Thermophilic bacterium comprising a surface-displayed carbonic anhydrase

Abstract

The invention relates to a thermophilic bacterium comprising a carbonic anhydrase, wherein the carbonic anhydrase is attached to and / or expressed on the surface of the thermophilic bacterium. The invention also relates to specific variants of a carbonic anhydrase, and it relates to a method of capturing carbon dioxide using the thermophilic bacterium or the carbonic anhydrase variants of the invention.

Description

[0001] THERMOPHILIC BACTERIUM COMPRISING A SURFACE-DISPLAYED CARBONIC ANHYDRASE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of carbon capture and storage, especially post-combustion capture. In particular, the present invention relates to a thermophilic bacterium comprising a carbonic anhydrase, wherein the carbonic anhydrase is attached to and / or expressed on the surface of the thermophilic bacterium. The invention also relates to specific variants of a carbonic anhydrase with properties suitable for the present purpose. Finally, the invention relates to a method of capturing carbon dioxide using the thermophilic bacterium or the carbonic anhydrase variants of the invention.

[0004] BACKGROUND OF THE INVENTION

[0005] In light of the challenges presented by climate change and global warming, various efforts are being made to combat the rapid rise in global average temperatures. The atmospheric concentrations of greenhouse gases, such as carbon dioxide (CO2), have been steadily increasing since the Industrial Revolution and the current global average concentration of CO2 in the atmosphere is 421 ppm as of May 2022 (0.04%) (carbon dioxide now more than 50% higher than pre-industrial levels, www.noaa.gov, 3 June 2022). To meet the 2050 net zero emission goal, various strategies have been proposed, including carbon capture and storage, which is a sustainable and effective method for reducing CO2 emissions.

[0006] Carbon capture and storage is a process in which a relatively pure stream of carbon dioxide from industrial sources is separated, treated and transported to a long-term storage location in an effort to reduce greenhouse gas emissions and thus mitigate the effects of climate change.

[0007] Post-combustion capture is directed to removing carbon dioxide after combustion of fossil fuels. In this method, carbon dioxide is captured from flue gases at power stations or other point sources. The method may be coupled to other short- or long-term storage solutions, such as carbon sequestration. However, carbonation is very slow, limiting the efficiency of carbon dioxide mineralization, because the rate constant of carbon dioxide hydration is below 10-1s1at ambient conditions.

[0008] Carbonic anhydrases (CA, EC 4.2. 1.1) are a family of enzymes that catalyse the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid (i.e., bicarbonate and hydrogen ions). Several CA families exist (a, p, y, 6 and ) and various isoforms have been identified in each of these families. The families have no significant amino acid sequence similarity, however the active site of most CAs contains a zinc iron. Mammalian, plant and prokaryotic CAs (a-CA and p-CA) generally function at physiological temperatures (around 37°C) or lower temperatures. The temperatures of combustion gases or flue gases, or the liquids they may be dissolved in, however, are high.

[0009] The most efficient CA reported to date is that of Sulfurihydrogenibium azorense (SazCA), with a reported Kcat of 4.4 x 105 / s (Luca, V. D., et al. An a-carbonic anhydrase from the thermophilic bacterium Sulphurihydrogenibium azorense is the fastest enzyme known for the CO2 hydration reaction. Bioorganic & medicinal chemistry, 21, 1465-1469 (2013)). The final product, CaCCh, is thermodynamically stable, environmentally benign and has a low solubility in water.

[0010] However, the utilization of purified CA enzyme is constrained by its high purification cost, poor stability, susceptibility to inactivation and lack of recycl ability. To overcome some of these limitations, various advancements have been proposed. Enzyme-based solutions using CAs for the capture and / or conversion of carbon dioxide have previously been described in the art. For example, WO 2008 / 095057 discloses heat-stable CAs and their use. WO 2012 / 025577 discloses heat-stable Persephonella CAs and their use.

[0011] However, despite these advances, the overall catalytic activity in these investigations remained comparatively low. Also, the instability of CA continues to pose challenges to its practical implementation in industrial carbonic mineralization processes.

[0012] To date, the most efficient biocatalyst reported involves the display of Sulfurihydrogenibium azorense (SazCA) on the outer membrane of E. coli, resulting in an activity of 11.43 U / mL / ODeoo (Zhu, Y., Liu, Y., Ai, M. & Jia, X. Surface display of carbonic anhydrase on Escherichia coli for 681 CO2 capture and mineralization. Synthetic and systems biotechnology 7, 460-473 (2022)). And the highest achieved thermal stability remains approximately 40% at 70°C for a duration of 12 hours.

[0013] OBJECT OF THE INVENTION

[0014] It is an object of embodiments of the invention to provide thermophilic bacteria displaying a carbonic anhydrase on their surface, thereby providing better heat-stability for capturing carbon dioxide at high temperatures. It is also an object to provide variants of carbonic anhydrase having both improved heat-stability and activity, and which can be used to capture carbon dioxide and generate commercially useful compounds or components.

[0015] SUMMARY OF THE INVENTION

[0016] It has been found by the present inventors that rational engineering of carbonic anhydrases (CAs) can provide for highly active and resilient CAs. It has also been found by the present inventors that any sufficiently thermostable CAs can be attached to and / or expressed on the surface of a thermophilic bacterium, such as a thermophilic bacterium which has been engineered and optimised for enhanced CO2 biomineralization, in order to provide a solution for carbon capture with an improved activity and thermostability. It was found advantageous to express a CA in bacteria for surface expression, since the resulting whole-cell catalyst was stable, and using it saved the step of isolating and purifying the CA. The inventors observed that a whole-cell catalyst according to their invention had markedly higher CO2 hydration activity compared to any previous studies' most active E. coli (tdCA) system by over 5-fold, and that it also surpassed the performance of the yeast CA bCAl and the C. glutamicum CA ngCA by 2-5 orders of magnitude (Jo, B. H. & Hwang, I. S. Characterization and high-level periplasmic expression of thermostable a-carbonic anhydrase from Thermosulfuri monas dismutans in escherichia coli for CO2 capture and utilization. International Journal of Molecular Sciences 21, (2020); Barbero, R. et al. Engineered yeast for enhanced CO2 mineralization. Energy & Environmental Science 6, 660 (2013); Koo, B. I. et al. Robust Biocatalysts Displayed on Crystalline Protein-Layered Cells for Efficient and Sustainable Hydration of Carbon Dioxide-Supplementary materials. Advanced Functional Materials 31, (2021)).

[0017] So, in a first aspect the present invention relates to a thermophilic bacterium comprising a carbonic anhydrase, wherein the carbonic anhydrase is attached to and / or expressed on the surface of the thermophilic bacterium.

[0018] In a second aspect, the invention relates to a carbonic anhydrase having at least 72% sequence identity with SEQ ID NO: 1 and having an amino acid selected from A, H, Y, and Q in the position corresponding to position 88 of SEQ ID NO: 1.

[0019] In a third aspect, the invention relates to a use of the thermophilic bacterium according to the first aspect or the carbonic anhydrase according to the second aspect for capturing carbon dioxide from a gaseous mixture, such as flue gas.

