Bacteria

Abstract

A modified cyanobacterium comprising one or more genetic modifications for improving expression of aromatic amino acids through overexpression of shikimate pathway to enhance the rate of carbon dioxide fixation relative to a cyanobacterium which does not comprise said genetic modifications. Also disclosed is use of AroF D174N and / or TesA for increasing the rate of carbon dioxide fixation by a modified cyanobacterium. The cyanobacterium can be Synechococcus sp., e.g. Synechococcus 11901 or a variant thereof. The modified cyanobacterium can comprise one or more genetic modifications to over-express shikimate kinase (SK) in the modified cyanobacterium.

Description

Field of the Invention The invention relates to modified photosynthetic bacteria, such as cyanobacteria, for carbon dioxide fixation, and methods of preparing said bacteria and uses thereof. Background to the Invention Anthropogenic carbon dioxide (CO2) emissions from industrial activities amount to 41 billion metric tonnes per annum globally. The search for effective carbon capture technologies is, therefore, a crucial step towards achieving Net Zero. Conventional carbon capture technologies have been developed, but their implementation has been limited due to their high energy requirements. Cyanobacteria are a group of photosynthetic microorganisms that can convert atmospheric CO2 into organic compounds through the process of photosynthesis. These microscopic organisms have been shown to have a high rate of CO2 uptake at ambient temperatures and ambient outdoor light conditions making them a promising option for mass-scale low-cost carbon capture. Cyanobacteria have been genetically modified with an aim to enhance their capabilities for industrial-scale carbon capture. However, challenges persist, such as achieving stable and consistent genetic modifications, ensuring efficient expression of introduced genes, and maintaining long-term viability of engineered strains under industrial conditions. Further challenges include the dependency on continuous lighting and the slow nature of carbon dioxide fixation. For example, the maximum photosynthetic efficiency of cyanobacteria is widely accepted to be between 1.5-2%, with the reaction kinetics in the light-independent carbon dioxide fixation step of the Calvin Benson cycle being a bottleneck in the flux of carbon in the cell’s metabolism (Oliver et al., 2013, PNAS 110(4): 1249-54). This is despite the quantum efficiency of the light harvesting complex (LHCs) in the thylakoids being upwards of 83%. Attempts to improve the activity of the key CO2 fixation enzyme, Rubisco, have had limited success (Whitney et al., 2011, Plant Physiol. 155(1):27-35), and many studies indicated that Rubisco catalytic efficiency may already be optimized to its maximum (Tcherkez et al., 2006, PNAS 103(19):7246-51). Therefore, alternative or improved strategies are needed to relieve the bottlenecks in CO2 fixation. Accordingly, it is an object of the invention to develop a further or improved modified photosynthetic bacteria, such as cyanobacteria, for improving carbon capture. Summary of the Invention The inventors generated modified cyanobacteria strains that have an enhanced rate of carbon dioxide fixation. To achieve this, cyanobacteria were engineered to create a ‘carbon sink’ at the expense of the cyanobacteria’s own energy balance by genetic mutations that result in overexpression and efflux of a carbon-dense compound such as aromatic amino acids. Interestingly, this created a perpetual reduction in the inhibitory feedback on the central metabolic pathways, thus alleviating some of the bottlenecks to the carbon flux and forcing the cell to fix more carbon dioxide into biomass and stable hydrocarbons as a compensation for the perpetual loss in fixed carbon species from the cell. In particular, the inventors modified the cyanobacterium Synechococcus 11901 strain to target the shikimate pathway, the Calvin Benson cycle, and fatty acid metabolism. The resulting modified cyanobacteria strains had deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes. The modified cyanobacteria strains provided improved growth rates (see Example 1) and improved CO2 fixation rate relative to the wild-type strain (see Example 2). In particular, modified cyanobacteria strains which encouraged the production of aromatic amino acids, e.g. strains B and C which over-expressed AroF D174N and shikimate kinase (SK), respectively, provided improved CO2 fixation rate. Accordingly, the invention provides a modified cyanobacterium comprising one or more genetic modifications for improving expression of aromatic amino acids through overexpression of shikimate pathway to enhance the rate of carbon dioxide fixation relative to a cyanobacterium which does not comprise said genetic modifications. The invention also provides a composition comprising one or more modified cyanobacteria of any one of the preceding claims for fixing carbon dioxide. The invention also provides a method of increasing the rate of carbon dioxide fixation of a cyanobacterium, comprising genetically modifying the cyanobacterium to generate a modified cyanobacterium according a method described herein. The invention also provides a method of fixing carbon dioxide, comprising exposing the modified cyanobacterium or composition as described herein to a source of carbon dioxide. The invention also provides the use of the modified cyanobacterium or composition as described herein for fixing carbon dioxide. The invention also provides the use of AroF D174N and / or tesA for increasing the rate of carbon dioxide fixation by a modified cyanobacterium. Brief Description of the Figures Figure 1 shows the growth of cyanobacteria strains over 9 days, as indicated by optical density (OD730) of the culture. Cyanobacteria strains tested: 11901 (wild type); strain A (AfadD::pJS23119-tesA), strain B (pcpt-aroF D174N), and strain E (AfadD::pJS23119-tesA, pJS23119-CA). Figure 2 shows the growth of cyanobacteria strains over 9 days, as indicated by optical density (OD730) of the culture. Cyanobacteria strains tested: 11901 (wild type); strain C (PpsbA2-SK), and strain D (Acpl2). Figure 3 shows the CO2 fixation rate of cyanobacteria strains: 11901 (wild type) and strain B (pcpt-aroF D174N) over 30 minutes. Figure 4 shows the CO2 fixation rate of cyanobacteria strains: 11901 (wild type) and strain C (PpsbA2-SK) over 30 minutes. Brief Description of the Sequence Listing SEQ ID NO: 1 is the nucleotide sequence of AroF. SEQ ID NO: 2 is the amino acid sequence of AroF. SEQ ID NO: 3 is the nucleotide sequence of AroF D174N. SEQ ID NO: 4 is the amino acid sequence of AroF D174N. SEQ ID NO: 5 is the nucleotide sequence of tesA. SEQ ID NO: 6 is the amino acid sequence of tesA. SEQ ID NO: 7 is the nucleotide sequence of carbonic anhydrase (CA). SEQ ID NO: 8 is the amino acid sequence of carbonic anhydrase (CA). SEQ ID NO: 9 is the nucleotide sequence of a carbonic anhydrase (CA) comprising PilA leader sequence. SEQ ID NO: 10 is the amino acid sequence of a carbonic anhydrase (CA) comprising PilA leader sequence. SEQ ID NO: 11 is the nucleotide sequence of Synechococcus 11901 shikimate kinase. SEQ ID NO: 12 is the amino acid sequence of Synechococcus 11901 shikimate kinase. SEQ ID NO: 13 is the nucleotide sequence of the PJS23119 promoter. SEQ ID NO: 14 is the nucleotide sequence of the Pcpt promoter. SEQ ID NO: 15 is the nucleotide sequence of the PpsbA2 promoter. SEQ ID NO: 16 is the nucleotide sequence of the Pcpc560 promoter. SEQ ID NO: 17 is the nucleotide sequence of UTEX 3222 shikimate kinase. SEQ ID NO: 18 is the amino acid sequence of UTEX 3222 shikimate kinase. SEQ ID NO: 19 is the nucleotide sequence of the luxA terminator. SEQ ID NO: 20 is the amino acid sequence of the trrnB terminator. SEQ ID NO: 21 is the amino acid sequence of E. coli AroF. SEQ ID NO: 22 is the amino acid sequence of E. coli AroF D146N. SEQ ID NO: 23 is the amino acid sequence of E. coli AroG. SEQ ID NO: 24 is the amino acid sequence of E. coli AroGD146N. SEQ ID NO: 25 is the amino acid sequence of Corynebacterium glutamicum AroF. SEQ ID NO: 26 is the amino acid sequence of Corynebacterium glutamicum AroF E154Q. SEQ ID NO: 27 is the amino acid sequence of Corynebacterium glutamicum AroF E154N. Detailed Description of the Invention The invention relates to a modified cyanobacterium comprising modifications to enzymes associated with the shikimate pathway, the Calvin Benson cycle, and / or fatty acid metabolism, resulting in the creation of a ‘carbon sink’ at the expense of the cyanobacteria’s own energy balance. The modifications described herein may be combined. Shikimate pathway A modified cyanobacterium of the invention may comprise one or more genetic modifications for improving expression of aromatic amino acids through overexpression of shikimate pathway to enhance the rate of carbon dioxide fixation relative to a cyanobacterium which does not comprise said genetic modifications. These genetic modifications may comprise increasing the quantity or activity of an enzyme associated with the shikimate pathway. Methods of over-expression of a gene are known in the art, and as described herein. For example, the gene may be placed under a constitutive promoter, and / or the copy number of the gene may be increased in the modified cyanobacterium. The resulting modified cyanobacterium would thus express a higher quantity of the enzyme compared to a cyanobacterium without said modifications. The modified cyanobacterium of the invention may comprise one or more genetic modifications to over-express shikimate kinase (SK) in the modified cyanobacterium. The enzyme associated with the shikimate pathway may be shikimate kinase (SK). SK is an enzyme that is central to the governance of many synthetic functions in the cell which is conserved across a diverse range of prokaryotic organisms. SK plays a pivotal role in the transformation of shikimate into 3-phosphoshikimate which enables the synthesis of aromatic amino acids such as tryptophan, phenylalanine, and