions can provide different functionality and advantages similar to nozzle placement.

[0069] Introducing gas via nozzles requires the gas to be first pressurized with a compressor, which can be expensive to operate. Therefore, in an alternative embodiment, gases are mixed via ejectors (Figure 3E). As used herein, the term “ejector” shall be given its usual meaning in the art and refers to a vacuum pump which uses velocity to mix separate streams of fluids. For example, two separate streams of fluids may be mixed using an ejector. Ejectors are a preferred means for mixing gases since they do not have moving parts, and thus allow for a more efficient process of gas mixing. Further, mixing that occurs naturally inside of an ejector results in efficient mass transfer.

[0070] Figure 3E shows the process of gas mixing via ejectors. Introduction of gases via nozzles that generate bubbles requires the gas to be pressurized with a compressor, which are expensive to operate. Another approach is to use an ejector, which is a vacuum pump that uses Bernoulli’s principle (Pressure Energy and Bernoulli's Principle. George A. Lindsay. American Journal of Physics. 20, 86-88 (1952)). Ejectors, also known as eductors or Venturi’s, use velocity to mix two different streams of liquids / fluids and are advantageous because they do not have moving parts. In the present case, the liquid is pumped at high pressure through the nozzle of an ejector converting the pressure into velocity and generating a region of low pressure thereby drawing in low pressure gas connected to the side inlet of the ejector. The liquid and gas streams travel through the diffuser section of the ejector where velocity is decreased, pressure is increased, and the two streams are mixed together.

[0071] A bioreactor according to the invention may, when working to convert one or more inputs into one or more outputs (i.e., desirable products), contain a microorganism. As used herein, the term “microorganism” in the context of the present invention, refers to, but is not limited to, acetogenic microorganisms and non-sulphur purple bacteria. In particular, acetogenic microorganisms may include, but are not limited to, Acetobacterium woodii (A. woodii), Clostridium ljungdahlii, C. carboxidivorans, C. autoethanogenum, Eubacterium limosum and Moorella thermoacetica. However, the preference is C. autoethanogenum. In particular, non-sulphur purple bacteria may include, but are not limited to, Rhodobacter sphaeroides and Rhodospirillum rubrum. Preferably, the microorganism is an acetogenic microorganism. One or more strains of microorganisms can be used, for example, a mixture of one or more, two or more, three or more or four or more microorganisms may be utilised to mediate a bioprocess (or a number of bioprocesses) within the chamber of the bioreactor. The preference of the present invention is to use a single genetically modified bacterial strain. The bioreactor may be configured to control two or more microorganisms in a bioprocess, for example using different wavelengths of light to control distinct aspects of different microorganisms. The use of two or more microorganisms may result in a synergistic improvement of bioreactor efficiency, however, the preference of the present invention is to use different wavelengths of light to control different mechanisms / switches in the engineered microorganism.

[0072] The bioreactor can be used in both domestic and industrial settings. Preferably, the bioreactor is attached to a chimney or waste outlet. In a particularly preferred embodiment, a bioreactor is attached to an industrial chimney or waste outlet. This will provide a rich source of waste gases, in particular CO2. Industrial CO2 can be sourced from the combustion of fossil fuels, biomass, or other carbonaceous materials such as household waste and can be fed directly into a bioreactor or concentrated via a carbon capture technology (e.g., amine-based solvents), and then introduced into the bioreactor. Atmospheric CO2 can be captured and concentrated using direct air capture (DAC) devices, which includes thermochemical, electrochemical, and photochemical based technologies, which generate a high purity stream of CO2 that can be fed directly into a bioreactor.

[0073] It is envisaged that the bioreactor of the present invention will not operate in isolation, i.e., the bioreactor will operate as part of a larger system. In addition to the CO2, the bioprocess requires electricity to operate the system as well as produce hydrogen, which is preferably produced using renewable electricity. Other sources of renewable hydrogen include hydrogen produced from biomass through gasification, or the decomposition of biomethane. The appropriate process technology is required to deliver the hydrogen that is required by the proposed process. For example, an electrolyser is required to produce hydrogen from water and electricity. Electricity will also be required to generate light.

[0074] The required CO2 and renewable hydrogen would need to be processed to remove impurities, mixed to the appropriate ratio, and pressurised in preparation for injection into the bioreactor. A molar ratio of at least 2:1 of H2:CO2 is required to drive growth of the microorganism and consequently favourable product formation. The components described compose the upstream gas processing system and which is required for to feed the bioreactor with the appropriate gas feedstock. The bioreactor will operate at a temperature between 35 and 37 degrees Celsius (°C) at a pressure of at least 0.3 barg.

[0075] In one embodiment, the bioreactor is used as a single bioreactor. In another embodiment, two or more bioreactors can be used together, e.g., in a “stack” or series configuration, or in parallel, preferably utilising the same inputs. Such a configuration could then be attached to the same chimney waste outlet. In one embodiment, a stack or series of bioreactors contains a number of bioreactors each directed to a different purpose, i.e. , configured to control distinct aspects of microorganisms such as to control different phases of growth in order to produce different desirable products (output). In another embodiment, a stack or series of bioreactors contains a number of bioreactors each directed to the same purpose i.e., producing the same desirable product and are configured as a stack or in series to increase throughput.

[0076] Over time, the microorganisms which catalyse the conversion of gases into desirable products will lose their activity and will need to be replenished. In one embodiment, the bioprocess is restarted by pumping out the majority of the contents (for example four fifths), leaving the remaining amount (i.e., one fifth) to be used to re-seed the bioreactor and generate the required microbial catalyst. In a preferred embodiment, a continuous process is operated in which a purge continuously removes a small fraction of overall culture while the bioreactor is replenished continuously with a seed train to maintain the concentration of microorganism in the production bioreactor.

[0077] Acetogenic microorganisms and non-sulphur purple bacteria may be genetically modified such that they express additional proteins and protein complexes, e.g., for carbon fixation, which can be manipulated or controlled via several means. For example, light-dependent or “light-responsive” proteins may be incorporated into said microorganism in order to control the expression of certain genes or operons using light, preferably different wavelengths of light. For example, redoxdependent ion pumps such as Rnf or Ech complexes are already present in acetogenic microorganisms. Therefore, introduction of a light-dependant ion pump, such as a rhodopsin, results in acetogenic microorganisms that can use both redox-dependent and light-dependent mechanisms to generate an electrochemical ion gradient to drive ATP synthesis. Further, photosynthetic machinery is already present in non-sulphur purple bacteria. Therefore, introduction of a redox-dependant ion pump, such as an Rnf or Ech complex, results in non-sulphur purple bacteria that can use both light-dependent and redox-dependent mechanisms to generate an electrochemical ion gradient to drive ATP synthesis. Additionally, microorganisms may be genetically modified such that photosynthetic machinery is present with a light-dependant ion pump. Finally, microorganisms may be genetically modified such that a combination of photosynthetic machinery, light-dependent ion pumps and redox-based ion pumps is present to generate an electrochemical ion gradient to drive ATP synthesis. Finally, microorganisms that have native photosynthetic machinery such as purple non-sulphur bacteria may be genetically modified to improve their ability to fix carbon by introducing the ATP efficient Wood-Ljungdahl pathway. Such a recombinant microorganism would in essence have the same metabolic and carbon fixation capabilities as an acetogen that has been genetically engineered to express a light-harvesting rhodopsin or the photosynthetic machinery of a purple non-sulphur bacteria.

[0078] As used herein, the term “photosynthetic machinery” in the context of the present invention, refers to the biological or non-biological components necessary for the capture and storage of light energy in the form of electrochemical potential energy and / or chemical potential energy. . The former is achieved through the translocation (pumping) of ions across a membrane against their electrochemical gradient. The latter is achieved through the reduction of a substrate, reducing equivalent or redox mediator. The system may be a single, multiple proteins or a protein complex that generates an electrochemical gradient by using light energy to transport ions from areas of low electrochemical potential to areas of high electrochemical potential, independently from the generation or net consumption of reducing equivalents. The system may also include non-protein components such as organic compounds including electron transfer components (e.g., quinones). Components comprising the photosynthetic machinery may include, but are not limited to, the photosynthetic machinery (cytochrome bc1 complex, cytochrome c, quinone, Type II reaction centre, peripheral antennae and / or chlorosomes) of purple sulphur bacteria, ion pumps that require the action of all- trans-retinal (microbial rhodopsins), and organic or inorganic semiconducting materials. Examples of photosynthetic machinery include, but are not limited to, cyclic electron transfer in purple non-sulphur bacteria and the action of microbial rhodopsins, both of which use light energy to pump ions across a membrane generating an electrochemical ion gradient independently from the generation or net consumption of reducing equivalents.

[0079] In accordance with the invention, the bioreactor is configured to provide an input of CO2, H2as well as light. As regards the provision of light, within the bioreactor may be a light source. The light source may be an internal light source, an external light source, and / or a combination thereof. In a preferred embodiment, the light source is an artificial light source. Examples of an artificial light source include, but are not limited to, a light bulb, light strips, a fluorescent light, or a lightemitting diode (LED), or combinations thereof. Preferably, the light source is an LED. One or more light sources may be present in the bioreactor, for example one or more LEDs, two or more LEDs, three or more LEDs, four or more LEDs, five or more LEDs, or more than five LEDs may be utilised. More preferably, the LED is an LED bulb or an LED strip. The light source may be internal to the chamber, or alternatively, external to the chamber. In the latter embodiment, the bioreactor may comprise a window such that an external light source may illuminate the interior. Light (external or internal to the bioreactor) may be introduced from the the top, bottom, or any other side of the bioreactor, such as on the riser or on the external loop. Using an externally mounted light source light can introduce light using a light guide. Internal light sources and light guides may be inserted from the top of the bioreactor, or may be horizontal or incorporated into internal components of the bioreactor, such as inner tubes (risers). The light source or light guides may be present within an external loop of the bioreactor and / or within the chamber of the bioreactor.

[0080] Figure 4A shows the process of introducing light into a bioreactor. Light can be introduced using external light sources which are mounted externally and illuminate the interior via a window (Figure 4A, bioreactor designs F1 and F2). Light can be introduced from the top, bottom, or any other side. Light sources of various types can also be put inside the bioreactor to illuminate from within (Figure 4A, bioreactor design F3). Using an externally mounted light source light can also be introduced using a light guide (Figure 4A, bioreactor design F4). Internal light sources and light guides do not always have to be vertical or inserted from the top, they can be horizontal or incorporated into internal components of the bioreactor, such as inner tubes (risers). Figure 4B shows numerous arrangements of light sources and light guides within an external loop bioreactor, in which there may be light sources and light guides present in the bioreactor chamber (Figure 4B, bioreactor design F5), in the external loops (Figure 4B, bioreactor designs F6 and F7), and / or in both the chamber and the external loops of the bioreactor.

[0081] As would be appreciated by the skilled person, several different bioreactor designs may be utilised in the present invention, including internal loop and external loop bioreactors. The bioreactor of the present invention may comprise one or more external loops, for example one, two, three, four, five or more external loops. Further, a plug-flow bioreactor design may be utilised. Further, a bubble column bioreactor design may be used. However, the preference of the present invention is to utilise a unique geometry / configuration / design of bioreactor which allows for the efficient fermentation of gases as disclosed herein.

[0082] Figures 5A and 5B show preferred embodiments of the bioreactor of the present invention. It is envisaged that the bioreactor will feature an external loop (or more than one external loop) with one or more inputs and outputs for liquids and gases. The locations of the inputs and outputs for liquids and gases shown in Figure 5A and Figure 5B are for illustrative purposes only, and the skilled person will be aware that these inputs and outputs may be positioned at various locations on the bioreactor or external loops. The positioning of the inputs and outputs will induce concurrent or countercurrent through introduction of gases or liquids or both using any combination of nozzles, spargers and or ejectors, with light introduced internally using repeating sections of baffles and lighting systems (internal lights, external lights through windows or external lights through light guides) that are built into either the main chamber or an external loop. A preferred bioreactor design of the present invention will have one or more baffles for agitation which may be positioned within the external loop and / or within the chamber of the bioreactor. The preferred bioreactor design of the present invention will also feature the introduction of liquids from the top of the bioreactor and will use an ejector to mix in gases either from the side or from the bottom. Further, the preferred bioreactor design of the present invention will be illuminated with light. Illumination with light will be external and / or internal. Internal illumination with light will involve internal light sources, such as LEDs or LED strips, and / or wave guides. External illumination of light will involve windows through with external light sources such as LED strips can illuminate the interior of the bioreactor. As such, in this embodiment the bioreactor would be partially opaque featuring a window or multiple windows to allow for the provision of light from the external light source. The light sources may be present within the chamber of the bioreactor and / or within the external loops of the bioreactor. In accordance with the invention, the genetically engineered microorganisms comprised within a chamber of the bioreactor will comprise the components necessary for the biochemical conversion of an inorganic carbon source into acetyl-CoA. In one embodiment, the components may be modified such that their enzymatic capacities are enhanced to increase the productivity of carbon fixation (i.e., the process of converting an inorganic carbon source into acetyl-CoA). Based on synthetic biology, the carbon fixation pathway, the module for ATP generation, and the module for reducing equivalent generation may be redesigned to provide a system which can achieve highly efficient carbon fixation.

