bacteria

Abstract

The invention relates to modified cyanobacterial strains comprising one or more modifications to enhance the rate of carbon dioxide fixation relative to a cyanobacterial strain which does not comprise said modifications.

Description

[0001] Bacteria

[0002] Field of the Invention

[0003] The invention relates to modified photosynthetic bacteria, such as cyanobacteria, for carbon dioxide fixation, and methods of preparing said bacteria and uses thereof.

[0004] Background to the Invention

[0005] 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.

[0006] 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.

[0007] 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(l):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.

[0008] Accordingly, it is an object of the invention to develop a further or improved modified photosynthetic bacteria, such as cyanobacteria, for improving carbon capture.

[0009] Summary of the Invention

[0010] The inventors generated modified cyanob acteri al strains that have an enhanced rate of carbon dioxide fixation. To achieve this, cyanob acteri al strains 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. The resulting cyanob acteri al strains were metabolically stable, had faster growth rates and had increased carbon fixation. Whilst it was predicted that CO2 fixation may increase as a result, it was expected to be at the expense of biomass productivity, surprisingly, there was an increase in carbon fixed as cellular biomass in addition to additional carbon fixed from the carbon sink. Hence, the inventors found that surprisingly the combination of enhanced carbon fixation with the engineering of a “carbon sink” in one or more unrelated metabolic pathways results in particularly advantageous cyanob acteri al strains.

[0011] In particular, the inventors modified the cyanobacterium Synechococcus PCC 11901 strain to target the shikimate pathway, the Calvin Benson cycle, fatty acid metabolism, and / or carbon uptake pathway. The resulting modified cyanob acteri al 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 cyanob acteri al strains provided improved growth rates (e.g. see Example 1) and improved CO2 fixation rate relative to the wild-type strain (e.g. see Example 2). In particular, modified cyanob acteri al strains which encouraged the production of aromatic amino acids, e.g. strains B and C which overexpressed AroF D174N and shikimate kinase (SK), respectively, provided improved CO2 fixation rate. Furthermore, the modified cyanob acteri al strains having a combination of modifications (e.g. genetic modifications), e.g. for encouraging the production of free fatty acids (e.g. deletion of fadD and high expression of acyl-CoA thioesterase TesA) and for improving bioavailability of dissolved inorganic carbon species (e.g. high expression of carbonic anhydrase), are advantageous, e.g. improved growth rate as shown in Example 1.

[0012] Accordingly, the invention provides a modified cyanob acteri al strain 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 cyanobacterial strain which does not comprise said genetic modifications.

[0013] The invention also provides a modified cyanobacterial strain comprising one or more modifications to enhance the rate of carbon dioxide fixation relative to a cyanobacterial strain which does not comprise said modifications, wherein the one or more modifications is for (a) improving expression of aromatic amino acids through overexpression of the shikimate pathway, (b) encouraging production of free fatty acids, (c) dysregulation of the Calvin Benson cycle, and / or (e) improving bioavailability of dissolved inorganic carbon species.

[0014] The invention also provides a composition comprising one or more modified cyanobacterial strains described herein for fixing carbon dioxide.

[0015] The invention also provides a method of increasing the rate of carbon dioxide fixation of a cyanobacterial strain, comprising genetically modifying the cyanobacterial strain to generate a modified cyanobacterial strain according to a method described herein.

[0016] The invention also provides a method of increasing the rate of carbon dioxide fixation of a cyanobacterial strain, comprising modifying the cyanobacterial strain to: (a) improve expression of aromatic amino acids through overexpression of the shikimate pathway as described herein, (b) encourage production of free fatty acids as described herein, (c) dysregulate the Calvin Benson cycle as described herein; and / or (e) improve bioavailability of dissolved inorganic carbon species as described herein.

[0017] The invention also provides a method of fixing carbon dioxide, comprising exposing the modified cyanobacterial strain or composition as described herein to a source of carbon dioxide, e.g. having a concentration of 5% CO2 or more.

[0018] The invention also provides the use of the modified cyanobacterial strain or composition as described herein for fixing carbon dioxide.

[0019] The invention also provides the use of AroF D174N and / or tesA for increasing the rate of carbon dioxide fixation by a modified cyanobacterial strain. Brief Description of the Figures

[0020] Figure 1 shows the growth of cyanob acteri al strains over 9 days, as indicated by optical density (OD730nm) of the culture. Cyanobacterial strains tested: 11901 (wild type); strain A (AfadD::pJS23119-tesA), strain B (pcpt-aroF D174N), and strain E (AfadD: :pJS23119-tesA, pJS23119-CA).

[0021] Figure 2 shows the growth of cyanobacterial strains, as indicated by optical density (OD730nm) of the culture. Cyanobacterial strains tested: 11901 (wild type); strain C (PpsbA2-SK), and strain D (Acpl2).

[0022] Figure 3 shows the CO2 fixation rate of cyanobacterial strains: 11901 (wild type) and strain B (pcpt-aroF D174N) over 30 minutes.

[0023] Figure 4 shows the CO2 fixation rate of cyanobacterial strains: 11901 (wild type) and strain C (PpsbA2-SK) over 30 minutes.

[0024] Figure 5 shows the growth of cyanobacterial strains, as indicated by optical density (OD730nm) of the culture. Cyanobacterial strains tested: 11901 (wild type); strain G (pcpc560-CA), strain H (pcpc560-TesA; AfadD) and strain I (AfadD, pcpc560-TesA, pcpc560-CA).

[0025] Brief Description of the Sequence Listing

[0026] SEQ ID NO: 1 is the nucleotide sequence of AroF.

[0027] SEQ ID NO: 2 is the amino acid sequence of AroF.

[0028] SEQ ID NO: 3 is the nucleotide sequence of AroF D174N.

[0029] SEQ ID NO: 4 is the amino acid sequence of AroF D174N.

[0030] SEQ ID NO: 5 is the nucleotide sequence of tesA.

[0031] SEQ ID NO: 6 is the amino acid sequence of tesA.

[0032] SEQ ID NO: 7 is the nucleotide sequence of carbonic anhydrase (CA).

[0033] SEQ ID NO: 8 is the amino acid sequence of carbonic anhydrase (CA).

[0034] SEQ ID NO: 9 is the nucleotide sequence of a carbonic anhydrase (CA) comprising PilA leader sequence.

[0035] SEQ ID NO: 10 is the amino acid sequence of a carbonic anhydrase (CA) comprising PilA leader sequence.

[0036] SEQ ID NO: 11 is the nucleotide sequence of Synechococcus PCC 11901 shikimate kinase. SEQ ID NO: 12 is the amino acid sequence of Synechococcus PCC 11901 shikimate kinase. SEQ ID NO: 13 is the nucleotide sequence of the PJS23119 promoter.

[0037] SEQ ID NO: 14 is the nucleotide sequence of the Pcpt promoter.

[0038] SEQ ID NO: 15 is the nucleotide sequence of the PpsbA2 promoter.

[0039] SEQ ID NO: 16 is the nucleotide sequence of the Pcpc560 promoter.

[0040] SEQ ID NO: 17 is the nucleotide sequence of UTEX 3222 shikimate kinase.

[0041] SEQ ID NO: 18 is the amino acid sequence of UTEX 3222 shikimate kinase.

[0042] SEQ ID NO: 19 is the nucleotide sequence of the luxA terminator.

[0043] SEQ ID NO: 20 is the amino acid sequence of the trrnB terminator.

[0044] SEQ ID NO: 21 is the amino acid sequence of E. coli AroF.

[0045] SEQ ID NO: 22 is the amino acid sequence of E. coli AroF D146N.

[0046] SEQ ID NO: 23 is the amino acid sequence of E. coli AroG.

[0047] SEQ ID NO: 24 is the amino acid sequence of E. coli AroG D146N.

[0048] SEQ ID NO: 25 is the amino acid sequence of Corynebacterium glutamicum AroF.

[0049] SEQ ID NO: 26 is the amino acid sequence of Corynebacterium glutamicum AroF E154Q.

[0050] SEQ ID NO: 27 is the amino acid sequence of Corynebacterium glutamicum AroF E154N.

[0051] SEQ ID NO: 28 is the 16S rRNA sequence of Synechococcus sp. PCC 11901.

[0052] SEQ ID NO: 29 is the amino acid sequence of Synechococcus sp. PCC 11901 ppc.

[0053] SEQ ID NO: 30 is the amino acid sequence of Synechococcus sp. PCC 11901 petB.

[0054] SEQ ID NO: 31 is the amino acid sequence of Synechococcus sp. PCC 11901 rnpB.

