A photosynthetic system for catalyzing carbon dioxide mineralization

Abstract

The present invention provides for a photosynthetic cell, such as a cyanobacteria cell, comprising a nucleic acid encoding a heterologous carbonic anhydrase (CA) operatively linked to a promoter capable of expression in the photosynthetic cell. The present invention also provides for a method for mineralizing carbon dioxide comprising: (a) culturing or growing a photosynthetic cell of the present invention in a medium under conditions such that the photosynthetic cell expresses the CA, (b) converting carbon dioxide into the H2CO3 through the enzymatic activity of the CA, (c) optionally complexing the carbonate of the H2CO3 with a metal to form a metal carbonate, (d) optionally precipitating the metal carbonate from the medium, (e) optionally removing or separating the metal carbonate from the medium and / or the photosynthetic cell.

Description

A photosynthetic system for catalyzing carbon dioxide mineralizationInventor: Peter AgboRELATED PATENT APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 63 / 552,451, filed February 12, 2024, which is incorporated by reference in its entirety.STATEMENT OF GOVERNMENTAL SUPPORT

[0002] The invention was made with government support under Contract Nos. DE-AC02- 05CH11231 and DE-SC0004993 awarded by the U.S. Department of Energy. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] Reserved.FIELD OF THE INVENTION

[0004] The present invention is in the field of carbon dioxide mineralization.BACKGROUND OF THE INVENTION

[0005] The concept of using carbonic anhydrase (CA) enzymes in CO2 capture applications is not new1 8. This idea, inspired by the observation that fast rates of enzyme-catalyzed CO2 hydration catalyzed by the Zn(His)s2+motifs comprising CA active sites, may be used to overcome the kinetic limitations of aqueous CO2 capture, has been explored extensively. To date, various groups have tested the utility of the isolated enzyme in driving CO2 conversion to aqueous carbonates (CO32‘), bicarbonates (HCCb ) and mineral carbonates when acting in the presence of alkali-earth cations1 3-5 7-9.SUMMARY OF THE INVENTION

[0006] The present invention provides for a photosynthetic cell comprising a nucleic acid encoding a heterologous carbonic anhydrase (CA) operatively linked to a promoter capable of expression in the photosynthetic cell.

[0007] The present invention provides for a nucleic acid encoding a heterologous carbonic anhydrase (CA) operatively linked to a promoter capable of expression in the photosynthetic cell. In some embodiments, the nucleic acid comprises a nucleotide sequence described herein.

[0008] The photosynthetic cell is any microbe capable of photosynthesis, or any autotrophic microbe. In some embodiments, the photosynthetic cell is a cyanobacteria cell. In some embodiments, the cyanobacteria cell is a cell from the class Cyanophyceae. In some embodiments, the cyanobacteria cell is a cell from the order Synechococcales. In some embodiments, the cyanobacteria cell is a cell from the family Merismopediaceae. In some embodiments, the cyanobacteria cell is a cell from the genus Synechocystis. In some embodiments, the cyanobacteria cell is a cell from the genus Synechococcus. In some embodiments, the cell from the genus Synechococcus is Synechococcus ambiguus, Synechococcus arcualus. Synechococcus bigranulatus, Synechococcus brunneolus, Synechococcus caldctrius. Synechococcus capilalus. Synechococcus carcerarius. Synechococcus elongatus, Synechococcus endogloeicus. Synechococcus epigloeicus. Synechococcus ferrunginosus. Synechococcus intermedins, Synechococcus koidzumii, Synechococcus lividus, Synechococcus marinus, Synechococcus minulissimus, Synechococcus mundulus, Synechococcus nidulans, Synechococcus rayssae, Synechococcus rhodobaklron, Synechococcus roseo-persicinus, Synechococcus roseo-purpureus, Synechococcus salinarum, Synechococcus salinus, Synechococcus sciophilus, Synechococcus sigmoideus, Synechococcus spongiarum, Synechococcus subsalsus, Synechococcus sulphuricus, Synechococcus vantieghemii, Synechococcus violaceus, Synechococcus viridissimus, Synechococcus vulcanus, Synechococcus sp. PCC 11901, Synechococcus sp. PCC 7002, Synechococcus elongatus PCC 7942, or Synechococcus elongatus UTEX 2973.

[0009] In some embodiments, the CA is a Sulfurohydrogenibium azorense CA or human CA, or any wild-type CA. In some embodiments, the CA has an amino acid sequence is any engineered or synthetic sequence having CA enzymatic activity, and having an amino acid sequence at least 70%, 80%, 90%, 95%, 99%, or 100% amino acid residue identity with any of wild-type CA, or CA polypeptide described herein, such as S. azo CA or S. azo ORF, and any conserved amino acid residues (conserved among the CA, such as those associated and / or essential for enzymatic activity or the active site).

[0010] In some embodiments, the nucleic acid is on a plasmid stably residing in the photosynthetic cell or is stably integrated unto a chromosome of the photosynthetic cell. In some embodiments, the promoter is a constitutive promoter or an inducible promoter. In some embodiments, the CA is expressed and accumulates or transports to a periplasm, membrane, cell wall, or a surface of the cell.

[0011] The present invention provides for a method for mineralizing carbon dioxide comprising: (a) culturing or growing a photosynthetic cell of the present invention in a medium under conditions such that the photosynthetic cell expresses the CA, (b) converting carbon dioxide into the H2CO3 through the enzymatic activity of the CA.

[0012] In some embodiments, the method further comprising (c) complexing the carbonate of the H2CO3 with a metal to form a metal carbonate. In some embodiments, the method further comprising (d) precipitating the metal carbonate from the medium. In some embodiments, the method further comprising (e) removing or separating the metal carbonate from the medium and / or the photosynthetic cell.

[0013] The present invention provides for a method for catalyzing mineralization of carbon dioxide or dissolution of a carbonate comprising: (a) culturing or growing a photosynthetic cell of claim 1 in a medium under conditions such that the photosynthetic cell expresses the CA, and (b) converting carbon dioxide into carbonic acid at acidic pH or bicarbonate and carbonate at neutral and alkaline pH via the enzymatic activity of the CA.

[0014] In some embodiments, the method further comprising (c) complexing soluble carbonates and bicarbonates of the with a metal to form a metal carbonate or metal bicarbonate. In some embodiments, the method further comprising (d) precipitating the metal carbonate or metal bicarbonate from the medium.

[0015] In some embodiments, the carbonate is a mineral carbonate. In some embodiments, the mineral carbonate is a calcium carbonate or magnesium carbonate, or mixture thereof. In some embodiments, the carbonate is a soluble carbonate, bicarbonate, carbonic acid, carbon dioxide, or mixture thereof.

[0016] The present invention provides for a method for catalyzing the hydration and / or dehydration of carbon dioxide comprising: (a) culturing or growing a photosynthetic cell of claim 1 in a medium under conditions such that the photosynthetic cell expresses the CA ineither the cell periplasm, membrane or wall or cell surface, and (b) converting carbon dioxide into carbonic acid at acidic pH or bicarbonate and carbonate at neutral and alkaline pH via the enzymatic activity of the CA.

[0017] The present invention provides for a method for mineralizing carbon dioxide comprising: (a) culturing or growing a photosynthetic cell of the present invention in a medium under conditions such that the photosynthetic cell expresses the CA, (b) converting carbon dioxide into the H2CO3 through the enzymatic activity of the CA, (c) optionally complexing the carbonate of the H2CO3 with a metal to form a metal carbonate, (d) optionally precipitating the metal carbonate from the medium, (e) optionally removing or separating the metal carbonate from the medium and / or the photosynthetic cell.

[0018] In some embodiments, the metal is a valence +2 metal, such as calcium or magnesium. In some embodiments, the metal carbonate is insoluble. In some embodiments, the medium is seawater or salt water.

[0019] The ability of carbonic anhydrase (CA) metalloenzymes to catalyze the otherwise slow hydration of CO2 into carbonic acid (H2CO3), suggests their utility in using seawater to drive the rapid mineralization of dissolved CO2 into limestones (Mg2+ / Ca2+carbonates). Complexation of CCh2' with the Ca2+and Mg2+ions found in ocean water results in the formation of limestone precipitates. The potential for rapidly converting dissolved CO2 into these insoluble metal carbonates, by accelerating the rate-limiting, CO2 hydration step, represents a promising mode of sequestering atmospheric CO2 via using seawater. A key bottleneck towards implementation of enzymatic CO2 capture involves the cost of sourcing carbonic anhydrase in sufficient quantities; realizing a megaton-scale CO2 capture facility would be cost prohibitive if CA were supplied exogenously. However, as a biosynthetic product, CA could be sourced renewably at steady-state by a photosynthetic bacterial expression system, engineered to produce the enzyme during the course of cellular photometabolism. This invention aims to address the problem of achieving economi cally- viable capture of carbon dioxide from marine waters using CA. The approach advanced here is a self-sustaining, biochemical system that uses an engineered cyanobacterium growing on sunlight and CO2 as a “factory” for renewably generating carbonic anhydrase by expressing it on the cell surface. This is achieved here by engineering cyanobacterial mutants through integration of chimeric carbonic anhydrase genes into the genomes of Synechococcus PCC 7002 cyanobacteria.