[0083] As used herein, the term “system” in the context of the present invention, refers to an assembly of the components necessary for the biochemical conversion of an inorganic carbon source into acetyl-CoA. In one embodiment, the system is an in vivo system. In another embodiment, the system is an in vitro system.

[0084] As used herein, the terms “in vivo" and “cellulai1’ may be used interchangeably and refer to a system in which the components necessary for the biochemical conversion of an inorganic carbon source into acetyl-CoA are enclosed by the membrane of a living cell, such as a bacterial cell. Components of each module can be freely combined or modified to develop increasingly productive systems for carbon fixation that offer higher efficiencies and productivities than those found in nature. Typical modifications include targeting low turnover, rate-limiting enzymes. For example, carbon fixation pathways employed by autotrophic organisms are mainly limited by carboxylase enzymes. Therefore, strategies to improve productivity of carbon fixation include replacing rate-limiting enzymes with more efficient homologs, reconstructing hybrid enzymes, or selecting more efficient mutant versions of the rate-limiting enzyme. Additional modifications can include overexpression of key enzymes. Further, creating novel systems for carbon fixation by combining the components of other pathways provides several avenues for increasing the productivity of carbon fixation. In a preferred embodiment, flavin based electron bifurcation (FBEB) is used to couple the endergonic reduction of a low potential electron carrier (such as ferredoxin) with a higher potential electron carrier (such as nicotinamide adenine dinucleotide (NADH)) to the exergonic reduction of an intermediate in the carbon fixation pathway with NADH (or similar).

[0085] As used herein, the terms “in vitro", “acellulaf’ and “cell-free" may be used interchangeably and refer to the assembly of the described components into a system in which the components necessary for the biochemical conversion of an inorganic carbon source into acetyl-CoA are provided to the system. In an in vitro system, the components necessary may be outside their normal biological context. Examples of an in vitro system include, but are not limited to, systems enclosed by a vesicle (including but not limited to exosomes, microvesicles, giant unilamellar vesicles), liposome or microdroplet, which may be composed of carbohydrates, peptides or fatty acids, phospholipids, polymers, or combinations thereof. Further, systems may be enclosed by non-naturally occurring organic compounds or inorganic compounds that may or may not self-assemble and enclose soluble components of the system while also providing a hydrophobic region such that integral or transmembrane proteins or complexes can be inserted into the wall of the membrane. Further, some components may be enclosed in vesicles, exosomes etc. while other components (enzymes) may be free floating.

[0086] As used herein, the term “inorganic carbon source” in the context of the present invention, refers to any simple compound wherein the carbon atom is chemically bonded to an element or elements other than hydrogen. For example, an inorganic carbon source may include, but is not limited to, atmospheric carbon generated by natural or industrial manufacturing processes, transportation, or fossil fuel combustion. Examples of inorganic carbon according to the invention may include, but are not limited to, CO2, CO, carbides, carbonates, and cyanides. However, the preference is for the system of the present invention to convert CO2 to acetyl-CoA. In particular, it is envisaged that CO2 is converted into acetyl-CoA. In another embodiment, the system may utilise carbon dioxide and hydrogen.

[0087] In order for the present invention to convert an inorganic carbon source into acetyl-CoA, a source of reducing equivalents is required. Reducing equivalents are necessary for the process of carbon fixation to occur. For example, a microorganism of the present invention will require reducing equivalents or a source thereof. As used herein, the term “reducing equivalent” in the context of the present invention, refers to any number of chemical species capable of donating its electrons to an electron acceptor. The terms “reducing equivalent”, “reducing agent”, “electron carrier” and “electron donor” may be used interchangeably and refer to the formal transfer of electrons from one species to another, such that the electron donor becomes oxidised, and the electron acceptor becomes reduced. For example, reducing equivalents that may be generated according to the invention include, but are not limited to, NADH, nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FADH2), H2, Fd2-, and reduced flavodoxin (FldHq) (Table 2). However, the preference is for the system of the present invention to generate NADH, NADPH Fd2_and FldHq. A “source” of reducing equivalents refers to a species or component capable of generating said reducing equivalents or a pool thereof, as described herein.

[0088] As used herein, the term “net change” in the context of the present invention, refers to the pool of reducing equivalents, such that there has been an increase or decrease in the total number of reducing equivalents. In the context of a redoxdependant ion pump, the term “net change” specifically refers to ion pumps that generate an electrochemical ion gradient by mechanisms that are independent of the consumption or generation of reducing equivalents, such that there is no net change in the number of reducing equivalents. For example, the Rnf complex consumes reduced ferredoxin (Fd2-) while also generating NADH.

[0089] able 2. Cellular cofactors, coenzymes, proteins, and organic compounds acting as redox mediators or involved in redox reactions, flavin based electron bifurcating (FBEB) redox reactions or quinone based electron bifurcating (QBEB) redox reactions.

[0090] In accordance with the invention, the module for generating reducing equivalents consists of biotic and / or abiotic components that facilitate the transfer of electrons from an external source to a reducing equivalent. External electron sources can take the form of organic compounds such as but not limited to formate, inorganic compounds such as but not limited to hydrogen or ammonia, as well as electrical systems providing a current via a cathode, the use of the latter is an example of microbial electrosynthesis (MES). As used herein, the term “biotic” in the context of the present invention, refers to any naturally occurring, recombinant, or synthetic biochemical component. Examples of biotic components that may be used to generate reducing equivalents include, but are not limited to, electron bifurcating enzymes, oxygen-tolerant hydrogenases, formate dehydrogenases, carbon-monoxide dehydrogenases, protein nanowires, and redox mediators.

[0091] In another embodiment, the invention incorporates an electron bifurcating enzyme to generate reducing equivalents. As used herein, the term “electron bifurcating enzyme” in the context of the present invention, refers to enzymes that oxidise one electron donor and deliver the electrons simultaneously to two different electron acceptors, whereby reduction of one accepter is exergonic and is tightly coupled to the endergonic reduction of the second acceptor. In a preferred embodiment, the source of reducing equivalents may be an electron bifurcating enzyme selected from Table 3 that the system can make use of for the generation of reducing agents. For example, Fd2-is generated through the activity of an intracellular electron bifurcating hydrogenase, which oxidises hydrogen gas to generate Fd2_and NADH. However, the preferred electron bifurcating enzymes of the invention are the NADP+and ferredoxin dependent [FeFe] hydrogenase, HytA-E, native to C. autoethanogenum and the NAD+and ferredoxin dependent [FeFe] hydrogenase, HydABCD, native to A. woodii to generate reducing equivalents. In another embodiment, the reducing equivalents may be generated by an oxygen-tolerant hydrogenase. As used herein, the term “oxygen-tolerant hydrogenase” in the context of the present invention, refers to oxygen-insensitive enzymes that are capable of catalysing H2oxidation to water (H2O) to generate reducing equivalents, under aerobic conditions while avoiding oxygenation and destruction of the active site. In one mechanism accounting for this property, membrane-bound hydrogenases accommodate a pool of electrons that allows an oxygen molecule to be converted rapidly to H2O. Examples of oxygen-tolerant hydrogenases include the [NiFe]-hydrogenase from Ralstonia eutropha H16 and the NAD+-reducing [NiFe]-hydrogenase from Hydrogenophilus thermoluteolus.

[0092] In another embodiment, the reducing equivalents may be generated by a formate dehydrogenase. As used herein, the term “formate dehydrogenase” in the context of the present invention, refers to enzymes capable of catalysing the oxidation of formate to CO2with the concomitant reduction of NAD+to NADH or NADP+to NADPH. Examples of formate dehydrogenases include the NADP(H)-dependent [FeFe]-hydrogenase (Hyt) complex from C. autoethanogenum, the NADH- dependent formate dehydrogenase / heterodisulfide reductase (Fdh) complex from Methanococcus maripaludis (HdrABC / FdhAB) and the NADH-dependent formate dehydrogenase (Hyl) complex from Clostridium acidurici.

[0093] In another embodiment, the reducing equivalents may be generated by a carbonmonoxide dehydrogenase. As used herein, the term “carbon-monoxide dehydrogenase” in the context of the present invention, refers to enzymes capable of catalysing the reversible oxidation of CO to CO2. Two classes of carbon-monoxide dehydrogenases exist: Cu,Mo- carbon-monoxide dehydrogenases and Ni,Fe-containing carbon-monoxide dehydrogenases.

[0094] In another embodiment, the reducing equivalents may be generated by a protein nanowire. As used herein, the term “protein nanowire” in the context of the present invention, refers to electrically conductive appendages produced by a number of bacteria most notably from, but not exclusive to, the Geobacter and Shewanella genera. Protein nanowires are used for generating reducing equivalents from electrical energy as in MES.

[0095] In another embodiment, the reducing equivalents may be generated by a redox mediator. As used herein, the term “redox mediator” in the context of the present invention refers to macromolecules such as proteins and organic compounds involved in the transfer of electrons from external electrochemical sources to reducing equivalents. Redox mediators may be soluble, or membrane bound and are involved in both indirect and direct extracellular electron transfer (EET). Examples of redox mediators includes but is not limited to heme proteins such as cytochromes, flavin based proteins, iron-sulfur proteins, and FMN or quinone based coenzymes (Table 2).

[0096] In another embodiment, the reducing equivalents may be generated by abiotic components. As used herein, the term “abiotic” in the context of the present invention, refers to any non-biologically relevant organic compound or inorganic compound. Abiotic compounds may be used for the generation of reducing equivalents from electrical or electromagnetic (light) energy and may be used to facilitate direct or indirect EET. Examples of abiotic components that may be used to generate reducing equivalents include, but are not limited to, allotropic carbon, inorganic compounds, inorganic semiconducting materials, and redox mediators. Abiotic components may be manufactured through a variety of approaches including by not limited to chemical approaches or through deposition such as in 3D printing.

[0097] In another embodiment, the reducing equivalents may be generated by an allotropic carbon component. As used herein, the term ‘allotropic carbon’ in the context of the present invention, refers to carbon-based materials. Examples of allotropic carbon include but are not limited to single-wall carbon nanotubes (SWCNTs), multi-wall carbon nanotubes (MWCNTs), graphene, and carbon felt. Allotropic carbon is frequently used for the generation of reducing equivalents by facilitating direct EET in MESs. In another embodiment, the reducing equivalents may be generated by an inorganic compound. As used herein, the term “inorganic compound” in the context of the present invention, refers to precious and non-precious metals typically used as cathodic materials in MES. Examples of precious metals include but are not limited to palladium and silver. Examples of non-precious metals include but are not limited to cobalt-phosphate, copper and nickel. Inorganic compounds are frequently used for the generation of reducing equivalents in the form of hydrogen, facilitating indirect EET in MES.

[0098] In another embodiment, the reducing equivalents may be generated by an inorganic semiconducting material. As used herein, the term “inorganic semiconducting material” in the context of the present invention, refers to a non- carbon-based materials such as silicon, gallium or arsenide with an intermediate level of conductivity between that of an insulator and that of most metals. Examples of inorganic semiconducting materials include, but are not limited to, cobalt phosphate (CoP) Inorganic semiconducting materials may also be used as light-harvesting semiconducting materials. The term “light-harvesting semiconducting material” in the context of the present invention, refers to materials with an intermediate level of conductivity between that of an insulator and that of most metals that generate reducing equivalents when illuminated with light. Examples include, but are not limited to, cadmium sulphide semiconducting nanoparticles, silicon nanowires, quantum dots, indium phosphate, and composites of perylene diimide derivative (PDI) and poly(fluorene-co-phenylene) (PFP). These technologies which use these materials to generate reducing equivalents can be used to generate reducing equivalents in a system that is independent from the bioreactor (ex situ), after which reducing equivalents are fed into the bioreactor. For example, there may also be a feeding vessel separate to the bioreactor in which reducing equivalents are generated and delivered into the bioreactor. These technologies can also be incorporated into the bioreactor to generate reducing equivalents internally (in situ). For example, any of the semiconducting materials described herein may be incorporated into the materials (i.e. , the metal, plastic, glass, rubber etc) from which the bioreactor is constructed.