tyrosine, along with a variety of related metabolites. In the context of higher plants and eukaryotic microalgae, the shikimate pathway is a source of precursors for the synthesis of significant plant hormones such as indole-3-acetic acid. The shikimate pathway's enhancement may also affect photosynthesis by impacting the synthesis of plastoquinone (PQ) which is a critical part of the photosynthetic and respiratory electron transport chains, as well as numerous other metabolic activities. PQ acts as an electron carrier in various energy metabolism processes, including linear and cyclic electron flows. PQ's availability is critical in acclimating photosynthesis to conditions of high light and temperature. An analysis of 2657 proteins in Synechococcus, classified by the KEGG pathway and protein solubility, revealed the proportions of shikimate pathway-derived aromatic amino acids (tyrosine, phenylalanine, and tryptophan). The total proportions in all proteins, soluble proteins, and membrane proteins were 7.7%, 7.3%, and 9.3%, respectively. Notably, the proportion was highest in the photosynthesis pathway (10.2%), particularly in photosystem II (14.5%), compared to all other annotated pathways. Without wishing to be bound by theory, shikimate kinase pathways may potentially improve CO2 fixation in two ways: first through the upregulated synthesis of key proteins involved in photosynthesis and secondly by relieving the metabolic bottlenecks by increasing the demand for carbon flux from the Calvin Benson cycle. To over-express shikimate kinase (SK), or a variant thereof, a cyanobacterium may be modified to insert a gene encoding SK, or a variant thereof, into the cyanobacterial genome. Hence, a modified cyanobacterium of the invention may comprise a gene encoding SK, or a variant thereof, in the cyanobacterial genome. The SK may be expressed at a high quantity, e.g. by being placed under a constitutive promoter and / or increasing the copy number of the gene per cell, as described herein. The modified cyanobacterium over-expressing SK may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. The SK may be from a prokaryote. The SK may be from Synechococcus 11901, which has the Locus tag: FEK30 022I0, and proteinJd: QCS48346.1. The SK may have an amino acid sequence as set out in SEQ ID NO: 12. For example, a modified cyanobacterium of the invention may comprise a nucleotide sequence as set out in SEQ ID NO: 11, which encodes a SK having an amino acid as set out in SEQ ID NO: 12. In the embodiment where the cyanobacterium is Synechococcus 11901, the modified cyanobacterium of the invention may comprise an increased copy number of the Synechococcus 11901 SK gene. For example, the modified cyanobacterium may comprise multiple copies, such as 2, 3, 4, or 5 copies of the Synechococcus 11901 SK gene per cell. For example, the modified cyanobacterium may comprise one copy of the endogenous Synechococcus 11901 SK gene that is present in wild-type Synechococcus 11901 and one or more (e.g. 1, 2, 3, or 4) copies of an exogenous Synechococcus 11901 SK gene that is inserted into the cyanobacterial genome. For example, the modified cyanobacterium comprises 2 copies of the Synechococcus 11901 SK gene per cell. The additional copy or copies of Synechococcus 11901 SK gene (i.e. additional to the endogenous Synechococcus 11901 SK gene that is present in wild-type Synechococcus 11901) may be inserted into the cyanobacterial genome by known techniques in the art, e.g. as described herein. A modified cyanobacterium of the invention may comprise a gene encoding a variant of Synechococcus 11901 SK. For example, the variant may comprise or consist of an amino acid sequence having >70%, >80%, >90%, >95%, >96%, >97%, >98%, or >99% sequence identity with SEQ ID NO: 12. The variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues that are substituted, deleted or added, in any combination, relative to SEQ ID NO: 12. The SK may be from UTEX 3222. The SK may have an amino acid sequence as set out in SEQ ID NO: 18. The SK from UTEX 3222 has the Locus tag: EAOMGH 05420 and proteinjd: gnl Bakta EAOMGH 05420. For example, a modified cyanobacterium of the invention may comprise a nucleotide sequence as set out in SEQ ID NO: 17, which encodes a SK having an amino acid as set out in SEQ ID NO: 18. A modified cyanobacterium of the invention may comprise a gene encoding a variant of UTEX 3222 SK. For example, the variant may comprise or consist of an amino acid sequence having >70%, >80%, >90%, >95%, >96%, >97%, >98%, or >99% sequence identity with SEQ ID NO: 18. The variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues that are substituted, deleted or added, in any combination, relative to SEQ ID NO: 18. A modified cyanobacterium of the invention may comprise one or more genetic modifications that encourage synthesis of aromatic amino acids by removing inhibitory feedback on the shikimate pathway. Hence, a modified cyanobacterium of the invention may comprise one or more genetic modifications to remove inhibitory feedback on the shikimate pathway in the modified cyanobacterium. 3-Deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS) is the first enzyme of the shikimate pathway and is responsible for catalysing the reaction of PEP and E4P to 3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) and inorganic phosphate. DAHPS controls the carbon flow into the shikimate pathway of bacteria by allosteric regulation of the enzyme resulting in a feedback inhibition exerted by the aromatic amino acids produced downstream. DAHPS may be modified such that it is unsusceptible to allosteric modulation, so it is defective in providing inhibitory feedback on the shikimate pathway. A modified cyanobacterium of the invention may comprise a gene encoding a modified DAHPS that is unsusceptible to allosteric modulation. This would remove inhibitory feedback on the shikimate pathway in the modified cyanobacterium. The modified DAHPS may be expressed at a high quantity, e.g. by being placed under a constitutive promoter and / or increasing the copy number of the gene per cell, as described herein. The modified cyanobacterium over-expressing a modified DAHPS that is unsusceptible to allosteric modulation may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. The DAHPS may be from a prokaryote, e.g. Synechococcus 11901, E. coli or Corynebacterium glutamicum. The DAHPS may be from Synechococcus 11901. The DAHPS may be AroF, which is a tyrosine sensitive isoenzyme of DAHPS. The DAHPS may be AroG, which is a phenylalanine and tyrosine sensitive isoenzyme of DAHPS. The AroF may be from Synechococcus 11901 (SEQ ID NO: 2), and the modified AroF that is unsusceptible to allosteric modulation may comprise a substitution of the aspartic acid residue at position 174, e.g. D174N (SEQ ID NO: 4). A modified cyanobacterium of the invention may comprise a nucleotide sequence encoding Synechococcus 11901 AroF D174N as set out in SEQ ID NO: 4. A modified cyanobacterium of the invention may comprise a nucleotide sequence as set out in SEQ ID NO: 3. The AroF may be from E. coli (SEQ ID NO: 21), and the modified E. coli AroF that is unsusceptible to allosteric modulation may comprise a substitution of the aspartic acid residue at position 146, e.g. D146N (SEQ ID NO: 22). A modified cyanobacterium of the invention may comprise a nucleotide sequence encoding E. coli AroF D146N as set out in SEQ ID NO: 22. The AroG may be from E. coli (SEQ ID NO: 23), and the modified E. coli AroG that is unsusceptible to allosteric modulation may comprise a substitution of the aspartic acid residue at position 146, e.g. D146N (SEQ ID NO: 24). A modified cyanobacterium of the invention may comprise a nucleotide sequence encoding E. coli AroG D146N as set out in SEQ ID NO: 24. The AroF may be from Corynebacterium glutamicum (SEQ ID NO: 25), and the modified AroF that is unsusceptible to allosteric modulation may comprise a substitution of the glutamic acid residue at position 154 e.g. E154Q (SEQ ID NO: 26) or E154N (SEQ ID NO: 27). A modified cyanobacterium of the invention may comprise a nucleotide sequence encoding Corynebacterium glutamicum AroF E154Q as set out in SEQ ID NO: 26. A modified cyanobacterium of the invention may comprise a nucleotide sequence encoding Corynebacterium glutamicum AroF E154N as set out in SEQ ID NO: 27. A modified cyanobacterium of the invention may comprise a gene encoding a variant of modified AroF that is unsusceptible to allosteric modulation and comprises a substitution at the amino acid position corresponding to 174 in Synechococcus 11901 AroF. For example, the modified AroF variant may comprise or consist of an amino acid sequence having >70%, >80%, >90%, >95%, >96%, >97%, >98%, or >99% sequence identity with SEQ ID NO:4, provided that asparagine (N) is present at position 174. The modified AroF variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues that are substituted, deleted or added, in any combination, relative to SEQ ID NO: 4, provided that asparagine (N) is present at position 174. The invention also provides the use of a modified DAHPS, e.g. AroF or AroG, that is unsusceptible to allosteric modulation, or a variant thereof, for increasing carbon dioxide fixation by a modified cyanobacterium. The invention also provides the use of Synechococcus 11901 AroF D174N (SEQ ID NO: 4), or a variant thereof, for increasing carbon dioxide fixation by a modified cyanobacterium. Fatty acid metabolism A modified cyanobacteria of the invention may comprise one or more genetic modifications that encourage production of free fatty acids, or one or more genetic modifications that discourage the synthesis of long chain fatty acids, or a combination thereof. Genetic modifications that encourage production of free fatty acids may comprise increasing the quantity or activity of an enzyme associated with synthesis of free fatty acids in the modified cyanobacterium. Methods of over-expression of a gene are known in the art and as described herein. For example, the gene may be placed under a constitutive promoter, e.g. as