[0020] In some embodiments, the method comprises dilute CO2 capture involves coupling the fast rates of enzyme-catalyzed CO2 mineralization to a self-sustaining expression system for the enzyme itself. Zinc-dependent, carbonic anhydrase metalloenzymes, which catalyze the conversion of CO2 into carbonic acid (H2CO3), provide a promising route for accelerating the typically slow mineralization processes that transform CO2 into insoluble carbonates. For uncatalyzed reactions, the generation of insoluble metal carbonates from dissolved CO2 is impeded by its rate-limiting hydration to yield the initial H2CO3 (H++ HCO3 ) species. At pH > 8.5, this equilibrium yields a significant CCh2' fraction in the dissolved inorganic carbon (DIC) equilibrium; complexation of CCh2' with alkaline-earth metals, such as the Ca2+and Mg2+ions found at a cumulative concentration of ca. 62 mM in ocean water, results in the formation of limestone precipitates (Figure 2). While the peak solubility of CO2 in water is low (34 mM at 25 °C), this dissolved carbon dioxide (CO2 (aq)) remains in equilibrium with atmospheric carbon dioxide, CO2 (g). As a result, a solution-based process that can rapidly siphon off CO2 (aq) and ultimately sequester it in an insoluble form such as calcium and magnesium limestones, will ultimately force the continuous drawdown of CO2 from the gas phase (atmosphere) via Le Chatelier’s principle).

[0021] The present invention provides for a photosynthetic cell comprising a genomic or plasmid DNA modification that encodes a recombinant carbonic anhydrase and associated gene elements for achieving either induced or constitutive expression of the carbonic anhydrase (CA) in either the cell periplasm, membrane or wall or on the cell surface.

[0022] The present invention provides for a method for catalyzing the mineralization of carbon dioxide or catalyzing the dissolution of mineral carbonates, such as but not limited to, calcium carbonate and magnesium carbonate, to form soluble carbonate, bicarbonate, carbonic acid and carbon dioxide comprising: (a) culturing or growing a photosynthetic cell of the present invention under conditions such that the photosynthetic cell expresses the CA in either the cell periplasm, membrane or wall or cell surface, (b) converting carbon dioxide into carbonic acid at acidic pH or bicarbonate and carbonate at neutral and alkaline pH (pH 0- 14), through the enzymatic activity of the CA, (c) optionally complexing soluble carbonates and bicarbonates of the with a metal to form a metal carbonate or metal bicarbonate, and (d) optionally precipitating the metal carbonate or metal bicarbonate from the medium

[0023] The present invention provides for a method for catalyzing the hydration and / or dehydration of carbon dioxide comprising: (a) culturing or growing a photosynthetic cell ofthe present invention under conditions such that the photosynthetic cell expresses the CA in either the cell periplasm, membrane or wall or cell surface, (b) converting carbon dioxide into carbonic acid at acidic pH or bicarbonate and carbonate at neutral or alkaline pH, through the enzymatic activity of the CA, and / or (c) converting carbonic acid or soluble carbonates into carbon dioxide.

[0024] A key bottleneck towards implementation of enzymatic CO2 capture involves the cost of sourcing the enzyme in sufficient quantities; realizing a megaton-scale CO2 capture facility would be cost prohibitive if CA were supplied exogenously. A possible way around this problem involves generating CA continuously in bacteria.

[0025] However, non-photosynthetic bacterial growth has its own associated cost demands, including the significant quantities of exogenous nutrients that would be necessary to support a sufficiently dense bacterial population for generating CA at volumes relevant to CO2 capture at a megaton-plus scale. In some embodiments, the method comprises solving a nutrient supply constraint through the use of a bacterial expression system based on a photosynthetic, marine cyanobacterium. As a biosynthetic product, CA could be sourced renewably at steady-state by a photosynthetic bacterial expression system, engineered to produce the enzyme during the course of cellular growth and photometabolism. Such biochemical modifications are readily achieved using established molecular biology / genetic engineering tools for protein overexpression. In this use case, the inorganic carbon and nutrients required for sustaining the bacterial population naturally exist in seawater at the necessary concentrations, as evidenced by the long-documented problem of dense algal / cyanobacterial ocean blooms persisting in natural aquatic environments. Similarly, the energy driving cell photosynthesis and CA biosynthesis is supplied using solar illumination as an energy source, making it unnecessary to support the bacterial population through externally-supplied, electron-donor compounds, as required by nonphotosynthetic bacteria. In addition, Zn2+exists at a mean concentration of 200 nM in seawater, a large excess relative to what is needed for stable saturation of CA zinc active sites (kd = 10-12 - 10-9 M)

[0026] Key milestones:1. Design of chimeric gene constructs for inducing mutations in the genomes of Synechococcus PCC 7002 cyanobacteria that will result in cell surface expression of carbonic anhydrase (CA) enzymes.2. Gene construction3. Construction and confirmation of Syn PCC 7002 mutants with chimeric CA genes4. Testing of Syn PCC 7002 mutants for enhanced mineralization5. Descriptive model for simulating CA-catalyzed mineralization kinetics6. Design of an efficient reactor system for maintaining Syn PCC 7002 populations under mineralization conditions

[0027] The present invention has one or more of the following advantages: The system is self-sustaining and self-replicating, by virtue of its biological design. Since the mineralization catalyst is produced continuously by cells that do not require feeding (cyanobacteria are photosynthetic), and can utilize CO2 itself and sunlight as carbon and energy sources respectively, this will carry the potential for significantly reducing capital expenses of dilute CO2 capture from the atmosphere. Most CO2 capture technologies at the moment are pointcapture systems; they are not useful for collecting CO2 from the air, only sources such as plant exhaust where CO2 concentration is high. The invention would be able to function in either modality. Principal energetic inputs into a system using these genetically engineered bacterial would be the energy required to mix air to sustain a target threshold of dissolved CO2, a requirement for the mineralization catalysis. Calculations suggest thresholds would require between 100-200 micromolar CO2 to be maintained in the mineralization medium at steady state, in order for achieving megaton-quantities of CO2 capture for a realistic plant size. Given that the equilibrium concentration of CO2 in water is approximately 15 uM, this implies an energy cost associated with mixing, for this scheme to work. The necessary regeneration of the metal ions and base consumed in the reaction will also represent the other key energy cost required for this process.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

[0029] Figure 1. (A) Aqueous carbonate equilibrium speciation. For pH > 8, hydration kinetics are dominated by OH' attack on CO2 , rather than H2O. At pH > 8.5, appreciableprecipitation of solid carbonates may be observed, with the extent of {Mg / Ca}C03(s) generated at moderately alkaline pH significantly influenced by the buffering capacity of the medium (buffer not shown). (B) PSW068 parent plasmid used for integration of constructs 1- 9 into the Fad gene locus of WT Synechococcus PCC 7002. (C) Structures of S. azorense carbonic anhydrase (blue, PDBID 4X5 S) and human carbonic anhydrase II (gray, PDBID 6DH2). Positions of the Zinc active sites (red spheres) with respect to potential anchoring at either N or C termini (shown in turquoise) were used to determine anchoring considerations for CA fusions to ice nucleation protein, antigen 43, somA and putative S-layer protein surface anchor domains.

[0030] Figure 2. Colony PCR of S. PCC 7002 genomic mutants. Lanes: L = Ladder; 1 = PCC 7002-C1; 2 = PCC 7002-C2; 3 = PCC 7002C3; 4 = PCC 7002-C4; 5 = PCC 7002-C5; 6 = PCC 7002-C6; 7 = PCC 7002-C7; 8 = HCAAg43; 9 = sAzoCA-Ag43; 10 = PSW068-C1 vector (+) control; 11 = PCC 7002-WT (-) control. All mutant genes were amplified using the same PSW068-UP-LAC / PSW068DOWN-T7TERM primer set, which initiates amplification from common lac operator (5’) and T7term (3’) hybridization sites, adding 287 bp to each gene.

[0031] Figure 3. CO2 mineralization in ambient air. Samples include 1 ml of cells (O.D. 730 = 1.3) added to 9 ml of pH 9.0 100 mM Tris and 50 mM CaC12 . A no cell control is amended with an additional 1 ml of cell suspension buffer (pH 7.1 20 mM MOPS + 300 mM NaCl. Flasks are stirred at 400 rpm for 1 hour. Mutants C4 and C8 are shown to be top performers at early times. Within two hours, mineralization has approached equilibrium in all flasks, resulting in a plateau in all treatments

[0032] Figure 4. (a) Calculations of steady-state mineralization, (b) competitive metal hydroxide formation and (c) enzyme reaction rates under buffered conditions as functions of pH and [CO2(aq)]. (d, e, f) Contours of carbonic acid (H2 CO3 ), bicarbonate (HCO3 ) and soluble carbonate (CO32‘) accumulation. Calculations assume an idealized system at constant pH and CO2 concentration (9.0 and 10-20 pM, respectively), operating over a 1.04 million liter reactor volume, representing an upper bound on physically accessible conversion rates. For reference, 0.1 MT on CO2 yr-1 of CO2 corresponds to 6.2xl06mol day'1.

[0033] Figure 5. (a) Calculated effects of carbonic anhydrase CO2 binding constants (km) and catalytic rate constants (kcat) for CO2 hydration, on steady-state CO2 mineralization.Calculations assume an idealized system at constant pH and CO2 concentration (9.0 and 15 pM, respectively), operating over a 1.04 million liter reactor volume, representing an upper bound on physically accessible conversion rates. For reference, 1 MTon CO2 yr’1of CO2 corresponds to 6.2 x 107mol day’1, (b) Relationship between enzyme efficiency (kcat / km) and mineralization.