[0099] In another embodiment, the reducing equivalents may be generated by a redox mediator. As used herein, the term “redox mediator” in the context of the present invention, refers to organic compounds which can be reduced by external electron sources, or facilitate direct electron transfer to be used to shuffle electrons from an external environment to a cell, enabling electron uptake into cells. Redox mediators can increase the availability of reducing equivalents in the system by enabling the uptake of electrons directly from a cathode via EET as in MES. An example of an abiotic redox mediator includes but is not limited to methyl viologen.

[0100] In accordance with the invention, reducing equivalents may be utilised by proteins dependent on them for their activity. For example, NADH-dependent reductase and an NADPH-dependent reductase couple the respective oxidation of NADH and NADPH to the reduction of a substrate. In the methyl branch of the WLP, formyl-THF is dehydrated to methenyl-THF and then sequentially reduced via methylene-THF to yield methyl-THF. The reduction of methenyl-THF to methylene-THF is carried out by both a NADH-dependent methylene-THF dehydrogenase AND an NADPH-dependent methylene-THF dehydrogenase. Further, the reduction of methylene-THF to methyl-THF is preferentially carried out by an electron bifurcating methylene-THF reductase or by an NADH- dependent methylene reductase.

[0101]

[0102]

[0103] Table 3. Enzymes exhibiting flavin-based electron bifurcation.

[0104] The system of the present invention requires energy encapsulated in the form of ATP for carbon fixation to occur. ATP generation can occur using membrane- bound ATP synthases (ATPases), which are driven by the flow of ions down an electrochemical gradient spanning the membrane. This gradient is maintained by the action of ion pumps, which store energy in the ions’ electrochemical potential by transferring them up their electrochemical gradient. The flow of ions forms a circuit which transfers energy from the ion pumps to the ATPases, where the energy is stored by phosphorylation of adenosine diphosphate (ADP) to ATP. The electrochemical gradient is therefore utilised to generate ATP. The electrochemical gradient may be used to generate ATP independently of the generation or consumption of reducing equivalents. ATP generation module may occur via one or more ion pumps to generate an electrochemical gradient independently of the generation or consumption of reducing equivalents. As used herein, the term “ion pump” in the context of the present invention, refers to at least two proteins or protein complexes that pump ions across a membrane to generate an electrochemical gradient through mechanisms that are independent of the net generation or consumption of reducing equivalents. Specifically, it refers to proteins or protein complexes that require energy to transport ions against an electrochemical gradient, from areas of low electrochemical potential to areas of high electrochemical potential.

[0105] Ion pumps are selective and dependent on ions of a specific species. For example, proton pumps are optimally adapted to drive the passage of hydrogen ions (protons) across a membrane. Other varieties of ion pump include, but are not limited to, sodium ion (Na+) pumps, calcium ion (Ca2+) pumps, chloride ion (Cl’ ) pumps, potassium ion (K+) pumps, sodium / potassium (Na+ / K+) ion pumps, sodium / hydrogen ion (Na+ / H+) pumps, and potassium / hydrogen ion (K+ / H+) pumps. However, it is known in the art that ion pumps are also capable of pumping ions other than their dependent ion, albeit less efficiently.

[0106] In one embodiment, the system of the present invention comprises at least two or more ion pumps that are dependent on ions of the same species, and which the electrochemical ion gradient of that same ion is used by the ATP synthase in the system. For example, at least two or more Na+ion pumps, at least two or more K+ion pumps, or at least two or more H+ion pumps.

[0107] In another embodiment, the system of the present invention comprises at least two or more ion pumps that are dependent on ions of different species. For example, a Na+ion pump in combination with an H+ion pump. In the context of a system using ion pumps dependent on ions of different species, an antiporter ion pump is present to convert the electrochemical gradient of one ion species into the electrochemical ion gradient of another species. As used herein, the terms “antiporter”, “exchanger” and “counter-transporter” may be used interchangeably, and in the context of the present invention refer to a protein involved in secondary active transport of two or more different ions across a membrane in opposite directions. The term “secondary active transport” refers to one ion species being moved down its concentration gradient, from an area of high electrochemical potential to an area of low electrochemical potential, providing energy for the transport of a second ion species which is moved against its concentration gradient, from an area of low electrochemical potential to an area of high electrochemical potential. Examples of antiporters may include, but are not limited to, Na+ / K+ion antiporter and Na+ / H+ion antiporter. Other types of antiporters will be well known in the art.

[0108] In a preferred embodiment, the ion pump is selected from the group comprising light-dependent ion pumps and redox-dependent ion pumps.

[0109] The term “light-dependent ion pump” refers to any ion pump in which the mechanism of ion transport is controlled by light, for example via conformational changes in the protein structure caused by absorption of a photon, allowing for precisely regulated flow of ions in response to light stimuli. Examples of lightdependent ion pumps include, but are not limited to, retinal-pigmented rhodopsins, such as bacteriorhodopsins, proteorhodopsins, deltarhodopsins, xanthorhodopsins, halorhodopsins, channelrhodopsins, archaerhodopsins, and bacterial sensory rhodopsins. However, the preference is for the invention to use a bacteriorhodopsin (also known as a rhodopsin).

[0110] The term “redox-dependent ion pump” refers to any ion pump in which the transport of an ion against its electrochemical gradient is driven by the free energy released from a redox reaction, such that there is not a net change in the number of reducing equivalents. Examples of redox-dependent ion pumps include, but are not limited to, Rnf complexes and Ech complexes. However, the preference is for the invention to use an Rnf complex.

[0111] In accordance with the invention, a genetically modified microorganism or system may comprise a light-dependent ion pump and a redox-dependent ion pump. Light-dependent and redox-dependent ion pumps may be selected from those known in the prior art, including those listed in Table 4. However, the preference is for the invention to use a bacteriorhodopsin and an Rnf or Ech complex.

[0112] In accordance with the invention, the microorganism contained within a chamber of the bioreactor further comprises biochemical components necessary for converting the generated acetyl-CoA into a biochemical product.

[0113] As used herein, the term “biochemical product” in the context of the present invention, refers to any desired end product generated from the acetyl-CoA with commercially or industrially relevant applications. Preferably, desirable biochemical end products may be selected from the compound classes comprising alcohols, aldehydes, alkaloids, alkanes, alkenes, alkynes, natural or synthetic amino acids, amines, aromatics, carboxylic acids, dicarboxylic acids, dienes, diols, esters, ethers, polymeric (e.g., polyhydroxyalkanoates) and monomeric (ex. ethylene glycol) chemicals, isoprenoids, polyketides, surfactants, terpenes, terpenoids, sugars, proteins, fats and other secondary metabolites, or any combination thereof. These biochemical products can be directed into other processes for the purpose of generating, for example, renewable materials or products.

[0114] In one embodiment, industrial CO2 emissions from a manufacturing plant may be fed directly into the bioreactor such that a desired product can be fed directly into the manufacturing plant, thus providing a renewable source of precursor or intermediate for manufacturing processes. In another embodiment, renewable energy can be stored in chemical bonds through the conversion of CO2 into highly reduced and longer chain organic compounds.

[0115] In another embodiment, industrial CO2 emissions from a manufacturing plant may be fed directly into the bioreactor such that a desired product is produced, wherein the desired product is already an input or substrate of the manufacturing process. For example, a manufacturing process which converts an alcohol, such as ethanol, into another product and emits CO2 as part of this process would benefit from increased yield and efficiency of the process where a bioreactor of the present invention converts this waste CO2 into additional ethanol. The resulting ethanol could be fed back into the manufacturing process as a substrate for further processing.

[0116] In yet another embodiment, renewable energy can be stored in chemical bonds through the conversion of CO2into highly reduced and longer chain organic compounds.

[0117]

[0118]

[0119] Table 4. Enzymes and protein complexes involved in generating an electrochemical ion gradient via ion translocating reactions.

[0120] The system of the present invention requires ATP for carbon fixation to occur. Membrane-bound ATP synthases utilise the electrochemical ion gradient generated by the light-dependent and redox-dependent ion pumps to drive the generation of ATP. Electrochemical potential energy is transferred to the ATP synthase as ions flow down their electrochemical gradient, providing energy for the phosphorylation of ADP to ATP.

[0121] In accordance with the invention, the ATP generation module makes use of an ATP synthase to drive ATP synthesis at the expense of an electrochemical gradient. As used herein, the term “electrochemical ion gradient” in the context of the present invention, refers to the change in Gibbs energy associated with the transfer of 1 mol of a membrane-permeable ion across that membrane. It comprises both a chemical (or concentrative) part that accounts for the difference in chemical potential between regions of different ion concentrations, and an electrical part that represents the change in electrostatic potential energy due to a difference in electric potential. The terms “chemical gradient” and “concentration gradient” may be used interchangeably. The direction and rate of passive ion transport across a membrane is determined by the electrochemical gradient of the ion. Accordingly, an electrochemical gradient can be used for the synthesis of ATP by an ATP synthase. The present invention therefore provides a system in which the electrochemical gradient generated by ion pumps (redoxdependent and light-dependent) is utilised to generate ATP.

[0122] ATP synthases are multi-subunit protein complexes found in the inner mitochondrial membrane, bacterial plasma membrane and thylakoid membrane. ATP synthases are classified as F-type (phosphorylation factor), V-type (vacuole), A-type (archaea), P-type (proton) or E-type (extracellular) ATPases based on their functional differences. ATP synthases do not necessarily use H+ions as the coupling ion, and can use the electrochemical ion gradient of other ions, including but not limited to, protons (H+) and Na+ions.

[0123] To achieve carbon fixation, the present invention may utilise components of a linear carbon fixation pathway. The preferred system is the WLP (Figure 1), or a modified version thereof. In one embodiment, the WLP is modified such that a recombinant rhodopsin is introduced into an acetogenic microorganism, resulting in a recombinant acetogenic microorganism capable of using light to produce ATP and desirable biochemical products via the conversion of an inorganic carbon source. Preferably, the acetogenic microorganism is C. autoethanogenum, an acetogen which naturally produces ethanol. The acetogenic microorganism may be A. woodii, Clostridium ljungdahlii or Clostridium autoethanogenum. However, the skilled person would be aware of other suitable microorganisms which could be utilised to achieve carbon fixation. Preferably, the acetogenic microorganism is C. autoethanogenum. Clostridium ljungdahlii and Clostridium autoethanogenum are acetogenic microorganisms which naturally produce ethanol. As described herein, such microorganisms would be useful in a bioreactor configured to convert CO2 into ethanol (i.e., for manufacturing processes which utilise ethanol and produce CO2 as a result of the process). The inventors of the present invention have surprisingly found that that the production of desired biochemical products via microorganisms genetically engineered to utilise both redox-dependent ion pumps and light-dependent ion pumps can be modulated at an increasingly efficient level using synthetic biocircuitry which responds to light as an inducer. Using light as an inducer allows for the concentration and presence of light-responsive proteins of interest to be modified according to the wavelength of light used and the function of the light- responsive protein. Accordingly, different wavelengths of light, or different durations of exposure to light, can be utilised in combination with unique biocircuitries within microorganisms to increase or decrease a concentration of a protein. Increased protein concentrations may thus be achieved for example by inducing or increasing protein expression, reducing protein degradation, and / or increasing protein stability. Similarly, decreased protein concentrations may be achieved for example by abolishing or decreasing protein expression, increasing protein degradation, and / or decreasing protein stability. Using light as a means for modulating the production of desired biochemical products via microorganisms is beneficial in that it is direct, non-toxic to the cell, and easily programmable.

[0124] Accordingly, in a second aspect of the invention, there is provided a method for controlling the production of a microbially-produced biochemical product, wherein the product is derived from the conversion of acetyl-CoA, comprising culturing a genetically engineered microorganism under suitable conditions, said microorganisms comprising:

[0125] (i) a light-dependent ion pump that generates an electrochemical ion gradient independently of the generation of reducing equivalents; and

[0126] (ii) a redox-dependent ion pump that generates an electrochemical ion gradient independently of a net change in the number of reducing equivalents; and wherein said culturing comprises modulating the exposure of the microorganism to light to drive the light-dependent ion pump to control the generation of the electrochemical ion gradient.