[0055] SEQ ID NO: 32 is the amino acid sequence of Synechococcus sp. PCC 11901 rpoA.

[0056] SEQ ID NO: 33 is the amino acid sequence of Synechococcus sp. PCC 11901 secA.

[0057] SEQ ID NO: 34 is the DNA sequence of Synechococcus sp. PCC 11901 rpaA.

[0058] SEQ ID NO: 35 is the amino acid sequence of Synechococcus sp. PCC 11901 atpA.

[0059] SEQ ID NO: 36 is the amino acid sequence of Synechococcus sp. PCC 11901 ppnK.

[0060] Detailed Description of the Invention

[0061] The invention relates to a modified cyanob acteri al strain 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

[0062] A modified cyanob acteri al strain of the invention may comprise one or more modifications (e.g. 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 cyanobacterial strain which does not comprise said modifications (e.g. genetic modifications). These modifications (e.g. 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 cyanobacterial strain. The resulting modified cyanobacterial strain would thus express a higher quantity of the enzyme compared to a cyanobacterial strain without said modifications.

[0063] The modified cyanobacterial strain of the invention may comprise one or more modifications (e.g. genetic modifications) to over-express shikimate kinase (SK) in the modified cyanobacterial strain. 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.

[0064] 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.

[0065] To over-express shikimate kinase (SK), or a variant thereof, a cyanob acteri al strain may be modified to insert a gene encoding SK, or a variant thereof, into the cyanob acteri al genome. Hence, a modified cyanob acteri al strain of the invention may comprise a heterologous gene encoding SK, or a variant thereof, in the cyanob acteri al genome. Alternatively, SK may be expressed from a vector-based expression system (e.g. from a non-integrative plasmid).

[0066] 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. For example, the modified cyanob acteri al strain may comprise a gene encoding SK operably linked to a constitutive promoter. The modified cyanob acteri al strain may comprise multiple copies (e.g. 2, 3, 4, 5) of a gene encoding SK per cell. The modified cyanob acteri al strain may comprise one or more copies (e.g. 1, 2, 3, 4, 5) of a heterologous gene encoding SK per cell.

[0067] The modified cyanob acteri al strain over-expressing SK may further comprise one or more modifications (e.g. 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.

[0068] The SK may be from a prokaryote. The SK may be from Synechococcus PCC 11901, which has the Locus tag: FEK30 02210, and protein id: QCS48346.1. The SK may have an amino acid sequence as set out in SEQ ID NO: 12. For example, a modified cyanob acteri al strain 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.

[0069] In the embodiment where the cyanob acteri al strain is Synechococcus PCC 11901, the modified cyanob acteri al strain of the invention may comprise an increased copy number of the Synechococcus PCC 11901 SK gene. For example, the modified cyanob acteri al strain may comprise multiple copies, such as 2, 3, 4, or 5 copies of the Synechococcus PCC 11901 SK gene per cell. For example, the modified cyanob acteri al strain may comprise one copy of the endogenous Synechococcus PCC 11901 SK gene that is present in wild-type Synechococcus PCC 11901 and one or more (e.g. 1, 2, 3, or 4) copies of an exogenous Synechococcus PCC 11901 SK gene that is inserted into the cyanob acteri al genome. For example, the modified cyanob acteri al strain comprises 2 copies of the Synechococcus PCC 11901 SK gene per cell. The additional copy or copies of Synechococcus PCC 11901 SK gene (i.e. additional to the endogenous Synechococcus PCC 11901 SK gene that is present in wild-type Synechococcus PCC 11901) may be inserted into the cyanob acteri al genome by known techniques in the art, e.g. as described herein.

[0070] A modified cyanob acteri al strain of the invention may comprise a gene encoding a variant of Synechococcus PCC 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 variant retains the SK activity of catalysing the transformation of shikimate into 3-phosphoshikimate. Methods of measuring SK activity are known in the art, e.g. by measuring the conversion of [14C]shikimate to [14C] shikimate 3-phosphate, or by coupling the release of ADP to the pyruvate kinase and lactate dehydrogenase reactions.

[0071] 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 protein id: gnl Bakta EAOMGH 05420. For example, a modified cyanob acteri al strain 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.

[0072] A modified cyanob acteri al strain 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. The variant retains the SK activity of catalysing the transformation of shikimate into 3-phosphoshikimate. Methods of measuring SK activity are known in the art, as described herein.

[0073] A modified cyanob acteri al strain of the invention may comprise one or more modifications (e.g. genetic modifications) that encourage synthesis of aromatic amino acids by removing (e.g. reducing) inhibitory feedback on the shikimate pathway. Hence, a modified cyanob acteri al strain of the invention may comprise one or more modifications (e.g. genetic modifications) to remove (e.g. reduce) inhibitory feedback on the shikimate pathway in the modified cyanob acteri al strain.

[0074] 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. Methods of generating modified DAHPSs that are unsusceptible to allosteric modulation are known in the art, e.g. by site directed mutagenesis and the resulting protein is assessed in enzyme activity assays in the presence or absence of aromatic amino acids produced further downstream, such as tyrosine, tryptophan, and / or phenylalanine. For example, DAHPS activity (and the lack thereof) may be determined by measuring the decrease in the substrate phosphoenolpyruvate by a spectrophotometric assay, or the increase in the product DAHP by a colorimetric method, e.g. as described in Jayaraman et al. (Applied Microbiology and Biotechnology, 2022, 106:6505-6517).

[0075] A modified cyanob acteri al strain of the invention may comprise a heterologous gene encoding a modified DAHPS that is unsusceptible to allosteric modulation. This would remove (e.g. reduce) inhibitory feedback on the shikimate pathway in the modified cyanob acteri al strain.

[0076] A cyanob acteri al strain may be modified to insert a gene encoding the modified DAHPS into the cyanob acteri al genome. Alternatively, the modified DAHPS may be expressed from a vector-based expression system (e.g. from a non-integrative plasmid).

[0077] 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. For example, the modified cyanobacterial strain may comprise a gene encoding a modified DAHPS that is unsusceptible to allosteric modulation operably linked to a constitutive promoter. The modified cyanobacterial strain may comprise multiple copies (e.g. 2, 3, 4, 5) of a gene encoding a modified DAHPS that is unsusceptible to allosteric modulation per cell. The modified cyanobacterial strain over-expressing a modified DAHPS that is unsusceptible to allosteric modulation may further comprise one or more modifications (e.g. 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.

[0078] 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. AroF is particularly advantageous for use with the invention because it acts early in the shikimate pathway, and hence targeting AroF may provide optimal impact on the shikimate pathway.

[0079] The AroF may be from Synechococcus PCC 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 cyanob acteri al strain of the invention may comprise a nucleotide sequence encoding Synechococcus PCC 11901 AroF D174N as set out in SEQ ID NO: 4. A modified cyanob acteri al strain of the invention may comprise a nucleotide sequence as set out in SEQ ID NO: 3.

[0080] 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 cyanobacterial strain of the invention may comprise a nucleotide sequence encoding E. coli AroF D146N as set out in SEQ ID NO: 22.

[0081] 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 cyanobacterial strain of the invention may comprise a nucleotide sequence encoding E. coli AroG D146N as set out in SEQ ID NO: 24.

[0082] 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), E154N (SEQ ID NO: 27), or E154S, and / or at position 155 e.g. P155L. A modified cyanobacterial strain of the invention may comprise a nucleotide sequence encoding Corynebacterium glutamicum AroF E154Q as set out in SEQ ID NO: 26. A modified cyanob acteri al strain of the invention may comprise a nucleotide sequence encoding Corynebacterium glutamicum AroF E154N as set out in SEQ ID NO: 27.

[0083] A modified cyanob acteri al strain of the invention may comprise a gene encoding a variant of modified AroF that is unsusceptible to allosteric modulation and comprises a mutation (e.g. substitution) at the amino acid position corresponding to 174 in Synechococcus PCC 11901 AroF. For example, the modified AroF may comprise a mutation at a position corresponding to position 174 of the AroF from Synechococcus PCC 11901 (SEQ ID NO: 2). For example, the aspartic acid residue at position 174 in SEQ ID NO: 2 may be substituted with an amino acid having an uncharged polar side chains (e.g. asparagine, glycine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), or an amino acid having a basic side chain (e.g. lysine, arginine, histidine).

[0084] 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 modified AroF variant is capable of catalysing the reaction of PEP and E4P to DAHP and inorganic phosphate and is unsusceptible to allosteric modulation.