described herein, and / or the copy number of the gene may be increased in the modified cyanobacterium. The resulting modified cyanobacterium would thus express a higher quantity of the enzyme compared to a cyanobacterium without said modifications. The modified cyanobacterium of the invention may comprise one or more genetic modifications to over-express a thioesterase, which is an enzyme associated with the synthesis of free fatty acids, in the modified cyanobacterium. The thioesterase may be from a prokaryote, e.g. E. coli. The thioesterase may be an acyl-CoA thioesterase. The thioesterase may be a truncated E. coli acyl-CoA thioesterase, also referred to herein as TesA, having an amino acid sequence as set out in SEQ ID NO: 6. TesA is an enzyme capable of hydrolysing the Coenzyme A group from an ester to produce a free acid. To over-express acyl-CoA thioesterase, TesA, or a variant thereof, a cyanobacterium may be modified to insert a gene encoding TesA, or a variant thereof, into the cyanobacterial genome. Hence, a modified cyanobacterium of the invention may comprise a gene encoding TesA, or a variant thereof, in the cyanobacterial genome. The SK may be expressed at a high quantity, e.g. by being placed under a constitutive promoter and / or increasing the copy number of the gene per cell, as described herein. The modified cyanobacterium over-expressing TesA may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. For example, a modified cyanobacterium of the invention may comprise a nucleotide sequence as set out in SEQ ID NO: 5, which encodes TesA having an amino acid sequence as set out in SEQ ID NO: 6. A modified cyanobacterium of the invention may comprise a gene encoding a variant of E. coli TesA. For example, the variant may comprise or consist of an amino acid sequence having >70%, >80%, >90%, >95%, >96%, >97%, >98%, or >99% sequence identity with SEQ ID NO: 6. The variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues that are substituted, deleted or added, in any combination, relative to SEQ ID NO: 6. The invention also provides the use of TesA for increasing carbon dioxide fixation by a modified cyanobacterium. A modified cyanobacterium of the invention may comprise one or more genetic modifications that discourage the synthesis of long chain fatty acids. Such genetic modifications may comprise reducing or eliminating the quantity or activity of an enzyme associated with the synthesis of long chain fatty acids. Methods of reducing or eliminating the expression of a gene are known in the art, and described herein. For example, the gene may be deleted from the cyanobacterial genome. The gene may be truncated such that a functional protein is not expressed. The resulting modified cyanobacterium would thus express a lower quantity of the enzyme compared to a cyanobacterium without said modifications. A modified cyanobacteria of the invention may not express a functional long chain fatty acyl-CoA ligase. The long chain fatty acyl-CoA ligase is an enzyme associated with the synthesis of long chain fatty acids, and catalyses the oxidation of free fatty acids to form fatty acyl-CoA and adenosine monophosphate and pyrophosphate. The long chain fatty acyl-CoA ligase may be FadD. A modified cyanobacteria of the invention may not express a functional FadD. The gene encoding FadD may be deleted from the cyanobacterial genome. The entire FadD gene may be deleted from the cyanobacterial genome. Hence, a modified cyanobacteria of the invention may not comprise a gene encoding FadD. The modified cyanobacterium which does not express a functional FadD may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. For example, a modified cyanobacteria of the invention may comprise a combination of genetic modifications that encourage synthesis of free fatty acids as described herein, and genetic modifications that discourage the synthesis of long chain fatty acids as described herein. Without wishing to bound by theory, this combination of genetic modifications may increase carbon flux towards the synthesis of free fatty acids from carbon dioxide as well as reducing the metabolism of free fatty acids for energy. For example, a modified cyanobacteria of the invention may comprise the deletion of the gene encoding FadD, wherein the gene encoding FadD is replaced with a gene encoding TesA. Carbon anhydrase The invention also relates to improving the bioavailability of dissolved carbon species to the carboxysome by favouring the assimilation of HCO3- over dissolved CO2 This is possible by upregulating the expression of the carbonic anhydrase (CA) enzyme which shifts the direction of the reversible reaction in favour of HCO3- production, which can be taken up more efficiently by the cell which already expresses a native bicarbonate transporter at the cell surface membrane. Hence, a modified cyanobacterium of the invention may comprise one or more genetic modifications to improve bioavailability of dissolved carbon species. A cyanobacterium may be modified to insert a gene encoding carbonic anhydrase (CA), or variant thereof. Hence, a modified cyanobacterium of the invention may comprise a gene encoding carbonic anhydrase (CA), or a variant thereof, in the cyanobacterial genome. The SK may be expressed at a high quantity, e.g. by being placed under a constitutive promoter and / or increasing the copy number of the gene per cell, as described herein. The modified cyanobacterium expressing CA may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. The gene encoding CA may comprise a nucleotide sequence as set out in SEQ ID NO: 7, which encodes a CA having an amino acid sequence as set out in SEQ ID NO :8. Hence, a modified cyanobacterium of the invention may comprise a nucleotide sequence as set out in SEQ ID NO: 7. The CA may be coupled to a PilA leader sequence in the N terminus. This would translocate the resulting protein to the plasma membrane as opposed to carboxysome. Hence, this would increase speciation of dissolved inorganic carbon (DICs) into the form of HCO3-, which is more readily taken up by the cell. Hence, a modified cyanobacterium of the invention may comprise a gene encoding a carbonic anhydrase (CA) enzyme, or variant thereof, which is coupled to a pilA leader sequence in the N terminus. The gene may comprise a nucleotide sequence as set out in SEQ ID NO: 9, which encodes pilA-CA having an amino acid sequence as set out in SEQ ID NO: 10. Hence, a modified cyanobacterium of the invention may comprise a nucleotide sequence as set out in SEQ ID NO: 9. Calvin Benson (CB) cycle A modified cyanobacteria of the invention may comprise one or more genetic modifications that encourage the conversion of carbon dioxide into organic compounds. Such genetic modifications may comprise deletion of an inhibitory regulator of photosynthesis. Methods of reducing or eliminating the expression of a gene are known in the art and as described herein. For example, the gene may be deleted from the cyanobacterial genome. The gene may be truncated such that a functional protein is not expressed. The resulting modified cyanobacterium would thus express a lower quantity of the enzyme compared to a cyanobacterium without said modifications. A modified cyanobacteria of the invention may not express a functional inhibitory regulator of photosynthesis. The inhibitory regulator of photosynthesis may be CP 12. The modified cyanobacteria of the invention may not express a functional CP12. The gene encoding CP12 may be deleted from the cyanobacterial genome. The entire CP12 gene may be deleted from the cyanobacterial genome. Hence, a modified cyanobacteria of the invention may not comprise a gene encoding CP12. CP12 is known to suppress the activity of two crucial enzymes in the CB cycle: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). Under normal conditions, activation of CP12 is triggered by a rise in the NAD(H) / NADP(H) ratio, a scenario commonly seen under photomixotrophic and dark environmental conditions. Therefore, the CP12 protein acts as a regulatory mechanism that controls the rate of CO2 fixation based on the cell's energy state or redox balance. Upon activation, CP 12 impedes GAPDH, which is pivotal for directing carbon flow towards the lower EMP pathway in cyanobacteria. Additionally, CP12 exerts its inhibitory effect on PRK which is a key enzyme that is responsible for converting ribulose-5-phosphate (Ru5P) into ribulose-l,5-bisphosphate (R15P), a crucial substrate for carbon dioxide fixation by RuBisCO. The modified cyanobacterium which does not express a functional CP 12 may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. Cyanobacteria The invention relates to modified cyanobacterium. Any cyanobacteria strain is a suitable starting point for the invention. For example, the cyanobacterium may be Synechocystis sp. or Synechococcus sp. The Synechococcus sp. may be PCC 11901 (Wlodarczyk et al., 2020, Commun Biol 3, 215). The cyanobacterium may be PCC 11901, PCC 11802, PCC 6301, or UTEX 2973. The cyanobacterium may be a variant of PCC 11901, UTEX 3154 (Mills et al., 2022, Biomolecules 12(7):872). UTEX 3154 has two point mutations 320 bp upstream of the start codon for the metE gene in 11901, conferring viability in the absence of vitamin B12 in the culture medium. Having reduced vitamin B12 auxotrophy is advantageous, and hence UTEX 3154 is particularly useful with the invention. The cyanobacterium may be UTEX 3222 (Schubert et al., 2023, bioRxiv 2023.10.30.564770). UTEX 3222 is particularly useful with the invention because it is thermotolerant and able to grow fast in a range of conditions. The optimal growth condition for the modified cyanobacterium is under industrial carbon emission settings, such as light intensity mimicking outdoor natural sunlight, 30 °C and 5% CO2 (v / v). For example, the modified cyanobacterium may be grown from about 25°C to about 50 °C. The modified cyanobacterium may be grown from about 0.04% CO2 (v / v) to up to 10% CO2 (v / v). The modified cyanobacterium may be grown in artificial or natural light, e.g. ranging