[0034] Figure 6. Constructs 1-9 (C1-C9).DETAILED DESCRIPTION OF THE INVENTION

[0035] Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0036] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0038] As used in the specification and the appended claims, the singular forms “a”, “an”,and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a “polysaccharide” includes a single polysaccharide molecule, and a plurality of polysaccharide molecules having the same, or similar, chemical formula, chemical and / or physical properties.

[0039] The terms “optional” or “optionally” as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0040] These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.

[0041] The constructs used are shown as follows:

[0042] Construct 1, Antigen 43 Fusion: NcoI-PCC 6301 / 7942 / 7002 somA signal peptide-6X- HisTag (N-term)- S. azo ORF-(GGGGS)3 Linker- Recomb. Ag43 beta chain- PCC 7002 STOP codon (67%)- BsrGI.

[0043] Construct 2, S-layer Fusion (7002): Ncol- S-Layer (B1XN72_SYNP2)-(GGGGS)3 Linker-S. azo ORF-6X-HisTag (C-term)- PCC 7002 STOP codon (67%)- BsrGI.

[0044] Construct 3, S-layer Fusion (7002): Ncol- 7002 / 11901 S-layer signal peptide-6X- HisTag (N-term)- S. azo ORF-(GGGGS)3 Linker- S-Layer (B1XN72_SYNP2)- PCC 7002 STOP codon (67%)- BsrGI.

[0045] Construct 4, INP Fusion: Ncol- PCC 6301 / 7942 / 7002 somA signal peptide- Ice Nucleation Protein (inaK-NC)- (GGGGS)3 Linker- S. azo ORF-6X-HisTag (N-term)- PCC 7002 STOP codon (67%)- BsrGI.

[0046] Construct 5, SomA Fusion: NcoI-PCC 6301 / 7942 / 7002 somA signal peptide-6X-HisTag (N-term)- S. azo ORF-(GGGGS)3 Linker-SomA- PCC 7002 STOP codon (67%)-BsrGI.

[0047] Construct 6, SomA Fusion: Ncol- SomA (+signal peptide)- (GGGGS)3 Linker-S. azo ORF-6X-HisTag (N-term)- PCC 7002 STOP codon (67%)- BsrGI.

[0048] Construct 7, Native Human CA IX: Ncol- 7002 / 11901 S-layer signal peptide- 6X- HisTag (N-term)- HCA IX enzyme domain (38-459)- PCC 7002 STOP codon (67%)-BsrGI.

[0049] Construct 8: Ncol- E. coli signal Ag43 signal peptide- 6X-HisTag (N-term)-HCA II- (GGGGS)3 Linker- Recomb. Ag43 [truncated] alpha chain- Recomb. Ag43 beta chain- PCC 7002 STOP codon (67%)- BsrGI.

[0050] Construct 9: Ncol- E. coli signal Ag43 signal peptide- 6X-HisTag (N-term)-S. azo ORF-(GGGGS)3 Linker- Recomb. Ag43 [truncated] alpha chain- Recomb. Ag43 beta chain- PCC 7002 STOP codon (67%)- BsrGI.

[0051] Construct 10: Ncol- PCC 6301 / 7942 / 7002 somA signal peptide- 6X-HisTag (N- term)-S. azo ORF-(GGGGS)3 Linker- Recomb. Ag43 alpha chain- Recomb. Ag43 beta chain- PCC 7002 STOP codon (67%)- BsrGI.

[0052] Construct 11 : Ncol- E. coli signal Ag43 signal peptide- 6X-HisTag (N-term)-S. azo ORF-(GGGGS)3 Linker- Recomb. Ag43 alpha chain- Recomb. Ag43 beta chain- PCC 7002 STOP codon (67%)- BsrGI.

[0053] The nucleotide sequence of the Ncol restriction site (“Ncol”) is: CCA TGG.

[0054] The nucleotide sequence of the BsrGI restriction site (“BsrGI”) is: TGT ACA.

[0055] The nucleotide sequence of “PCC 6301 / 7942 / 7002 somA signal peptide” is: ATG AAG CGC CTC TTT AGC GCC TTG TTA TTG GCT CCA GCT ATC GCG GGT GTA GCT GCG GGG GCT GCA AAT GCT (SEQ ID NO: 1).

[0056] The nucleotide sequence of “6X-HisTag (N-term)” and “6X-HisTag (C-term)” are: CAC CAT CAC CAC (SEQ ID NO:2).

[0057] The nucleotide sequence of “S. azo ORF” is: GGG AGT CAC ATG GCA GAA GTT CAC CAT TGG AGT TAT GAA GGT GAA AAT GGC CCT GAG AAC TGG GCT AAA CTC AAC CCT GAA TAC TTT TGG TGT AAT CTG AAA AAT C AA TCG CCA GTAGAC ATC TCG GAT AAC TAT AAG GTG CAC GCA AAA TTA GAA AAG TTA CAC ATT AAT TAT AAC AAA GCA GTA AAT CCT GAG ATC GTT AAT AAC GGA CAT ACC ATC CAA GTG AAT GTC CTG GAG GAT TTT AAA CTG AAC ATT AAG GGA AAG GAG TAT CAC TTA AAA CAG TTC CAT TTC CAC GCT CCT AGT GAA CAC ACA GTT AAC GGG AAA TAC TAT CCT CTG GAA ATG CAT TTA GTG CAC AAG GAC AAG GAC GGT AAC ATC GCA GTG ATC GGC GTC TTT TTT AAG GAA GGC AAA GCT AAC CCG GAA TTG GAT AAG GTC TTC AAG AAC GCT CTG AAA GAG GAG GGG TCT AAG GTC TTC GAC GGC TCT ATC AAC ATT AAC GCC CTG CTG CCA CCG GTT AAA AAT TAC TAT ACT TAC AGT GGA AGT TTG ACC ACG CCC CCA TGC ACT GAA GGT GTT TTA TGG ATC GTT CTG AAG CAG CCC ATT ACG GCC TCG AAG CAA CAA ATC GAA CTG TTC AAA AGT ATT ATG AAG CAT AAC AAT AAT CGG CCT ACG CAA CCG ATT AAC AGT CGG TAC ATT TTG GAA AGC AAC (SEQ ID N0:3).

[0058] The nucleotide sequence of “(GGGGS)3 Linker” is: GGA GGT GGA GGA TCT GGT GGT GGG GGT TCG GGA GGA GGA GGA TCC (SEQ ID NO:4).

[0059] The nucleotide sequence of “Recomb. Ag43 beta chain” is: CCA ACG AAT GTA ACG TTG GCG TCG GGC GCA ACT TGG AAT ATC CCT GAT AAT GCG ACA GTG CAA TCG GTC GTC GAT GAT TTG TCC CAC GCA GGC CAA ATC CAT TTC ACC TCT ACA CGC ACG GGA AAG TTT GTG CCT GCA ACG CTC AAA GTG AAA AAC CTC AAC GGA CAG AAC GGA ACG ATC AGT CTC CGT GTC CGT CCT GAC ATG GCG CAG AAC AAT GCA GAC CGC TTG GTT ATT GAC GGG GGA CGC GCA ACC GGT AAA ACA ATC CTG AAC CTC GTT AAT GCG GGC AAC TCG GCC TCC GGC CTC GCT ACT TCT GGT AAA GGG ATC CAG GTT GTC GAG GCC ATC AAC GGA GCC ACT ACA GAG GAG GGA GCC TTC GTT CAG GGA AAC CGG CTG CAG GCG GGC GCT TTT AAT TAC TCC TTA AAT CGT GAT AGT GAT GAA TCC TGG TAC TTG CGC AGC GAA AAT GCT TAT CGC GCT GAG GTG CCG CTC TAT GCC AGT ATG TTA ACT CAA GCG ATG GAT TAC GAC CGG ATC GTC GCT GGA AGC CGC TCC CAC CAA ACG GGC GTG AAT GGT GAA AAT AAC TCT GTG CGG TTG TCT ATT CAA GGC GGT CAT CTC GGC CAT GAC AAT AAT GGA GGA ATT GCT CGT GGT GCA ACA CCT GAG TCG TCG GGG TCT TAT GGC TTT GTA CGT TTA GAA GGA GAT CTC ATG CGG ACC GAA GTC GCC GGT ATG TCT GTA ACC GCG GGC GTT TAC GGT GCC GCG GGG CAT TCC AGT GTC GAC GTT AAG GAT GAC GACGGG AGT CGT GCA GGA ACA GTG CGC GAC GAC GCG GGT TCC TTG GGA GGT TAC CTC AAC CTC GTT CAT ACA TCC TCC GGG CTG TGG GCT GAC ATT GTG GCT CAG GGC ACT CGC CAT AGT ATG AAG GCC TCT TCT GAT AAT AAT GAC TTT CGC GCT CGT GGG TGG GGT TGG TTG GGG AGC CTC GAA ACC GGT CTG CCG TTT TCT ATC ACG GAT AAT CTC ATG TTA GAG CCG CAA CTC CAA TAC ACT TGG CAG GGG CTC TCT CTG GAC GAT GGC AAA GAC AAT GCA GGG TAC GTT AAA TTT GGA CAT GGG TCC GCC CAG CAT GTA CGT GCC GGA TTC CGC CTG GGA TCT CAC AAT GAC ATG ACT TTC GGA GAA GGC ACC TCT AGT CGC GCC CCA CTG CGT GAC AGT GCC AAA CAT TCT GTT TCT GAG TTA CCA GTT AAT TGG TGG GTG CAA CCG TCC GTA ATC CGT ACC TTC AGC AGT CGC GGC GAT ATG CGG GTG GGC ACA AGC ACG GCT GGG AGC GGG ATG ACA TTC TCG CCC AGC CAA AAC GGA ACA TCG TTA GAC TTA CAA GCC GGC TTG GAA GCT CGG GTT CGC GAA AAT ATC ACG CTC GGG GTC CAG GCT GGA TAT GCT CAT TCT GTG TCT GGC AGT AGT GCG GAG GGA TAC AAT GGT CAG GCG ACG TTG AAT GTA ACC TTC (SEQ ID N0:5).