[0085] 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 cyanobacterial strain. The invention also provides the use of Synechococcus PCC 11901 AroF D174N (SEQ ID NO: 4), or a variant thereof, for increasing carbon dioxide fixation by a modified cyanobacterial strain.

[0086] Fatty acid metabolism

[0087] A modified cyanobacterial strain of the invention may comprise one or more modifications (e.g. genetic modifications) that encourage production of free fatty acids, or one or more modifications (e.g. genetic modifications) that discourage the synthesis of long chain fatty acids, or a combination thereof.

[0088] Modifications (e.g. 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 cyanob acteri al strain. Methods of overexpression 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 cyanob acteri al strain. The resulting modified cyanob acteri al strain would thus express a higher quantity of the enzyme compared to a cyanob acteri al strain without said modifications.

[0089] The modified cyanob acteri al strain of the invention may comprise one or more modifications (e.g. genetic modifications) to over-express a thioesterase, which is an enzyme associated with the synthesis of free fatty acids, in the modified cyanob acteri al strain. 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.

[0090] To over-express acyl-CoA thioesterase, TesA, or a variant thereof, a cyanob acteri al strain may be modified to insert a gene encoding TesA, or a variant thereof, into the cyanob acteri al genome. Hence, a modified cyanob acteri al strain of the invention may comprise a heterologous gene encoding TesA, or a variant thereof, in the cyanob acteri al genome. Alternatively, TesA may be expressed from a vector-based expression system (e.g. from a non-integrative plasmid).

[0091] The TesA 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. For example, the modified cyanob acteri al strain may comprise a gene encoding TesA operably linked to a constitutive promoter. The modified cyanob acteri al strain may comprise multiple copies (e.g. 2, 3, 4, 5) of a gene encoding TesA per cell. The modified cyanob acteri al strain may comprise one or more copies (e.g. 1, 2, 3, 4, 5) of a heterologous gene encoding TesA per cell.

[0092] The modified cyanob acteri al strain over-expressing TesA may further comprise one or more modifications (e.g. 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 cyanob acteri al strain 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.

[0093] A modified cyanob acteri al strain 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 variant retains the TesA activity of hydrolysing the Coenzyme A group from an ester to produce a free acid.

[0094] The invention also provides the use of TesA for increasing carbon dioxide fixation by a modified cyanob acteri al strain.

[0095] A modified cyanob acteri al strain of the invention may comprise one or more modifications (e.g. genetic modifications) that discourage the synthesis of long chain fatty acids. Such modifications (e.g. 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 cyanob acteri al genome. The gene may be truncated such that a functional protein is not expressed. The resulting modified cyanob acteri al strain would thus express a lower quantity of the enzyme compared to a cyanob acteri al strain without said modifications.

[0096] A modified cyanob acteri al strain 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.

[0097] A modified cyanob acteri al strain of the invention may not express a functional FadD. Hence, the modified cyanob acteri al strain is not capable of catalysing the oxidation of free fatty acids to form fatty acyl-CoA and adenosine monophosphate and pyrophosphate. The gene encoding FadD may be deleted from the cyanob acteri al genome. The entire FadD gene may be deleted from the cyanob acteri al genome. If multiple copies (e.g. 2. 3 or 4) of the gene encoding fadD are present in the cyanob acteri al genome, all of which may be deleted. Hence, a modified cyanob acteri al strain of the invention may not comprise a gene encoding FadD in the cyanob acteri al genome. In the embodiment where the cyanob acteri al strain is Synechococcus PCC 11901, the gene encoding fadD at FEK30 07485 may be deleted from the cyanob acteri al genome.

[0098] The modified cyanob acteri al strain which does not express a functional FadD may further comprise one or more modifications (e.g. 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.

[0099] For example, a modified cyanob acteri al strain of the invention may comprise a combination of modifications (e.g. genetic modifications) that encourage synthesis of free fatty acids as described herein, and modifications (e.g. genetic modifications) that discourage the synthesis of long chain fatty acids as described herein. Without wishing to bound by theory, this combination of modifications (e.g. 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 cyanob acteri al strain of the invention may comprise the deletion of the gene encoding FadD, wherein the gene encoding FadD is replaced with a gene encoding TesA.

[0100] Carbonic anhydrase

[0101] The invention also relates to improving the bioavailability of dissolved carbon species (e.g. dissolved inorganic 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 catalyses the conversion of CO2 to HCCh', and shifts the direction of the reversible reaction in favour of HCCh' production, which can be taken up more efficiently by the cell which already expresses a native bicarbonate transporter at the cell surface membrane.

[0102] Hence, a modified cyanob acteri al strain of the invention may comprise one or more modifications (e.g. genetic modifications) to improve bioavailability of dissolved inorganic carbon species. A cyanob acteri al strain may be modified to insert a gene encoding carbonic anhydrase (CA), or variant thereof. Hence, a modified cyanob acteri al strain of the invention may comprise a heterologous gene encoding carbonic anhydrase (CA), or a variant thereof, in the cyanobacterial genome. Alternatively, CA may be expressed from a vector-based expression system (e.g. from a non-integrative plasmid). The CA 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. For example, the modified cyanob acteri al strain may comprise a gene encoding CA operably linked to a constitutive promoter. The modified cyanob acteri al strain may comprise multiple copies (e.g. 2, 3, 4, 5) of a gene encoding CA per cell. The modified cyanob acteri al strain may comprise one or more copies (e.g. 1, 2, 3, 4, 5) of a heterologous gene encoding CA per cell.

[0103] The modified cyanob acteri al strain expressing CA may further comprise one or more modifications (e.g. 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.

[0104] 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 cyanob acteri al strain of the invention may comprise a nucleotide sequence as set out in SEQ ID NO: 7.

[0105] A modified cyanob acteri al strain of the invention may comprise a gene encoding a variant of CA. 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: 8. 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: 8. The variant is capable of catalysing the interconversion of CO2 and HCOs' in solution.

[0106] 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 cyanob acteri al strain 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 cyanobacterial strain of the invention may comprise a nucleotide sequence as set out in SEQ ID NO: 9. Calvin Benson (CB) cycle

[0107] A modified cyanob acteri al strain of the invention may comprise one or more modifications (e.g. genetic modifications) that encourage the conversion of carbon dioxide into organic compounds. Such modifications (e.g. genetic modifications) may comprise deletion of an inhibitory regulator of photosynthesis, resulting in dysregulation of the Calvin Benson (CB) cycle (also known as the Calvin Benson Bassham (CBB) cycle). 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 cyanob acteri al genome. The gene may be truncated such that a functional protein is not expressed. The resulting modified cyanob acteri al strain would thus express a lower quantity of the enzyme compared to a cyanob acteri al strain without said modifications.

[0108] A modified cyanob acteri al strain of the invention may not express a functional inhibitory regulator of photosynthesis. The inhibitory regulator of photosynthesis may be CP12.

[0109] CP12 is a master regulator of the CB cycle, controlling central metabolism in response to light-dark cycles. A modified cyanob acteri al strain of the invention may not express a functional master regulator protein of central metabolism and circadian rhythm by sensing redox balance. The master regulator protein of central metabolism and circadian rhythm by sensing redox balance may be CP12.

[0110] A modified cyanob acteri al strain of the invention may comprise one or more modifications to prevent CP12 expression from the modified cyanob acteri al strain.

[0111] The modified cyanob acteri al strain of the invention may not express a functional CP12. The gene encoding CP12 may be deleted from the cyanob acteri al genome. The entire CP12 gene may be deleted from the cyanob acteri al genome. If multiple copies (e.g. 2. 3 or 4) of the gene encoding CP12 are present in the cyanob acteri al genome, all of which may be deleted. Hence, a modified cyanob acteri al strain of the invention may not comprise a gene encoding CP12. In the embodiment where the cyanob acteri al strain is Synechococcus PCC 11901, the gene encoding cpl2 at FEK30 05070 may be deleted from the cyanob acteri al genome.

[0112] 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, CP12 impedes GAPDH, which is pivotal for directing carbon flow towards the lower EMP pathway in cyanobacteria. Additionally, CP 12 exerts its inhibitory effect on PRK which is a key enzyme that is responsible for converting ribulose-5-phosphate (Ru5P) into ribulose- 1,5 -bisphosphate (R15P), a crucial substrate for carbon dioxide fixation by RuBisCO.

[0113] The modified cyanob acteri al strain which does not express a functional CP 12 may further comprise one or more modifications (e.g. 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.

[0114] Cyanobacteria

[0115] The invention relates to modified cyanob acteri al strains. Any cyanob acteri al strain is a suitable starting point for generating the modified cyanob acteri al strains of the invention. Hence, a modified cyanob acteri al strain of the invention may be derived from a cyanob acteri al strain described herein.