from 200 to 1500 micro Einsteins of solar irradiation. The modified cyanobacterium may be grown at 0 to about 3.5 % salinity. Modifications The cyanobacterium may be modified to express a protein described herein in high quantity or activity. The quantity or activity of the protein may be increased by >20%, >40%, >60%, >80%, >100%, >200%, or >300%. The increase may be measured by any standard techniques in the art, such as western blot or appropriate functional assays. The increase in the quantity of a protein, in other words over-expression of a protein, may be achieved by introducing an expression cassette containing the proteinencoding gene into a cyanobacterium, such that expression of the protein by the modified cyanobacterium is increased. Introduction may take place through transformation of the expression cassette into the host cell, as described herein. Increased gene expression includes both augmentation of native production of the protein as well as production of a protein in a cyanobacterium that lacks native production. For example, in some instances production may be increased from a measurable initial value whereas in other instances the initial value may be zero. The increase in the quantity of a protein may be achieved by an increased copy number of the protein-encoding gene present in the modified cyanobacterium, such that the modified cyanobacterium comprises >1, >2, >3, >4, >5, >6 or >7 copies of the gene per cell. For example, a gene described herein may be integrated into the cyanobacterial genome. The gene may be integrated into the cyanobacterial chromosome or an endogenous cyanobacterial plasmid. The endogenous cyanobacterial plasmid may be present in a higher copy number than the cyanobacterial chromosome, resulting in gene expression at a higher level than if it were chromosomally integrated. Alternatively, the gene may be integrated in a recombinant plasmid which is retained in a high copy number in the modified cyanobacterium. The gene encoding a protein described herein is typically introduced into a cyanobacterium in an expression cassette. Thus, the invention also provides an expression cassette comprising a gene encoding a protein described herein. The expression cassette may comprise any suitable selection marker for selecting transformed bacterial cells. The selection marker may be an antibiotic resistance selection marker, such as streptomycin, spectinomycin, kanamycin, gentamycin, erythromycin, neomycin, rifampin, ampicillin, and zeomycin resistance. Other types of selection markers may be used, such as any marker enabling the cyanobacteria to grow on media that it would not be able to grow on but for the expression of the selection marker. The expression cassette typically comprises a promoter operably linked to the gene encoding a protein described herein. The promoter may be any suitable promoter. The promoter may be a prokaryotic (e.g. bacterial) promoter or an artificial promoter. The promoter may be a constitutive promoter, e.g. as listed in Table 1. The promoter may be an inducible promoter. Table 1. Examples of promoters useful with the invention. SEQID Promoter Type of promoter Promoter origin Reference 14 Pcpt Constitutive truncated cpcB (c-phycocyanin beta subunit) upstream region Markley et al., ACS Synth. Biol. 4, 595 603 (2015). 15 pPsbA2 Constitutive psbA2 (redundant PSII reaction center protein) upstream region Lindberg et al., Metabolic Engineering, Volume 12, Issue 1 (2010). 16 pcpc560 Constitutive cpcB (c-phycocyanin beta subunit) upstream region Zhou et al., Sci Rep 4, 4500 (2014). 13 BBa_JS23 119 Constitutive synthetic g70 consensus promoter from BioBricks Markley et al., ACS Synthetic Biology, 4(5), 595-603 (2014). The expression cassette typically comprises a terminator operably linked to the 3’-end of the gene encoding a protein described herein. Appropriate terminators are well known to a person skilled in the art. For example, the terminator may be a luxA terminator, e.g. SEQ ID NO: 19 (Chen et al., 2013, Nat. Methods, 10, 659-664) or a trrnB terminator, e.g. SEQ ID NO: 20 (Liu and Pakrasi, 2018, Microb Cell Fact 17: 48; Wang et al., 2018, ACS Synth Biol 7: 276-286). The expression cassette may be integrated into a locus of interest in the cyanobacterial genome, such as in the endogenous cyanobacterial plasmids or chromosomes. Typically, the integration is a permanent, stable integration. Integration of the expression cassette into the locus of interest may be achieved by homologous recombination. For example, it may involve transforming the host cyanobacterium cell with a non-replicative vector or a linear DNA fragment containing the expression cassette flanked by sequences that are homologous to sequences upstream and downstream of the locus of interest, so that the expression cassette can be integrated into the endogenous cyanobacterial plasmid through homologous recombination. The expression cassette disclosed herein can be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3 SR). Amplified nucleotide sequences may be fused using ligation methods. Amplified nucleotide sequences may be fused by Gibson Assembly to produce an expression cassette. A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to the skilled person. Transformation of a cyanobacterium cell with expression cassettes can be carried outby conventional techniques, e.g. ssDNA transformation. The cyanobacterium may be modified to express a bacterial protein described herein having reduced quantity or activity, including no expression and no functional expression of said protein. The quantity or activity of the protein may be reduced by >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, or 100%. The reduction may be measured by any standard techniques in the art, such as western blot or appropriate functional assays. Modifications to reduce the expression of a bacterial gene may include one or more mutations in the open reading frame of the gene, one or more mutations in the coding sequence of the gene, one or more mutations in the promoter that controls the gene. Modifications may include missense mutations, nonsense mutations and silent mutations. The cyanobacterium may be modified such that it does not express the gene by various techniques known in the art, such as via CRISPR-mediated interference, by disrupting the gene via gene editing, by modifying an endogenous CRISPR RNA which is designed to guide a Cas enzyme to the gene, by expressing a recombinant CRISPR RNA which is designed to guide a Cas enzyme to the gene. The cyanobacterium may be modified such that the gene is rendered nonfunctional. For example, the gene may be rendered non-functional by mutating the gene itself or the control sequences flanking the gene, for example the promoter sequence. Deletions may remove one or more portions of the gene, the entire gene, and all or some of the control sequences, for example the promoter sequence. For example, deletion of only one nucleotide within the gene may be made, resulting in a frame shift. However, a larger deletion may be made, for example at least about 25%, or at least about 50% of the total coding and / or non-coding sequence. Where two or more copies of the gene are present in the cyanobacterium, both copies of the gene may be rendered non-functional. The entire gene may be deleted from the endogenous plasmid or chromosome. Where two or more copies of the gene are present in the cyanobacterium, all copies of the gene are deleted. The gene in the cyanobacterium may be replaced. The entire gene may be replaced, or portions of it may be replaced. The replacement gene may be inserted into the locus of the gene to be excised by homologous recombination. The replacement gene may be comprised within an expression cassette, e.g. as described herein. The invention also provides a method of preparing a modified cyanobacterium described herein. The modified cyanobacterium of the invention is constructed using methods well known in the art. It is within the abilities of the skilled person to determine a method of preparing a modified cyanobacterium of the invention, based on well-established molecular biology and microbiology techniques. Exemplary modified cyanobacterium strains The invention provides a modified cyanobacterium wherein the endogenous gene encoding long-chain fatty acid CoA ligase fadD in the cyanobacteria genome is replaced with a gene encoding the truncated acyl-CoA thioesterase (tesA). The TesA-encoding gene may be operably linked to a constitutive promoter, e g. JS23119. The modified cyanobacterium may be derived from Synechococcus 11901, UTEX 3154 or UTEX 3222. The modified cyanobacterium may comprise may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. The invention also provides a modified cyanobacterium comprising a gene encoding AroF D174N. The AroF D174N-encoding gene may be operably linked to the constitutive promoter, e.g. JS23119. The modified cyanobacterium may be derived from Synechococcus 11901, UTEX 3154 or UTEX 3222. The modified cyanobacterium may comprise may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. The invention also provides a modified cyanobacterium comprising overexpression of shikimate kinase (SK). The SK may be expressed in a high quantity by increasing the copy number of a SK-encoding gene in the cyanobacterium. The modified cyanobacterium may comprise two or more (e.g. 2, 3 or 4) copies of SK gene in the cyanobacterium genome per cell. The SK-encoding gene may be operably linked to the constitutive promoter, e.g. JS23119. The modified cyanobacterium may be derived from Synechococcus 11901, UTEX 3154 or UTEX 3222. The modified cyanobacterium may comprise may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. The invention also provides a modified cyanobacterium wherein CP 12 is knocked out from the cyanobacteria genome. The modified cyanobacterium may be derived from Synechococcus 11901, UTEX3154 or UTEX 3222. The modified cyanobacterium may comprise may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. The invention also provides a modified