[0020] In a fourth aspect, the invention relates to a method of capturing carbon dioxide and forming an alkaline earth metal carbonate, the method comprising a step of: contacting carbon dioxide with the thermophilic bacterium according to the first aspect or the carbonic anhydrase according to the second aspect and alkaline earth metal ions, so as to form one or more alkaline earth metal carbonate(s), and optionally, extracting the alkaline earth metal carbonate(s).

[0021] In a fifth aspect, the invention relates to a polynucleotide comprising a nucleotide sequence encoding the carbonic anhydrase according to the second aspect of the invention or any embodiments thereof disclosed herein.

[0022] In a sixth aspect, the invention relates to a vector comprising the polynucleotide according to the fifth aspect of the invention.

[0023] In a seventh aspect, the invention relates to a cell comprising the polynucleotide according to the fifth aspect of the invention or the vector according to the sixth aspect of the invention. In embodiments of the seventh aspect of the invention, the cell is the thermophilic bacterium according to the 1staspect of the invention or any embodiments thereof described herein.

[0024] LEGENDS TO THE FIGURES

[0025] Fig. 1 shows a bacterial cell factory model according to embodiments of the invention.

[0026] Fig. 2 shows the CO2 hydration activity of carbonic anhydrase from various origins expressed in P. thermoglucosidasius. (A) MICA, CA from Micrococcus luteus; hCAII, human; SazCA, Sulfurihydrogenibium azorense; TaCA, Thermovibrio ammonificans; ApCA, Aerbaciiius paiiidus,- Cab, Methanobacterium thermoautotrophicum. MICA, hCAII, TaCA, ApCA and Cab were produced as fusions with a secretion signal peptide, expressed in P. thermoglucosidasius. Activity was measured in two fractions: the supernatant, detecting secreted CA activity (no pattern), and cellular extracts, detecting non-secreted CA activity (with pattern). Specific CO2 hydration activity is defined as the ratio of enzyme activity (measured by Wilbur-Anderson assay) to the volume of enzyme solution, denoted as U / mL. Each candidate was normalized by ODeoo, and each fraction was normalized by volume. Data are presented as mean ± s.d. for three biological replicates with error bars. (B) pH stability of SazCA and TaCA supernatant shown in specific CO2 hydration activity within the pH range of 4-12, with no treatment serving as the control. Data are presented as mean ± s.d. for three biological replicates with error bars. (C and D) Temperature stability of SazCA (C) or TaCA (D) supernatant shown in specific CO2 hydration activity. Temperature scale is coded using the patterns as shown in the figure inset. Duration of exposure is shown in days (1-15). Data are presented as mean ± s.d. for three biological replicates with error bars.

[0027] Fig. 3 shows the establishment of a cell surface display platform. Western blot images, using anti- 6X His tag® antibodies, of extracellular, cell wall and pellet fractions of different P. thermoglucosidasius (Pt) strains expressing anchored SazCA. M, Protein ladder; S, supernatant; CW, cell wall; P, cell pellet; Pt-backbone, control strain containing an empty plasmid backbone; Pt- SazCA_Sec, control strain lacking any anchoring motif. The arrowheads represent the molecular weight of LysM-SazCA and secreted SazCA.

[0028] Fig. 4 shows optimization of anchor motif and linker positioning in SazCA constructs. (A) A schematic illustration of the plasmids used for surface expression of SazCA. The anchoring motif can be positioned at either the N- or C-terminus of SazCA, optionally with a linker employed between the anchoring domain and SazCA. (B) The CO2 hydration activity of P. thermoglucosidasius harboring the above plasmids. Each strain was normalized by ODeoo, then the overall cell was tested with Wilbur-Anderson assay. Statistical significance between various LysM fusions (light grey) was analyzed using an ordinary one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test. Data are presented as mean ± s.d. for three biological replicates with error bars. Asterisks denote significance for P values. *P < 0.05; **P < 0.01; ***p < 0.001; ****p < 0.0001. Fig. 5 shows effects of natural protease inactivation on SazCA CO2 hydration activity. Seamless inactivation of a specific protease-encoding gene is indicated with an "X". CO2 hydration activity of different protease-deficient strains with surface-displayed SazCA, expressed using a high copy plasmid, is presented. Each strain was grown in SPY medium and normalized by ODeoo, and then the whole-cell catalyst was tested by the Wilbur-Anderson assay. Data are presented as mean ± s.d. for three biological replicates with error bars. Statistical significance between multiple treatment groups was analyzed using an ordinary one-way ANOVA with Dunnett's multiple comparisons test, comparing each group to WT. Asterisks denote significance for P values. *P < 0.05; **P < 0.01; ***P < 0.001; ****p < 0.0001.

[0029] Fig. 6 shows a stability test of the engineered P. thermoglucosidasius with surface-displayed SazCA in harsh conditions. Each strain was normalized by ODeoo, then exposed to harsh conditions, and the overall cell was tested with the Wilbur-Anderson assay. Data are presented as mean ± s.d. for three biological replicates with error bars. (A) CO2 hydration activity of the whole-cell biocatalyst after exposure to high temperatures over time for 20 days. (B) CO2 hydration activity of the whole-cell biocatalyst after exposure to different pH for 1 or 24 hours. Control was set as no treatment but diluted to the same volume as the experimental group. (C) CO2 hydration activity of the whole-cell biocatalyst after exposure to CaCh treatments (10-1000 mM) for 24 hours. Control was set as mixed with CaCl2 to achieve final concentrations of 0 mM.

[0030] Fig. 7 shows the specific activity (bars) and the residual activity (dotted lines) of CA from Persephonella hydrogeniphila - PhyCA, Persephonella atlantica - PaCA, Persephonella sp. KM09- Lau-8 - PlauCA, and Sulfurihydrogenibium azorense - SazCA, at various temperatures. 30°C is the reference temperature for the residual activity.

[0031] Fig. 8 shows the specific activity (top) and the residual activity (bottom) of CA from Persephonella hydrogeniphila - PhyCA, its mutants K88Q, K88A, K88H and K88Y, as well as from Sulfurihydrogenibium azorense - SazCA, at 30°C to 90°C in 10°C increments.

[0032] DETAILED DISCLOSURE OF THE INVENTION

[0033] Definitions

[0034] The term 'thermophilic bacterium' refers to a bacterium that thrives at relatively high temperatures, typically between 40°C and 122°C, such as between 50°C and 122°C. Thermophilic bacteria can survive at high temperatures, where other bacteria or microorganisms would be damaged and / or killed if exposed to the same temperatures. They are classified as extremophiles, organisms that are adapted to survive in extreme environments. Thermophilic bacteria are found in a variety of habitats, including hot springs, volcanic vents, compost piles, and even the human gut.

[0035] Thermophilic bacteria may be classified according to their optimal growth temperatures. By 'optimal growth temperature' is meant the temperature at which the growth-rate of the bacterium is at its highest. The optimal growth temperature may be determined by standard techniques known to the person skilled in the art, including measurement of optical density (OD) at 600 nm. Continuous measurements at different temperatures can be used to make growth curves, from which the growth-rates can be determined and compared (see, e.g., Doran, Elsevier (2013), Bioprocess engineering principles, 2ndedition, chapter 12.8). Thermophilic bacteria typically have an optimal growth temperature of at least 50°C. More specifically, a simple thermophile has an optimal growth temperature in the range of 50-64°C, an extreme thermophile has an optimal growth temperature in the range of 65-79°C, and a hyperthermophile has an optimal growth temperature of 80°C and beyond (and typically not below 50°C). The thermophilic bacterium as used herein may also be a combination or mixture of one or more bacteria belonging to different classes. The thermophilic bacterium can be a Gram-positive or Gram-negative bacterium.