[0116] In other words, a modified cyanob acteri al strain of the invention may be a cyanob acteri al strain described herein which has been modified to comprise modifications (e.g. genetic modifications).

[0117] The modified cyanob acteri al strain of the invention is capable of fixing carbon dioxide at a higher rate compared to a cyanob acteri al strain which does not comprise the respective modifications (e.g. genetic modifications). For example, the modified cyanob acteri al strain of the invention may be capable of fixing carbon dioxide at an increased rate of >10%, >20%, >30%, >40%, >50%, >60%, >70%, >90%, >100%, >150% or >200% compared to a cyanob acteri al strain which does not comprise the respective modifications (e.g. genetic modifications) when grown under appropriate condition, e.g. as shown in the Examples (e.g. at 30°C and light intensity at 750 uE of light, 5 vol% CO2 gas). The modified cyanob acteri al strain of the invention may be capable of fixing carbon dioxide at a rate of: >0.5 g / L / day, >0.6 g / L / day, >0.7 g / L / day, >0.8 g / L / day, >0.9 g / L / day, >1 g / L / day, >2 g / L / day, or >3 g / L / day. The rate of carbon dioxide fixation may be measured by monitoring the amount of CO2 uptake by a cyanob acteri al strain when grown under appropriate condition, e.g. as shown in the Examples (e.g. at 30°C and light intensity at 750 uE of light, 5 vol% CO2 gas).

[0118] The cyanob acteri al strain useful with the invention or the modified cyanob acteri al strain of the invention is typically a strain that can double in a period less than 3 hours, less than 2.5 hours or around 2 hours. The modified cyanob acteri al strain of the invention may have a similar growth rate (e.g. about ±5%, about ±10%, or about ±20%) to a cyanob acteri al strain which does not comprise the modifications (e.g. genetic modifications) described herein when grown under appropriate condition, e.g. as shown in the Examples (e.g. at 30°C and light intensity at 750 uE of light, 5 vol% CO2 gas).

[0119] The cyanob acteri al strain or the modified cyanob acteri al strain may have an optimal growth condition under industrial carbon emission settings, such as light intensity mimicking outdoor natural sunlight, about 30 °C (such as from about 25 °C to about 30 °C) and about 5% CO2 (v / v) (such as from about 1% to about 10% CO2 (v / v)).

[0120] For example, the cyanob acteri al strain or the modified cyanob acteri al strain may be grown from about 25°C to about 50 °C. The cyanob acteri al strain or the modified cyanob acteri al strain may be capable of tolerating high temperatures, e.g. up to 35 °C, up to 40 °C, up to 43 °C, up to 45 °C, or up to 50°C.

[0121] The cyanob acteri al strain or the modified cyanob acteri al strain may be grown from about 0.04% CO2 (v / v) to up to 10% CO2 (v / v). The cyanob acteri al strain or the modified cyanob acteri al strain may be capable of tolerating high atmospheric carbon dioxide concentrations, e.g. up to 5% CO2 (v / v), 6% CO2 (v / v), 7% CO2 (v / v), 8% CO2 (v / v), 9% CO2 (v / v), or 10% CO2 (v / v).

[0122] The cyanob acteri al strain or the modified cyanob acteri al strain may be grown in artificial or natural light, e.g. ranging from 200 to 1500 micro Einsteins of solar irradiation. The cyanob acteri al strain or the modified cyanob acteri al strain may be capable of tolerating high light intensities, such as >700 pmol photons m’2s-1, >800 pmol photons m’2s-1, >900 pmol photons m’2s-1, or >1000 pmol photons m’2s-1.

[0123] The cyanob acteri al strain or the modified cyanob acteri al strain may be grown at 0 to about 3.5 % salinity. The cyanob acteri al strain or the modified cyanob acteri al strain may be capable of tolerating salinities over 1.5-fold, 2-fold, or 2.5-fold higher than sea water. The cyanob acteri al strain or the modified cyanob acteri al strain may have the capacity for sustained growth to high densities, e.g. up to 10 g / L dry cell weight, up to 20 g / L dry cell weight, up to 30 g / L dry cell weight, or up to 40 g / L dry cell weight.

[0124] The invention also provides a method of determining the optimal growth condition of a modified cyanob acteri al strain of the invention, e.g. by testing a range of each growth condition described herein. The method may further comprise selecting a modified cyanob acteri al strain of the invention that has a desired property, e.g. can double in a period less than 3 hours, or has the capacity for sustained growth to high densities, under the industrial carbon emission settings as describe herein.

[0125] For example, the cyanob acteri al strain may be Synechocystis sp. or Synechococcus sp. The cyanob acteri al strain or the modified cyanob acteri al strain may be a fresh water strain, such as Synechococcus elongatus UTEX 2973 (Yu et al., Sci Rep 2015 : 5(1): 8132), Synechococcus elongatus PCC 11801 (Jaiswal et al., Sci Rep, 2018, 8(1): 16632), Synechococcus elongatus PCC 11802 (Jaiswal et al., Sci Rep 2020, 10(1): 191), Synechococcus elongatus PCC 7942, or Synechocystis sp. PCC 6803. The cyanob acteri al strain or the modified cyanob acteri al strain may be a marine strain, such as PCC 7002 (Batterton and Van Baal en, Archiv Mikrobiol. 1971 :76(2): 151-165), o Synechococcus sp. PCC 11901 (Wlodarczyk et al., 2020, Commun Biol 3, 215).

[0126] The Synechococcus sp. may be PCC 11901. Synechococcus sp. PCC 11901, also known as Picosynechococcus sp. PCC 11901, or PCC 11901, deposited at the Pasteur Cultures of Cyanobacteria (PCC) under 11901, having NCBI Taxonomy ID 2579791, and genome assembly ASM557713vl (22 May 2019). Its complete genome is set out in NCBI Reference Sequence: NZ_CP040360.1. PCC 11901 is particularly useful with the invention because it is capable of doubling every ~2 hours in optimal conditions, has the capacity for sustained growth to high densities, e.g. up to 30g / L dry cell weight, and can tolerate high light intensities, such as >900 pmol photons m'1, high temperatures, e.g. up to 43 °C, and salinities over 2-fold higher than sea water. Hence, a modified cyanob acteri al strain of the invention may be derived from Synechococcus sp. PCC 11901. In other words, a modified cyanob acteri al strain of the invention may be a variant of Synechococcus sp. PCC 11901, which comprises genetic modifications, e.g. as described herein. The cyanob acteri al strain may be C. aponinum 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.

[0127] The cyanob acteri al strain may be Synechococcus sp. PCC 7002. Hence, a modified cyanob acteri al strain of the invention may be derived from Synechococcus sp. PCC 7002.

[0128] The cyanob acteri al strain may be Synechocystis sp. PCC 6803. Hence, a modified cyanob acteri al strain of the invention may be derived from Synechocystis sp. PCC 6803.

[0129] The cyanob acteri al strain may be Synechococcus elongatus PCC 7942. Hence, a modified cyanob acteri al strain of the invention may be derived from Synechococcus elongatus PCC 7942.

[0130] The cyanob acteri al strain may be PCC 11901, PCC 11802, PCC 6301, or UTEX 2973.

[0131] The cyanob acteri al strain 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.

[0132] The optimal growth condition for the cyanob acteri al strain or the modified cyanob acteri al strain is under industrial carbon emission settings, such as light intensity mimicking outdoor natural sunlight, 30 °C and 5% CO2 (v / v).

[0133] The cyanob acteri al strain or the modified cyanob acteri al strain may comprise in its genome a gene encoding a 16S rRNA sequence having >95% (i.e. equal or greater than 95%), >96%, >97%, >98%, >99% or 100% sequence identity with the 16S rRNA sequence of a cyanob acteri al strain described herein, such as Synechococcus sp. PCC 11901. For example, the 16S rRNA sequence of the cyanob acteri al strain or the modified cyanob acteri al strain may have >99.6%, >99.7%, >99.8%, >99.9% or 100% sequence identity with SEQ ID NO: 28. The 16S rRNA of the cyanob acteri al strain or the modified cyanob acteri al strain may differ from SEQ ID NO: 28 by <2 (i.e. equal to or less than 2), <3, <4, <5, <6 or <7 bases. In particular, the cyanobacterial strain or the modified cyanob acteri al strain may comprise in its genome a gene encoding a 16S rRNA sequence having >99.6% sequence identity with SEQ ID NO: 28. The cyanobacterial strain or the modified cyanobacterial strain may comprise in its genome a gene encoding a 16S rRNA sequence having >99.8% sequence identity with SEQ ID NO: 28. The cyanobacterial strain or the modified cyanob acteri al strain may comprise in its genome a gene encoding a 16S rRNA sequence that differs from SEQ ID NO: 28 by <3 bases. The 16S rRNA sequence of the cyanob acteri al strain or the modified cyanob acteri al strain may be identical to SEQ ID NO: 28.