[0060] The nucleotide sequence of “PCC 7002 STOP codon (67%)” is: TAA.

[0061] The nucleotide sequence of “S-Layer (B1XN72 SYNP2)” is: CAA TCT GGA GGC TCT GAA GTA ACA CCG GAA GAC CTG GCG GCG TTA CGG CGC CTG ATC AAT GAG TTC GAG GCC GAG CTG GCA ACG TTA GGA GCC CGT GTA GAC GAC TTG GAG GGA CGT GTT GAA TTT CTC GAG GAC AAC CAG TTC TCG ACT ACT ACC AAA TTA AAA GGC GAG GTA GTT TTC GCT ATT GGA GAT TCG TTC GGA GAC GTT AAC GCT CTC GAC TTT CGC ATT GCT GAG GAT ACT ACT ATC GAT GAT GTG GTG TTT ACC CCC GAT TTT TTA CGG GAC GGA GTT GCG TCT GGA TTC GGC GGC GCC ACG ATT GTC GCT AAC GAC GAT GAC GAT ACG CAG ACG ACA TTT ACT GAT CGT GTC CGG CTG ACG TTG GAG ACT TCT TTT ACT GGA AAG GAC ACG CTG ATC ACA CGC TTG ACT GCG GGT AAT AGT TCT TCC TTT TCT CCT ACC GCT GGA GCG GTG GTT GAC GAT GGA CCC GAT TTA GGA AGC GGC GTG GGG TTT CAA ACC TTC AAT TTA GAT ACT GGT AAC GAT GTT GTT ATT GAT TGG TTA GCG TAC TAC TTT CCA TTA CGC CGC GCA AAT GTA TAC CTG GCA GCC TTC GGC GGA TTG CAC TCG GAT TAC ACT TCG ACT ACT TTT GGG GCC ACA GGC TTA GAG GAT TTT ACC GGT GGT TCC GGC TCT CTC ACA CAA CTG GCA GCA AGT TCG CCT ATT TAC ACT ATT GGT GGT GGT AGC GGT CTCGGG TTT AAC ATG CCT TTA GGA CCA GTA GAC ATC AGC CTC GGC TAT TTA GCA GAT GAA GCT GCT GAT TCT ACT GAG AGC AAT GGA CTG TTC AAC GGA GAA TAT GGT GCG TTG GCT CAG ATC GCA TTT AAT TTG GGA GAC CGG GCG GAG TTG GGG GTA ACC TAC GTT AAC AGC TAT CAC GAT AGC GGC GCC ATT TAC GAC TTT GGC GGG GGC TCT GCC GTA AAC GGA ACT GCC TGG GCA AAT GCA CTC GGC TTG TTT GGA ACA GAG GCT AAC TCT TAT GGG GTC CAA GGA AAG TTC GAC ATT ACC GAC CGG ATC TCT CTG GCC GCT TAC GGA ATG TAT ACG GAC GCG AAA GTA TCG GGA TCT TCG GAC GAA TTC GAT ATC TGG AGC TAT GGA CTG GGT GTT GCT TTT AAT GAC TTG GGG AAG GAG GGT AAC GTT CTG GGT TTA TTC GCA GGA GCC CCC CCA TAT CTC GCC GAA GGA GAT CTC AAG ACT CCC TTA CAA GTG GAG GGG TTC TAT AAG TAC CAA CTC ACA GAC GGC ATC AGT ATC ACT CCA GGG GTC ATC TGG TTG AAA GAC GCC GCC CAG GGC GTT TTA GGT GAA GAG GAT GCT ATC ATT GGC ACT CTC CGG ACA ACA TTT ACG TTC (SEQ ID N0:6).

[0062] The nucleotide sequence of “7002 / 11901 S-layer signal peptide” is: ATGAAAACATCCTTGTCGCTCTGGAAAAGCTTATCTATCGCTAGCGCAGCAGTGG GTGTCTCTGTTGCGACCGCGGGCACGGCACAAGCC (SEQ ID NO:7).

[0063] The nucleotide sequence of “SomA” is:CCGCTCAGCCGGTACGAATTCGCGGCAGGGCTGAATGCTTGCTTGGATAAGGTG ATCGAGTTCGCGGCTAGTAAAGAGGACCTGGATACGTTGAAGCGTCTCACAGAA GAATTTCAAGCGGAGTTAGCCACTCTGCGTGGACGGGTAGACAGCTTAGAAGCC CGGGTGAAAGAACTCGAAGCGACACGGTTTAGTACAACCACGAAGCTCCAGGGG GAGGTTATCTTCTCCCTCGATGCTGTAGCCAACACTGCAGGGAACGAGCGGAAC CAGGACGGAGCGGTATCGTTCGGGAATCGTGTCTCCTTAAACCTGAACACGAGC TTTACAGGGAAAGACCTGCTCTTGACCCGTCTGCGGGCCCGGAACATCGAAACA ATCCAACAGCGTCTCTCGCCTGGGTTTAACCCGTCGGGATCGCGCTTGGACTACG ATGGGACCGGTTCCCCGGGGGTACCAAACTCTGCCAATACATTCTTCCTCGATAA GTTGTTATACCGGTTTCCGGTCGGAGATGTATCTTTTACAGTAGGTACTGCGGGA GTCCAGCCGCAAGATTACGGATTAAGTGATGCGACCTTTTTCAGTGGGCCGGCAA ATACCAAGGCCTTCAAGTATGTAGGCGCGGGAGTCTACGCCGATACACGTGATG CGGATACTGCAGGGGTCGGTTTTAATTGGAAGGCAAGCAAAAACTTCAGCTTTC AAGCCGGCTACATCAATCGTAATAGTGCTGATGTCTCGACCGTGAACTCCGGCGGCGTCTTTGGTTTCACACCCACCGGAACGGGAACTAACTCCTGGGACGTAAACGCGCAAGTGAAATATCAAACCGACAACAATAAATTCCGTGTTGCATTGGCGTATGCA CTGCGGAAT (SEQ ID NO:8).

[0064] The nucleotide sequence of “SomA (+signal peptide)” is: CTG CAA TCC TTG GTT GAA CGC TAT GGT TGT ATC GTG GGC TAC CCG GAT CGC ACT TAT CGC GGA TCT CGC CCG CTC AGC CGG TAC GAA TTC GCG GCA GGG CTG AAT GCT TGC TTG GAT AAG GTG ATC GAG TTC GCG GCT AGT AAA GAG GAC CTG GAT ACG TTG AAG CGT CTC ACA GAA GAA TTT CAA GCG GAG TTA GCC ACT CTG CGT GGA CGG GTA GAC AGC TTA GAA GCC CGG GTG AAA GAA CTC GAA GCG ACA CGG TTT AGT ACA ACC ACG AAG CTC CAG GGG GAG GTT ATC TTC TCC CTC GAT GCT GTA GCC AAC ACT GCA GGG AAC GAG CGG AAC CAG GAC GGA GCG GTA TCG TTC GGG AAT CGT GTC TCC TTA AAC CTG AAC ACG AGC TTT ACA GGG AAA GAC CTG CTC TTG ACC CGT CTG CGG GCC CGG AAC ATC GAA ACA ATC CAA CAG CGT CTC TCG CCT GGG TTT AAC CCG TCG GGA TCG CGC TTG GAC TAC GAT GGG ACC GGT TCC CCG GGG GTA CCA AAC TCT GCC AAT ACA TTC TTC CTC GAT AAG TTG TTA TAC CGG TTT CCG GTC GGA GAT GTA TCT TTT ACA GTA GGT ACT GCG GGA GTC CAG CCG CAA GAT TAC GGA TTA AGT GAT GCG ACC TTT TTC AGT GGG CCG GCA AAT ACC AAG GCC TTC AAG TAT GTA GGC GCG GGA GTC TAC GCC GAT ACA CGT GAT GCG GAT ACT GCA GGG GTC GGT TTT AAT TGG AAG GCA AGC AAA AAC TTC AGC TTT CAA GCC GGC TAC ATC AAT CGT AAT AGT GCT GAT GTC TCG ACC GTG AAC TCC GGC GGC GTC TTT GGT TTC ACA CCC ACC GGA ACG GGA ACT AAC TCC TGG GAC GTA AAC GCG CAA GTG AAA TAT CAA ACC GAC AAC AAT AAA TTC CGT GTT GCA TTG GCG TAT GCA CTG CGG AAT (SEQ ID NO: 9).