[0036] Thermophilic bacteria typically have enzymes that are adapted to function at high temperatures. These enzymes have special structural features that help to stabilize them at high temperatures and prevent them from denaturing. Thermophilic bacteria also have membrane lipids that are adapted to high temperatures. These lipids have a higher melting point than the lipids found in mesophilic bacteria, which allows them to maintain their fluidity at high temperatures. Thermophilic bacteria also typically have a high tolerance for acid and alkali. As such, many thermophilic bacteria can survive in environments with a pH as low as 1 or as high as 12.

[0037] The thermophilic bacterium can be, for example, selected from the genus Parageobacillus, preferably from the species P. thermoglucosidasius.

[0038] The term 'carbonic anhydrase' or 'CA', also known as 'carbonate dehydratase', (EC 4.2.1.1), refers to a member of a family of enzymes that catalyse the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid (i.e., bicarbonate and hydrogen ions).

[0039] The term covers variants of naturally occurring CAs. By 'variant' is meant that the polypeptide comprises one or more mutations, such as amino acid substitutions, insertions and deletions, as compared to the parent or reference protein. Variants comprise both naturally occurring and non- naturally occurring variants. Furthermore, the polypeptide disclosed herein preferably comprises proteinogenic amino acids, although non-proteinogenic amino acids are not excluded. A variant may be the product of rational mutagenesis, where one or more mutations are introduced into the DNA encoding the polypeptide. Standard recombinant DNA and molecular cloning techniques are well known in the art and are described by, e.g., Sambrook, J., Fritsch, E. F., and Maniatis, T. (2012). Molecular cloning: A laboratory manual, 4th ed. Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, and by Silhavy, T. J., Bennan, M. L., and Enquist, L. W. (1984). Experiments with gene fusions. Cold Spring Harbor Laboratory: Cold Spring Harbor, New York. Typically, the variant has a high sequence identity to the amino acid sequence of the parent or reference protein, e.g., at least about 70%, such as at least about 80%, such as at least 85%, such as at least about 90%, such as at least about 95%, and such as at least about 99%. The term 'carbonic anhydrase' furthermore covers fragments of naturally occurring CAs or of CA variants. By 'fragment' is meant a polypeptide which consists of less than the full-length protein. Usually, the fragments of interest herein include the catalytically active portion and have carbonic anhydrase activity, as described below. A fragment of a CA may, e.g., consist of at least or exactly or at most 50 contiguous amino acid residues present in the parent or reference protein, such as at least or exactly or at most 75, at least or exactly or at most 100, at least or exactly or at most 125, at least or exactly or at most 150, at least or exactly or at most 175, and at least or exactly or at most 200, contiguous amino acid residues, depending on the length of the reference protein.

[0040] The CA as defined herein, in particular in the context of the 2ndaspect of the invention, may be an "isolated" CA, meaning that the polypeptide is removed from its natural environment and / or that the polypeptide is substantially free from components that normally accompany the polypeptide when it is present in its natural environment. For example, an isolated polypeptide may be substantially free of cellular material, such as other proteins. The isolated polypeptide may be a "purified" polypeptide. A purified polypeptide is a polypeptide which is substantially pure. For example, a preparation of protein may be substantially pure if it comprises less than or exactly 20% by dry weight of contaminating protein, such as less than or exactly 10%, such as less than or exactly 5%, and such as less than or exactly 1%, contaminating protein.

[0041] A polypeptide having the ability to catalyse the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid is also referred to herein as a polypeptide having 'carbonic anhydrase activity'. Thus, a CA according to the present invention is a polypeptide having carbonic anhydrase activity. In some cases, a 'CA' according to the invention may have a lower carbonic anhydrase activity than a naturally occurring CA. However preferably, the CA according to the first or the second aspect of the invention has at least 90% carbonic anhydrase activity as compared to the reference / parent CA. Thus, in some embodiments, the carbonic anhydrase has at least 90% of the CO2 hydration activity of the parent CA, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, and such as at least 100%, as determined by the Wilbur- Anderson assay at 50°C, pH 7, after 1 hour of incubation. As such, it may have an increased CO2 hydration activity compared to the parent CA.

[0042] The active site of most CAs contains a zinc iron. The CA according to the first aspect of the present invention preferably originates from a thermophilic bacterium, that can be the same or a different thermophilic bacterium than the thermophilic bacterium on the surface of which the CA is attached and / or expressed. The CA thus may be obtained or derived from a thermophilic bacterium which is different from the thermophilic bacterium comprising the CA according to the first aspect of the invention.

[0043] The phrase 'wherein the carbonic anhydrase is attached to and / or expressed on the surface of the thermophilic bacterium' can refer to a CA that is attached to the surface of the thermophilic bacterium by, e.g., a linker. The linker can be a short linker or a long linker, for example a Fibronectin-binding protein B (FnBPB) linker. The CA can be directly attached to the Promoter-RBS- signal peptide motif, or a single or multiple, for example 2, 3, 4 or 5, LysM domains can be positioned before or after the signal peptide and before or after the attachment of the CA. Other components, such as a tag, may also be incorporated before or after the CA. Alternatively, or additionally, the CA is expressed on the surface of the thermophilic bacterium. The CA can be in- or out-oriented, preferably, the CA is out-oriented so as to facilitate easier access to carbon dioxide in the surroundings.

[0044] The term 'Parageobacillus' and 'Parageobacillus thermoglucosidasius' (NCBI Taxonomy ID: 1426), previously known as Geobacillus thermoglucosidasius, refers to thermophilic Gram-positive, rodshaped bacteria, which are members of the Bacillota phylum. The native P. thermoglucosidasius is a thermophile, having an optimal growth temperature at around 60°C. Parageobacillus are catalasepositive, which means that they can produce hydrogen peroxide (H2O2) and then break it down into oxygen and water. This is an important property for survival in hot environments, as H2O2 can be toxic to cells. As used herein, the term 'Parageobacillus species' refers to any member of the group of species classified as belonging to the bacterial genus Parageobacillus. K non-limiting list of Parageobacillus species can be found at the National Center for Biotechnology Information (World- Wide Web (www) address ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id = 1906945; accessed on 19 February 2024, hereby incorporated by reference in its entirety) and elsewhere herein.

[0045] The term 'lysine motif domain' or 'LysM' refers to a small, globular protein domain found in a variety of extracellular proteins and receptors. It is typically about 40-70 amino acids long and is characterized by a p-a(2)-p fold with antiparallel strands. The LysM domain is named after the lysine residue that is often found at its N-terminus.