[0134] The cyanob acteri al strain or the modified cyanob acteri al strain may comprise a genome having >90% (i.e. equal or greater than 90%), >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, >99.1%, >99.2%, >99.3%, >99.4%, >99.5%, >99.6%, >99.7%, >99.8%, >99.9% or 100% sequence identity with the genome sequence of a cyanob acteri al strain described herein, such as Synechococcus sp. PCC 11901 having NCBI Reference Sequence: NZ CP040360.1. In particular, the cyanob acteri al strain or the modified cyanob acteri al strain may comprise a genome having >97% sequence identity with the genome sequence of a cyanob acteri al strain described herein, such as Synechococcus sp. PCC 11901 having NCBI Reference Sequence: NZ_CP040360.1.

[0135] The cyanob acteri al strain or the modified cyanob acteri al strain may comprise >1 (i.e. one or more), >2, >3, >4, >5, >6, >7, >8 or >9 reference genes or variants thereof from a cyanob acteri al strain described herein, such as PCC 11901. Each of the reference gene variants may have >98% (i.e. equal or greater than 98%), >99% or 100% sequence identity with the corresponding reference gene. The reference gene may be a housekeeping gene in a cyanob acteri al strain described herein, such as PCC 11901, such as a gene encoding ppc (e.g. SEQ ID NO: 29), petB (e.g. SEQ ID NO: 30), mpB (e.g. SEQ ID NO: 31), rpoA (SEQ ID NO: 32), or secA (SEQ ID NO: 33). The reference gene may be a gene that confers a fast growth rate phenotype in a cyanob acteri al strain described herein, such as PCC 11901, such as rpaA, atpA (e.g. SEQ ID NO: 35) or ppnK (e.g. SEQ ID NO: 36). For example, the reference gene may be rpaA (e.g. having a DNA sequence as set out in SEQ ID NO: 34).

[0136] The cyanob acteri al strain or the modified cyanob acteri al strain may contain modifications (e.g. genetic modifications) relative to a cyanob acteri al strain described herein (e.g. Synechococcus sp. PCC 11901). The genetic modification may comprise deletions, insertions, inversions, repeats and substitutions in the genome of the bacteria. Hence, a modified cyanob acteri al strain of the invention may be derived from any cyanob acteri al strain described herein.

[0137] Using known methods in the art, variants may be generated to improve or alter the characteristics of the cyanob acteri al strains or the modified cyanobacterial strain described herein. Such variants include deletions, insertions, inversions, repeats and substitutions in the genome of the bacteria selected according to general rules known in the art. For example, the variants may contain modified genes with codon optimisation, modified genes with increased or reduced expression levels and / or activities, deletion of native genes, or addition of heterologous genes. These modifications may allow for improved growth rates, improved carbon dioxide fixation, and / or improve production of carbon-rich compounds (such as aromatic amino acids). For example, modifications to modify 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. The modification may comprise increasing or decreasing the copy number of a gene.

[0138] Alternatively, the modifications optionally do not substantially alter the biological activity of the cyanob acteri al strain or the modified cyanob acteri al strain. For example, a protein variant may contain conservative amino acid changes which are less likely to perturb the structure and / or function of the polypeptide. For example, the cyanob acteri al strain or the modified cyanobacterial strain may comprise a gene encoding a protein having 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), betabranched 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. Modifications

[0139] The cyanob acteri al strain may be modified to express a protein described herein (e.g. SK, TesA, CA, or AroF D174N) 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.

[0140] 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 cyanob acteri al strain, such that expression of the protein by the modified cyanob acteri al strain is increased. Introduction may take place through transformation of the expression cassette into the host cell, as described herein. In embodiments where the gene of interest, e.g. SK is present endogenously, increasing the quantity of the protein encoded by said gene may be achieved by replacing the endogenous promoter of said gene with a stronger promoter, or replacing the nucleotide sequence of said gene with a codon optimised nucleotide sequence.

[0141] Increased gene expression includes both augmentation of native production of the protein as well as production of a protein in a cyanob acteri al strain 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.

[0142] The increase in the quantity of a protein (e.g. SK, TesA, CA, or AroF D174N) may be achieved by an increased copy number of the protein-encoding gene present in the modified cyanob acteri al strain, such that the modified cyanob acteri al strain comprises >1, >2, >3, >4, >5, >6 or >7 copies of the gene per cell. Hence, the modified cyanob acteri al strain may comprise one or more copies (e.g. 1, 2, 3, 4, 5) of the heterologous gene per cell. For example, a gene described herein may be integrated into the cyanob acteri al genome. The gene may be integrated into the cyanob acteri al chromosome or an endogenous cyanob acteri al plasmid. The endogenous cyanob acteri al plasmid may be present in a higher copy number than the cyanob acteri al 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 cyanob acteri al strain.

[0143] The gene encoding a protein described herein is typically introduced into a cyanobacterial strain 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.

[0144] 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.

[0145] Table 1. Examples of promoters useful with the invention.

[0146] 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 cyanob acteri al genome, such as in the endogenous cyanob acteri al 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 cyanob acteri al strain 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 cyanob acteri al plasmid through homologous recombination.

[0147] 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.

[0148] Transformation of a cyanob acteri al strain cell with expression cassettes can be carried out by conventional techniques, e.g. ssDNA transformation.

[0149] The cyanob acteri al strain may be modified to express a bacterial protein described herein (e.g. FadD or CP12) 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.

[0150] Modifications to reduce the expression of a bacterial gene (e.g. FadD or CP12) 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 cyanob acteri al strain 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 cyanob acteri al strain may be modified such that the gene (e.g. FadD or CP12) is rendered non-functional. 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 copies of the gene are present in the cyanobacterial strain, both copies of the gene may be rendered nonfunctional (e.g. deleted). The entire gene may be deleted from the endogenous plasmid or chromosome. Where two or more copies of the gene are present in the cyanobacterial strain, all copies of the gene may be rendered non-functional (e.g. deleted).

[0151] The gene (e.g. FadD or CP12) in the cyanobacterial strain 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.

[0152] The invention also provides a method of preparing a modified cyanobacterial strain described herein. The modified cyanobacterial strain 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 cyanobacterial strain of the invention, based on well- established molecular biology and microbiology techniques.

[0153] Exemplary modified cyanobacterial strains

[0154] The invention provides a modified cyanobacterial strain comprising a combination of the modifications described herein. For example, the modified cyanobacterial strain may comprise one or more modifications for (a) improving expression of aromatic amino acids through overexpression of the shikimate pathway, (b) encouraging production of free fatty acids, (c) dysregulation of the Calvin Benson cycle, and / or (e) improving bioavailability of dissolved inorganic carbon species.

[0155] The modified cyanobacterial strain may comprise one or more modifications for improving expression of aromatic amino acids through overexpression of the shikimate pathway as described herein, and one or more modifications for dysregulation of the Calvin Benson (CB) cycle as described herein. Whilst not being bound to theory, it is considered that if the CB cycle is less prone to negative feedback / inhibition due to the CP 12 knock out, combining this with engineering carbon sinks may be complementary in improving CO2 fixation rate. Furthermore, since deletion of CP 12 does not involve a new gene insertion, there is little risk of an additional metabolic burden on the cell. For example, a modified cyanob acteri al strain of the invention may comprise: (a) a gene encoding AroF D174N, or a variant thereof, capable of catalysing the reaction of PEP and E4P to DAHP and inorganic phosphate, and is unsusceptible to allosteric modulation; and (b) deletion of the gene encoding CP12 from the cyanob acteri al genome. As a further example, a modified cyanob acteri al strain of the invention may comprise: (a) overexpression of SK, or a variant thereof, capable of catalysing the transformation of shikimate into 3-phosphoshikimate; and (b) deletion of the gene encoding CP12 from the cyanob acteri al genome.

[0156] The invention provides a modified cyanob acteri al strain wherein the endogenous gene encoding long-chain fatty acid CoA ligase fadD in the cyanob acteri al 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 or pcpc560. The modified cyanob acteri al strain may be derived from Synechococcus 11901, UTEX 3154 or UTEX 3222. The modified cyanob acteri al strain may further comprise one or more modifications (e.g. 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.