[0065] The nucleotide sequence of “HCA IX enzyme domain (38-459)” is:CGGACGCCCCGGGGGTCTGGCGGTCTTAGCGGCCTTCTTGGAAGAGGGACCTGA GGAGAATTCTGCGTACGAACAGTTATTGTCTCGGTTGGAAGAGATCGCGGAAGA GGGGTCGGAAACCCAAGTCCCTGGCTTAGACATCTCGGCTTTGCTCCCATCTGAT TTTAGCCGGTACTTCCAGTATGAAGGAAGCCTCACCACTCCACCCTGCGCCCAGG GTGTAATTTGGACAGTTTTCAATCAAACGGTGATGTTGTCCGCCAAACAGCTGCA CACACTCTCTGACACACTGTGGGGACCAGGAGACTCGCGTTTGCAATTGAACTTT CGCGCTACACAACCGTTGAACGGTCGGGTGATCGAAGCGTCGTTTCCTGCCGGTGTTGACTCCTCTCCTCGTGCTGCTGAGCCTGTCCAGTTAAATTCCTGTTTAGCCGCT GGAGACATCCTCGCGTTAGTGTTTGGATTGCTGTTCGCGGTGACCTCTGTGGCAT TTCTCGTGCAAATGCGTCGCCAACATCGCCGTGGAACGAAAGGTGGCGTCAGCT ATCGTCCAGCTGAGGTTGCAGAAACAGGCGCA (SEQ ID NO: 10).

[0066] The nucleotide sequence of “E. coli signal Ag43 signal peptide” is: ATG AAG CGT CAC CTC AAC ACG TGT TAC CGC CTG GTA TGG AAT CAC ATG ACG GGC GCT TTT GTT GTG GCA AGC GAA TTA GCT CGC GCT CGT GGA AAA CGT GGT GGA GTC GCC GTG GCC CTC TCG TTA GCT GCT GTT ACG TCT TTA CCC GTA CTG GCC (SEQ ID NO: 11).

[0067] The nucleotide sequence of “HCA II” is: CGG ATT CTG AAT AAT GGT CAC GCT TTT AAT GTA GAA TTT GAC GAT TCC C AA GAT AAA GCC GTT TTG AAA GGC GGT CCC CTG GAC GGT ACT TAT CGG CTC ATC CAA TTC CAT TTT CAC TGG GGG AGC TTG GAC GGA CAA GGA AGC GAA CAT ACC GTC GAT AAG AAA AAG TAT GCT GCT GAG TTA CAT CTC GTA CAC TGG AAC ACG AAA TAC GGT GAC TTC GGG AAA GCC GTA CAA CAA CCC GAT GGC TTG GCA GTC CTG GGC ATC TTT CTG AAA GTC GGG AGC GCA AAG CCG GGG TTG CAG AAA GTC GTT GAT GTG CTC GAT TCG ATT AAA ACA AAA GGT AAA TCT GCT GAC TTT ACG AAC TTT GAT CCC CGG GGT CTC TTG CCG GAG TCT CTG GAT TAC TGG ACC TAC CCT GGA TCG CTC ACG ACC CCG CCA CTG TTG GAG TGT GTA ACC TGG ATT GTC CTG AAA GAG CCC ATT AGT GTA TCG TCC GAA CAG GTT CTG AAA TTC CGG AAG CTC AAC TTT AAT GGT GAA GGC GAA CCA GAA GAA CTC ATG GTT GAT AAC TGG CGT CCG GCC CAG CCG TTA AAA AAT CGC CAG ATC AAA GCC TCG TTC AAG (SEQ ID NO: 12).

[0068] The nucleotide sequence of “Recomb. Ag43 [truncated] alpha chain” is: GGGGCCTTCAGCGTGGTCGAGGGAAAAGCTGACAATGTTGTACTGGAGAACGGA GGCCGCCTCGATGTTTTAACGGGTCATACAGCGACCAATACGCGGGTCGACGAT GGCGGGACGTTAGACGTTCGGAATGGTGGTACGGCGACTACTGTCAGTATGGGG AACGGTGGTGTGTTACTGGCAGACAGCGGCGCGGCGGTTTCCGGCACGCGGTCC GATGGCAAAGCTTTTTCCATTGGCGGAGGCCAAGCAGACGCCCTGATGCTGGAA AAGGGCAGCTCTTTTACACTCAATGCAGGAGACACCGCGACCGACACCACTGTT AATGGCGGACTCTTCACAGCTCGTGGTGGGACGTTGGCAGGGACTACTACGCTG AACAACGGCGCGATTCTCACTCTGAGCGGAAAGACCGTAAACAATGATACTCTGACGATCCGGGAGGGAGATGCTCTGCTCCAAGGAGGAAGCCTGACTGGTAATGGA TCCGTTGAGAAGAGCGGATCCGGAACACTCACAGTTTCGAACACAACACTCACT CAAAAGGCAGTAAACTTAAACGAAGGGACGTTGACCCTGAATGATAGCACTGTG ACCACTGATGTTATTGCCCAACGGGGAACGGCGCTCAAACTGACGGGATCCACG GTGCTGAACGGAGCCATTGAT (SEQ ID NO: 13).

[0069] The nucleotide sequence of “Recomb. Ag43 alpha chain” is: GGG GGA GTA GCG GGG AAC ACC ACT GTT AAT CAG AAA GGA CGT TTG CAA GTC GAT GCT GGA GGG ACG GCA ACC AAT GTG AC A TTA AAA CAG GGG GGC GCA CTG GTC ACA TCT ACA GCG GCC ACC GTA ACC GGA ATC AAT CGC CTG GGT GCG TTT TCG GTA GTA GAG GGT AAG GCA GAT AAC GTA GTC TTG GAG AAT GGA GGC CGG TTG GAC GTT CTG ACC GGT CAT ACT GCC ACA AAT ACC CGT GTG GAC GAT GGA GGA ACG CTC GAC GTG CGT AAC GGG GGA ACC GCT ACT ACT GTT AGT ATG GGA AAT GGG GGG GTT TTA TTG GCC GAT AGT GGA GCT GCC GTA TCT GGG ACG CGT AGC GAC GGC AAG GCC TTC TCG ATC GGC GGC GGC CAA GCT GAT GCC CTC ATG TTA GAG AAA GGT AGT TCG TTT ACC CTC AAC GCC GGT GAT ACG GCC ACC GAC ACC ACT GTA AAT GGC GGA CTG TTC ACT GCC CGT GGT GGG ACT TTG GCC GGC ACA ACT ACA TTG AAT AAC GGA GCT ATT TTA ACT TTG TCG GGA AAG ACA GTT AAT AAC GAT ACT TTA ACC ATC CGT GAA GGA GAT GCA CTG TTG CAG GGT GGC TCT TTG ACG GGG AAC GGA AGT GTC GAA AAA TCG GGC TCT GGT ACA TTG ACA GTC TCT AAC ACG ACA CTG ACC CAG AAG GCA GTT AAC CTG AAC GAG GGC ACA CTC ACT CTG AAC GAT TCT ACC GTG ACA ACA GAC GTT ATC GCG CAA CGG GGG ACA GCC CTG AAG CTG ACA GGC AGT ACA GTG TTG AAC GGG GCG ATC GAT (SEQ ID NO: 14).

[0070] References cited herein:(1)Miijafari, P.; Asghari, K.; Mahinpey, N. Investigating the Application of Enzyme Carbonic Anhydrase for CO2 Sequestration Purposes. Ind. Eng. Chem. Res. 2007, 46 (3), 921-926.(2)Yadav, R. R.; Krishnamurthi, K.; Mudliar, S. N.; Devi, S. S.; Naoghare, P. K.; Bafana, A.; Chakrabarti, T. Carbonic Anhydrase Mediated Carbon Dioxide Sequestration: Promises, Challenges and Future Prospects. Journal of Basic Microbiology 2014, 54 (6), 472-481.(3)Bhagat, C.; Dudhagara, P.; Tank, S. Trends, Application and Future Prospectives ofMicrobial Carbonic Anhydrase Mediated Carbonation Process for CCUS. Journal of Applied Microbiology 2018, 124 (2), 316-335.(4)Power, I. M.; Harrison, A. L.; Dippie, G. M.; Southam, G. Carbon Sequestration via Carbonic Anhydrase Facilitated Magnesium Carbonate Precipitation. International Journal of Greenhouse Gas Control 2013, 16, 145-155.(5) Jo, B. H.; Kim, I. G.; Seo, J. H.; Kang, D. G.; Cha, H. J. Engineered Escherichia Coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration. Appl. Environ. Microbiol. 2013, 79 (21), 6697-6705.(6) da Costa Ores, J.; Sala, L.; Cerveira, G. P.; Kalil, S. J. Purification of Carbonic Anhydrase from Bovine Erythrocytes and Its Application in the Enzymic Capture of Carbon Dioxide. Chemosphere 2012, 88 (2), 255-259.(7)Sundaram, S.; Thakur, I. S. Induction of Calcite Precipitation through Heightened Production of Extracellular Carbonic Anhydrase by CO2 Sequestering Bacteria. Bioresource Technology 2018, 253, 368-371.(8)Del Prete, S.; Perfetto, R.; Rossi, M.; Alasmary, F. A. S.; Osman, S. M.; AlOthman, Z.; Supuran, C. T.; Capasso, C. A One-Step Procedure for Immobilising the Thermostable Carbonic Anhydrase (SspCA) on the Surface Membrane of Escherichia Coli. J Enzyme Inhib Med Chem 2017, 32 (1), 1120-1128.(9) da Costa Ores, J.; Sala, L.; Cerveira, G. P.; Kalil, S. J. 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[0071] It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0072] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.