[0046] The term 'CO2 hydration activity' refers to the rate at which carbon dioxide (CO2) is converted into bicarbonate (HCO3) and hydrogen ions (H+) by a carbonic anhydrase (CA). The rate of CO2 hydration is determined by the activity of the CA. The activity of the CA can be measured in several ways, including spectrophotometry, colorimetric assays, such as the Wilbur-Anderson method described in Example 1 section 1.3, and enzyme-linked immunosorbent assays (ELISA).

[0047] Unless otherwise stated, 'sequence identity', as used for nucleotide and amino acid sequences herein, is determined by comparing two optimally aligned sequences of equal length according to the following formula: (Nref - Ndif)- 100 / Nref, wherein Nref is the number of nucleotides or amino acid residues in one of the two sequences and Ndif is the number of residues which are non-identical in the two sequences when they are aligned over their entire lengths and in the same direction. Hence, the nucleotide sequence ATACGGTT will have a sequence identity of 75% with the sequence ATTCGATT ridif=2 and nref=8).

[0048] The sequence identity can be determined by conventional methods, e.g., Smith and Waterman (Adv. Appl. Math.;2:482 1981), by the 'search for similarity' method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA;85: 2444 1988), using the CLUSTAL W algorithm of Thompson et al. (Nucleic Acids Res.;22:467380 1994), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group), or the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol.;48:443-453 1970) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., Trends Genet.; 16: 276-277 2000), e.g., as provided at the European Bioinformatics Institute website (www.ebi.ac.uk). The BLAST algorithm (Altschul et al., Mol. Biol.;215:403-410 1990), for which software may be obtained through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), may also be used. When using any of the mentioned algorithms, the default parameters for "Window" length, gap penalty, etc., may be used.

[0049] A residue in one amino acid sequence which 'corresponds to' a specific reference residue in a reference amino acid sequence is the residue which aligns with the reference residue, e.g., as determined by use of sequence alignment software described in the preceding paragraph.

[0050] The term 'expression', as used herein, refers to the process in which a gene is transcribed into mRNA, and may optionally include the subsequent translation of the mRNA into an amino acid sequence, i.e., a protein or polypeptide.

[0051] As used herein, a 'genetic modification' refers to the introduction of a genetically inherited change in the host cell genome. Examples of changes include mutations in genes and regulatory sequences, mutations in coding and non-coding DNA sequences. 'Mutations' include deletions, substitutions and insertion of nucleic acids or nucleic acid fragments in the genome.

[0052] As used herein, 'reduced expression' of a gene in a host cell means that the levels of the mRNA or protein encoded by the gene are significantly reduced in the host cell, typically by at least 25%, such as at least 50%, such as at least 75%, such as at least 90%, such as at least 95%, as compared to a control. Similarly, the term 'overexpression' of a gene in a host cell means that the levels of the mRNA or protein encoded by the gene are significantly increased in the host cell, typically by at least a factor 2, such as at least a factor 3, such as at least a factor 5, such as at least a factor 10, such as at least a factor 50, and such as at least a factor 100, as compared to a control. Typically, when the reduced expression or the overexpression is obtained by a genetic modification in the host cell, the control is the unmodified parent strain.

[0053] The term 'unmodified parent strain' refers to the original, wild-type strain of a bacterium before it has been genetically modified in a specific way, such as in a way described herein.

[0054] By 'abolished expression' of a gene in a host cell is meant that mRNA or protein encoded by that gene is essentially absent, absent or undetectable in the host cell.

[0055] The term 'activity' or 'function', as used herein and when referring to the activity or function of a protein, can, when nothing more is specified, mean any activity or function of that protein - such as catalytic activity, etc. As used herein, 'reduced activity' of a protein in a bacterium means that one or more specific activities of that protein are significantly reduced in the host cell, typically by at least 25%, such as at least 50%, such as at least 75%, such as at least 90%, such as at least 95%, as compared to a control. Typically, when the reduced activity is obtained by a genetic modification in the host cell, the control is the unmodified parent strain. By 'abolished activity' of a protein in a bacterium is meant that one or more specific activities of that protein are essentially absent, absent or undetectable in the bacterium.

[0056] Genetic modifications resulting in reduced or abolished activity of a target protein can include a mutation or deletion in the coding sequence of that protein which results in the expression of nonfunctional or less functional protein. Furthermore, genetic modifications resulting in reduced or abolished expression and / or activity of a target gene, as used herein, may be indirect, meaning that they are not genetic modifications in the gene itself. Such genetic modifications may for example include the introduction of a nucleic acid sequence that reduces the expression of the target gene, e.g., a repressor that inhibits expression of the target gene.

[0057] Standard recombinant DNA and molecular cloning techniques useful for carrying out embodiments of the present invention are well known in the art and are described by, e.g., Sambrook, J., Fritsch, E. F., and Maniatis, T. (2012). Molecular cloning: A laboratory manual, 4th ed. Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, and by Silhavy, T. J., Bennan, M. L., and Enquist, L. W. (1984). Experiments with gene fusions. Cold Spring Harbor Laboratory: Cold Spring Harbor, New York. Techniques for targeted genome editing, such as knock-out of a target gene in a bacterial genome, include Clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, such as CRISPR-Cas9.

[0058] Specific embodiments of the invention

[0059] 1staspect of the invention and embodiments thereof

[0060] The invention, in a first aspect, relates to a thermophilic bacterium comprising a carbonic anhydrase, wherein the carbonic anhydrase is attached to and / or expressed on the surface of the thermophilic bacterium.

[0061] By attaching and / or expressing the CA on the surface of the thermophilic bacterium, the sequestration and carbonation process can be localised and greatly improved.

[0062] Fig. 1 shows a thermophilic bacterium according to embodiments of the invention.

[0063] The thermophilic bacterium may have an optimal growth temperature of at least 50°C, such as in a range between 50°C and 64°C (i.e., it may be a simple thermophile), in a range between 65°C and 79°C (i.e., it may be an extreme thermophile), or such as at least 80°C (i.e., it may be a hyperthermophile); preferably it is in a range between 50°C and 64°C, such as around 60°C. The thermophilic bacterium preferably belongs to a Parageobacillus species, more preferably to Parageobacillus thermoglucosidasius .

[0064] Parageobacillus and P. thermoglucosidasius bacteria have several advantages, for example they exhibit fast growth and are naturally heat-tolerant at alkaline pH (optimum growth pH of the native species is around pH 8.0). Further, P. thermoglucosidasius utilizes multiple glycogens and shows potential for biomineralization.

[0065] In some embodiments, the thermophilic bacterium belongs to a species selected from any one of the following: Thermus thermophilus, Caldicellulosiruptor bescii, Thermoanaerobacter kivui, Sulfolobus isiandicus Thermotoga maritima, Thermococcus barophilus, and Pyrococcus furiosus.

[0066] In some embodiments, the thermophilic bacterium belongs to a Parageobacillus species selected from any one of the following: P. caidoxyiosiiyticus, P. galactosidasius, P. genomosp. 1, P. thermantarcticus, P. toebii, and P. yumthangensis.

[0067] In some embodiments wherein the bacterium belongs to the Parageobacillus thermoglucosidasius species, it may belong to the DSM2542 strain; a strain which was previously deposited with DSM no. 2542 at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, and which was described in Suzuki, Y., et al. (1983) Bacillus thermoglucosidasius sp. nov., a new species of obligately thermophilic bacilli. Syst. Appl. Microbiol. 4: 487-495.