[0157] The invention also provides a modified cyanob acteri al strain comprising a gene encoding AroF D174N. The AroF D174N-encoding gene may be operably linked to the constitutive promoter, e.g. JS23119. The modified cyanob acteri al strain may be derived from Synechococcus 11901, UTEX 3154 or UTEX 3222. The modified cyanob acteri al strain may further comprise one or more modifications (e.g. 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.

[0158] The invention also provides a modified cyanob acteri al strain 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 cyanob acteri al strain. The modified cyanob acteri al strain may comprise two or more (e.g. 2, 3 or 4) copies of SK gene in the cyanob acteri al strain genome per cell. The SK-encoding gene may be operably linked to the constitutive promoter, e.g. PpsbA2, pcpc560 or JS23119. The modified cyanob acteri al strain may be derived from Synechococcus 11901, UTEX 3154 or UTEX 3222. The modified cyanob acteri al strain may further comprise one or more modifications (e.g. 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.

[0159] The invention also provides a modified cyanob acteri al strain wherein CP12 is knocked out from the cyanob acteri al genome. The modified cyanob acteri al strain may be derived from Synechococcus 11901, UTEX3154 or UTEX 3222. The modified cyanob acteri al strain may further comprise one or more modifications (e.g. 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.

[0160] The invention also provides a modified cyanob acteri al strain 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 cyanob acteri al genome. The TesA-encoding gene may be operably linked to the constitutive promoter, e.g. pcpc560 or JS23119. The modified cyanob acteri al strain may be derived from Synechococcus 11901, UTEX3154 or UTEX 3222. The modified cyanob acteri al strain may further comprise one or more modifications (e.g. 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.

[0161] Protein variants

[0162] 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.

[0163] 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.

[0164] 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.

[0165] A protein variant may be codon optimized to increase expression levels of the respective protein in host cells as compared to the unaltered sequence. Methods for codon optimisation are known in the art, e.g. GeneScript OptimumGene™ algorithm can be used.

[0166] Composition and kits

[0167] The invention also provides a composition comprising one or more of the modified cyanob acteri al strains described herein.

[0168] The invention also provides a kit comprising the modified cyanob acteri al strain or the composition described herein for fixing carbon dioxide. Uses

[0169] The modified cyanob acteri al strains described herein are particularly effective in fixing carbon dioxide. Thus, the invention also provides a method of fixing carbon dioxide, comprising exposing a modified cyanob acteri al strain described herein to a source of carbon dioxide (e.g. at a concentration of 5% or more). The carbon dioxide source may be supplied from flue gas. The amount of carbon dioxide provided to the modified cyanob acteri al strain may be 0.03-5.0% of a total volume of the flue gas.

[0170] The invention also provides the use of a modified cyanob acteri al strain 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.

[0171] The invention also provides the use of a modified cyanob acteri al strain described herein as a high productivity platform strain for the overexpression of high value compounds of interest to nutraceuticals, pharmaceuticals and biomanufacturing.

[0172] The invention also provides the use of AroF D174N and / or tesA for increasing the rate of carbon dioxide fixation by a modified cyanobacterial strain.

[0173] Embodiments of the invention

[0174] 1. A modified cyanobacterial strain 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 cyanobacterial strain which does not comprise said genetic modifications.

[0175] 2. The modified cyanobacterial strain of embodiment 1, wherein the cyanobacterial strain is Synechococcus sp., e.g. Synechococcus PCC 11901 or a variant thereof.

[0176] 3. The modified cyanobacterial strain of embodiment 1 or 2, comprising one or more genetic modifications to over-express shikimate kinase (SK) in the modified cyanobacterial strain.

[0177] 4. The modified cyanobacterial strain of embodiment 3, wherein the modified cyanobacterial strain comprises a gene encoding SK, or a variant thereof, in the cyanobacterial genome. 5. The modified cyanob acteri al strain of embodiment 4, wherein the gene encoding SK comprises a nucleotide sequence as set out in SEQ ID NO: 11.

[0178] 6. The modified cyanob acteri al strain of any one of the preceding embodiments, further comprising one or more genetic modifications to remove (e.g. reduce) inhibitory feedback on shikimate pathway in the modified cyanob acteri al strain.

[0179] 7. The modified cyanob acteri al strain of embodiment 6, wherein the modified cyanob acteri al strain comprises a gene encoding AroF D174N, or variant thereof, in the cyanob acteri al genome.

[0180] 8. The modified cyanob acteri al strain of embodiment 7, wherein the gene encoding AroF D174N comprises a nucleotide sequence as set out in SEQ ID NO: 3.

[0181] 9. The modified cyanob acteri al strain of any one of the preceding embodiments, further comprising one or more genetic modifications to encourage production of free fatty acids in the modified cyanob acteri al strain, optionally wherein the modified cyanob acteri al strain comprises a gene encoding acyl-CoA thioesterase, TesA (e.g. SEQ ID NO: 5), or a variant thereof, in the cyanobacterial genome.

[0182] 10. The modified cyanobacterial strain of any one of the preceding embodiments, further comprising one or more genetic modifications to improve bioavailability of dissolved carbon species (e.g. dissolved inorganic carbon species), optionally wherein the modified cyanobacterial strain comprises a gene encoding carbonic anhydrase (CA) (e.g. SEQ ID NO: 7), or a variant thereof, in the cyanobacterial genome.

[0183] 11. The modified cyanobacterial strain of embodiment 10, wherein the CA is coupled to a PilA leader sequence (e.g. SEQ ID NO: 9) in its N terminus.

[0184] 12. The modified cyanobacterial strain of any one of embodiments 3 to 11, wherein the gene is operably linked to a constitutive promoter.

[0185] 13. The modified cyanobacterial strain of embodiment 12, wherein the constitutive promoter is selected from the group consisting of: JS23119, Pcpt, PpsbA2, Pnbl A, and pcpc560.

[0186] 14. The modified cyanobacterial strain of any one of embodiments 3 to 13, wherein the modified cyanobacterial strain comprises multiple copies of the gene in the cyanobacterial genome, optionally wherein the modified cyanobacterial strain comprises multiple copies of a gene encoding SK in the cyanobacterial genome.

[0187] 15. The modified cyanobacterial strain of any one of the preceding embodiments, further comprising one or more genetic modifications to remove an inhibitory regulator of photosynthesis, optionally wherein the gene encoding CP12 is deleted from the cyanob acteri al genome.

[0188] 16. The modified cyanob acteri al strain of any one of the preceding embodiments, 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 cyanob acteri al genome.

[0189] 17. The modified cyanob acteri al strain of embodiment 16, wherein the gene encoding FadD is replaced with a gene encoding TesA.

[0190] 18. The modified cyanob acteri al strain of any one of the preceding embodiments, 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).

[0191] 19. A composition comprising one or more modified cyanob acteri al strains of any one of the preceding embodiments for fixing carbon dioxide.

[0192] 20. A method of increasing the rate of carbon dioxide fixation of a cyanob acteri al strain, comprising genetically modifying the cyanobacterial strain to generate a modified cyanobacterial strain according to any one of the methods of embodiments 1 to 18.

[0193] 21. A method of fixing carbon dioxide, comprising exposing the modified cyanobacterial strain of any one of embodiments 1 to 18 or the composition of embodiment 19 to a source of carbon dioxide.

[0194] 22. The method of embodiment 21, wherein the carbon dioxide source is supplied from flue gas.

[0195] 23. The method of embodiment 21 or 22, wherein the amount of carbon dioxide provided to the modified cyanobacterial strain is 0.03-5.0% of a total volume of the flue gas.

[0196] 24. Use of the modified cyanobacterial strain of any one of embodiments 1 to 18 or the composition of embodiment 19 for fixing carbon dioxide.

[0197] 25. Use of AroF D174N and / or TesA for increasing the rate of carbon dioxide fixation by a modified cyanobacterial strain. Other

[0198] 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.

[0199] It is to be understood that different applications of the modified cyanob acteri al 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.

[0200] 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.

[0201] 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.

[0202] In some embodiments of the invention, the word “comprising may be replaced with the phrase “consisting essentially o . 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.

[0203] 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.

[0204] In addition, as used in this specification and the appended claims, the singular forms “d “ari and “the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “a modified cyanobacterial strain" includes two or more modified cyanobacterial strains.

[0205] 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 than y.

[0206] 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 (z.e., % identity = number of identical positions / total number of positions in the reference sequence x 100).

[0207] 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.

[0208] 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.

[0209] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0210] The following examples illustrate the invention. Examples

[0211] Example 1 - Generation of modified cyanobacterial strains

[0212] The Synechococcus PCC 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 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).