[0127] The invention comprises modulating the exposure of light to the specific microorganisms. As used herein, the term “modulating the exposure of light” refers to a process of actively varying or changing the wavelength and / or intensity of light, or the duration of exposure to light (or certain wavelengths and / or intensities of light). The wavelength of light, intensity of light and duration of exposure to light are herein referred to collectively as “parameters” of light. In the context of the present invention, the light applied to microorganisms in culture will not be constant or passively applied. In one embodiment, the wavelength of light applied to microorganisms will be approximately between 380 nm and 800 nm (i.e., visible light), preferably between 400 and 700 nm. For example, the wavelength of light may be 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm,

[0128] 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm,

[0129] 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm,

[0130] 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm,

[0131] 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, 790 nm, or 800 nm. The wavelength of light applied to microorganisms may correspond to red light, orange light, yellow light, green light, cyan light, blue light, or violet light. In particular, the wavelength of light applied to microorganisms may be approximately between 625 nm to 800 nm, approximately between 590 nm to 625 nm, approximately between 570 nm to 590 nm, approximately between 495 nm to 570 nm, approximately between 450 nm to 495 nm, approximately between 380 nm to 450 nm.

[0132] In another embodiment, the intensity of light applied to microorganisms will be approximately between 50-100 lumens per square meter (lux), 100-250 lux, 250- 1000 lux, 1000-5000 lux, 5000-10000 lux, or greater than 10000 lux.

[0133] In another embodiment, the duration of exposure to light will be between 0.5-1 second, 1-2 seconds, 2-3 seconds, 3-5 seconds, 5-10 seconds, 10-20 seconds, 20-60 seconds, 60-300 seconds, 300-1800 seconds, 1800-3600 seconds, 3600- 7200 seconds, or greater than 7200 seconds.

[0134] Modulating the exposure of light to the specific microorganisms may therefore involve varying a single parameter. For instance, only the wavelength of light may be modulated, while the intensity of light and duration of exposure to light remains constant. Alternatively, only the intensity of light may be modulated, while the wavelength of light and duration of exposure to light remains constant. Alternatively, only the duration of exposure to light may be modulated, while the intensity of light and wavelength of light remains constant. However, modulating the exposure of light to the specific microorganisms may involve varying multiple parameters. For instance, modulating the wavelength of light, intensity of light, and / or duration of exposure to light in any combination. The wavelength of light and intensity of light may be modulated, while the duration of exposure to light remains constant. The wavelength of light and duration of exposure to light may be modulated, while the intensity of light remains constant. The intensity of light and duration of exposure to light may be modulated, while the wavelength of light remains constant. Alternatively, the wavelength of light, intensity of light and duration of exposure to light may be modulated together. Modulating all multiple parameters together could involve simultaneously or sequentially modulating any of the parameters.

[0135] In addition to modulating the exposure of light to the microorganisms, it is envisaged that the microorganisms of the method of the present invention will be cultured under suitable conditions. Microorganisms are cultured in a defined media containing appropriate salts, trace metals, additives and the gases required for autotrophic growth, preferably CO2 and H2gases with a molar ratio greater than 1 :2 at a sufficient flow rate, in an external loop bioreactor. Light is to be supplied internally or externally at a required intensity and wavelength that match those required by light responsive systems engineered into the recombinant microorganism. Culturing microorganisms under suitable growth conditions will allow for the production of acetyl-Coenzyme A (acetyl-CoA) which can be used in the production of a desired biochemical product. Acetyl-CoA in microbes is produced via carbon fixation. The process of carbon fixation requires ATP.

[0136] In any aspect of the invention, the term “independently” refers to the separation of mechanisms by which ATP and reducing equivalents are generated simultaneously, or whereby ATP is generated by a net consumption of reducing equivalents. In particular, the generation of an electrochemical ion gradient can occur either concomitantly or independently from the generation of reducing equivalents or consumption of reducing equivalents. An example of the former is the electron transport chain in plants, wherein photosystems use light energy to oxidise water while simultaneously generating an electrochemical ion gradient and the generation of reducing equivalents in the form of NADPH. The mitochondrial electron transport chain is an example of where reducing equivalents are consumed (oxidised) to generate an electrochemical ion gradient. The multi-subunit ferredoxin-NAD+ oxidoreductase (Rnf) complex found in acetogens is an example of how an electrochemical ion gradient can be generated independently from the generation of new reducing equivalents, or net consumption of reducing equivalents. The Rnf complex couples the transfer of electrons from Fd2- to NAD+ with the transport of ions across the membrane. Although a redox reaction is used to generate an electrochemical ion gradient, there is no net increase or decrease in reducing equivalents. Further examples include cyclic electron transfer in purple non-sulphur bacteria and the action of microbial rhodopsins, both of which use light energy to pump ions across a membrane generating an electrochemical ion gradient independently from the generation or net consumption of reducing equivalents.

[0137] In addition to ATP, the process of carbon fixation requires the components necessary for the fixation of an inorganic carbon source into acetyl-CoA. As used herein, the term “components” and “biochemical components” are used interchangeably, and in the context of the present invention, refer to any biological macromolecule such as but not limited to proteins or protein complexes that form constituent elements of a biochemical pathway, responsible for the catalysis of enzymatic steps within said biochemical pathway. For example, components necessary for the conversion of an inorganic carbon source into acetyl-CoA may include, but are not limited to, formate dehydrogenase, formyl-tetrahydrofolate (formate-THF) synthase, methenyl- tetrahydrofolate (methenyl-THF) cyclohydrolase, methylene-tetrahydrofolate (methylene-THF) dehydrogenase, methylene-THF reductase, methyl transferase, and carbon monoxide dehydrogenase / acetyl-CoA synthase (CODH / ACS) via the WLP (Figure 1). The term “components” and “biochemical components” may also refer to any biological macromolecule such as but not limited to proteins or protein complexes, non-biological organic compounds or inorganic compounds that form constituent elements of the module for ATP generation or the module for reducing equivalent generation.

[0138] In one embodiment, components may be modified such that their enzymatic capacities are enhanced to increase the productivity of carbon fixation. Based on synthetic biology, the carbon fixation pathway, the module for ATP generation and the module for reducing equivalent generation may be redesigned. As described herein, components of each module can be freely combined or modified to develop increasingly productive systems for carbon fixation that offer higher efficiencies and productivities than those found in nature.

[0139] As described herein, a “system” may be an in vitro or in vivo system. In the second aspect of the invention, the system is an in vivo system. Examples of an in vivo system include, but are not limited to, systems enclosed by a semi-permeable barrier, for example, a lipid bi-layer such as a cell membrane. In one embodiment, the system may be enclosed by the membrane of a living cell, such as a microorganism (i.e. , a bacterial cell). As used herein, the term “membrane” in the context of the present invention, refers to a semi-permeable barrier. In one embodiment, the term “membrane” may refer to a lipid bi-layer such as a cell membrane. For example, the membrane of a microorganism, such as a bacterial cell, may enclose components of the system. In the second aspect of the invention, the system for carbon fixation may be contained within a recombinant microorganism. In one embodiment, the recombinant microorganism is capable of self-replication. In another embodiment, the recombinant microorganism is incapable of self-replication. In yet another embodiment, the recombinant microorganism is attenuated. As used herein, the term “attenuated” in the context of the present invention, refers to the alteration of a microorganism to reduce its pathogenicity, whilst maintaining its viability. Such an attenuated microorganism is preferably a live attenuated microorganism, although non-live attenuated microorganisms are also disclosed.

[0140] A microorganism of the present invention may feature any of the suitable components as described herein for the first aspect of the invention. For example, a microorganism of the present invention may utilise any of the components listed in any of Tables 2, 3, and / or 4.

[0141] As described herein, microorganisms may use ion pumps (redox-dependent, light-dependent) to drive ATP synthesis. The resulting ATP is key for carbon fixation to occur. Accordingly, the microorganisms present invention provides for a method of producing acetyl-CoA in carbon fixation using both redox-dependent ion pumps and light-dependent ion pumps. As such, the present invention may utilise provides a dual layered means for controlling microorganisms which are capable of utilising both light-dependent and redox-dependent ion pumps are amenable to control at both the light and redox level. Exposure of microorganisms with a light-dependent ion pump to light will drive the light-dependent ion pump to control the generation of an electrochemical ion gradient independently of the generation of reducing equivalents in response to illumination with light. Specifically, an acetogenic microorganism modified to comprise a light-dependant ion pump, such as a rhodopsin (in addition to any already present redoxdependent ion pumps) will therefore be both redox-responsive and light- responsive (i.e. , using both redox-based and light-based mechanisms to generate ATP for enhanced carbon fixation). As used herein, the terms “light-responsive", “light sensitive”, “light-reactive”, “light-inducible”, “light-activated”, “light-active” and “photoactive” are used interchangeably and refer to the ability of system (i.e., a microorganism) or components which may form part of or the whole of a system (i.e., proteins, molecules, nucleic acids) to respond to changes in light (i.e., wavelengths of light, intensities of light, duration of exposure to light or different wavelengths and / or intensities thereof.). The skilled person will understand that the terms “light” and “photo” may be used interchangeably, and refer to electromagnetic radiation with wavelengths between 380 nm and 800 nm which is visible to the eye. Responses to changes in light (i.e., wavelengths of light, intensities of light, duration of exposure to light or wavelengths and intensities thereof) may involve systems or components undergoing chemical or physical change in response to illumination with light. For example, a light-responsive protein may undergo a conformational change upon illumination with light, where the change in conformation either activates or inactivates the protein. Further, the expression of certain genes under the control of a promoter which is modulated by a light-responsive protein may change upon illumination with light (Figures 8-10). Further, light-responsive biocircuitry can be engineered by combining light-responsive components to simultaneously modulate activity of proteins and enzymes in parallel to modulating the expression or downstream genes. Light-responsive systems (recombinant microorganisms) or components are therefore amenable or susceptible to modulation, manipulation or control using light (different wavelengths of light, intensities of light, duration of exposure to light or wavelengths and intensities thereof).

[0142] In modulating the exposure of light to microorganisms to control the production of a biochemical product, the microorganisms must be light-responsive, i.e., comprise a light-dependent ion pump. In one embodiment, the microorganisms may be engineered such that they comprise one light-dependent ion pump (in addition to any already present, i.e., endogenous, redox-dependent ion pumps). In one embodiment, the microorganisms may be engineered such that they comprise multiple light-dependent ion pumps (in addition to any already present, i.e., endogenous, redox-dependent ion pumps). In a preferred embodiment, the microorganisms may be engineered such that they comprise multiple different light-dependent ion pumps (in addition to any already present, i.e. , endogenous, redox-dependent ion pumps). Preferably, the microorganisms will be engineered to comprise multiple different rhodopsins such that the microorganism can utilise light at different wavelengths to thereby control regulation of the rhodopsin- generated electrochemical ion gradient. For example, a single acetogenic microorganism which ordinarily uses redox-dependent ion pumps may be engineered to comprise multiple differed light-dependent ion pumps, such as multiple different rhodopsins. However, the microorganism may also be engineered to comprise multiple different kinds of light-dependent ion pumps, such as rhodopsins (bacteriorhodopsins), proteorhodopsin, deltarhodopsin, xanthorhodopsin, halorhodopsins, channelrhodopsins, archaerhodopsins, bacterial sensory rhodopsins, or any combination thereof. Accordingly, it is envisaged that a single microorganism may comprise multiple different lightdependent ion pumps which absorb different wavelengths of light or ranges thereof. The wavelength of light absorbed by rhodopsins may be between 400 to 600 nm.

[0143] A single microorganism may be engineered to comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten light-dependent ion pumps, where the light-dependent ion pumps may be the same kind of light-dependent ion pump or different kinds of light-dependent ion pumps, or a combination thereof (i.e., two rhodopsins together with a proteorhodopsin and a deltarhodopsin, for example). Preferably, a single microorganism may be engineered to comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten rhodopsins, where rhodopsins may be the same kind of rhodopsins or different kinds of rhodopsins, or a combination thereof. There may be some microorganisms engineered to comprise certain rhodopsins which absorb a certain wavelength of light or range thereof, together in culture with other microorganisms engineered to comprise a different set or number of rhodopsins which absorb a different wavelength of light or range thereof. There may also be present in culture some microorganisms engineered to comprise a certain set of light-dependent ion pumps together with other microorganisms engineered to comprise a different set of light-dependent ion pumps (for example, some microorganisms comprising rhodopsins with other microorganisms comprising proteorhodopsins). Accordingly, it is envisaged that, by introducing a variety of different light-dependent ion pumps into microorganisms (i.e., engineering rhodopsins into acetogenic microorganisms), the genetically engineered microorganisms can be controlled or modulated using multiple different wavelengths of light. As such, the production of desirable biochemical products via the conversion of inorganic carbon sources can be modulated at the level of light and redox in microorganisms engineered to utilise both light-dependent and redox-dependent mechanisms of carbon fixation.