[0068] Thermophilic hosts have other benefits than remaining stable under harsh conditions. They also reduce the risk of contamination during the fermentation process and enhance the efficient expression of thermal heterologous proteins. The latter minimizes the risk of misfolding and contributes to a broader spectrum of biologically functional thermo-enzymes, typically being overexpressed in their active forms.

[0069] In a preferable embodiment, the carbonic anhydrase is obtained or derived from a second thermophilic bacterium selected from: Sulfurihydrogenibium azorense and a Persephonella species, wherein the Persephonella species is optionally selected from any one of: Persephonella hydrogeniphila, Persephonella atlantica, Persephonella marina, and Persephonella sp. KM09-Lau-8, preferably from Persephonella hydrogeniphila, or is a variant or fragment thereof.

[0070] The 'second' thermophilic bacterium, in contrast to the thermophilic bacterium according to the first aspect of the invention, refers to the thermophilic bacterium from which the CA as described in the first aspect of the invention may be derived, and the second thermophilic bacterium may either belong to the same species as the thermophilic bacterium according to the first aspect of the invention, or it may belong to a different species of thermophilic bacteria.

[0071] In some embodiments, the thermophilic bacterium according to the 1staspect of the invention belongs to the Parageobacillus thermoglucosidasius species, and the CA which is attached to and / or expressed on its surface is also derived from Parageobacillus thermoglucosidasius (i.e., the second thermophilic bacterium).

[0072] In other, preferred, embodiments, the thermophilic bacterium according to the 1staspect of the invention belongs to a Parageobacillus species, such as Parageobacillus thermoglucosidasius, and the second thermophilic bacterium belongs to a Persephonella species, such as Persephonella hydrogeniphila.

[0073] S. azorense (Saz) is a bacterium naturally found in hot springs and hydrothermal vents, and the carbonic anhydrase (SazCA) obtained from Saz is one of the most efficient thermostable CAs discovered to date. Bacteria belonging to the Persephonella genus are Aquificales, which are Gram- negative bacteria that oxidize hydrogen and sulphur in hydrothermal vent systems. These bacteria are known to grow at high temperatures between 60°C to about 90°C, however certain species have been isolated from harsh, excessive temperature conditions.

[0074] By obtaining or deriving the CA from a thermophilic bacterium such as Saz or a Persephonella species, the CA may naturally be able to metabolise carbon dioxide at high temperature conditions. Table 1. Selected carbonic anhydrases from different thermophilic bacteria

[0075] In the table above, PhyCA is from Persephonella hydrogeniphila, PaCA is from Persephonella atlantica, PlauCA is from Persephonella sp. KM09-Lau-8, TaCA is from Thermovibrio ammonificans, and SazCA is from Sulfurihydrogenibium azorense.

[0076] In preferred embodiments, the carbonic anhydrase is PhyCA.

[0077] As such, in some embodiments, the carbonic anhydrase comprises or consists of a polypeptide with the amino acid sequence of SEQ ID NO: 1.

[0078] In further embodiments, the carbonic anhydrase comprises or consists of a polypeptide having at least 60% sequence identity with SEQ ID NO: 1, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, and such as at least 99%.

[0079] In further embodiments, the carbonic anhydrase has at least 90% of the CO2 hydration activity of the carbonic anhydrase consisting of a polypeptide with the amino acid sequence of SEQ ID NO: 1, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, and such as at least 100%, as determined by a Wilbur-Anderson assay at 50°C, after 1 hour of incubation.

[0080] In other preferred embodiments, the carbonic anhydrase is SazCA.

[0081] As such, in some embodiments, the carbonic anhydrase comprises or consists of a polypeptide with the amino acid sequence of SEQ ID NO: 5.

[0082] In further embodiments, the carbonic anhydrase comprises or consists of a polypeptide having at least 60% sequence identity with SEQ ID NO: 5, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least

[0083] 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least

[0084] 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least

[0085] 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, and such as at least 99%.

[0086] In further embodiments, the carbonic anhydrase has at least 90% of the CO2 hydration activity of the carbonic anhydrase consisting of a polypeptide with the amino acid sequence of SEQ ID NO: 5, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, and such as at least 100%, as determined by the Wilbur-Anderson assay at 50°C, after 1 hour of incubation.

[0087] In other embodiments, the carbonic anhydrase comprises or consists of a polypeptide with an amino acid sequence selected from any one of SEQ ID NOs: 2, 3 and 4.

[0088] In further embodiments, the carbonic anhydrase comprises or consists of a polypeptide having at least 60% sequence identity with an amino acid sequence selected from any one of SEQ ID NOs: 2, 3 and 4, such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, and such as at least 99%.

[0089] In further embodiments, the carbonic anhydrase has at least 90% of the CO2 hydration activity of the carbonic anhydrase consisting of a polypeptide with an amino acid sequence selected from any one of SEQ ID NOs: 2, 3 and 4, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, and such as at least 100%, as determined by the Wilbur-Anderson assay at 50°C, after 1 hour of incubation.

[0090] In some embodiments, the carbonic anhydrase maintains at least 90% of its specific CO2 hydration activity at 70°C as compared to at 50°C, as measured by a Wilbur-Anderson assay after 1 hour of incubation. In preferred embodiments, the carbonic anhydrase comprises or consists of a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-5 or a polypeptide having at least 60% sequence identity with any one of these sequences.

[0091] The Wilbur-Anderson assay may, e.g., be performed as described in Example 1, section 1.3. In short, a CCh-saturated solution is mixed with a solution comprising the carbonic anhydrasecomprising bacteria and with a pH indicator, and the time taken for the pH to change from 8.4 to 6.4 is measured and used to calculate Wilbur-Anderson Units (WAU) as a measure of the carbonic anhydrase activity.

[0092] The CA as described in the first aspect of the invention may be genetically modified, engineered or improved, so as to improve the CO2 hydration activity and / or the thermal stability.

[0093] As will be described below, the present inventors have identified CA mutants with improved properties, and CA variants comprising any of these mutations may be attached to and / or expressed on the surface of the thermophilic bacterium according to the first aspect of the invention.

[0094] Thus, in embodiments of the first aspect of the invention, the carbonic anhydrase is the carbonic anhydrase according to the second aspect of the invention or embodiments thereof.

[0095] In yet another embodiment or aspect of the first aspect of the invention, the carbonic anhydrase is attached to the surface of the thermophilic bacterium as a part of a fusion protein comprising at least one lysine motif (LysM) domain.

[0096] By providing a highly active CA from a thermophilic bacterium, such as a Persephonella species bacterium, and attaching or expressing the CA on the surface of a different genus and / or species of thermophilic bacteria than the one the CA was derived from, such as a Parageobacillus genus, such as Parageobacillus thermoglucosidasius , the carbon dioxide hydration activity may be maintained at high temperatures.

[0097] LysM was found by the present inventors to be the most efficient anchoring motif for displaying CA on the surface of a thermophilic bacterium, such as P. thermoglucosidasius (see Example 1, section 2.2). LysM, which originates from bacterial lysins, can bind directly to peptidoglycan and can be located either at the N- or C-terminus of the target domain, exhibiting notable specificity and stability.