[0213] A non-exhaustive set of example strains constructed according to this invention are summarised in Table 2.

[0214] Table 2. Cyanobacterial strains tested in the Examples.

[0215] 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 or PCC 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.

[0216] 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.

[0217] 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 cyanob acteri al metabolic efficiency.

[0218] Strain D - To create this strain, a knockout strategy was used to delete the cpl2 gene, which is a master regulator protein of the Calvin Benson cycle, aiming to deregulate the Calvin Benson cycle and enhance carbon fixation and photosynthetic efficiency by dysregulation of central metabolism, redox state sensing and circadian control. 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.

[0219] 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 cyanob acteri al metabolic efficiency.

[0220] 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.

[0221] 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.

[0222] 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 expression of TesA and C A was under a stronger promoter (pcpc560) in Strain I compared to in Strain E (JS23119). It was considered that whilst overexpression of these enzymes may yield a higher CO2 fixation rate, too much production may be unnecessary and impose an unnecessary metabolic burden if the enzyme concentration is no longer rate limiting in the pathway.

[0223] 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 S1000 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'1, increased after one day to 750 pmol photons m’1. Daily compensation for water loss was achieved by adding 700 pL of sterile MilliQ 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.

[0224] The OD730 of these strains at day 9 are shown in Tables 3 and 4. 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.

[0225] Table 3. Growth rate of strain B (AroFD174N) compared to WT strain 11901.

[0226] Table 4. Growth rate of strain C (SK) and strain D (Acpl2) compared to WT strain 11901.

[0227] The growth of the strains, as monitored by measuring OD730, are shown in Figures 1 and 2.

[0228] Compared to the wild-type strain (11901), it can be seen that the growth of the modified strains was increased, providing fast growth rates.

[0229] Example 2 - Modified cyanobacterial strains showed improvements in rate of carbon capture

[0230] The cyanobacterial strains from Example 1 were tested for the rate of carbon dioxide absorption.

[0231] 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.

[0232] 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.

[0233] 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).

[0234] Example 3

[0235] The Synechococcus PCC 11901 strain is modified to express tesA and CA.

[0236] To generate the modified cyanob acteri al strain, 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. coh. and synthesised in a codon-optimised form to maximise expression in Synechococcus PCC 11901. The plasmid is transformed into an exponentially growing culture of Synechococcus PCC 11901 by natural transformation.

[0237] 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.

[0238] The rate of carbon dioxide absorption is measured according to the method in Example 2. The CO2 fixation rate of modified cyanob acteri al strain expressing tesA and CA is superior to that of the wild-type strain.

[0239] Example 4

[0240] Strains G, H and I were prepared and their growth were monitored according to Example 1. The growth of these strains, as monitored by measuring OD730, are shown in Figure 5.

[0241] Compared to the wild-type strain (11901), the growth of the modified strains was not affected. Hence, the modified bacteria strains are able to maintain the fast growth rate of the wild-type strain.

[0242] Summary and Discussion

[0243] The data show that these strains showed both higher growth rates and CO2 fixation rates compared to the wild type strains:

[0244] Strain B: in which the shikimate pathway was enhanced by reducing the inhibition mediated by AroF. Strain C: in which the shikimate pathway was enhanced by overexpressing the shikimate kinase enzyme.

[0245] Strain D: in which the master regulator protein (cpl2) of the Calvin Benson cycle was deleted, causing a dysregulation of central metabolism, redox state sensing and circadian control.

[0246] These data show that under high light, high temperature and constant illumination, engineering carbon sinks (e.g. enhanced aromatic amino acids synthesis) into fast-growing cyanob acteri al strains have resulted in higher CO2 fixation rates than in the wild type strains. The data also show that deletion of a master regulator protein, CP12, unexpectedly increases CO2 fixation rate.

[0247] The rationale is that by making the cell increase production of aromatic amino acids, carbon flux in the metabolism can be directed towards less bottlenecked pathways and would confer an improvement in overall CO2 fixation. This may improve CO2 fixation as a result of the additional carbon fixed from the products, but would be expected to reduce biomass productivity (cell density measured by OD?3onm as a proxy). Hence, biomass productivity would be compromised slightly, but CO2 fixation rates would be overall improved.

[0248] However, the data show that, despite having engineered carbon sinks, the biomass growth of the modified strains was not compromised at all, but had improved, to the inventors’ surprise.

[0249] The inventors also found that by combining carbon sinks with an upstream improvement in CO2 fixation, such as by overexpressing carbonic anhydrase (CA), metabolic bottlenecks that hamper growth can be alleviated. Carbonic Anhydrase (CA) is an enzyme that converts CO2 into HCCh', enabling more carbon (inorganic) to enter the cell.

[0250] Sequence listing

Claims

Claims1. A modified cyanob acteri al strain comprising one or more modifications to enhance the rate of carbon dioxide fixation relative to a cyanob acteri al strain which does not comprise said modifications, wherein the one or more modifications is for (a) improving expression of aromatic amino acids through overexpression of the shikimate pathway, (b) encouraging production of free fatty acids, (c) dysregulation of the Calvin Benson cycle, and / or (e) improving bioavailability of dissolved inorganic carbon species.

2. The modified cyanob acteri al strain of claim 1, wherein the one or more modifications for improving aromatic amino acids through overexpression of shikimate pathway comprises one or more modifications to over-express shikimate kinase (SK) in the modified cyanob acteri al strain.

3. The modified cyanob acteri al strain of claim 1 or 2, wherein the modified cyanob acteri al strain comprises a heterologous gene encoding SK, or a variant thereof, in the cyanob acteri al genome.

4. The modified cyanob acteri al strain of claim 3, wherein the SK, or variant thereof, comprises an amino acid sequence having >80%, 90%, 95% or 100% sequence identity to SEQ ID NO: 12 or 18, and wherein the SK, or variant thereof, is capable of catalyzing the transformation of shikimate into 3-phosphoshikimate.

5. The modified cyanob acteri al strain of claim 3 or 4, wherein the gene encoding SK is from Synechococcus PCC 11901, optionally wherein the SK comprises a nucleotide sequence as set out in SEQ ID NO: 11.

6. The modified cyanob acteri al strain of claim 3 or 4, wherein the gene encoding SK is from UTEX 3222, optionally wherein the gene encoding SK comprises a nucleotide sequence as set out in SEQ ID NO: 17.

7. The modified cyanob acteri al strain of any one of claims 3 to 6, wherein the gene encoding SK is operably linked to a constitutive promoter.

8. The modified cyanob acteri al strain of claim 7, wherein the constitutive promoter is selected from the group consisting of JS23119, Pcpt, PpsbA2, PnblA, and pcpc560, optionally wherein the constitutive promoter is PpsbA2.

9. The modified cyanob acteri al strain of any one of claims 4 to 8, wherein the modified cyanob acteri al strain comprises multiple copies of the gene encoding SK per cell.

10. The modified cyanob acteri al strain of any one of the preceding claims, wherein the one or more modifications for improving aromatic amino acids through overexpression of shikimate pathway comprises one or more modifications to reduce inhibitory feedback on the shikimate pathway in the modified cyanob acteri al strain.

11. The modified cyanob acteri al strain of any one of the preceding claims, wherein the modified cyanob acteri al strain comprises a heterologous gene encoding a modified 3- Deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS) that is unsusceptible to allosteric modulation.

12. The modified cyanob acteri al strain of claim 11, wherein the DAHPS is AroF.

13. The modified cyanob acteri al strain of claim 12, wherein the modified AroF comprises a mutation at a position corresponding to position 174 of the AroF from Synechococcus PCC 11901 (SEQ ID NO: 2), optionally wherein the aspartic acid residue at position 174 in SEQ ID NO: 2 is substituted with an amino acid having a basic side chain, such as lysine, arginine, or histidine.

14. The modified cyanob acteri al strain of any one of the preceding claims, wherein the modified cyanobacterium comprises a gene encoding AroF D174N, or variant thereof, in the cyanob acteri al genome, and optionally wherein the gene encoding AroF D174N comprises a nucleotide sequence as set out in SEQ ID NO: 3.

15. The modified cyanob acteri al strain of claim 12 or 13, wherein the modified DAHPS is AroF D146N (SEQ ID NO: 22), or a variant thereof.

16. The modified cyanob acteri al strain of claim 12 or 13, wherein the modified DAHPS is AroF E154N (SEQ ID NO: 27), or a variant thereof.

17. The modified cyanob acteri al strain of any one of claims 11 to 16, wherein the gene encoding the modified DAHPS is operably linked to a constitutive promoter.