cyanobacterium comprising overexpression of CA and a truncated acyl-CoA thioesterase (tesA), and wherein the endogenous gene encoding long-chain fatty acid CoA ligase fadD is knocked out from the cyanobacteria genome. The modified cyanobacterium may be derived from Synechococcus 11901, UTEX3154 or UTEX 3222. The modified cyanobacterium may comprise may further comprise one or more genetic modifications, such as deletions and / or alterations of the expression of one or more bacterial genes, and / or expressed one or more heterologous genes, as described herein. Protein variants As described herein, the invention relates to variants of proteins described herein. A variant may comprise or consist of an amino acid sequence having >70%, >80%, >90%, >95%, >96%, >97%, >98%, or >99% sequence identity with the wild-type sequence. Such variants may contain modifications, such as amino acid substitutions, additions or deletions relative to the wild-type sequence. For example, the variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues that are substituted, deleted or added, in any combination. For example, the variants may have additions, deletions or substitutions of amino acid residues which do not substantially alter the biological activity of the protein described herein. Those individual sites or regions of the protein described herein, which can be altered without affecting biological activity, can be determined by examination of the protein structure, for example. Alternatively, the regions which would tolerate amino acid substitutions may be determined by alanine scanning mutagenesis. In this method, selected amino acid residues are individually substituted with a neutral amino acid (e.g. alanine) in order to determine the effects on biological activity. A protein variant may contain conservative amino acid changes which are least likely to perturb the structure and / or function of the polypeptide. For example, the variant may comprise one or more conservative amino acid changes to the wild-type sequence. Conservative amino acid changes generally involve substitution of one amino acid with another that is similar in structure and / or function (e.g. amino acids with side chains similar in size, charge and shape). Amino acid residues having similar side chains are known in the art. These include amino acids with basic side , chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residue within a protein can be replaced with other amino acid residues having similar side chains and the altered protein can be tested for retained function using the functional assays described herein. Modifications can be introduced by standard techniques known in the art, such as site-specific mutagenesis and PCR-mediated mutagenesis, provided that activity of the protein is retained. A protein variant may be codon optimized to increase expression levels of the respective protein in host cells as compared to if the unaltered sequence. Methods for codon optimisation are known in the art, e.g. GeneScript OptimumGene™ algorithm can be used. Composition and kits The invention also provides a composition comprising one or more of the modified cyanobacteria described herein. The invention also provides a kit comprising the modified cyanobacterium or the composition described herein for fixing carbon dioxide. Uses The modified cyanobacteria described herein are particularly effective in fixing carbon dioxide. Thus, the invention also provides a method of fixing carbon dioxide, comprising exposing a modified cyanobacterium described herein to a source of carbon dioxide. The carbon dioxide source may be supplied from flue gas. The amount of carbon dioxide provided to the modified cyanobacterium may be 0.03-5.0% of a total volume of the flue gas. The invention also provides the use of a modified cyanobacterium described herein for carbon dioxide fixation in open raceway ponds or closed photobioreactor systems. This may be for industrial flue gas sources such as power stations, cement factories, steel factories, anaerobic digestion, fermenters, fertiliser factories, combined heat and power, biogenic emission sites, waste incineration, water treatment, or CO2 removal from the air. The invention also provides the use of a modified cyanobacterium described herein as a high productivity platform strain for the overexpression of high value compounds of interest to nutraceuticals, pharmaceuticals and biomanufacturing. The invention also provides the use of AroF D174N and / or tesA for increasing the rate of carbon dioxide fixation by a modified cyanobacterium. Other Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. It is to be understood that different applications of the modified cyanobacteria strains described herein may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing the particular embodiments of the invention only, and is not intended to be limiting. In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “an amino acid sequence comprising SEQ ID NO: X” should be interpreted to mean that the amino acid sequence comprises SEQ ID NO: X, but the amino acid sequence may comprise further sequences. In some embodiments of the invention, the word “comprising” may be replaced with the phrase “consisting of. The term “consisting of is intended to be limiting. For example, the phrase “an amino acid sequence consisting of SEQ ID NO: X” should be understood to mean that the amino acid sequence has SEQ ID NO: X and no further sequences. In some embodiments of the invention, the word “comprising” may be replaced with the phrase “consisting essentially of. The term “consisting essentially of means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter. The term “about” or “around” when referring to a value refers to that value but within a reasonable degree of scientific error. Optionally, a value is “about x” or “around x” if it is within 10%, within 5%, or within 1% of x. In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “a modified cyanobacterium" includes two or more modified cyanobacteria. Furthermore, when referring to “>x” herein, this means equal to or greater than x. When referring to “<y” herein, this means equal to or less thany For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotides at each position are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the nucleotides are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100). Typically the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 2, SEQ ID NO: 2 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 2 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 2, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 2. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 2. If the sequence is shorter than SEQ ID NO: 2, the gaps or missing positions should be considered to be non-identical positions. The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In an embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleic acid sequences is determined by the Smith- Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty = 12 and gap extension penalty = 2. All publications, patents and patent applications cited herein, whether supra or 5 infra, are hereby incorporated by reference in their entirety. The following examples illustrate the invention. Examples Example 1 - Generation of modified cyanobacteria strains 10 The Synechococcus 11901 strain was modified in various ways. The modifications include upregulation (if gene endogenously present in bacteria) or expression (if gene not endogenously present in bacteria) of genes encoding enzymes associated with the shikimate kinase pathway (such as AroF D174N or shikimate kinase (SK)) or fatty acid metabolism (such as the truncated E. coli acyl-CoA thioesterase (tesA)), and 15 downregulation of genes encoding enzymes associated with negative regulation of the Calvin Benson cycle, such as cpl2, or genes encoding enzymes associated with fatty acid metabolism (such as the long-chain fatty acid CoA ligase fadD). In summary, the strains were tested in this experiment are summarised in Table 2. Table 2. Cyanobacterium strains tested in the Examples. Strain ID Mutation 11901 wild type (i.e. no mutations) A Deletion of fadD High expression of tesA under promoter pJS23119 B High expression of AroF D174N under promoter pcpt (with naturally evolved methionine synthase mutation) C High expression of shikimate kinase (SK) under promoter PpsbA2 D Deletion of Cpl2 (with naturally evolved methionine synthase mutation) E Deletion of fadD High expression of TesA under promoter pJS23119 High expression of CA under promoter pJS23119 F High expression of shikimate kinase (SK) under promoter pcpc560 G High expression of Carbon Anhydrase (CA) under promoter pcpc560 TT rl Deletion of fadD High expression of tesA under promoter pcpc560 I Deletion of fadD High expression of TesA under promoter pcpc560 High expression of CA under promoter pcpc560 Strain A: To generate a free-fatty acid overproducing strain, a plasmid containing fadD knockout flanks, an expression cassette containing tesA (SEQ ID NO: 5) under the JS23119 promoter (SEQ ID NO: 13), and a spectinomycin resistance marker was assembled. The coding sequence tesA gene was obtained from E. coh. and synthesised in a codon-optimised form to maximise expression in UTEX 3154 orPCC 11901. The constitutive promoter JS23119 was synthesised. The plasmid was transformed into an exponentially growing culture of UTEX 3154 or PCC 11901 by natural transformation. Strain B - To generate this strain, a plasmid containing the AroF D174N variant (SEQ ID NO: 3) under the Pcpt promoter (SEQ ID NO: 14), and a spectinomycin resistance marker was assembled. The AroF D174N variant, designed to be resistant to feedback inhibition by tyrosine, enhances the flux through the shikimate pathway for increased production of aromatic compounds. The plasmid was transformed into an exponentially growing culture of UTEX 3154 or PCC 11901 by natural transformation, aiming to improve metabolic flow towards aromatic amino acid biosynthesis. Strain C - This strain was