[0098] Preferably, the LysM domain is located C-terminally to the CA in the fusion protein. This was found to increase CO2 hydration activity, as compared to the N-terminal placement relative to the CA, by a notable 1.79-fold (44.05U / mL / OD6oo) (see Example 1, Fig. 4B).

[0099] The inventors of the present invention found that the successful establishment of a LysM-based cell surface display platform for a CA not only proves the effectiveness of immobilizing the enzyme and enhancing its stability under extreme conditions, but also harnesses the potential of the bacterial surface to facilitate CaCCh crystallization, a crucial stage in the process of carbon capture and storage.

[0100] The fusion protein may further comprise a long, flexible linker, such as a Fibronectin-binding protein B (FnBPB) linker, between the carbonic anhydrase and the LysM domain. The long, flexible linker was found to contribute to an improvement in activity (Example 1, Fig. 4B).

[0101] In an embodiment, the thermophilic bacterium has been genetically modified to reduce or abolish the expression and / or activity of at least one serine protease in the bacterium, as compared to an unmodified parent strain.

[0102] Parageobacillus strains, and strains of other thermophilic bacteria, may produce proteases that reduce the yield and stability of heterologous proteins, both secreted and cell-wall associated. This may cause a challenge for displaying CA on the cell surface, especially with long linker structures. Therefore, by reducing or abolishing the expression and / or activity of protease-encoding genes, the CA-display on the cell surface may become more efficient and stable. One way to achieve this beneficial effect is to knock-out or delete one or more serine protease-encoding genes in the bacterium, thus providing a protease-deficient strain. For example, a markerless approach may be used, which does not introduce antibiotic resistance determinants into the mutated strains.

[0103] Exemplary proteases which may be knocked-out or deleted are, e.g., extracellular 'feeder' proteases, such as the proteases with NCBI references AOT13_01245 and AOT13_01525, intracellular 'feeder' proteases, such as the proteases with NCBI references AOT13_01905 and AOT13_10345, and 'quality control' or other types of proteases, such as the proteases with NCBI references AOT13_03220 and AOT13_06160. The genes encoding these proteases may be deleted individually or in combination. In preferred embodiments, the at least one serine protease comprises AOT13_01245, AOT13_01525, or a combination thereof, such as a combination comprising AOT13_01245, AOT13_01525, AOT13_01905, AOT13_10345, AOT13_03220, and AOT13_06160.

[0104] By deleting at least one serine protease-encoding gene, an increase, such as an increase of about 2.5-fold, in carbon dioxide hydration activity may be achieved for cell surface-displayed CAs and an increase of about 5.6-fold may be achieved for secreted CAs. The higher impact on the secreted CA may be, without being bound by theory, because the secreted enzyme is more exposed to proteolytic cleavage.

[0105] The knockout of serine proteases appears to significantly enhance the rate of CaCCh formation in comparison to the wild type. Interestingly, in both the wild type and the protease-deficient host, the cell surface display led to a precipitous slope. This suggests that bacterial surface display promotes the creation of nucleation sites for calcium carbonate, thereby accelerating the generation of CaCCh. As described in more detail in Example 1, section 2.5, there already exists a steep gradient of Ca2+ions between the intracellular and extracellular compartment in bacteria, where extracellular free Ca2+concentrations can be as much as 100-1000 times higher than their intracellular counterparts. However, a further accumulation of calcium ions outside of the cell, particularly in the extracellular space, may lead to an improvement in the calcium carbonate formation rate.

[0106] An increased extracellular Ca2+concentration may be achieved by overexpressing calcium binding proteins (CaBPs). This kind of protein is characterized by a helix-loop-helix structural motif, where Ca2+ions are bound within the loop. CaBPs which are compatible with the host system at elevated temperatures may beneficially be chosen. The CaBP may, e.g., be the dockerin Docl24A, such as Docl24A from Acetivibrio thermocellus (structure PDB code 4DH2). As shown in Example 1, section 2.6, surface display of Docl24A increased the CaCCh formation rate of the whole-cell biocatalyst.

[0107] Another strategy involves accelerating calcium influx and efflux by overexpressing the Ca2+pumping system. Calcium is exported by Ca2+exchangers, such as Ca2+ / H+or Ca2+ / Na+antiporters. For this purpose, Ca2+exchangers which are compatible with the host system at elevated temperatures may beneficially be chosen.

[0108] Thus, in an embodiment, the thermophilic bacterium has been genetically modified to overexpress and anchor at least one calcium binding protein on the surface of the bacterium, and / or the thermophilic bacterium has been genetically modified to overexpress at least one calcium exchanger-encoding gene, wherein the overexpression is compared to an unmodified parent strain.

[0109] In some further embodiments, the calcium binding protein is a dockerin, such as a dockerin sourced from Acetivibrio thermocellus, which thrives in temperatures ranging from 50°C to 68°C. This dockerin consists of two subunits: 4dh2, which can bind two Ca2+ions, and 2xqo, which can bind one Ca2+ion. Although this protein has not undergone extensive biochemical or functional studies regarding calcium binding, it has been reported that Ca2+binding induces folding of the dockerin domain, thereby increasing its stability.

[0110] The at least one calcium exchanger-encoding gene may be, e.g., chaA and / or yloB, which may boost the influx and efflux of calcium.

[0111] 2ndaspect of the invention and embodiments thereof

[0112] In a second aspect, the invention relates to a carbonic anhydrase having at least 72% sequence identity with SEQ ID NO: 1 and having an amino acid selected from A, H, Y, and Q in the position corresponding to position 88 of SEQ ID NO: 1.

[0113] In one embodiment or aspect, the sequence identity is at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least

[0114] 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least

[0115] 97%, such as at least 98%, and such as at least 99%.

[0116] SEQ ID NO: 1 refers to the amino acid sequence of the Persephonella hydrogeniphila carbonic anhydrase (PhyCA) with NCBI Reference Sequence: WP_096999253.1.

[0117] Proton transfer has been identified as a rate-limiting step in the hydration of carbon dioxide in CAs, with a number of residues lined up in the catalytic cavity to carry out this reaction. Without being bound by theory, K88 present in the native CA of Persephonella hydrogeniphila (PhyCA; SEQ ID NO: 1) is considered to be involved in proton shuttling together with residues Y26, N83, T196, and T197. Interestingly, all these residues, except from K88, were found to form hydrogen bonds as both proton donors and acceptors, whereas K88 acts merely as a donor. A lysine (K) in the position corresponding to position 88 in PhyCA also appears to be semi-conserved across CAs, while some CAs, including SazCA, have a glutamine (Q) in that position.

[0118] As shown herein by the present inventors (see Example 2 and Fig. 8), by using rational mutagenesis on PhyCA, the CO2 hydration activity could be increased to match or surpass that of SazCA, the most active bacterial CA to date. The first investigated mutation in PhyCA was K88Q, since SazCA has a Q residue in the position corresponding to position 88 in PhyCA. K88 was also mutated to either histidine (H), tyrosine (Y) or alanine (A). The rationale for choosing H and Y was that these were found to be possible proton shuttling residues, while the non-reactive A was chosen to investigate the effect of the absence of this proton shuttle.