[0144] In another embodiment, the microorganisms may be engineered such that they comprise at least one modified rhodopsin. The rhodopsin may be modified in order to achieve responsiveness to a variety of different wavelengths. In particular, rhodopsins may be modified such that the wavelength of light at which their peak absorption occurs may be increased or decreased. Substitution of amino acids involved in the capture of photons can be used to alter the binding kinetics of the ion being translocated by the rhodopsin. Therefore, it is envisaged that microorganisms may comprise multiple different rhodopsins, multiple different kinds rhodopsins, multiple different kinds of light-dependent ion pumps, multiple modified rhodopsins, or any combination thereof. A single microorganism may be engineered to comprise multiple different modified rhodopsins modified to absorb different wavelengths of light. A single microorganism may be engineered to comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten modified rhodopsins (which may have the same or different modifications). A single microorganism may be engineered to comprise a modified rhodopsin (or multiple different modified rhodopsins) together with unmodified rhodopsins (or multiple different unmodified rhodopsins) and / or different kinds of light-dependent ion pumps. In addition to microorganisms being engineered to comprise different light-dependent ion pumps such as rhodopsins, microorganisms may also be engineered to comprise other light-responsive proteins. Changes in light or wavelengths of light can be transmitted to impact to gene expression in microorganisms. Therefore, in one embodiment, the microorganism is engineered to comprise a recombinant light-sensitive DNA binding protein that targets a promoter for a gene of interest, such that the promoter is controlled by the defined modulation of light. As used herein, the term “light-sensitive DNA binding protein” refers to a protein which is capable of binding or unbinding a DNA sequence in response to light. In particular, a light-sensitive DNA binding protein may be activated or repressed in response to light.

[0145] As will be understood by the skilled person, DNA binding proteins can have specific affinity for a certain DNA sequence or general affinity for DNA sequences, and can bind single stranded DNA or double stranded DNA. DNA binding proteins can be generally classified into four groups: homeodomain proteins, zinc finger proteins, leucine zipper proteins, and helix loop helix proteins. DNA binding proteins can bind or interact with DNA via structural motifs which will be known in the art, and can act to stimulate or repress transcription of DNA into messenger RNA (mRNA). A light-responsive DNA binding protein may act on DNA in response to light. For example, a light-responsive DNA binding protein may undergo activation upon illumination by light at a specific wavelength such that it dimerises enabling the dimerised protein to bind a specific DNA sequence, such as a promoter.

[0146] In the context of the present invention, the production of desirable biochemical products can be controlled using light in microorganisms engineered to comprise a recombinant light-sensitive DNA binding protein. In particular, the components necessary for the production of desirable biochemical products may be encoded downstream of a promoter controlled or targeted by a light-responsive DNA binding protein. If the DNA binding protein controlling said promoter is an activator of transcription, light can be used to manipulate the binding of the DNA binding protein to the promoter such that transcription of the genes encoding the components necessary for the production of desirable biochemical products is activated. For instance, if the DNA binding protein is activated by a specific wavelength of light, application of light at this this wavelength to a culture of microorganisms expressing said DNA binding protein would activate or increase production of the desirable biochemical products. Likewise, limitation of light at this wavelength would reduce or prevent the production of the desirable biochemical products. Similarly, if the DNA binding protein controlling said promoter is a repressor of transcription, activation of the DNA binding protein at light at its activating wavelength to a culture of microorganisms expressing said DNA binding protein would reduce or prevent production of the desirable biochemical products. Likewise, limitation of light at this wavelength would activate or increase the production of the desirable biochemical products. Examples of light-sensitive DNA binding proteins include, but are not limited to, proteins with DNA binding domains, recombinases, transcription factors, polymerases, nucleases, histones, and DNA repair proteins.

[0147] As would be readily understood by the skilled person, light-responsive RNA targeting proteins as well as light-responsive DNA targeting proteins (such as light-responsive DNA binding proteins) may also be used to modulate the production of desirable biochemical products in microorganisms engineered to comprise recombinant light-responsive proteins. A light-sensitive DNA or RNA targeting enzyme may be activated or repressed in response to light.

[0148] Accordingly, in another embodiment of the invention, the microorganism comprises a recombinant DNA or RNA targeting enzyme that is activated upon application of light. As used herein, the term “DNA targeting enzyme” refers to an enzyme which acts on single stranded and / or double stranded DNA. In contrast to “DNA binding proteins”, “DNA targeting enzymes” do not only bind DNA, but also perform some form of catalytic activity on the DNA to which they become bound. Therefore, all DNA targeting enzymes are DNA binding proteins, but not all DNA binding proteins are DNA targeting enzymes. DNA targeting enzymes which are activated upon application of light are therefore enzymes which are prompted to act on DNA in response to certain wavelengths of light. DNA targeting enzymes may have several modes of ‘acting’ on DNA, such as DNA repair, recombination, replication, restriction, modification, topoisomerase action, cleavage, or recognition. In particular, the components necessary to produce desirable biochemical products may be encoded a sequence of DNA which can targeted by a light-activated DNA targeting enzyme. Thus, light can be used to control the expression of the DNA sequence and thus the expression of the components necessary to produce desirable biochemical products. For instance, if the light-activated DNA targeting enzyme is a nuclease activated by a specific wavelength of light, then application of light at this wavelength to a culture of microorganisms expressing said DNA targeting enzyme would decrease production of the desirable biochemical products by triggering the degradation of the DNA sequence. Likewise, limitation of light at this wavelength would increase production of the desirable biochemical products by decreasing the degradation of the DNA sequence. The preference of the present invention is EL222.

[0149] As used herein, the term “RNA targeting enzyme” refers to an enzyme which acts on RNA molecules (including, but not limited, to messenger RNA, ribosomal RNA, transfer RNA, and regulatory RNAs including but not limited to, microRNA, small interfering RNA etc). RNA targeting enzymes which are activated upon application of light are therefore enzymes which are prompted to act on RNA in response to certain wavelengths of light. RNA targeting enzymes include, but are not limited to, recombinases, nucleases (including programmable nucleases such as Cas enzymes), polymerases, translation factors, RNA processing enzymes etc. The components necessary to produce desirable biochemical products may be encoded a sequence of RNA which can targeted by a light-activated RNA targeting enzyme. Thus, light can be used to control the expression of the RNA sequence and thus the expression of the components necessary to produce desirable biochemical products. For instance, if the light-activated RNA targeting enzyme is a polymerase activated by a specific wavelength of light, then application of light at the activating wavelength to a culture of microorganisms expressing said RNA targeting enzyme would increase translation and thus increase production of the desirable biochemical products. Likewise, limitation of light at this wavelength would decrease or prevent translation and thus decrease or prevent production of the desirable biochemical products. In a preferred embodiment, the DNA or RNA targeting enzyme is a recombinase protein. As will be known in the art, recombinases are a superfamily of proteins which can be generally categorised either as tyrosine or serine recombinases, depending on the active amino acid present within the catalytic domain of the enzyme. Tyrosine recombinases can be either ‘bidirectional’ (such as Cre) or ‘unidirectional’ (such as Lambda). Serine recombinases are either ‘small’ (such as beta-six) or ‘large’ (such as Bxb1). Each recombinase is site specific and will act at specific recognition sites to cleave and reunite sequences leading to integration, deletion, or inversion of a nucleic acid fragment without gain or loss of nucleotides. A recombinase may be split into two parts, each part being linked to an inactive photo-dimer, known as WD. Upon activation by light, the two WD parts undergo a conformational change, resulting in the formation of a WD dimer which enables the recombinase to recover its function and target a DNA or RNA sequence.

[0150] As would be understood in the art, inducible promoters are a common tool that can be configured and engineered to induce a specific change to the expression of a gene or set of genes. Inducible promoters can be responsive to process parameters such as pH, temperature, or parameters internal to the cell such as stress, redox, pH, the concentration or presence of biochemical compounds such as but not limited to carbohydrates, proteins, short peptides, organic compounds and fatty acids (Figures 8 and 9). Heat shock inducible promoters are regulated by two alternative sigma factors (sigma-32 and sigma-E). These sigma factors direct RNA-polymerase to specific promoters. At low temperatures, the secondary structures of the mRNA of sigma-32 and sigma-E hide the Shine- Dalgarno sequence and start codon leading to low translational rates. At higher temperatures the secondary structures melt leading to higher translational rates of the sigma factors and subsequently activation of heat shock inducible promoters.

[0151] Inducible promoters can be engineered to implement Boolean logic gates to confer fine and tight control of transcription and therefore expression of genes (Figure 10). One way this can be accomplished is by tying together the outputs of one promoter to the inputs of other promoters. Another example consists of programming modulation of the inputs of one promoter by outputs of one or more promoters. Examples of Boolean logic gates that can be engineered include but are not limited to AND gates, NAND gates, OR gates, XOR gates, NOT gates and XNOR gates. Components that can be used to assemble Boolean logic gates, herein referred to as biocircuitry, includes but is not limited to, DNA binding molecules that induce or repress expression, RNA binding molecules that stabilise or destabilise RNA, protein molecules that promote degradation or stability of a protein, protein molecules that activate, deactivate or modulate the activity of an enzyme or protein complex.

[0152] In another embodiment, the microorganism comprises a target-specific light- activated protein degradation system, such that activation of the system is controlled by controlled exposure to light. As used herein, the term “protein degradation system” refers to a degron tag, an adapter protein and the native protein degradation system. Examples of a protein degradation system include, but are not limited to the degron tag SsrA native and the SspB adapter and the CIpX and CIpP protein degradation system, all of which are native to Escherichia coli. The protein degradation system of the present invention will preferably be based on an E. coli protein degradation system. As would be readily understood by the skilled person, to use components of a heterologous protein degradation system such as that of E. coli in a different target host microorganism requires characterisation of those components to validate and optimise the interaction of those components such that they behave and operate effectively, meaning there is limited cross-talk between the native and heterologous components that result in protein degradation of unintended proteins or that the target protein is efficiently degraded.

[0153] The microorganisms of the present invention may comprise both a lightdependent ion pump and a redox-dependent ion pump. The presence of both a light-dependent ion pump and a redox-dependent ion pump allows for dual layered control of microorganisms via the separate light-dependent and redoxdependent mechanisms. Accordingly, it will also be possible to control the microorganism using light as well as CO2 and H2. Controlling or modulating microorganisms using light in combination with other inputs (such as CO2 and H2) may be used to create Boolean logic circuits. For example, utilising two promoters within a recombinant microorganism wherein the first promoter is controlled via light-dependent mechanisms, and wherein the second promoter is controlled via light-dependent or non-light dependent mechanisms such as redox-dependent mechanisms including inputs such as CO2 and H2or pH, temperature, chemicals, and so on. A third promoter may be incorporated which is controlled via both the lightdependent and redox-dependent mechanisms. In such a microorganism, the first promoter would trigger the production of a first desirable biochemical product, and the second promoter would trigger the production of a second desirable biochemical product, with the third promoter triggering the production of a third biochemical product only during the presence of both inputs. In an alternative configuration, the first and second promoters lead to intermediate biochemical products required for activation of a third promoter, which, is only activated in the presence of products from both the first and second promoter. Therefore, in another embodiment, the metabolic function of the microorganism is regulated by controlled exposure to light, CO2, H2, pH, temperature, chemicals and the like, or any combination thereof. These gases can be provided to a culture of said microorganisms at a controlled rate. In one embodiment, light can be supplied to a culture of microorganisms via an artificial light source such as an LED or fluorescent light. The light source must be capable of emitting varying wavelengths of light such that the light-responsive components within the microorganism can be appropriately modulated. In another embodiment, CO2and H2can be supplied to a culture of microorganisms via the environment, i.e. , the culture of microorganisms may be present within a vessel which is configured to directly receive CO2, H2(or other waste gases) from the environment such as the atmosphere, or an industrial waste stream or outlet.

[0154] In a third aspect of the invention, there is provided a genetically modified acetogen comprising a recombinant rhodopsin and a recombinant nucleic acid molecule comprising a sequence encoding a light-activated protein, preferably wherein the light-activated protein is partitioned and then reconstituted through a light- activated partner, more preferably wherein the light-activated protein is a light- activated DNA or RNA binding protein.