developed by assembling a plasmid with the shikimate kinase (SK) gene (SEQ ID NO: 11) under the PpsbA2 promoter (SEQ ID NO: 15) along with a spectinomycin resistance marker. SK, crucial for the shikimate pathway leading to aromatic amino acid production. Plasmid was transformed in UTEX 3154 or PCC 11901. The transformation aimed at boosting the production of aromatic amino acids, thereby potentially enhancing the cyanobacteria! metabolic efficiency. Strain D -: To create this strain, a knockout strategy was used to delete the cpl2 gene, aiming to deregulate the Calvin cycle and enhance carbon fixation and photosynthetic efficiency. The deletion was confirmed through PCR and sequencing, and the modified cells were selected for spectinomycin resistance. This genetic modification was intended to improve growth rates and biomass production in UTEX 3154 or PCC 11901. Strain E - Engineering of this strain involved deleting the fadD gene and introducing a plasmid with the tesA gene (SEQ ID NO: 5) under the JS23119 promoter (SEQ ID NO: 13) and the carbonic anhydrase (CA) gene (SEQ ID NO: 7) under the same promoter, alongside a spectinomycin resistance marker. The dual expression system aimed at maximizing free fatty acid production and enhancing CO2 fixation efficiency in UTEX 3154 or PCC 11901 through natural transformation. Strain F - This strain was developed by assembling a plasmid with the shikimate kinase (SK) gene (SEQ ID NO: 11) under the pcpc560 promoter (SEQ ID NO: 16) along with a spectinomycin resistance marker. SK, crucial for the shikimate pathway leading to aromatic amino acid production. Plasmid was transformed in UTEX 3154 or PCC 11901. The transformation aimed at boosting the production of aromatic amino acids, thereby potentially enhancing the cyanobacterial metabolic efficiency. Strain G - To generate this strain, a plasmid harboring the carbonic anhydrase (CA) gene (SEQ ID NO: 7) under the control of the pcpc560 promoter (SEQ ID NO: 16) and a spectinomycin resistance marker was constructed. This genetic configuration was designed to enhance CO2 uptake and conversion, facilitating more efficient carbon fixation. The plasmid was transformed into UTEX 3154 or PCC 11901 strains through natural transformation, aiming at improved biomass production and carbon capture capabilities. Strain H - This strain was developed by knocking out the fadD gene and introducing a plasmid with the tesA gene (SEQ ID NO: 5) under the pcpc560 promoter (SEQ ID NO: 16), along with a spectinomycin resistance marker. The strategy aimed to enhance free fatty acid production by leveraging the strong expression driven by the pcpc560 promoter, with the transformation carried out in exponentially growing cultures of UTEX 3154 or PCC 11901 by natural transformation. Strain I - Engineering of this strain involved deleting the fadD gene and introducing a plasmid with the tesA gene (SEQ ID NO: 5) under the pcpc560 promoter (SEQ ID NO: 16) and the carbonic anhydrase (CA) gene (SEQ ID NO: 7) under the same promoter, alongside a spectinomycin resistance marker. The dual expression system aimed at maximizing free fatty acid production and enhancing CO2 fixation efficiency in UTEX 3154 or PCC 11901 through natural transformation. The growth of these strains was monitored. The bacteria strains were cultured in MAD media, with an initial optical density (OD730) of approximately 0.1. The cultures were agitated at 120 rpm. Illumination was provided by RGB SI000 panel lights, with R:G:B ratios of 1:1:1, at a CO2 concentration of 5% v / v. The initial light intensity was set to 200 pmol photons m’V1, increased after one day to 750 pmol photons m'2s-1. Daily compensation for water loss was achieved by adding 700 pL of sterile Mi 11 iQ water to each culture, quantified by weight difference. Cell growth was monitored by measuring the optical density at 730 nm (OD730). Each experiment was conducted in triplicate, with a 50 mL culture volume, and incubated at 30°C. The OD730 of these strains at day 9 are shown in Tables 1 and 2. The tables also show the results of a two-tailed unpaired Student’s T-test (df=4), which was carried out across three replicates each for the control strain (11901) and each modified strain individually for the batch cultivation growth assays in flasks on day 9 as the endpoint of the growth assays in the incubators. Table 3. Growth rate of strain B (AroFDl 74N) compared to WT strain 11901. Strain Day Mean OD730 SE OD730 Test statistic P Value 11901 9 74.4 0.767029 0 1 3020 9 87.375 3.64037 6.04074 0.003787 Table 4. Growth rate of strain C (SK) and strain I) (Acpl2) compared to WT strain 11901. Strain Day Mean OD730 SE OD730 Test statistic P Value 11901 9 81.15 1.92592 0 0.375 1011 9 88.15 2.45442 3.886224 0.007524 3023 9 89.8625 4.81064 2.912191 0.021806 The growth of the strains over 9 days, as monitored by measuring OD730, are shown in Figures 1 and 2. Compared to the wild-type strain (11901), it can be seen that the growth of the modified strains were increased, providing fast growth rates. Example 2 - Modified cyanobacteria strains showed improvements in rate of carbon capture The cyanobacteria strains from Example 1 were tested for the rate of carbon dioxide absorption. To measure CO2 absorption, a CO2 fixation assay was performed in 500ml volumes in a gas wash bottle. Prior to the assay, CO2 sensors were calibrated with a 5 vol% CO2 gas. The gas wash bottle was sterilized with bleach, followed by thorough rinsing with DI water prior to inoculation. Cyanobacterial cultures with an optical density at 730 nm (OD730) were inoculated into photobioreactors (PBRs). The gas wash bottle, fitted with a sparger lid and connected to a control valve, was used to regulate CO2 flow. The outlet system included a dehumidifier filled with CaCh flakes to manage moisture reduction. A controlled environment was maintained, setting the incubator temperature at 30°C and light intensity at 750 uE of light. A flow rate of approximately 0.01 LPM was established and a gas flow sensor on the outlet was used to measure the true flow rate of gases. The CO2 in the outlet gas was measured using a CO2 sensor. Data from flow and CO2 sensors were continuously recorded, ensuring accurate monitoring of CO2 absorption rates. The CO2 fixation rates were calculated by multiplying the mass flow (L / min) by the volumetric concentration difference of CO2 from the inlet and the outlet. This methodology ensured precise measurement of CO2 fixation by cyanobacteria. The results are shown in Figures 3 and 4. In particular, Figure 3 shows that the CO2 fixation rate of strain B (pcpt-aroF D174N) is superior to that of the wild-type strain 11901. Figure 4 shows that the CO2 fixation rate of strain C (PpsbA2-SK) is superior to that of the wild-type strain 11901. Therefore, these data demonstrate that cyanobacteria can be effectively modified to increase CO2 fixation by over-expression genes encoding enzymes associated with the shikimate pathway that encourage synthesis of aromatic amino acids, such as AroF D174N or shikimate kinase (SK). Example 3 The Synechococcus 11901 strain is modified to express tesA and CA. To generate the modified cyanobacterium, a plasmid containing an expression cassette containing tesA (SEQ ID NO: 5) under the PJS23119 promoter (SEQ ID NO: 13) and CA (SEQ ID NO: 7) under the PJS23119 promoter (SEQ ID NO: 13), and a spectinomycin resistance marker is assembled. The coding sequence tesA gene is obtained from E. coll, and synthesised in a codon-optimised form to maximise expression in Synechococcus 11901. The plasmid is transformed into an exponentially growing culture of Synechococcus 11901 by natural transformation. The growth of these strains was monitored according to the growth assay in Example 1. Compared to the wild-type strain (11901), the growth of the modified strains is not affected. Hence, the modified bacteria strain is able to maintain the fast growth rate of the wild-type strain. The rate of carbon dioxide absorption is measured according to the method in Example 2. The CO2 fixation rate of modified cyanobacteria strain expressing tesA and CA is superior to that of the wild-type strain. Sequence listing SEQ ID Brief description Sequence 1 AroF DNA sequence atgatcgtagtcatgaaagttggcactcctgaagccgaaatcaaccgtttaggg agtgagcttaaagagttaggcctgacccccgaaaaaattgttggggcccaca aagtggtcattggcttagtgggagataccgccacttttaacattgaactgatcca agaaatgagcccctggatcgaaaatgtactccgggtcgaaaagccctttaagc gggtcagtcttgagtaccgtcatggtcaatacagcgaggtcgttgtgcccactc cgaacggggatgtcaccttcggccccaatcatccggtggtcgtcgtcgctgg cccttgctccgttgaaaatgaagaaatgatcgttgaaactgccctccgggtcaa agcagccggggcaaaattcctccggggtggggcgtacaagccgagaacttc tccctatgcgttccagggccacggcgaaagtgctttggaattgttggcggcag cacgggaagcctccggtctagggattatcacagaagtaatggatacggcgga cgtagaaaaaattgctgaggttgccgatgtgctccaaatcggcgcgcgcaata tgcaaaactttgccctcttgaagaaggtaggagctcagaataagccggtgttgt taaagcgtgggatggccgccaccatcgacgactggttaatggctgcagaatat attttggcggaaggaaattctcaggtgatcctctgtgaacgggggattcgcacc ttcgatagtaagtacacacgcaatgttttggatctatcggtgattccggtgttgcg cagtttgacccacttaccgatgatgatcgatccgagccacggtacagggaagt ctgaatttgttctttcgctggctaaaggggcgatcgccattggcactgattcgct gatgatcgaagttcaccccaatccgaagaaagccctttctgatggcccccaat ccctcaccccagaagcgttcgaccaggtgatgcaagaactggcagaggttga aaaattaacgggacgtggccaaaaggaattggcaacagtttaa 2 AroF amino acid sequence MIVVMKVGTPEAEINRLGSELKELGLTPEKIVGA HKVVIGLVGDTATFNIELIQEMSPWIENVLRVEKP FKRVSLEYRHGQYSEVVVPTPNGDVTFGPNHPVV VVAGPCSVENEEMIVETALRVKAAGAKFLRGGA YKPRTSPYAFQGHGESALELLAAAREASGLG1ITE VMDTADVEKIAEVADVLQIGARNMQNFALLKKV GAQNKPVLLKRGMAATIDDWLMAAEYILAEGNS QVILCERGIRTFDSKYTRNVLDLSVIPVLRSLTHLP MMIDPSHGTGKSEFVLSLAKGAIAIGTDSLMIEVH PNPKKALSDGPQSLTPEAFDQVMQELAEVEKLTG RGQKELATV 3 AroF D174N DNA sequence atgatcgtagtcatgaaagttggcactcctgaagccgaaatcaaccgtttaggg agtgagcttaaagagttaggcctgacccccgaaaaaattgttggggcccaca aagtggtcattggcttagtgggagataccgccacttttaacattgaactgatcca agaaatgagcccctggatcgaaaatgtactccgggtcgaaaagccctttaagc gggtcagtcttgagtaccgtcatggtcaatacagcgaggtcgttgtgcccactc cgaacggggatgtcaccttcggccccaatcatccggtggtcgtcgtcgctgg cccttgctccgttgaaaatgaagaaatgatcgttgaaactgccctccgggtcaa agcagccggggcaaaattcctccggggtggggcgtacaagccgagaacttc tccctatgcgttccagggccacggcgaaagtgctttggaattgttggcggcag cacgggaagcctccggtctagggattatcacagaagtaatgaatacggcgga cgtagaaaaaattgctgaggttgccgatgtgctccaaatcggcgcgcgcaata tgcaaaactttgccctcttgaagaaggtaggagctcagaataagccggtgttgt taaagcgtgggatggccgccaccatcgacgactggttaatggctgcagaatat attttggcggaaggaaattctcaggtgatcctctgtgaacgggggattcgcacc ttcgatagtaagtacacacgcaatgttttggatctatcggtgattccggtgttgcg cagtttgacccacttaccgatgatgatcgatccgagccacggtacagggaagt ctgaatttgttctttcgctggctaaaggggcgatcgccattggcactgattcgct gatgatcgaagttcaccccaatccgaagaaagccctttctgatggcccccaat ccctcaccccagaagcgttcgaccaggtgatgcaagaactggcagaggttga aaaattaacgggacgtggccaaaaggaattggcaacagtttaa 4 AroF D174N amino acid sequence MIVVMKVGTPEAEINRLGSELKELGLTPEKIVGA HKVVIGLVGDTATFNIELIQEMSPWIENVLRVEKP FKRVSLEYRHGQYSEVVVPTPNGDVTFGPNHPVV VVAGPCSVENEEMIVETALRVKAAGAKFLRGGA YKPRTSPYAFQGHGESALELLAAAREASGLGIITE VMNTADVEKIAEVADVLQIGARNMQNFALLKKV GAQNKPVLLKRGMAATIDDWLMAAEYILAEGNS QVILCERGIRTFDSKYTRNVLDLSVIPVLRSLTHLP MMIDPSHGTGKSEFVLSLAKGAIAIGTDSLMIEVH PNPKKALSDGPQSLTPEAFDQVMQELAEVEKLTG RGQKELATV 5 tesA DNA sequence GATACCCTCCTCATTCTCGGCGATAGTCTCTCC GCCGGTTATCGCATGTCTGCCTCTGCCGCCTGG CCCGCCCTCCTCAATGATAAATGGCAAAGCAA AACCTCTGTGGTGAATGCCAGTATTTCCGGCGA TACCTCTCAACAAGGCCTCGCCCGTCTCCCCGC rpTppTr a a Ar A APApp A AfYYYViTT a—s X a—s 1 a— / £Az Az z Axa a^x xa\>x a^x xAz aa—a-J JL JL JL a-J JL JL nTHTrfrA ACTCGGCGGTA ATG ATGGCCTCCfGCG A—* x ax a,JL a-J a-J x A—•• £ x £.