[0119] The results for mutant K88A were quite unexpected. Not only was this mutant very active but actually more active than wildtype (WT) PhyCA. Specific activity for this mutant was 3-fold higher than the WT at 90°C. The inventors also detected a surprising temperature activation, as the activity increased from 60°C to 70°C.

[0120] Thermostability of K88H was comparable to that of the WT, maintaining a similar activity after incubation at temperatures from 30°C up to 70°C, followed by a decline at 80°C and a sharp decrease at 90°C. Specific activity for this mutant, however, was high, similarly to that of K88Q, and significantly higher than for the WT.

[0121] Compared to K, Q is a relatively medium sized and uncharged residue. For mutant K88Q, a similar thermostability trend was observed as for the WT. Incubation of the enzyme variant K88Q at temperatures up to 80°C did not affect residual activity, whereas incubation at 90°C resulted in a drastic drop in activity. This variant interestingly had a more than 3-fold higher activity compared to the WT at 30°C. K88Q had comparable activity to SazCA at higher temperatures, from 60°C to 80°C, but both are still surpassed by K88Y at 90°C. Mutant K88Y was less active than the other mutants, showing similar specific activity to the WT but exhibiting surprisingly higher thermal stability than all other three variants as well as the WT. Similarly to K88A, a temperature activation at 70°C was observed.

[0122] Thus, in some embodiments, the amino acid in the position corresponding to position 88 of SEQ ID NO: 1 is A. In some embodiments, the amino acid in the position corresponding to position 88 of SEQ ID NO: 1 is H. In some embodiments, the amino acid in the position corresponding to position 88 of SEQ ID NO: 1 is Y. In some embodiments, the amino acid in the position corresponding to position 88 of SEQ ID NO: 1 is Q.

[0123] In some embodiments, the carbonic anhydrase maintains at least 90% of its specific CO2 hydration activity at 70°C as compared to at 50°C, as measured by a Wilbur-Anderson assay after 1 hour of incubation.

[0124] The Wilbur-Anderson assay may, e.g., be performed as described in Example 2, section 1.3. In short, a CO2-saturated solution is mixed with a solution comprising the carbonic anhydrase and with a pH indicator, and the time taken for the pH to change from 8.4 to 6.4 is measured and used to calculate Wilbur-Anderson Units (WAU) as a measure of the carbonic anhydrase activity.

[0125] In some embodiments, the carbonic anhydrase is attached to and / or expressed on the surface of a thermophilic bacterium according to the first aspect of the invention or any embodiments thereof.

[0126] 3rdand 4thaspects of the invention and embodiments thereof

[0127] In a third aspect, the invention relates to a use of the thermophilic bacterium according to the first aspect or the carbonic anhydrase according to the second aspect for capturing carbon dioxide from a gaseous mixture, such as flue gas.

[0128] Advantageously, the presently disclosed thermophilic bacteria with surface-display of CA can withstand high temperatures and have a high activity.

[0129] In a fourth aspect, the invention relates to a method of capturing carbon dioxide and forming an alkaline earth metal carbonate, the method comprising a step of: contacting carbon dioxide with the thermophilic bacterium according to the first aspect or the carbonic anhydrase according to the second aspect and alkaline earth metal ions, so as to form one or more alkaline earth metal carbonate(s), and optionally, extracting the alkaline earth metal carbonate(s).

[0130] The alkaline earth metal carbonate(s) may comprise calcium carbonate and / or magnesium carbonate, and the alkaline earth metal ions may comprise calcium and / or magnesium ions. The alkaline earth metal ions may be provided in the form of a salt, such as CaCh or MgCh. The method may be carried out directly at the source, e.g., at an industrial power plant, or the flue gas may be directed to a bioprocessor, preferably on the site of the industrial power plant, for processing the flue gas. From the bioprocessor, carbon dioxide free gas may be released, whereas stable carbonates are produced to fix carbon dioxide into a stable, commercially useful form and product.

[0131] The metal carbonate produced may be directly used, e.g., in the cement industry. For example, calcium carbonate and / or magnesium carbonate are also valuable products in the pharmaceutical, glass, ceramics, steel, paints and inks, papermaking, plastics, rubber, adhesive, and sealant industries.

[0132] EXAMPLE 1

[0133] Surface engineering of thermostable Parageobacillus thermoglucosidasius for enhanced CO; biomineralization

[0134] Novel approaches are needed for capturing CO2 efficiently, particularly to create an effective catalyst suitable for industrial processes and capable of continuous capture and reuse. In this Example, the inventors explored the possibility of making a microbial cell that can be used to capture CO2 from flue gas via biomineralization. This would in essence comprise a whole-cell biocatalyst that expresses a CA on its surface, allowing for both expression and immobilization of CA. Such a whole-cell biocatalyst would cause conversion of CO2 to carbonate, that in presence of Ca2+would precipitate, using the cell as a nucleation point. Additionally, the inventors optimized the structure of the anchoring system, eliminated proteases that could disrupt the stable expression of cell-displayed carbonic anhydrase, and regulated the Ca2+transport mechanism to enhance the interaction between Ca2+and CCh2’.

[0135] Materials and methods

[0136] 1.1 Bacterial strains, culture conditions, and transformation

[0137] E. coll DH5a was used as a cloning strain and grown in lysogeny broth (LB; 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCI) or on LB agar plates at 37 °C, supplemented with kanamycin (50 pg / mL) or 100 pg / mL ampicillin when appropriate. The transformation of E. coll was conducted with the heat shock method.

[0138] P. thermoglucosidasius DSM2542 (CP012712.1) was used as the host strain for expression of the carbonic anhydrases herein. For routine cultivation, P. thermoglucosidasius was grown in 2SPY / 2SPYNG medium (16 g / L soy peptone, 10 g / L yeast extract, NaCI 5 g / L) with 250 rpm shaking or plating on TSA agar (40 g / L TSA) with 250 rpm shaking or plating on TSA agar plates at 52-66°C when appropriate. 12.5 pg / mL kanamycin was added for plasmid selection. For promoter screening and calcium transporter tuning, minimal medium TMM or TMM agar was used (Pogrebnyakov et al., 2017). Transformation of P. thermoglucosidasius by electroporation was carried out as described previously by Cripps, R. E., et al. (Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production. Metabolic engineering 11, 398-408 (2009)).

[0139] 1.2 DNA manipulation and plasmid construction

[0140] In an exploration of carbonic anhydrase candidates' expression, MICA (WP_036384960.1), hCAII (NP_000058.1), SazCA (WP_012674680.1), TaCA (WP_013538320.1), ApCA (WP_094245285.1) and Cab (WP_048061095.1) underwent the removal of the native signal peptide and were codon- optimized. Then each gene was cloned with a modular shuttle vector (pGlAK) for Geobacillus and E. coll under the control of a strong promoter, Prpis, and a signal peptide derived from endo-l,4-b- xylanase in P. thermoglucosidasius C56-YS93. The assembly process employed Gibson assembly techniques for efficient construction.