[0155] The genetically modified acetogen of the third aspect of the invention may be any of Acetobacterium woodii (A. woodii), Clostridium ljungdahlii, C. carboxidivorans, C. autoethanogenum, Eubacterium limosum and Moorella thermoacetica. However, the preference is C. autoethanogenum. The genetically modified acetogen may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten recombinant rhodopsins. As used herein, the term “recombinant rhodopsin” refers to a rhodopsin (a light-dependent ion pump) that is non-naturally occurring or not ordinarily present in an acetogen. For example, a single acetogenic microorganism which ordinarily uses redox-dependent ion pumps may be engineered to comprise multiple recombinant rhodopsins. The rhodopsins may absorb the same wavelengths of light or ranges thereof, or different wavelengths of light or ranges thereof. The wavelength of light absorbed by rhodopsins may be between 400 to 700 nm.

[0156] An acetogen modified to comprise a recombinant rhodopsin or other light- responsive proteins may therefore be termed an “light-responsive acetogen” or a “photolithoautotroph”. A photolithoautotroph may or not be synthetic.

[0157] In addition to the genetically modified acetogen comprising a recombinant rhodopsin, the genetically modified acetogen will also comprise a recombinant nucleic acid molecule comprising a sequence encoding a light-activated DNA or RNA binding protein. As used herein, the term “recombinant nucleic acid molecule” refers to a sequence, segment of fragment of a nucleic acid, such as DNA or RNA, that is non-naturally occurring or not ordinarily present in an acetogen. As used herein, the terms “DNA” and “deoxyribonucleic acid” are used interchangeably, and refer to (single stranded and double stranded) nucleic acids composed of thymine, adenine, guanine, and cytosine deoxyribonucleic acid bases. As used herein, the terms “RNA” and “ribonucleic acid” are used interchangeably, and refer to nucleic acids composed of uracil, adenine, guanine, and cytosine ribonucleic acid bases. Types of RNA molecules include, for example, mRNA, siRNA, shRNA, miRNA, tRNA, and rRNA. These terms and concepts will be well known to those in the art.

[0158] The recombinant nucleic acid molecule in the context of the present invention will comprise a sequence encoding a light-activated protein. As used herein, the term “light-activated protein”, refers to a protein with an ability to switch from an inactive to an active state in response to illumination by light or wavelengths of light. Light- activated proteins may be activated by specific wavelengths of light or ranges thereof. In one embodiment, light-activated proteins are partitioned and reconstituted through a light-activated partner. For example, light-responsive domains are attached to the two halves of partitioned enzyme, which are brought into proximity upon illumination by light allowing for reconstitution of the enzyme and therefore recover its activity. In another embodiment, an enzyme or protein has one or more intein tags and a light responsive domain inserted into the middle of its amino acid sequence. Upon illumination the light-responsive domains activate the intein domains which self-splice and remove the inserted amino acid sequences and returning the protein to its native sequence and therefore activating it. In a preferred embodiment, two separate transcriptional units are indued by light responsive proteins that are activated by light of different wavelengths, with each transcriptional unit controlling the expression of separate components of a protein degradation system, and thereby upon illumination of the required wavelengths of light targets a preprogrammed protein for degradation by the host’s native system. Light-responsive systems (recombinant microorganisms) or components are therefore amenable or susceptible to modulation, manipulation or control using light or different wavelengths of light.

[0159] Light-activated proteins may be light-activated DNA binding proteins, light- activated RNA binding proteins, light-activated recombinase enzymes, and so on. In a preferred embodiment, the light activated protein is a light-activated DNA binding protein. As used herein, the term “light-activated DNA binding protein” refers to a protein which is capable of binding DNA sequence in response to light. The DNA binding protein will switch from an inactive to an active state upon illumination with a specific wavelength of light or range thereof. In its inactive state (i.e., in the absence of light) the light-activated DNA binding protein is unable to bind DNA. In its active state, the light-activated DNA binding protein is capable of binding DNA. The active state which is capable of binding DNA may involve a conformational change, such as the revealment of a DNA binding sequence which was previously sequestered. As will be understood by the skilled person, DNA binding proteins can have specific affinity for a certain DNA sequence or general affinity for DNA sequences, and can bind single stranded DNA or double stranded DNA. DNA binding proteins can be generally classified into four groups: homeodomain proteins, zinc finger proteins, leucine zipper proteins, and helix loop helix proteins. DNA binding proteins can bind or interact with DNA via structural motifs which will be known in the art, and can act to stimulate or repress transcription of DNA into messenger RNA. A light-activated DNA binding protein will become activated to bind or interact with DNA in response to light. For example, a light-activated DNA binding protein may switch from being unbound from (or unable to bind to) a DNA sequence, such as a promoter, to being bound to the DNA sequence upon illumination by light at a specific wavelength. In the context of the present invention, the production of desirable biochemical products can be controlled using light in microorganisms engineered to comprise a recombinant rhodopsin and a recombinant nucleic acid molecule comprising a sequence encoding a light-activated DNA binding protein.

[0160] In another preferred embodiment, the light activated protein is a light-activated RNA binding protein. As used herein, the term “light-activated RNA binding protein” refers to a protein which is capable of binding RNA sequence in response to light. The RNA binding protein will switch from an inactive to an active state upon illumination with a specific wavelength of light or range thereof. In its inactive state (i.e., in the absence of light) the light-activated RNA binding protein is unable to bind RNA. In its active state, the light-activated RNA binding protein is capable of binding RNA. The active state which is capable of binding RNA may involve a conformational change, such as the revealment of an RNA binding sequence which was previously sequestered. An RNA sequence may include, but is not limited to messenger RNA, ribosomal RNA, transfer RNA, and regulatory RNAs including but not limited to, microRNA, small interfering RNA. Light-activated RNA binding proteins are therefore enzymes which are prompted to bind or interact with RNA in response to certain wavelengths of light. In the context of the present invention, the production of desirable biochemical products can be controlled using light in microorganisms engineered to comprise a recombinant rhodopsin and a recombinant nucleic acid molecule comprising a sequence encoding a light- activated RNA binding protein.

[0161] In a preferred embodiment, the light-activated DNA or RNA binding protein is EL222. EL222 is a light-dependent DNA binding protein naturally occurring in the marine bacterium Erythrobacter litoralis HTCC2594. EL222 has reversible DNA binding activity depending on illumination with light. Once illuminated with blue light at a wavelength of around 450 nm, an internal flavin mononucleotide (FMN)- protein adduct is formed which triggers a change in the structural conformation of EL222. This conformational change reveals a previously sequestered C-terminal DNA binding domain (helix-turn-helix domain), which becomes free to bind DNA. Thus, in the dark or in the absence of the correct wavelength of light, EL222 is inactive and loses its DNA binding capacity (Nash, A.I. et al. (2011) ‘Structural basis of photosensitivity in a bacterial light-oxygen-voltage / helix-turn-helix (LOV- HTH) DNA-binding protein’, Proceedings of the National Academy of Sciences, 108(23), pp. 9449-9454).

[0162] In a more preferred embodiment, the acetogen comprises a target polynucleotide comprising a DNA binding site for said light-activated DNA binding protein. As used herein, the terms “target polynucleotide”, “recognition site”, “consensus sequence”, “binding site” and “motif” may be used interchangeably, and refer to a DNA sequence which will be recognised by a light-activated DNA binding protein. DNA binding proteins often require structural motifs within sequences of DNA which must be recognised in order for them to bind the correct sequence of DNA. The target polynucleotide may range from a short DNA sequence (e.g., 4 to 30 nucleotides in length) to a longer sequence (e.g., 200 nucleotides in length or more). Each DNA binding protein will recognise and bind to a certain target polynucleotide, or a polynucleotide with a certain level of sequence similarity to the target polynucleotide. In this embodiment, upon illumination with an activating wavelength of light or range thereof, the light-activated DNA binding protein will become activated (i.e., capable of binding DNA), and will thus bind the target polynucleotide comprising its DNA binding site.

[0163] Genetically modified acetogens comprising a recombinant rhodopsin and a recombinant nucleic acid molecule comprising a sequence encoding a light- activated protein will therefore be able to make use of light to generate ATP via light-dependent mechanisms and also to control the expression of genes with promoters under the control of light-activated proteins.

[0164] In another embodiment, the genetically modified acetogen further comprises a recombinant light-activated protein. For the avoidance of doubt, the term “light- activated protein”, refers to a protein with an ability to switch from an inactive to an active state in response to illumination by light or wavelengths of light. Light- activated proteins may be activated by specific wavelengths of light or ranges thereof. The genetically modified acetogen may comprise a recombinant light- activated protein (i.e., a light-activated protein which is not naturally present within the acetogen). In a preferred embodiment, the light-activated protein is partitioned and then reconstituted through a light-activated partner. In a more preferred embodiment, the light-activated protein is a recombinase enzyme.

[0165] As will be known in the art, recombinases are a superfamily of proteins which can be generally categorised either as tyrosine or serine recombinases, depending on the active amino acid present within the catalytic domain of the enzyme. Tyrosine recombinases can be either ‘bidirectional’ (such as Cre) or ‘unidirectional’ (such as Lambda). Serine recombinases are either ‘small’ (such as beta-six) or ‘large’ (such as Bxb1). Each recombinase is site specific and will act at specific recognition sites to cleave and reunite sequences leading to integration, deletion, or inversion of a nucleic acid fragment without gain or loss of nucleotides. A recombinase may be split into two parts, each part being linked to an inactive photo-dimer, known as WD. WD domains are light-activated protein domains. Upon activation by light, the two WD domains undergo a conformational change, resulting in the formation of a WD dimer which enables the recombinase to recover its function and target a DNA or RNA sequence.

[0166] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component.

[0167] As used herein, "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" can mean a range of up to 20%. When particular values are provided in the application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

[0168] EXAMPLES

[0169] Example 1 - evaluating the effect of introducing a bacteriorhodopsin into C. autoethanogenum growing on a gas mixture of CO2 H2and light.

[0170] The wild-type acetogen (DSM 10061) Clostridium autoethanogenum (C. autoethanogenum) was utilised to evaluate the effect of introducing a bacteriorhodopsin into an acetogen. However, the skilled person would understand that any suitable acetogen could be utilised, such as A. woodii. C. autoethanogenum (DSM 10061) contains a H+ion dependent ferredoxin: NAD+oxidoreductase (Rnf) complex which oxidises ferredoxin and reduces NAD+, with the concomitant transfer of H+ions across the membrane, creating an electrochemical gradient that is used to drive a H+ion specific ATP synthase, for the generation of ATP. To overcome the bioenergetic limitations of the endogenous metabolism (carbon fixation system), it is necessary to bioengineer the host to generate additional ATP for increased growth and biomass formation, or to increase formation of commercial products of interest beyond acetate. One way to achieve this is to increase the generation of the electrochemical gradient. Typically, this is generated by the above mentioned Rnf complex. However, it is also possible to introduce a light-based ion pump such as a bacteriorhodopsin. A H+ion specific bacteriorhodopsin (BR1) from Krokinobacter eikastus (K. eikastus) was recently reported, which if introduced into C. autoethanogenum should increase the generation of the electrochemical gradient, and ultimately ATP formation. Increased ATP formation should result in improved growth / biomass formation, or similar biomass formation with less acetate formation, as BR1 should supplement ATP required for growth, thereby reducing the need for acetate production.

[0171] The bactehorhodopsin-1 (br1) gene from K. eikastus was cloned into the vector pMTL-81151 , containing a Cas9 protein under the control of a weak native promoter of C. autoethanogenum. The br1 gene was cloned between two regions of homology that would allow for insertion of the gene downstream of the pyrE gene through homologous recombination. An insertion site downstream of pyrE site was chosen as it has been reported in the literature to be stable for expression of exogenous proteins. A constitutive promoter was cloned upstream of the br1 gene on the plasmid. The vector pMTL-83151 also contained a transcriptional unit consisting of an arabinose inducible promoter that would express the required trans-activating crRNA (tracrRNA) and a 20-nucleotide sequence (guide RNA or gRNA) identical to the target in the genome. The assembled plasmid was named pMTL-83151_pyrE::dsBR1. The plasmid pMTL-83151_pyrE::dsBR1 was electroporated into C. autoethanogenum using methods reported in the literature. Following a published recombination protocol, mutants with br1 integrated into the genome were isolated by screening the region of insertion by colony PCR. Suspected mutants were cured of the modified plasmid, and successful integration was verified by sequencing. Three different biological clones were isolated and stored following confirmation of sequencing results and were named Ca-BR1 .