[0073] The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.EXAMPLE 1Engineering cyanobacterial carbonic anhydrase surface mutations for fast CO2 hydration and capture

[0074] Aqueous carbonate speciation is well understood to provide routes for capturing gaseous CO2 as insoluble minerals in the presence of alkaline-earth metals such as calcium and magnesium at alkaline pH. However, equilibration between CO2 and the other forms of dissolved inorganic carbon (DIC) is slow under ambient conditions, bottlenecked by the hydration of dissolved CO2 to form bicarbonate and a proton. This step is rate-limiting for carbonate mineralization under ambient temperature and pressure, and is precisely the chemistry catalyzed by the family of carbonic anhydrase enzymes. As a result, incorporation of carbonic anhydrases (CAs) into CO2 mineralization schemes can be expected to accelerate the mineralization process. This has been well-studied, with extension of these basic chemical principles to real applications being hampered by practical questions of how to economically source carbonic anhydrase enzymes at mass scale. This work explores the use of cyanobacterial expression hosts as a potential resolution to that problem. In particular, engineering the surface display of carbonic anhydrases in fast-growing, marine cyanobacterial would yield a self-sustaining mineralization catalyst, requiring only nutrients freely available in seawater, sunlight and CO2 itself, as inputs for passively generating carbonic anydrases on the cell envelope. Each of these inputs are freely available, suggesting the potential of cyanobacterial CA surface display mutants in easing the economic constraintsof scaling carbonic anhydrases for CO2 capture applications.

[0075] Under ambient conditions, aqueous CO2 mineralization proceeds in as a process ratelimited by this hydration step, with associated pseudo-first order rate constants of 0.03 s'1around pH 7 and 0.15 s'1at pH > 8 for the uncatalyzed reaction (Figure 1). This runs in stark comparison with the famously quick hydration kinetics for members of the CA family, with rate constants of catalysis ranging from 105M-1s to as high as 4.4 x 106s'1for Sulfurohydrogenibium azorense carbonic anhydrasel 0' . In the case of S. azorense carbonic anhydrase, this rate constant results in a rate enhancement as high as 200 fold, relative to the uncatalyzed case, for an enzyme concentration of 10 nM, operating over a CO2 substrate concentration of 15 pM, the level of CO2 dissolved in water under quiescent conditions as given by Henry’s law.

[0076] These favorable kinetics offer the promise of a meaningful role for CAs in future CO2 remediation efforts. This is a future that has yet to be realized, however, due, in large part, to the challenges of economically sourcing carbonic anhydrases at scales relevant to large-scale CO2 capture applications. Realizing a megaton-scale CO2 capture facility would employing CA-mediated hydration chemistry becomes rapidly cost-prohibitive if supplying CA as an exogenous additive. An intuitive resolution to this problem involves generating CA continuously in bacteria. However, non-photosynthetic bacterial growth has its own associated cost demands, including the significant quantities of exogenous nutrients that would prove necessary for supporting the dense bacterial populations needed for generating CA at volumes relevant to CO2 remediation efforts. Recombinant protein expression in marine, cyanobacterial host cells is proposed as the ultimate workaround this problem, with the ability of these photosynthetic hosts to grow on CO2, sunlight, and the nutrients available in seawater offering the potential of a self-replicating system for CA production at significantly reduced expense. In addition, the metallation requirement for CA activity - Zn2+incorporation of its conserved tris-histidine motif - may also be met through the use of seawater media, given an average ocean concentration of zinc cations (100 nM) that meets or exceeds the binding constant of typical CA active centers (kd = 10-12 - 10-9 M). These considerations form the underlying rationale for this report, which details the design, construction and validation of a series of mutants for the extracellular expression of carbonic anhydrase enzymes immobilized on the cellular envelope. Each mutant variant represents the extension of a previously-developed homologous recombination platform for the genomicmutation of Synechococcus PCC 7002 (S. PCC 7002) bacteria12

[0077] As opposed to cytosolic enzyme expression, surface display represents a number of difficulties, namely the matters of how to target the export of recombinant proteins to the cell surface. Here a number of strategies area incorporating fusions of CA to membraneanchoring domains, are explored. These include the better-known, ice nucleation protein surface display methods demonstrated in E. coh. native and modified domains of antigen 43 proteins. More exotic display strategies, based on the fusion of CA domains to putative PCC 7002 s-layer proteins and the exploration of human carbonic anhydrase IX, an integral membrane isoform of CA, were also tested as candidates for efficient CA surface display in S. PCC 7002. Efficacy of these mutant cyanobacterial constructs are inspected through the use of legacy CA activity assays involving hydrolysis of the model substrate 4-nitrophenol acetate (4-NPA), as well as through their performance in driving CO2 mineralization to CaCCh. Discussion is provided on the practical limits of this strategy, its relevant use cases for scalable CO2 drawdown, key assumptions and go / no-go thresholds of the outlined approach, and future prospects for its optimization.METHODSMutant Construction & Isolation

[0078] Mutants of S. PCC 7002 incorporating constructs C1-C9 were constructed by adding 3.3 pl of each CA construct plasmid (plasmid stocks of these constructs were at similar concentrations, ranging from 142 to 157 ng pl as measured by nanodrop) to 500 pl of freshly-grown PCC 7002 cells in sterile 2 ml Eppendorf tubes. These were left to incubate for 24 hours at 30 °C with moderate agitation, under LED illumination. Spectral data for the illumination source are available in the Supporting Information. Afterwards, cells were plated by first pelleting the cell masses in each tube via centrifugation, and resuspending the pellets in 20 pl of A+ media with 50 pg ml-1 kanamycin (A+ kan). 10 pl of each resuspended cell sample was then plated on solid A+ kan agarose media using sterile technique, and then left to grow at 30 °C under illumination. Following days of growth at the stated conditions, colonies were observed. Colonies were picked from for each mutant and restreaked a second time on A+ kan (100 pg ml ) agarose media. Liquid cultures were established by looping plated cultures using sterile technique, and transferring to sterile A+ kan liquid media.Colony PCR

[0079] PCR amplification of gene inserts from genomic DNA purified from each S. 7002 mutant was used as a diagnostic to confirm successful gene integration. DNA was purified from modified cells using Zymogen’s cell lysis kit. Primers binding to 5’ upstream and 3’ downstream sequences common to all constructs cloned into PSW068 were used to amplify the homologous recombination products from the purified genomic DNA samples via PCR. Reactions were setup as follows:Component Volume (pl) Final ConcentrationPCR millipore H2O (sample / +ctrl / -ctrl) 35 / 39.5 / 40 -Buffer (5x HF; supplier) 10 lxTaq polymerase (supplier) 0.5 0.04 U / ml dNTP mix (supplier) 1 200 pMMgCh (supplier) 0.5 0.5 mMFwd primer (Sigma) 0.5 1 pMRev primer (Sigma) 0.5 1 pMTemplate gDNA / PSW068 (+) Ctrl 5 / 0.5DMSO 2.5 5% v / v

[0080] The forward DNA primer (PSW068_UP_Lac) sequence was: 5’- GTGAGCGCTCACAATTCGGAATTC-3’ (SEQ ID NO: 15). The reverse DNA (PSW068_DOWN_Lac) primer sequence used for amplification was 5’- CCGTCAAGACCCGTTTAGAGGCCCC-3’ (SEQ ID NO: 16).

[0081] Following PCR amplification, 5 pl of each sample + 2 pl of DNA loading dye (supplier) was loaded onto a 1% agarose gel containing a 1 / 104dilution of ethidium bromide (17 pl into 170 pl of 1% agarose gel solution) prepped from and an immersed in lx TAE buffer. Three microliters of DNA ladder standard (Rockland MB-204-500) was used for determination of PCR fragment sizes. Electrophoretic gel separation was run for 2 hours at 100 V (~5 V cm field strength) to ensure good band resolution, followed by DNA visualization under UV (365 nm) illumination.Sequencing

[0082] Sequencing of gene constructs were conducted by the UC Berkeley DNA Sequencing Facility and Sequetech Corporation.Western Blotting

[0083] Standard Western Blotting techniques know to the art are used.Cell Growth & Enzyme Expression

[0084] Mutant and WT cells of S. PCC 7002 were grown in sterile, A+ media, prepared according to UTEX protocols (the webpage for: UTEX.org), at 30 °C, under LED illumination and ambient atmosphere (-450 ppm CO2). Aeration was achieved through use of an orbit shaker (VWR) at speeds between 190-220 rpm. Starter cultures were grown by inoculating 5 ml of A+ culture media in 12 ml culture tubes using 1 / 20 cell volumes or through direct inoculation from plate media by looping cells from agarose. Illumination power was measured at 560 nm using a Newport power meter and the total photon flux from the LED source calculated by integrating over the entire LED spectrum, power-normalized at 560 nm (SI). LED lamp spectra were acquired using an Ocean optics UV-vis spectrometer equipped with a fiber optic positioned from the light source at the same position as the growth flasks.Cell Counting

[0085] Cell counting was performed on an Invitrogen Countess 3 FL automated cell counter equipped with a Cy5 filter (kexc = 635 nm, 18 nm bandwidth; km = 692 nm, 40 nm bandwidth). These illumination / emission settings were suited for detection of the native chlorophyll fluorescence of S. PCC 7002 with an emission centered at 705 nm upon 630 nm excitation (SI)13. Samples were prepped by loading 10 pl of cell sample (0.1 < OD730 < 1.5) into disposable Countess cell count chambers. Standard curves were generated for correlating cell density, as measured by Rayleigh scatter at 730 nm, to real cell counts using the automated cell counter, enabling rapid conversion of OD730 values to explicit cell counts.CA Activity Screening Assays