-z £. £ a_J x a, JL a»J a-J a—z £ a— GTTTTCAACCCCAACAAACCGAACAAACCCTCC GGCAAATTCTCCAAGATGTGAAAGCCGCCAAT GCCGAACCCCTCCTCATGCAAATTCGCCTCCCC GCCAATTATGGTCGCCGCTACAATGAAGCCTTT AGTGCCATTTATCCCAAACTCGCCAAAGAATTT GATGTGCCCCTCCTCCCCTTTTTCATGGAAGAA GTGTACCTCAAACCCCAATGGATGCAAGATGA TGGTATTCACCCCAATCGGGATGCCCAACCCTT TATTGCCGATTGGATGGCCAAACAACTCCAACC CCTCGTGAATCATGATAGTTGAtgaTAA 6 tesA amino acid sequence MDTLLILGDSLSAGYRMSASAAWPALLNDKWQS KTSVVNASISGDTSQQGLARLPALLKQHQPRWVL VELGGNDGLRGFQPQQTEQTLRQILQDVKAANA EPLLMQIRLPANYGRRYNEAFSAIYPKLAKEFDVP LLPFFMEEVYLKPQWMQDDGIHPNRDAQPFIAD WMAKQLQPLVNHDS 7 CA DNA sequence atgaaagacatagatacactcatcagcaacaatgcactatggtcaaaaatgct ggtggaagaggatcccgggttttttgagaaactggcacaagcgcaaaaaccg cgctttctatggattggatgttccgacagtcgcgttcctgcagaacgtttaaccg gtcttgagccgggcgaactctttgttcaccgtaatgttgctaacctggtcattcac actgacctgaactgcctttccgtggttcagtatgcagtggatgtactcgaagttg aacacattattatctgtggccactacggttgcggcggcgtacaagccgcagtt gaaaacccggaactggggcttatcaacaactggctgctgcatatccgcgatat ctggttcaaacatagctcattgctcggcgaaatgccgcaagagcgccgtctgg ataccttgtgtgaactgaacgtcatggaacaggtgtataacctgggccactcca ccattatgcaatcagcgtggaaacgcgggcagaaagttaccattcacggctg ggcctacggcattcacgacggcttgctgcgtgatctggatgttaccgccacca accgcgaaacccttgagcaacgttaccgtcacgggatttccaacctcaagctg aaacacgccaaccacaaacaccaccatcatcaccattaatga 8 CA amino acid sequence MKDIDTLISNNALWSKMLVEEDPGFFEKLAQAQ KPRFLWIGCSDSRVPAERLTGLEPGELFVHRNVA NLVIHTDLNCLSVVQYAVDVLEVEHIIICGHYGC GG VQ A A VEN P ELGLIN N W LLHI RD IW FK H S SLLG EMPQERRLDTLCELNVMEQVYNLGHSTIMQSAW KRGQKVTIHGWAYGIHDGLLRDLDVTATNRETL eqryrhgisnlklkhanhk 9 CA comprising PilA Leader Sequence -DNA sequence (PilA Leader Sequence bold and underlined) ATGgctagtaattttaaattcaaactcctctctcaactctccaaaaaacg ggcagaaggtggtatgaaagacatagatacactcatcagcaacaatgcacta tggtcaaaaatgctggtggaagaggatcccgggttttttgagaaactggcaca agcgcaaaaaccgcgctttctatggattggatgttccgacagtcgcgttcctgc agaacgtttaaccggtcttgagccgggcgaactctttgttcaccgtaatgttgct aacctggtcattcacactgacctgaactgcctttccgtggttcagtatgcagtgg atgtactcgaagttgaacacattattatctgtggccactacggttgcggcggcgt acaagccgcagttgaaaacccggaactggggcttatcaacaactggctgctg catatccgcgatatctggttcaaacatagctcattgctcggcgaaatgccgcaa gagcgccgtctggataccttgtgtgaactgaacgtcatggaacaggtgtataa cctgggccactccaccattatgcaatcagcgtggaaacgcgggcagaaagtt accattcacggctgggcctacggcattcacgacggcttgctgcgtgatctgga tgttaccgccaccaaccgcgaaacccttgagcaacgttaccgtcacgggattt ccaacctcaagctgaaacacgccaaccacaaacaccaccatcatcaccatta atga 10 CA comprising PilA Leader Sequence -amino acid sequence MASNFKFKLLSQLSKKRAEGGMKDIDTLISNNA LWSKMLVEEDPGFFEKLAQAQKPRFLWIGCSDSR VPAERLTGLEPGELFVHRNVANLVIHTDLNCLSV VQYAVDVLEVEHIIICGHYGCGGVQAAVENPELG linnwllhirdiwfkhssllgempqerrldtlcel (PilA Leader Sequence bold and underlined) NVMEQVYNLGHSTIMQSAWKRGQKVTIHGWAY GIHDGLLRDLDVTATNRETLEQRYRHGISNLKLK 11 Synechococcus 11901 shikimate kinase DNA sequence ATGGATGATTTGCTCAAAGGTTTAAATATTTAT C TC AT C GGC AT GAT GGGC AC GGGG A A A AGT AC CCTCGCAGAAATTATTGCGGAGTTGATGAATTA CCGCAGTTTGGATTCGGACAGCATCATTGAAAC CCTGGCAAAGCAGTCGATCAGTGAGATTTTTGC CGAGATGGGAGAGGCAGAATTCCGCAAACTCG AAACCCAAGTCCTCAAGCAAATCGCGGTACAA ACCCGGACGGTGGTGGCGACGGGGGGCGGTAT TGTGGTGTCCCAGGCCAATTGGTATTATCTGCG CC AGGGTTTGACC ATT AGT GCC A ATCTTGGTAGA ACGGCTGCGGAGTGATA zxiLxjC JL JL JL x—J x—J JL xx -Lx JL x—J Jx JL x 1L JL x jl CGTCGCGCCCCCTTTTGCAAAATGTTGATCTTG AGCGCAAATTAACGCAACTGCTCCAACAGCGG CGATCGCGTTATGCGGAGGCAGATTTACATATC CAAATTCCCGGCAATCGTCCCCCCCGGGCCGTG GCAGAGGACATCATCGCCCAAATTCCTTCGGTG CTGCGAAAACCGCCAGAATTTATTGGCGATCGC CCTTGA 12 Synechococcus 11901 shikimate kinase amino acid sequence MDDLLKGLNIYLIGMMGTGKSTLAEIIAELMNYR SLDSD SIIETL AKQ SISEIF AEMGEAEFRKLETQ VL KQIAVQTRTVVATGGGIVVSQANWYYLRQGLTI WLDVPVPILVERLRSDTSRPLLQNVDLERKLTQL LQQRRSRYAEADLHIQIPGNRPPRAVAEDIIAQIPS VLRKPPEFIGDRP 13 JS23119 promoter sequence GGAGTTGACAGCTAGCTCAGTCCTAGGTATAAT GCTAGCTATTGTGAGCGGATAACAATTTCACAC ATACTAGAGAAAGAGGAGAAATACTAAATG 14 Pcpt promoter sequence ttaacaaaaaagcaggaataaaattaacaagatgtaacagacataagtcccat caccgttgtataaagttaactgtgggattgcaaaag 15 PpsbA2 promoter sequence ccgccaggtaaactcttctcaacccccaaaacgccctctgtttacccatggaaa aaacgacaattacaagaaagtaaaacttatgtcatctataagcttcgtgtatatta acttcctgttacaaagctttacaaaactctcattaatcctttagactaagtttagtca gtttagtggaggttctagaatg 16 Pcpc560 promoter sequence cacctgtagagaagagtccctgaatatcaaaatggtgggataaaaagctcaaa aaggaaagtaggctgtggttccctaggcaacagtcctccctaccccactggaa actaaaaaaacgagaaaagttcgcaccgaacatcaattgcataattttagccct aaaacataagctgaacgaaactggttatcttcccttcccaatccaggacaatct gagaatcccctgcaacattacttaacaaaaaagcaggaataaaattaacaaga tgtaacagacataagtcccatcaccgttgtataaagttaactgtgggattgcaaa agcattcaagcctaggcgctgagctgtttgagcatcccggtggcccttgtcgct gcctccgtgtttctccctggatttatttaggtaatatctctcataaatccccgggta gttaacgaaagttaatggagatcagtaacaataactctagggtcattactttgga ctccctcagtttatccgggggaattgtgtttaagaaaatcccaactcataaagtc aagtaggagattaattca 17 UTEX 3222 shikimate kinase DNA sequence atgaaagaattattaaagggtttgaatatatacttaattgggatgatgggatcag gtaaaactactgtaggagagttattagcaaaagagttggaatatcgttttttagat acggatgcaattattagtgctgtcagtgagaaaagtattaatcagatttttgccga agatggagaagatgagtttcgtcagttagaaagtgatgttttgcaagaagtctct gcttacttatatacggtgattgctacaggagggggaattatccttcgtcaacaaa attggagtcatctcagagatggtatggtagtgtggttaaatgttcctattgatgttt tggtacaaagattaaaagatgatgacactcgccctttactaaagcgggaagaa ttgcaaacaaaattaatcagcttatatgaacaaagaaagtctctttatcaacaag ctgatattactattggtattgaggaaaatgataaccctaacgatattgtccaaaaa ataatggttgaaattcctcataaaattaggcaagaaaacagtgatctaaattaa 18 UTEX 3222 shikimate kinase amino acid sequence MKELLKGLNIYLIGMMGSGKTTVGELL AKELEY RFLDTDAIISAVSEKSINQIFAEDGEDEFRQLESDV LQEVSAYLYTVIATGGGIILRQQNWSHLRDGMVV WLNVPIDVLVQRLKDDDTRPLLKREELQTKLISL YEQRKSLYQQADIT1GIEENDNPNDIVQK1MVEIP HKIRQENSDLN 19 luxA terminator agagaatataaaaagccagattattaatccggcttttttattattt 20 trrnB terminator CAAATAAAACGAAAGGCTCAGTCGAAAGACTG GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAAC GCTCTCTACTAGAGTCACACTGGCTCACCTTCG GGTGGGCCTTTCTGCG 21 J—T J • A TT-1 E. coll AroF MQKDALNNVHITDEQVLMTPEQLKAAFPLSLQQ EAQIADSRKSISDI1AGRDPRLLVVCGPCSIHDPET ALEYARRFKALAAEVSDSLYLVMRVYFEKPRTT VGWKGLINDPHMDGSFDVEAGLQIARKLLLELV NMGLPLATEALDPNSPQYLGDLFSWSAIGARTTE SQTHREMASGLSMPVGFKNGTDGSLATAINAMR AAAQPHRF VGINQ AGQVALLQTQGNPDGHVILR GGKAPNYSPADVAQCEKEMEQAGLRPSLMVDCS HGNSNKDYRRQPAVAESVVAQIKDGNRSIIGLMI ESNIHEGNQSSEQPRSEMKYGVSVTDACISWEMT DALLREIHQDLNGQLTARVA 22 E. coll AroF D146N MQKDALNNVHITDEQVLMTPEQLKAAFPLSLQQ EAQIADSRKSISDIIAGRDPRLLVVCGPCSIHDPET ALEYARRFKALAAEV SDSLYLVMRVYFEKPRTT VGWKGLINDPHMDGSFDVEAGLQIARKLLLELV NMGLPLATEALNPNSPQYLGDLFSWSAIGARTTE SQTHREMASGLSMPVGFKNGTDGSLATAINAMR AAAQPHRFVGINQAGQVALLQTQGNPDGHVILR GGKAPNYSPADVAQCEKEMEQAGLRPSLMVDCS HGNSNKDYRRQPAVAESVVAQIKDGNRSIIGLMI ESNIHEGNQSSEQPRSEMKYGVSVTDACISWEMT DALLREIHQDLNGQLTARVA 23 E. coli AroG MNYQNDDLRIKEIKELLPPVALLEKFPATENAAN TVAHARKAIHKILKGNDDRLLVVIGPC SIHDP VA AKEYATRLLALREELKDELEIVMRVYFEKPRTTV GWKGLINDPHMDNSFQINDGLRIARKLLLDINDS GLPAAGEFLDMITPQYLADLMSWGAIGARTTESQ VHRELASGLSCPVGFKNGTDGTIKVAIDAINAAG APHCFLSVTKWGHSAIVNTSGNGDCHIILRGGKE PNYSAKHVAEVKEGLNKAGLPAQVMIDF SHANS SKQFKKQMDVCADVCQQIAGGEKAIIGVMVESH LVEGNQSLESGEPLAYGKSITDACIGWEDTDALL RQLANAVKARRG 24 E. coli AroG D146N MNYQNDDLRIKEIKELLPPVALLEKFPATENAAN TVAHARKAIHKILKGNDDRLLVVIGPCSIHDPVA AKEYATRLLALREELKDELEIVMRVYFEKPRTTV GWKGLINDPHMDNSFQINDGLRIARKLLLDINDS GLPAAGEFLNMITPQYLADLMSWGAIGARTTESQ VHRELASGLSCPVGFKNGTDGTIKVAIDAINAAG APHCFLSVTKWGHSAIVNTSGNGDCHIILRGGKE PNYSAKHVAEVKEGLNKAGLPAQVMIDFSHANS SKQFKKQMDVCADVCQQIAGGEKAIIGVMVESH LVEGNQSLESGEPLAYGKSITDACIGWEDTDALL RQLANAVKARRG 25 Corynebacterium glutamicum AroF MSSPVSLENAASTSNKRVVAFHELPSPTDLIAANP LTPKQASKVEQDRQDIADIFAGDDDRLVVVVGPC SVHDPEAAIDYANRLAPLAKRLDQDLKIVMRVYF EKPRTIVGWKGLINDPHLNETYDIPEGLRIARKVL IDVVNLDLPVGCEFLEPNSPQYYADTVAWGAIGA RTTESQVHRQLASGMSMPIGFKNGTDGNIQVAV DAVQAAQNPHFFFGTSDDGALSVVETAGNSNSHI ILRGGTSGPNHDAASVEAVVEKLGENARLMIDAS HANSGKDHIRQVEVVREIAEQISGGSEAVAGIMIE SFLVGGAQNLDPAKLRINGGEGLVYGQSVTDKCI DIDTTIDLLAELAAAVRERRAAAK 26 Corynebacterium glutamicum AroF E154Q MSSPVSLENAASTSNKRVVAFHELPSPTDLIAANP LTPKQASKVEQDRQDIADIFAGDDDRLVVVVGPC SVHDPEAAIDYANRLAPLAKRLDQDLKIVMRVYF EKPRTIVGWKGLINDPHLNETYDIPEGLRIARKVL IDVVNLDLPVGCEFL2PNSPQYYADTVAWGAIG ARTTESQVHRQLASGMSMPIGFKNGTDGNIQVA VDAVQAAQNPHFFFGTSDDGALSVVETAGNSNS HIILRGGTSGPNHDAASVEAVVEKLGENARLMID ASHANSGKDHIRQVEVVREIAEQISGGSEAVAGI MIESFLVGGAQNLDPAKLRINGGEGLVYGQSVTD KCIDIDTTIDLLAELAAAVRERRAAAK 27 Corynebacterium glutamicum AroF E154N MSSPVSLENAASTSNKRVVAFHELPSPTDLIAANP LTPKQASKVEQDRQDIADIFAGDDDRLVVVVGPC SVHDPEAAIDYANRLAPLAKRLDQDLKIVMRVYF EKPRTIVGWKGLINDPHLNETYDIPEGLRIARKVL IDVVNLDLPVGCEFLNPNSPQYYADTVAWGAIGA RTTESQVHRQLASGMSMPIGFKNGTDGNIQVAV DAVQAAQNPHFFFGTSDDGALSVVETAGNSNSHI ILRGGTSGPNHDAASVEAVVEKLGENARLMIDAS HANSGKDHIRQVEVVREIAEQISGGSEAVAGIMIE SFLVGGAQNLDPAKLRINGGEGLVYGQSVTDKCI DIDTTIDLLAELAAAVRERRAAAK