[0172] Wild type C. autoethanogenum, and wild type C. autoethanogenum harboring pMTL-83151 and Ca-BR1 were cultivated in a 2-litre bioreactor in a fed-batch fermentation for 72 hours. A defined media was used for the fermentation, which was completed at 37 °C and without pH control. A gaseous mixture of 25% CO2 and 75% H2was supplied with a total flow rate of 80 ml I min. The cultures were illuminated using external LED strips as the bioreactor vessel / chamber was made of glass and transparent. The wavelength of light was approximately 520-575 nm and the light was illuminated continuously from the 8 hour timepoint. As C. autoethanogenum does not natively produce all-trans retinal (ATR) and it is required for the BR1 protein to be functional, 10uM was added during the fermentation at time of inoculation. Ca-BR1 was grown with (denoted via the asterisk) and without the addition of ATR in parallel, the latter acting as negative control. The biomass was tracked by measuring the optical density (OD) at 600 nm using a spectrophotometer, the pH was measured using an appropriate probe and the products (primarily acetate) were measured using high pressure liquid chromatography (HPLC). Samples to measure biomass (Figure 6A), pH (Figure 6B) and acetate (Figure 6C) were taken at 0 hours (h), 8h, 24h, 48h and 72h and plotted.

[0173] Strain Ca-BR1 cultured in the presence of ATR (Ca-BR1*) exhibited a slower growth rate than the wildtype or the wildtype harboring the empty plasmid pMTL- 83151. Addition of ATR (Ca-BR1*) resulted in higher biomass formation relative to Ca-BR1 cultured without ATR and C. autoethanogenum harboring the empty plasmid pMTL-83151 (Figure 6A). Importantly, Ca-BR1* generated higher biomass while producing less acetate than pMTL-83151 which correlates with a higher ending pH. Ca-BR1*was also able to generate significantly more biomass (1 .67 vs 1.27) than Ca-BR1 even though final pH and final acetate concentrations were similar. Although Ca-BR1* generated slightly less biomass (1.67 vs 1.73) than the wildtype, it was able to do so with significantly less acetate (135 mM vs 181 mM) resulting in a higher ending pH (5.62 vs 5.08) (Figures 6B and 6C). These results suggest that the activity of BR1 can contribute to improving the bioenergetics of carbon fixation by increasing ATP generation which results in less acetate production per biomass formation.

[0174] Example 2 - measuring in vivo activity of bacteriorhodopsin (BR1) expressed by a modified acetogen illuminated by light during growth in an external loop bioreactor using different concentration ofATR.

[0175] C. autoethanogenum was utilised to evaluate the activity of the bacteriorhodopsin when illuminated by light during growth in a bioreactor. Again, the skilled person would understand that any suitable acetogen could be utilised, such as A. woodii.

[0176] When introducing a heterologous protein into a new host, it is unclear whether the protein is expressed and if the protein is functional. The activity of introduced light-based ion pumps such as bacteriorhodopsins can be measured in vivo by measuring the pH of a solution containing cells expressing the protein and illuminated with light. This is one of the methods that is typically used when evaluating the activity of various rhodopsins when expressed in heterologous hosts.

[0177] The strain Ca-BR1 was cultured in a 2-litre external loop bioreactor in a fed-batch fermentation. A defined media was used for the fermentation, which was completed at 30° and without pH control. A gaseous mixture of 25% CO2 and 75% H2was supplied (gas ratio of 3: 1 ) with a total flow rate of 80 ml I min. The cultures were illuminated using external LED strips wrapped around the main chamber of bioreactor which was made of glass and transparent. The wavelength of light was approximately 520-550 nm. As C. autoethanogenum does not natively produce all-trans retinal (ATR) and it is required for the BR2 protein to be functional. Three concentrations of ATR were tested, 10uM, 1 uM and 100 nM were added during the fermentation at time of inoculation. To evaluate the activity of the BR2 proteins, the light source was turned on for approximately 5-minute intervals and the pH of the solution was measured using a pH probe.

[0178] The cultures were inoculated and approximately 48 hours after inoculation, the pH of the cultures was between 5-5.3. At this time, the lighting system was turned on and the bioreactor was illuminated for approximately 5 minutes and then turned off for approximately 5 minutes. The pH of the cultures was measured (Figure 7).

[0179] Example 3 - Use of a photo-sensitive DNA binding protein for transcriptional regulation

[0180] EL222 is a photo-sensitive DNA-binding protein, which, upon activation by blue light, undergoes a structural change that allows the protein to dimerize with itself. Other photo-sensitive DNA-binding proteins that can be used for this system are WD and LEVI. The EL222 dimer subsequently binds to a specific DNA EL222 binding sequence. By adding a DNA EL222 binding sequence in a promoter or ribosome binding site (RBS) region, the expression of a set protein can be controlled using light. Those skilled in the art will be able to use this system for either activation or repression of a promoter of which the EL222 has been designed to bind.

[0181] Example 4 - A split recombinase protein activated by light

[0182] A recombinase (as with other DNA / R NA targeting enzymes) is split into two parts, each part being linked to an inactive photo-dimer (WD). Upon activation by light, the WD parts undergo a conformational change, through which they can form dimers and bring the two parts of the protein together, enabling it to recover its function and target DNA or RNA sequence. Example 5 - Protein degradation activated by light

[0183] In a similar strategy to that of Example 4, an adapter or regulator required for protein degradation is split into two parts where each part is linked to an inactive photo-dimer (WD). Upon activation by light the WD parts undergo a conformational change, through which they can form dimers and bring the two parts of the adapter together, allowing it to target the tags it recognizes and flagging the proteins with those tags for degradation.

[0184] Example 6 - Evaluating the effect of introducing a bacteriorhodopsin into A. woodii growing on gas.

[0185] The acetogen A. woodii contains a Na+ion dependent ferredoxin: NAD+oxidoreductase (Rnf) complex which oxidises ferredoxin and reduces NAD+, with the concomitant transfer of Na+ions across the membrane, creating an electrochemical gradient that is used to drive a Na+ion specific ATP synthase, for the generation of ATP. To overcome the bioenergetic limitations of the endogenous metabolism (carbon fixation system), it is necessary to bioengineer the host to generate additional ATP for increased growth and biomass formation, or to increase formation of commercial products of interest beyond acetate. One way to achieve this is to increase the generation of the electrochemical gradient. Typically, this is generated by the above mentioned Rnf complex. However, it is also possible to introduce a light-based ion pump such as a bacteriorhodopsin. A Na+ion specific bacteriorhodopsin (BR2) from Krokinobacter eikastus (K. eikastus) was recently reported, which if introduced into A. woodii should increase the generation of the electrochemical gradient, and ultimately ATP formation. Increased ATP formation should result in improved growth / biomass formation, and thus can be used as a proxy to evaluate if the strategy is working (Figure 11)

[0186] (A) Wild type (DSM 1030) Acetobacterium woodii (A. woodii) and the empty expression plasmids pMTL-84151 and pMLT-83151 were used as controls. (B) The bacteriorhodopsin-2 (br2) gene from K. eikastus was cloned into pMTL- 84151 , and the resultant recombinant plasmid was transformed into A. woodii. Bacteriorhodopsins requires all-trans retinal (ATR) to be functionally active. Proton-pumping rhodopsins are associated with a retinal pigment that is isomerised from the all-trans state to the 13-cis state after absorption of a photon. Therefore, exogenous addition of ATR to culture media is necessary, as ATR is not naturally synthesised by A. woodii. Strains of A. woodii expressing bacteriorhodopsin in the presence (+ATR) were compared to the wild type and to A. woodii expressing bacteriorhodopsin in the absence (-ATR) of all-trans retinal, which served as a negative control. Pre-cultures of each strain were grown on a defined medium with fructose as the substrate, and inoculated into defined media lacking a carbon source. A gaseous mixture of 50% CO2and 50% H2was supplied to strains. The light source was applied after 48 hours when maximal growth of the strains was reached. A final optical density at 600 nm (OD6oo) reading was taken after 72 hours.

[0187] Example 7 - Measuring in vivo activity of bacteriorhodoosin (BR2).

[0188] When introducing a heterologous protein into a new host, it is unclear whether the protein is expressed and if the protein is functional. The activity of introduced light-based ion pumps such as bacteriorhodopsins can be measured in vivo by measuring the pH change (ApH) of a solution containing cells expressing the protein and illuminated with light. This is one of the methods that is typically used when evaluating the activity of various rhodopsins when expressed in heterologous hosts (Figure 12).

[0189] Figure 12A shows the results of measuring BR2 activity in A. woodii. The br2 gene was cloned into a pMTL-84151 expression plasmid and transformed into A. woodii. ATR was exogenously added to media to enable functionality of the bacteriorhodopsin. Three clones of A. woodii expressing the BR2 protein were cultured, two of which (BR2i and BR2ii) were grown on media with exogenous ATR supplementation (+ATR). The third clone was cultured in the absence of exogenous ATR supplementation (-ATR), providing a negative control. Cultures were harvested in the mid-exponential growth phase (OD6oo ~ 0.3) by centrifugation and washed in a buffer (100 mM NaCI, pH 7.2 - 7.3), then resuspended in the wash buffer to an OD6oo of 2.0. The pH of the solution was measured continuously by submerging a pH probe in the solution. The solution was allowed to equilibrate for at least 10 minutes before the light source was applied. The light source emitted light in the range 500 - 550 nm, and had 5 power settings (0 - 5). The light power setting applied to BR2i and BR2ii clones was 5 and 4, respectively. Figure 12B shows the results of measuring BR1 activity in A. woodii. The same protocol as for br2was used to evaluate the activity of the br1 gene when introduced into A. woodii using the expression plasmid pMTL- 84151. The protein BR1 is proton (H+) specific and is have the opposite effect on the pH as BR2, which was validated.

[0190] Example 8 - Controlling the expression of desired products in response to different wavelengths of light

[0191] Three proteins were utilised to assess their expression in response to specific wavelengths of light. Briefly, a synthetic photolithoautotrophic microorganism was engineered such that expression of a desired protein or enzyme could be controlled externally using light, temperature, pH or phosphate as the modulator. This biocircuitry is used for increasing the pool of acetyl-CoA within the cell for improved product formation, or for reducing byproduct formation to enhance overall yield.

[0192] The microorganism was grown in the presence of gases CO2 and H2in a bioreactor with the required nutrient medium to sustain growth. The bioreactor had light sources such as LEDs that can deliver differing intensities of light at specific wavelengths into the bioreactor, which are used for controlling expression of proteins or enzymes within the cells by triggering the biocircuitry of the microorganism.

[0193] In short, inteins are proteins that can ligate the flanking external proteins through a process called splicing. By adding a target protein to the N-terminus of the N- intein and a degron-tag to the C-terminus of the C-intein we created a system in which a degron tag can be added to a target protein. The controlled expression of both N-intein and C-intein, using different modulators, functions like an AND-gate. By controlling the expression of the SspB adapter a second AND-gate is created. Upon expression of the adapter (SspB) the expressed degron-tagged proteins are targeted for degradation (Figure 10).

[0194] In the case of Figure 10A, the target protein is added to the N-terminus of the N- intein and the degron tag is added to the C-terminus of the C-intein. When both inteins are expressed, they undergo a process known as trans-splicing in which the intein is removed and the degron-tag is added to the target protein. Upon expression of the adapter (SspB) the degron-tagged proteins are targeted for degradation.

[0195] In the case of Figure 10B, The SspB adapter was split into two parts (SspB-Aand SspB-B). When both SspB-Aand SspB-B are expressed, they undergo a proteinprotein interaction leading to the formation of a biologically functional adapter. The controlled expression of both SspB parts, using different modulators, functions like an AND-gate. By controlling the expression of target degron-tagged proteins a second AND-gate is created. Upon expression of the adapter (SspB) the expressed degron-tagged proteins are targeted for degradation.

[0196] The engineered microorganism was grown in the bioreactor with samples taken once every hour to measure protein or organic product concentrations.

[0197] Metabolomics studies were conducted to confirm the increased or decreased levels of acetyl-CoA within the cell following induction of the biocircuitry described.

[0198] High pressure liquid chromatography was used to detect and track the concentrations of ethanol, lactate and acetate in the bioreactor medium following induction of the various biocircuitry. Figures 13A and 13B show the results of the synthetic photolithoautotrophic microorganism that was grown in the presence of carbon dioxide and hydrogen gases, engineered with biocircuitry which enables the expression of desired products in response to different wavelengths of light. The arrows in Figure 13 indicate when illumination begins.

[0199] In Figure 13A (A) the microorganism was engineered to express protein A in response to illumination of a 550 nm wavelength of light (wavelength A), where protein A is a NADH or NADPH-dependent formate dehydrogenase.

[0200] In Figure 13A (B) the microorganism was engineered to express protein B in response to illumination of a 650 nm wavelength of light (wavelength B), where protein B is a NADH or NADPH-dependent methylene-THF reductase.