18. The modified cyanob acteri al strain of claim 17, wherein the constitutive promoter is selected from the group consisting of JS23119, Pcpt, PpsbA2, PnblA, and pcpc560, optionally wherein the constitutive promoter is pcpt.

19. The modified cyanob acteri al strain of any one of claims 11 to 18, wherein the modified cyanob acteri al strain comprises multiple copies of the gene encoding the modified DAHPS per cell.

20. The modified cyanob acteri al strain of any one of the preceding claims, wherein the one or more modifications to encourage production of free fatty acids in the modified cyanob acteri al strain comprises one or more modifications to over-express acyl-CoA thioesterase in the modified cyanob acteri al strain.

21. The modified cyanob acteri al strain of any one of the preceding claims, wherein the modified cyanob acteri al strain comprises a heterologous gene encoding acyl-CoA thioesterase, TesA, or a variant thereof, in the cyanob acteri al genome.

22. The modified cyanob acteri al strain of claim 21, wherein the TesA or variant thereof comprises an amino acid sequence having an amino acid sequence having >80%, 90%, 95% or 100% sequence identity to SEQ ID NO: 6, and wherein the TesA, or variant thereof, is capable of hydrolysing the Coenzyme A group from an ester to produce a free acid.

23. The modified cyanob acteri al strain of claim 21 or 22, wherein the gene encoding TesA comprises a nucleotide sequence as set out in SEQ ID NO: 5.

24. The modified cyanob acteri al strain of any one of claims 20 to 23, wherein the gene encoding TesA, or a variant thereof, is operably linked to a constitutive promoter.

25. The modified cyanob acteri al strain of claim 24, wherein the constitutive promoter is selected from the group consisting of JS23119, Pcpt, PpsbA2, PnblA, and pcpc560, optionally wherein the constitutive promoter is pJS23119.

26. The modified cyanob acteri al strain of any one of claims 20 to 25, wherein the modified cyanob acteri al strain comprises multiple copies of the gene encoding TesA per cell.

27. The modified cyanob acteri al strain of any one of the preceding claims, wherein the one or more modifications to improve bioavailability of dissolved inorganic carbon species comprises one or more modifications to over-express carbonic anhydrase (CA) in the modified cyanob acteri al strain.

28. The modified cyanob acteri al strain of any one of the preceding claims, wherein the modified cyanob acteri al strain comprises a heterologous gene encoding CA, or a variant thereof, in the cyanob acteri al genome.

29. The modified cyanob acteri al strain of claim 27 or 28, wherein the CA comprises an amino acid sequence having an amino acid sequence having >80%, 90%, 95% or 100% sequence identity to SEQ ID NO: 8, and wherein the CA, or variant thereof, is capable of catalysing the interconversion of CO2 and HCOs' in solution.

30. The modified cyanob acteri al strain of any one of claims 27 to 29, wherein the CA is coupled to a PilA leader sequence (e.g. SEQ ID NO: 9) in its N terminus.

31. The modified cyanobacterium of any one of claims 28 to 30, wherein the gene encoding CA is operably linked to a constitutive promoter.

32. The modified cyanobacterium of claim 31, wherein the constitutive promoter is selected from the group consisting of JS23119, Pcpt, PpsbA2, PnblA, and pcpc560, optionally, wherein the constitutive promoter is pJS23119.

33. The modified cyanobacterium of any one of claims 28 to 32, wherein the modified cyanobacterium comprises multiple copies of the gene encoding CA in the cyanob acteri al genome.

34. The modified cyanob acteri al strain of any one of the preceding claims, wherein the one or more modifications to dysregulate the Calvin Benson cycle comprises one or more modifications to remove an inhibitory regulator of photosynthesis, such as prevention of CP 12 expression from the modified cyanob acteri al strain.

35. The modified cyanob acteri al strain of any one of the preceding claims, wherein the modified cyanob acteri al strain does not express a functional CP 12.

36. The modified cyanob acteri al strain of claim 35, wherein the gene encoding CP12 is deleted from the cyanob acteri al genome.

37. The modified cyanob acteri al strain of any one of the preceding claims, wherein the one or more modifications to encourage production of free fatty acids comprise one or more modifications to discourage synthesis of long chain fatty acids.

38. The modified cyanob acteri al strain of any one of the preceding claims, wherein the modified cyanob acteri al strain does not express a functional FadD.

39. The modified cyanob acteri al strain of claim 38, wherein the gene encoding FadD is deleted from the cyanob acteri al genome.

40. The modified cyanob acteri al strain of claim 38 or 39, wherein the gene encoding FadD is replaced with a gene encoding TesA.

41. The modified cyanob acteri al strain of any one of the preceding claims, comprising one or more modifications for improving expression of aromatic amino acids through overexpression of the shikimate pathway as defined in any one of claims 2 to 9 and 10 to 19, and one or more modifications for dysregulation of the Calvin Benson cycle as defined in any one of claims 34 to 36.

42. The modified cyanob acteri al strain of any one of the preceding claims, comprising: a gene encoding AroF D174N, or a variant thereof, capable of catalysing the reaction of PEP and E4P to DAHP and inorganic phosphate, and is unsusceptible to allosteric modulation; and deletion of the gene encoding CP 12 from the cyanobacterial genome.

43. The modified cyanobacterial strain of any one of claims 1 to 42, comprising: over-expression of SK, or a variant thereof, capable of catalysing the transformation of shikimate into 3-phosphoshikimate; and deletion of the gene encoding CP 12 from the cyanobacterial genome.

44. The modified cyanobacterial strain 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).

45. The modified cyanobacterial strain of any one of the preceding claims, which(a) is capable of doubling every approximately 2 hours in optimal conditions;(b) has the capacity for sustained growth to high densities, e.g. up to 30g / L dry cell weight; and / or(c) can tolerate high light intensities, such as >900 pmol photons m'1, high temperatures, e.g. up to 43 °C, and / or salinities over 2-fold higher than sea water.

46. The modified cyanob acteri al strain of any one of the preceding claims, wherein the modified cyanob acteri al strain is capable of fixing carbon dioxide at a higher rate compared to a cyanob acteri al strain that does not comprise said modifications.

47. The modified cyanob acteri al strain of any one of the preceding claims, wherein the modified cyanob acteri al strain is capable of fixing carbon dioxide at an increased rate of >10%, >20%, >30%, >40%, >50%, >60%, >70%, >90%, >100%, >150% or >200% compared to a cyanob acteri al strain which does not comprise said modifications.

48. The modified cyanob acteri al strain of any one of the preceding claims, wherein the modified cyanob acteri al strain is capable of fixing carbon dioxide at a rate of >0.5 g / L / day, >0.6 g / L / day, >0.7 g / L / day, >0.8 g / L / day, >0.9 g / L / day, >1 g / L / day, >2 g / L / day, or >3 g / L / day.

49. The modified cyanob acteri al strain of any one of the preceding claims, wherein the modified cyanob acteri al strain is capable of doubling in a period less than 3 hours, less than 2.5 hours or around 2 hours in optimal conditions.

50. The modified cyanob acteri al strain of any one of the preceding claims, wherein the cyanob acteri al strain is derived from Synechococcus sp., e.g. Synechococcus PCC 11901 or a variant thereof.

51. A composition comprising one or more modified cyanob acteri al strains of any one of the preceding claims for fixing carbon dioxide.

52. A method of increasing the rate of carbon dioxide fixation of a cyanob acteri al strain, comprising modifying the cyanob acteri al strain to: (a) improve expression of aromatic amino acids through overexpression of the shikimate pathway as defined in any one of claims 2 to 19, (b) encourage production of free fatty acids as defined in any one of claims 20 to 26 and 37 to 40, (c) dysregulate the Calvin Benson cycle as defined in any oneof claims 34 to 36, and / or (e) improve bioavailability of dissolved inorganic carbon species as defined in any one of claims 27 to 33.

53. A method of fixing carbon dioxide, comprising exposing the modified cyanob acteri al strain of any one of claims 1 to 50 or the composition of claim 51 to a source of carbon dioxide having a concentration of 5% CO2 or more.

54. The method of claim 53, wherein the carbon dioxide source is supplied from flue gas.

55. The method of claim 53 or 54, wherein the amount of carbon dioxide provided to the modified cyanob acteri al strain is 0.03-5.0% of a total volume of the flue gas.

56. Use of the modified cyanob acteri al strain of any one of claims 1 to 50 or the composition of claim 51 for fixing carbon dioxide.

57. Use of AroF D174N and / or TesA for increasing the rate of carbon dioxide fixation by a modified cyanob acteri al strain.

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