Claims

1. A modified cyanobacterium comprising one or more genetic modifications for improving expression of aromatic amino acids through overexpression of shikimate pathway to enhance the rate of carbon dioxide fixation relative to a cyanobacterium which does not comprise said genetic modifications.

2. The modified cyanobacterium of claim 1, wherein the cyanobacterium is Synechococcus sp., e.g. Synechococcus 11901 or a variant thereof.

3. The modified cyanobacterium of claim 1 or 2, comprising one or more genetic modifications to over-express shikimate kinase (SK) in the modified cyanobacterium.

4. The modified cyanobacterium of claim 3, wherein the modified cyanobacterium comprises a gene encoding SK, or a variant thereof, in the cyanobacterial genome.

5. The modified cyanobacterium of claim 4, wherein the gene encoding SK comprises a nucleotide sequence as set out in SEQ ID NO: 11.

6. The modified cyanobacterium of any one of the preceding claims, further comprising one or more genetic modifications to remove inhibitory feedback on shikimate pathway in the modified cyanobacterium.

7. The modified cyanobacterium of claim 6, wherein the modified cyanobacteriumcomprises a gene encoding AroF D174N, or variant thereof, in the cyanobacterial genome.

8. The modified cyanobacterium of claim 7, wherein the gene encoding AroF D174N comprises a nucleotide sequence as set out in SEQ ID NO: 3.

9. The modified cyanobacterium of any one of the preceding claims, further comprising one or more genetic modifications to encourage production of free fatty acids in the modified cyanobacterium, optionally wherein the modified cyanobacterium comprises a gene encoding acyl-CoA thioesterase, TesA (e.g. SEQ ID NO: 5), or a variant thereof, in the cyanobacterial genome.

10. The modified cyanobacterium of any one of the preceding claims, further comprising one or more genetic modifications to improve bioavailability of dissolved carbon species, optionally wherein the modified cyanobacterium comprises a gene encoding carbonic anhydrase (CA) (e.g. SEQ ID NO: 7), or a variant thereof, in the cyanobacterial genome.

11. The modified cyanobacterium of claim 10, wherein the CA is coupled to a PilA leader sequence (e.g. SEQ ID NO: 9) in its the N terminus.

12. The modified cyanobacterium of any one of claims 3 to 11, wherein the gene is operably linked to a constitutive promoter.

13. The modified cyanobacterium of claim 12, wherein the constitutive promoter is selected from the group consisting of: JS23119, Pcpt, PpsbA2, PnblA, and pcpc560.

14. The modified cyanobacterium of any one of claims 3 to 13, wherein the modified cyanobacterium comprises multiple copies of the gene in the cyanobacterial genome, optionally wherein the modified cyanobacterium comprises multiple copies of a gene encoding SK in the cyanobacterial genome.

15. The modified cyanobacterium of any one of the preceding claims, further comprising one or more genetic modifications to remove an inhibitory regulator of photosynthesis, optionally wherein the gene encoding CP12 is deleted from the cyanobacterial genome.

16. The modified cyanobacterium of any one of the preceding claims, further comprising one or more genetic modifications to discourage synthesis of long chain fatty acids, optionally wherein the gene encoding FadD is deleted from the cyanobacterial genome.

17. The modified cyanobacterium of claim 16, wherein the gene encoding FadD is replaced with a gene encoding TesA.

18. The modified cyanobacterium of any one of the preceding claims, wherein the optimal growth condition for the modified bacterium is under industrial carbon emission settings, such as light intensity mimicking outdoor natural sunlight, 30°C and 5% CO2 (v / v).

19. A composition comprising one or more modified cyanobacteria of any one of the preceding claims for fixing carbon dioxide.

20. A method of increasing the rate of carbon dioxide fixation of a cyanobacterium, comprising genetically modifying the cyanobacterium to generate a modified cyanobacterium according to any one of the methods of claims 1 to 18.

21. A method of fixing carbon dioxide, comprising exposing the modified cyanobacterium of any one of claims 1 to 18 or the composition of claim 19 to a source of carbon dioxide.

22. The method of claim 21, wherein the carbon dioxide source is supplied from flue gas.

23. The method of claim 21 or 22, wherein the amount of carbon dioxide provided to the modified cyanobacterium is 0.03-5.0% of a total volume of the flue gas.

24. Use of the modified cyanobacterium of any one of claims 1 to 18 or the composition of claim 19 for fixing carbon dioxide.

25. Use of AroF D174N and / or TesA for increasing the rate of carbon dioxide fixation by a modified cyanobacterium.

Images