[0201] In the case of Figure 13A (Aand B), enzymes within the Wood-Ljungdahl pathway (WLP) were overexpressed such that they lead to increased gas uptake and CO2conversion or enhanced driving force and through the pathway, respectively, for enhanced carbon flux and increasing the pool of acetyl-CoA within the cell. Therefore, it will be appreciated that other proteins, beyond NADH or NADPH- dependent formate dehydrogenases and NADH or NADPH-dependent methylene-THF reductases, which may be involved in carbon fixation pathways such as the WLP may be controlled as described herein and as shown in Figure 13.

[0202] In Figure 13B (C) the microorganism was engineered to express proteins A and B in response to illumination of a 550 nm and 650 nm wavelength of light, respectively, whereby proteins A and B are transcription factors (expression levels not shown), in which both must be expressed to induce expression of protein C, where protein C is a NADH or NADPH-dependent aldehyde dehydrogenase, alcohol dehydrogenase and / or an aldehyde ferredoxin oxidoreductase. In the case of Figure 13B (C), an AND gate was built using transcription factors that are each independently controlled by two separate light inputs, which activate a third promoter that leads to expression of the desired protein C.

[0203] In Figure 13B (D) the microorganism was engineered to express proteins A and B in response to illumination of a 550 nm and 650 nm wavelength of light, respectively, while expression of protein C is in response to illumination of both 550 nm and 650 nm wavelength of light, and whereby proteins A, B and C are any one of the aforementioned enzymes. In the case of Figure 13B (D), expression of proteins A and B was independently controlled by separate light inputs of different wavelengths, while expression of protein C was controlled by both light inputs that control proteins A and B and is only expressed in the presence of both light inputs.

[0204] Proteins A, B or C may be used to improve gas conversion and increase carbon flux through central carbon pathways such as the Wood-Ljungdahl pathway to increase pools of acetyl-CoA, or proteins involved in protein degradation pathways such as adapter proteins to reduce by-product formation or increase pool of acetyl-CoA, or to increase product formation, for example ethanol, where the proteins may be a NADH or NADPH-dependent aldehyde dehydrogenase, NADH or NADPH-dependent alcohol dehydrogenase, and / or an aldehyde ferredoxin oxidoreductase, which use the existing acetyl-CoA pool directly or indirectly for increased ethanol formation.

[0205] Example 9 - Controlling the degradation of target proteins in response to different wavelengths of light

[0206] Figure 14 shows the results of a synthetic photolithoautotrophic microorganism that was grown in the presence of carbon dioxide and hydrogen gases, engineered with biocircuitry which enables the concentration of a target protein to be controlled through tuneable degradation (i.e. , degradation in a tuned manner). The expression of an adaptor protein was controlled through process conditions such as the presence of phosphate or light - the expression of the adapter protein is dependent on the concentration of phosphate or the intensity of light provided by the system (e.g., a bioreactor), respectively.

[0207] In Figure 14, the target protein A was lactate dehydrogenase, which through its degradation lead to decreased lactic acid formation. The adapter protein can be, but is not limited to, CIpX and CIpP or the SspB adapter.

[0208] PstSCAB proteins form a membrane bound complex essential for the uptake of phosphate from the external environment. When this complex is bound to its substrate, it prevents the phosphorylation of a histidine kinase called PhoR. PhoR in turn plays a role in the phosphorylation of a transcriptional activator called PhoB. At low phosphate concentrations PhoB is phosphorylated and interacts with specific binding sites (PHO boxes) in target promoter regions. The PHO boxes can be used to create activatable or repressible promoters using phosphate limitation as the modulator.

[0209] The adaptor protein was selected to trigger the targeted degradation of the target protein. The presence of the adapter is designed such that its expression is modulated in response to changing phosphate conditions.

[0210] In Figure 14 (A), a microorganism was engineered with biocircuitry such that the concentration of protein A could be tuned through targeted degradation by expressing an adapter protein that leads to its degradation. The presence of the adapter was designed such that its expression could be modulated by changing process conditions, in this case in response to decreasing concentration of PO4.

[0211] In Figure 14 (B), a microorganism was engineered with biocircuitry such that the concentration of Protein X can be tuned through targeted degradation by expressing an adapter protein that leads to its degradation. The expression of the adapter protein was controlled by a light inducible promoter and its expression level was modulated through the intensity of light used (ranging from low light, medium light to high light). Light intensity was measured in ranges of lumens, with between 0-500 lumens per square meter (lux) as ‘low light’, 500-5000 lux as ‘medium light’, and above 5000 lux as ‘high light’.

Claims

CLAIMS1. 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. 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. The bioreactor according to claim 1 or claim 2, wherein the inorganic carbon source is CO2.

4. The bioreactor according to any preceding claim, wherein the components comprise those of a linear carbon fixation pathway.

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. The bioreactor according to claim 4 or 5, wherein said system comprises NADH- and NADPH-dependent reductases and an electron bifurcating reductase.

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. 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. 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. 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. 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. 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. The bioreactor according to any preceding claim, wherein the microorganism further comprises photosynthetic machinery of purple non-sulphur bacteria.

14. The bioreactor according to any preceding claim, wherein the lightdependant ion pump is a rhodopsin.

15. The bioreactor according to claim 14, wherein the rhodopsin is a bacteriorhodopsin, proteorhodopsin, deltarhodopsin, xanthorhodopsin, halorhodopsin, channelrhodopsin, archaerhodopsin, or bacterial sensory rhodopsin.

16. The bioreactor according to any preceding claim, wherein the redoxdependant ion pump is an Rnf or Ech protein complex.

17. The bioreactor according to any preceding claim, wherein the recombinant microorganism is an acetogen.

18. The bioreactor according to any of claims 1 to 16, wherein the recombinant microorganism is a purple non-sulphur bacterium.

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. 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.

21. The bioreactor according to any preceding claim, wherein the bioreactor is configured to provide input of carbon dioxide (CO2), hydrogen (H2), and light.

22. The bioreactor according to claim 21 , wherein the bioreactor comprises a means for creating turbulence in the bioreactor.

23. The bioreactor according to claim 22, wherein the means for creating turbulence is a fixed means for creating turbulence, preferably wherein the fixed means is a baffle.

24. The bioreactor according to any preceding claim, wherein the bioreactor comprises a light source, preferably wherein the light source is an artificial light source.

25. The bioreactor according to claim 24, wherein the light source is a light bulb, a fluorescent light, or a light-emitting diode (LED).

26. The bioreactor according to any preceding claim, wherein the bioreactor comprises an inlet, a chamber for retaining the microorganism in an aqueous environment, and an outlet.

27. The bioreactor according to any preceding claim, wherein the bioreactor comprises one or more spargers and / or nozzles.

28. The bioreactor according to any preceding claim, wherein the bioreactor comprises one or more ejectors to mix the contents of the bioreactor.

29. The bioreactor according to any preceding claim, wherein the bioreactor comprises one or more external loops.

30. The bioreactor according to any preceding claim, wherein the bioreactor comprises a gas compressor to introduce gases into the bioreactor.

31. The bioreactor according to any preceding claim, wherein the bioreactor comprises a liquid pump to introduce liquids into the bioreactor.

32. The bioreactor according to any preceding claim, comprising means to control the pH and / or temperature of a liquid environment within the bioreactor during fermentation.

33. A method for controlling the production of a microbially-produced biochemical product, wherein the product is derived from the conversion of acetyl- CoA, comprising culturing a genetically engineered microorganism under suitable conditions, said microorganisms comprising: i. a light-dependent ion pump that generates an electrochemical ion gradient independently of the generation of reducing equivalents; and ii. a redox-dependent ion pump that generates an electrochemical ion gradient independently of a net change in the number of reducing equivalents; and wherein said culturing comprises modulating the exposure of the microorganism to light to drive the light-dependent ion pump to control the generation of the electrochemical ion gradient.

34. The method according to claim 33, wherein the electrochemical gradient generated by the ion pumps of (i) and / or (ii) is utilised to generate ATP.

35. The method according to claim 33 or claim 34, wherein the microorganism is cultured in the presence of an inorganic carbon source, preferably CO2.

36. The method according to any of claims 33 to 35, wherein the microorganism comprises the components of a linear carbon fixation pathway.

37. The method according to claim 36, wherein the linear carbon fixation pathway comprises components of the Wood-Ljungdahl pathway (WLP) required to generate acetyl-CoA.

38. The method according to claim 36 or 37, wherein said microorganism comprises NADH- dependent reductases and NADPH-dependent reductases and an electron bifurcating reductase.

39. The method according to claim 38, wherein the NADH- dependent reductases and NADPH-dependent reductases are present together with a transhydrogenase that interchanges reducing equivalents from one species to another.

40. The method according to any of claims 33 to 39, wherein there is a source of reducing equivalents generated by biotic components.

41. The method according to claim 40, wherein the biotic components comprise one or more of: electron bifurcating hydrogenase, oxygen tolerant hydrogenase, formate dehydrogenase, or carbon monoxide dehydrogenase.

42. The method according to any of claims 33 to 41 , wherein the ion pumps of(i) and (ii) 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.

43. The method according to claim 42, wherein an ATP synthase uses said electrochemical ion gradient generated by the ion pumps of (i) and / or (ii) to generate ATP.

44. The method according to claim 42 or 43, wherein the ion pumps of (i) and(ii) and the ATP synthase depend preferentially on ions of the same species.

45. The method according to claim 42 or 43, wherein the ion pumps of (i) and (ii) 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.

46. The method according to any of claims 33 to 45, further comprising photosynthetic machinery of purple non-sulphur bacteria.

47. The method according to any of claims 33 to 46, wherein the lightdependent ion pump is a rhodopsin.

48. The method according to claim 47, wherein the rhodopsin is a bacteriorhodopsin, proteorhodopsin, deltarhodopsin, xanthorhodopsin, halorhodopsin, channelrhodopsin, archaerhodopsin, or bacterial sensory rhodopsin.

49. The method according to any of claims 33 to 48, wherein the redoxdependent ion pump is an Rnf or Ech protein complex.

50. The method according to any of claims 33 to 49, wherein the recombinant microorganism is an acetogen.

51. The method according to any of claims 33 to 49, wherein the recombinant microorganism is a purple non-sulphur bacterium.

52. The method according to any of claims 33 to 51 , further comprising biochemical components necessary for converting the generated acetyl-CoA into a biochemical product.

53. The method according to claim 52, wherein the biochemical product is selected from one of the following compound classes: alcohols, sugars aldehydes, alkaloids, alkanes, alkenes, alkynes, natural or synthetic 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.

54. The method according to claims 47 or 48, wherein the microorganism comprises multiple different rhodopsins, such that the microorganism can utiliselight at different wavelengths to thereby control regulation of the rhodopsin- generated electrochemical ion gradient.

55. The method according to claim 54, wherein the microorganism comprises at least one modified rhodopsin and at least one light-activated protein, such that different wavelengths of light enhance different functions within the microorganism.

56. The method according to any of claims 33 to 55, wherein the microorganism is engineered to comprise a recombinant light-sensitive DNA binding protein that targets a promoter for a gene of interest, such that the promoter is controlled by the defined modulation of light.

57. The method according to any of claims 33 to 56, wherein the microorganism comprises a recombinant DNA or RNA targeting enzyme that is activated upon application of light.

58. The method according to claim 57, wherein the DNA or RNA targeting enzyme is a recombinase protein.

59. The method according to any of claims 33 to 58, wherein the microorganism comprises a target-specific light-activated protein degradation system, such that activation of the molecule is controlled by controlled exposure to light.

60. The method according to any of claims 33 to 60, wherein the metabolic function of the microorganism is regulated by controlled exposure to light, CO2, and H2.

61. A genetically modified acetogen comprising a recombinant rhodopsin and a recombinant nucleic acid molecule comprising a sequence encoding a light- activated protein, preferably wherein the light-activated protein is partitioned and then reconstituted through a light-activated partner, more preferably wherein the light-activated protein is a light-activated DNA or RNA binding protein.

62. The genetically modified acetogen according to claim 61 , wherein the light- activated binding protein is EL222.

63. The genetically modified acetogen according to claim 61 or claim 62, wherein the acetogen comprises a target polynucleotide comprising a DNA binding site for said light-activated DNA binding protein.

64. The genetically modified acetogen according to any of claims 61 to 63, further comprising a recombinant light-activated protein, preferably wherein the light-activated protein is partitioned and then reconstituted through a light- activated partner, more preferably wherein the light-activated protein is a recombinase enzyme.

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