[0086] Assays for testing the CA activity of the cyanobacterial mutants were designed bymodifying classical 4-nitrophenyl acetate (4-NPA) hydrolysis assays. Cells were resuspended in an assay buffer comprised of pH 7.4 50 mM sodium phosphate + 33 mM Na2SO4, to a final O.D. of 1.0. Stock solutions of 10 mM 4-NPA were prepared by dissolving 4- nitrophenyl acetate to a concentration of 100 mM in DMSO, followed by dilution to 10 mM in millipore water. Reactions were setup in in 96 well plates, with each reaction featuring compositions of 125 pl buffer, 100 pl cells and 75 pl of 4-NPA. No-cell controls and a positive control consisting of 1 U ml’1bovine carbonic anhydrase (BCA) contained 225 pl buffer. Assays were run by monitoring evolution of 4-nitrophenol, the product of 4-NPA hydrolysis, by monitoring the spectral peak of the nitrophenolate ion absorption (pKa = 7.15) at 405 nm. At pH 7.4, the ratio of the nitrophenolate ion to the protonated form is to be determined. Time-dependent spectral evolution was monitored by sampling changes in absorption spectra over the course of at least two hours using a Molecular Devices SpectraMax M3 photospectrometer.RESULTSBioengineering - gene design, assembly & host mutagenesis

[0087] Nine distinct mutant constructions for CA fusion proteins were designed (Table 1), each incorporating gene elements for signaling, to drive protein export to the cell surface; a 6x His tag for potential protein tagging and identification on the cell envelope; a cell domain for anchoring CA, and a CA catalyst domain. CA variants considered in this study were Human carbonic anhydrase II (HCA 2), as a result of its extensive characterization, S. azorense carbonic anhydrase (5. azo CA), the fastest CA isoform to date, and human carbonic anhydrase IX (HCA 9), a variant that is unique among CAs as it natively forms an integral membrane protein, making it a prospective candidate for direct integration into cyanobacterial surfaces without the need for a separate anchoring domain.

[0088] Table 1. Recombinant Gene Construction.

[0089] Each of these constructs was codon-optimized for expression in S. PCC 7002, using IDT’s codon refinement tool (webpage for: idtdna.com). Gene syntheses for all constructs, and subsequent cloning of the inserts into PSW068 vector plasmids (Nixon Lab, Imperial College London via webpage for: Addgene. org / 140036) were conducted by Genscript. PSW068 was originally developed for driving the homologous recombination of flanked genes into the Fad locus of Synechococcus PCC 1190112. However, inspection of flanking recombination sites of PSW068 revealed significant sequence similarity with the FADd locus of S. PCC 7002. Blast identity scores of 460 / 469 for the upstream flank site and 491 / 494 for the downstream flank sites strongly suggested the utility of this plasmid for gene integration at the Fad locus in S. PCC 7002.

[0090] These nine CA fusion constructs were designed and tested to test the possibility that any one of them could facilitate the export and binding of carbonic anhydrase to the surfacesof S. PCC 7002. Constructs were integrated into the PSW068 parent vector originally developed by Nixon et al. for modification of Synechococcus PCC 11901, a close relative of S. PCC 7002 (Figure 1, panel B)12. Key elements of this vector, common to each of its derivatives generated in this study, include a clac43 promoter, T7 phage RNA polymerase terminator sequence, and gene cassettes for both kanamycin resistance (situated within the homologous recombination region) and ampicillin resistance. Constructs were designed with an eye towards optimizing protein orientation on the cell surface; enhanced mass transport of CO2 to CA active sites would be assisted through constructs that orient the CA active site away from the cell surface and towards open solution, a factor determined by whether the point of cell anchoring occurs at the N or C-terminus of the recombinant CA (Figure 1, panel C).

[0091] Construct Cl consists of a N-terminal fusion of Synechococcus outer membrane protein A (somA) signal peptide and a C-terminal fusion of the antigen43 beta chain to S. azorense carbonic anhydrase. The somA signal peptide functions as the cell export signal for the entire construct ! 3, while the beta chain of antigen 43 is known to function as an anchoring domain for surface-bound proteins13 15. Critically, work has shown that use of antigen 43 fusions can be used for surface display applications in cyanobacteria, despite their complex cell wall structure13. Linkage between the CA and anchoring domains in all fusion constructs are mediated by a flexible (GGGGGS)3 (SEQ ID NO: 17) linker to provide conformational flexibility to the mature fusion proteins.

[0092] Constructs C2 and C3 consists of CA fusions to a putative S-layer protein found in the S. PCC 7002 genome (B1XN72_SYNP2). As major components of cyanobacterial cell walls, S-layer proteins could serve as ideal anchors for surface expression system. Due to the lack of biochemical or structural characterization of this putative s-layer protein, both N-terminal (C2) and C-terminal (C3) fusions to CA were inspected. In the case of C3, an additional signal peptide was appended to the N-terminus of CA, in attempts to aid extracellular export. C4 consists of an N-terminal fusion of S. azo CA to the inaK domain of ice nucleation protein, based on previous work showing the efficacy of ice nucleation proteins in cell surface attachments16 l 9.

[0093] Constructs C5 and C6 represent C- and N-terminal fusions of S. azo CA to the somA protein a native surface protein in S. PCC 7002. In the case of C5, a separate N-terminal fusion of the somA signal peptide to CA was used (the somA C-terminal fusion excludes thesignal sequence in for construct C5). C7 is the only construct that uses human carbonic anhydrase IX (HCA 9), a known integral membrane protein. As a result of its native structure, fusions to anchoring domains were omitted for C7, with the only modification being an N-terminal fusion to a PCC 7002 s-layer signal peptide sequence to help direct export.

[0094] Constructs C8 and C9 represent identical gene constructions featuring incorporating S. azo CA and HCA 2, respectively. These constructs are characterized by N-terminal fusions of each CA to the E. coli antigen 43 signal sequence from the flu gene, which reportedly yields the counterintuitive result of improving cell export of Ag43-fused proteins in Synechocystis PCC 6803 relative to constructs using native somA signal sequencesl3. Plasmid integration of these constructs followed by their transformation into WT S. PCC 7002 cells resulted in the formation of discrete colonies when streaked on A+ media amended with 50 pg ml-1 kanamycin and grown under light at 30 °C. Secondary transfers of these colonies onto 100 pg ml-1 containing media were used to ensure complete segregation of genuine mutants from WT cells. Isolation of the genomic DNA of the generated mutants, followed by colony PCR revealed successful amplification of the genes, with bands corresponding to the predicted molecular weights of the PCR amplicons confirming incorporation of each construct into WT PCC 7002 (Figure 2).Enzyme Surface Display and Mineralization

[0095] Assay screens for CA activity using the model substrate 4-nitrophenyl acetate were used to down-select a handful of preferred mutants for extensive characterization. Absorption at 405 nm was monitored to track the evolution of 4nitrophenol, the product of 4-NPA hydrolysis that is catalyzed by carbonic anhydrase. Results of this first-pass screen suggested that mutant constructs Cl, C4, C8 and C9 were the best of the 9 candidate recombinant strains for accelerating mineralization. This was confirmed through mineralization assays using the Alexafluor dye Mag-fluo-4. Ca2+ chelation by this dye modifies the chromophore’s luminescence spectrum, enabling quantitative determination of the extent of Ca2+ either consumed or incorporated as CaCO3. These assays were run in triplicate, which demonstrated that these mutants yielded mineralization levels above that of the WT control, whose mineralization was below the assay’s detection limit.

[0096] Predictive Simulation of Steady-State CO2 Mineralization

[0097] As a result of the reversible nature of carbonic anhydrase enzymes, in which the conversion of carbonic acid to CO2 is also catalyzed, a standard Michaelis-Menten description, which assumes irreversibility in the catalytic step, was not sufficient. Explicit accounting for the reversibility of this enzyme’s activity was achieved by analytically deriving a modified Michaelis rate expression for carbonic anhydrase for the following chemical mechanism:

[0098] Using this mechanism, a rate expression for the interconversion of CO2 and carbonic acid is derived under steady-state conditions:where

[0099] The concentration of carbonic acid, H2CO3, can be written in terms of the [CO2] using the following definition: [H2 CO3 ] = ki kcat [CO2 ] k'1 k'cat (3).

[0100] Here, kcat is the rate of CO2 conversion to carbonic acid, kcat' is the rate for the reverse reaction; kl and kl ' refer to the forward and back rates of the rapid preequilibrium that forms prior to catalysis to yield the activated enzyme-substrate complex, E- CO2. Notably, the utility of this catalyst requires the use of buffers capable of stabilizing pH above about pH 8.5, as without it, the evolution of two proton equivalents for every equivalent of carbonate ion generated acidifies the medium, counteracting mineralization.

[0101] The resulting rate expression for CA turnover (eq. 1) was incorporated into to a system of ordinary differential equations expressing the fluxes of metal (Mg2+ / Ca2+)carbonates(MCO3), metal hydroxides (MOH) and dissolved CO2, CO23‘, HCO3', and H2CO3 species. An ODE solver was used to numerically determine the time-dependent concentrations of all species, including the formation of solid metal carbonates, at all points during the simulation time. Results of this effort are captured by Figure 4. As a way of illustrating the consequence that choice of CA isoform will have on catalytic limits for CO2 mineralization, contours of mineralization to CaCCh / MgCCh were produced as a function of CA kcat and km, parameters assuming a broad range of values within the large family of known CA enzymes. While much work in the domain of CA catalyzed mineralization has focused on the utility of CA isoforms with higher kcat values for enhancing carbonate precipitation, equal attention should be paid to the ability of low-km / low kcat enzymes to potentially compete with high kcat / low kmvariants. For example, as illustrated by Figure 5, a hypothetical CA variant with a kcat of about 2 x 106s'1and a CO2 binding constant of 3 mM would be expected to outperform S. azorense CA, the fastest CA known as of this writing, with a kcat more than twice that (4.4 x 106s'1) of our hypothetical enzyme. The traditional measure of catalytic efficiency, kcat / km, collapses the relevance of kcat and km into a single metric (Figure 5), providing the critical measure for any downstream attempts to optimize CAs themselves for CO2 capture applications.DISCUSSIONUse cases, limits of ideal calculations and requirements for practical use

[0102] Although calculations presented in this report are conducted at steady state, real implementation of carbonic anhydrases in mineralization processes will have to address the fact that CO2 mineralization proceeds with concomitant drops in solution pH. This works against the goal of mineral accumulation, as the gradually acidifying media will result in the slow re-dissolution of the generated mineral precipitates and the eventual evolution of CO2. Such effects become especially pronounced if the pH is allowed to fall below 8. This competing acidification process, and the infeasibility of counteracting it with exogenous base at the rates and quantities needed to capture gigatons of CO2 annually, will likely exclude the utility of any such system in the open ocean. In practice, the likeliest use case of carbonic anhydrase enzymes in CO2 mineralization will exploit the changing pH as a function of reaction time in a pH-swing process for regenerating concentrated CO2 (and therefore alkalinity), in a closed system. In such a case, CO2 evolution becomes a feature rather than bug, with thoughtful system design potentially allowing the use of mineralization as anintermediate step for generating concentrated CO2 streams from the atmosphere. A fully developed DAC process may be conceivably built by pairing the biocatalysts developed here along with either a vacuum cycle for driving CO2 out of the aqueous phase following a period of mineralization and base / buffer consumption, thereby reversing the carbonate equilibrium in a closed reactor back towards CChfe) for collection. Alternatively, the application of reversible photoacids20 22with appropriately-chosen dark and excited-state pKas to effect rapid drops in pH, offer a promising use case for these compounds and could also provide a practical step towards regenerating consumed metal ions, alkalinity and deprotonated buffer in preparation for another cycle of carbonate mineralization. However, each of these possibilities require separate lines of deep scientific exploration and development, work beyond the scope of this report.

[0103] While this work emphasizes CA-catalyzed CO2 hydration as an amendment to CO2 mineralization chemistry, it is important to note the applicability of this system - or any system capable of enhancing CO2 hydration kinetics - will conceivably have a potential role in any CO2 capture scheme operating in aqueous media. This especially applies for cases of low [CO2]aq and weakly alkaline conditions, where the overall rate of catalyzed CO2 hydration is rate-controlling of DIC equilibration in water. As a result, besides the applications in CO2 mineralization, catalyzing CO2 hydration, has the potential to augment capture technologies such as electrochemical DOC23 25, capture in water-based amine sorbents26, pH-swing and electro-swing27 29capture strategies dependent on the availability of dissolved CO2, and whose kinetics may be limited by the mass-flux and concentration of its hydrates. In addition, recent work as also suggested a role for carbonic-anhydrase catalysis in accelerating advanced weathering of olivine, an option for scalable CO2 capture representing a rapidly-growing area of intensive research30. As a result, the technology developed here can be seen as offering a generalized fix to a rate-limiting component that is common to several different capture strategies.

[0104] Finally, the given assumptions of steady-state in the calculations presented here - a situation unlikely to be maintained for any practical CA mineralization process for the reasons of pH instability discussed - should be taken to represent an upper limit on mineralization rates for a system of a given initial pH, and initial concentrations of CA, dissolved CO2, and dissolved alkali metal ions. Stabilizing near-equilibrium concentrations of dissolved CO2 point to the importance of efficient aeration (via stirring, sparging, etc.) tofacilitate rapid equilibration of CO2 between the gas and liquid phase. As a result, reactor designs that address the constraints of mass-transport at air-liquid interfaces will be an essential part of any practical implementation of this technology.

[0105] Target Thresholds and Limits of Immobilization - assumptions of this approach and implications: enzyme activity unchanged upon immobilization, etc.

[0106] Exploring the physical limits of a surface display strategy for CA-mediated CO2 capture suggests its physical possibility but also highlights significant engineering challenges to be overcome. Using EM micrographs of PCC 7002 as a guide informs their approximation as about 1 x 0.2 um ellipsoids. Decomposing the cell shape into a hemispherically-capped cylinder allows for the calculation of a cell surface area of 0.63 um2. Given the footprint of typical soluble carbonic anhydrases (about 35 A diameter, making for an areal footprint of 9.6 x 10'6um2) suggests a maximum surface immobilization density of 65000 enzymes cell'1. This best-case scenario marks the limit of close packed, surface display. At peak cell densities of 108 cells ml'1(1011 cells I'1), this yields an effective enzyme concentration of 10 nM, the enzyme concentration used for simulations in this study. In addition to these considerations, success of any strategy employing these CyCAM constructs would also require ensuring that cell populations remain viable under the stresses of pH cycling, and that the cell suspensions are not destabilized by mineral precipitation.CONCLUSIONS

[0107] Coupling the properties of rapid chemical CO2 catalysis, with the selfreplicating nature of photosynthetic bacteria, holds promise for yielding CO2 capture systems that are essentially biological Von Neumann machines, with freely available CO2 and sunlight providing the necessary carbon and energy sources required for their replication and operation. In contrast to the majority of uses cases for cellular expression of high-value materials, here the rate of CO2 capture is not tied to cell replication or respiratory rates. The non-redox nature of CO2 hydration chemistry effectively partitions the rate at which CO2 hydration can proceed from the much slower kinetics of cell growth and biosynthesis. As a result, cell-supported, CA mineralization chemistry provides a particularly effective use case for biological systems in addressing CO2 capture issues, enabling rates of CO2 conversion to operate in regimes typical of pure chemical and physical capture processes, rather than the comparatively slow rates of CO2 conversion common to biofuels synthesis, where cellrespiration comprises a fundamental limit on product formation rates. Optimizing mineralization rates through detailed analysis of gene expression profiles, promoter choices, construction of CA gene fusions for maximizing CA surface density, and choice of host organisms capable of growing at high cell densities, mark natural extensions of the investigation disclosed here, and offer significant opportunities for enhancing the kinetics of bioengineered CO2 mineralization for applications scale-up.

[0108] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

What is claimed is:

1. A photosynthetic cell comprising a nucleic acid encoding a heterologous carbonic anhydrase (CA) operatively linked to a promoter capable of expression in the photosynthetic cell.

2. The photosynthetic cell of claim 1, wherein the nucleic acid is on a plasmid stably residing in the photosynthetic cell or is stably integrated unto a chromosome of the photosynthetic cell.

3. The photosynthetic cell of claim 1, wherein the promoter is a constitutive promoter or an inducible promoter.

4. The photosynthetic cell of claim 1, wherein the CA is expressed and accumulates or transports to a periplasm, membrane, cell wall, or a surface of the cell.

5. The photosynthetic cell of claim 1, wherein the photosynthetic cell is a cyanobacteria cell.

6. A method for mineralizing carbon dioxide comprising: (a) culturing or growing a photosynthetic cell of claim 1 in a medium under conditions such that the photosynthetic cell expresses the CA, (b) converting carbon dioxide into the H2CO3 through the enzymatic activity of the CA.

7. The method of claim 6, further comprising (c) complexing the carbonate of the H2CO3 with a metal to form a metal carbonate.

8. The method of claim 7, further comprising (d) precipitating the metal carbonate from the medium.

9. The method of claim 8, further comprising (e) removing or separating the metal carbonate from the medium and / or the photosynthetic cell.

10. A method for catalyzing mineralization of carbon dioxide or dissolution of a carbonate comprising: (a) culturing or growing a photosynthetic cell of claim 1 in a medium under conditions such that the photosynthetic cell expresses the CA, and (b) converting carbon dioxide into carbonic acid at acidic pH or bicarbonate and carbonate at neutral and alkaline pH via the enzymatic activity of the CA.

11. The method of claim 10, further comprising (c) complexing soluble carbonates and bicarbonates of the with a metal to form a metal carbonate or metal bicarbonate.

12. The method of claim 11, further comprising (d) precipitating the metal carbonate or metal bicarbonate from the medium.

13. The method of claim 10, wherein the carbonate is a mineral carbonate.

14. The method of claim 13, wherein the mineral carbonate is a calcium carbonate or magnesium carbonate, or mixture thereof.

15. The method of claim 10, wherein the carbonate is a soluble carbonate, bicarbonate, carbonic acid, carbon dioxide, or mixture thereof.

16. A method for catalyzing the hydration and / or dehydration of carbon dioxide comprising: (a) culturing or growing a photosynthetic cell of claim 1 in a medium under conditions such that the photosynthetic cell expresses the CA in either the cell periplasm, membrane or wall or cell surface, and (b) converting carbon dioxide into carbonic acid at acidic pH or bicarbonate and carbonate at neutral and alkaline pH via the enzymatic activity of the CA.

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