[0141] For cell-surface display platform establishment and anchoring motifs selection, LytE (WP_003244816.1, amino acid 25-232), derived from the cell wall hydrolase protein in B. subtilis, can bind to the peptidoglycan layer of the cell wall without causing lysis of the cell; LysM (WP_013400400.1, P. thermoglucosidasius, amino acid 1-171) was identified as a homologous series with LytE, with a protein similarity of 91%; PG (YP_009010493.1, P. thermoglucosidasius, amino acid 164-215), derived from the endolysin protein in P. thermoglucosidasius bacteriophage, is responsible for binding to the cell wall through peptidoglycan binding; IPxTG (WP_185222476.1, P. thermoglucosidasius, amino acid 450-486) has been previously utilized in Lactobacillus plantarum for successful cell-surface display of chitosanase; SLH protein (WP_042384074.1, P. thermoglucosidasius, amino acid 30-213) can bind bacterial cell wall polysaccharides; and the flexible linker, FnBPB polypeptide (ABD31805.1, amino acid 770-880), has been used as a tool to connect different proteins and enable them to penetrate through the peptidoglycan layer of the bacterial cell wall in B. subtilis. All anchoring motifs were fused with SazCA and cloned in pGlAK utilizing Gibson assembly.

[0142] Secreted proteases were identified through a bioinformatics approach. In particular, ORFs for P. thermoglucosidasius DSM2542 (CP012712.1) annotated with "protease" or "peptidase" were selected from the NCBI CDS database and the Prosite web server. Further filters were applied based on sequence similarity with Bacillus spp. serine proteases, which accounted for 99% of the extracellular protease activity along with neutral metalloproteases. The resulting proteases were then analyzed with the SignalP 5.0 server to identify putative signal peptides and the TMHMM servers to predict the subcellular location. 6 proteases (AOT13_01245, AOT13_01525, AOT13_01905, AOT13_03320, AOT13_06160, AOT13_10345) were individually or jointly knocked out, leading to the development of mutant strains devoid of proteases. Homology cassettes were amplified using P. thermoglucosidasius DSM2542 genomic DNA as the template and spliced by overlap extension polymerase chain reaction (SOE-PCR). Subsequently, these cassettes were assembled into an improved shuttle vector called Pmm5 using the Gibson assembly technique. Integration of the constructed vectors into the P. thermoglucosidasius genome was achieved via the double-crossover method described previously (Liu, Y. et al. Towards next-generation model microorganism chassis for biomanufacturing. Appl Microbiol Biotechnol 104, 9095-9108 (2020)).

[0143] For calcium mechanism optimization, the calcium binding proteins CtCell24 (WP_011837830.1, amino acid 131-350, structure PDB code 2xqo) and Docl24A (WP_011837830.1, amino acid 32- 112, structure PDB code 4DH2), derived from Acetivibrio thermocellus, were tested. These proteins were codon-optimized and anchored on the cell wall in the pGlAK vector under the promoter Prpisand anchoring motif LysM through Gibson assembly. Ca2+ / H+antiporter ChaA (AOT13_05630, P. thermoglucosidasius') facilitates Ca2+efflux and H+influx as the pH increases. Ca2+transporting ATPase YloB (AOT13_08805, P. thermoglucosidasius') is responsible for the uptake of Ca2+. The expression level of both were regulated by swapping strong promoter Pidh and inducible promoter PtetR-p by using a CRISPR-Cas9 system according to the protocol described in Lau, M. S. H., et al. Development of a Suite of Tools for Genome Editing in Parageobacillus thermoglucosidasius and Their Use to Identify the Potential of a Native Plasmid in the Generation of Stable Engineered Strains- Supplemary. ACS Synthetic Biology 59, 1-14.

[0144] Genomic DNA was extracted using the PureLink™ Genomic DNA Mini Kit (Invitrogen) while plasmid extractions were performed using the NucleoSpin® Plasmid EasyPure kit (Macherey-Nagel). Phusion U Hot Start DNA Polymerase (Thermo Fisher Scientific) was used for amplification of USER cloning fragments, while other routine PCR reactions were done using Phusion II Hot Start DNA Polymerase (Thermo Fisher Scientific). To identify positive constructs for subsequent downstream analysis, colony PCR was carried out using OneTaq® DNA Polymerase (NEB) and DNA sequencing (Eurofins).

[0145] 1.3 CO? hydration assay

[0146] The Wilbur-Anderson assay was utilized to determine the catalytic activity of CA. The procedure involved adding 0.1 ml of cell culture to 3 ml of an ice-cold Tris-HCI buffer (20 mM, pH 8.3) with stirring at 0°C. Subsequently, 2 ml of a CO2-saturated water was promptly added to the mixture. The time for decrease in pH from 8.3 to 6.3 (as estimated by the colour change of indicator bromothymol blue) was recorded. The activity of CA was calculated using the following equation: where tCOntroi and ttest are the time for the pH drop in the control and experimental group, respectively, df represents the dilution factor of cell culture, and Vo is the volume of diluted cell culture adding into the reaction (0.1 mL in our study). 1.4 SDS-PAGE and Western blotting

[0147] The subcellular localization of SazCA was analyzed using SDS-PAGE followed by Western blotting. Extracellular medium, cell wall, and cytoplasm fractions were produced following the previously described method (Holland, A. T. N., Danson, M. J. & Bolhuis, A. Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius. BMC Biotechnology 19, 1-8 (2019)) with minor modifications. P. thermoglucosidasius was grown to an ODeoo of 1.5 at 60°C and harvested by centrifugation at 4000xg for 10 min at 4°C, with the supernatant filtered through a 0.22-pm membrane (Millipore) and stored as the extracellular medium fraction. The cell pellet was washed 3 times and resuspended in protoplast buffer (50 mM Tris-HCI, 20% sucrose, 1 mg / mL lysozyme, 0.1% PMSF, 0.1% protease inhibitor cocktail, 50 mM EDTA, pH 7.6). After incubation for 30 min at 37°C, protoplasts were centrifuged at 700xg for 10 min. The supernatant was collected as the cell wall fraction, and the pellet containing the protoplasts as cytoplasmic fraction. The fractions were then boiled, and the proteins separated by SDS-PAGE using 4-20% mini- Protean TGX stain-free gels (Bio-Rad Laboratories, Hercules, CA, USA). The protein bands were transferred to PVDF membrane using the iBIot™ 2 gel transfer device (Invitrogen). Detection of FLAG-tagged / His-tagged SazCA was performed using Monoclonal Anti-FLAG® BioM2 antibody produced in mouse (Sigma-Aldrich) / Monoclonal Anti-6X His tag® antibody (Sigma-Aldrich) produced in rabbit and HRP-conjugated Goat Anti-Mouse IgG H&L (Abcam) / HRP-conjugated Goat Anti-Rabbit IgG H&L (Abeam), according to the manufacturer's instructions. Total protein served as a loading control for the purpose of normalization and guantification in accordance with the manufacturer's instructions (Taylor, S. C. & Posch, A. The Design of a Quantitative Western Blot Experiment. BioMed Research International 2014, e361590 (2014)).

[0148] 1.5 Immunofluorescence microscopy

[0149] Strains harboring different plasmids were cultured to an ODeoo of 1.5 at 60°C and harvested though centrifugation with 4000xg for 10 min at 4°C, followed by washing with phosphate buffered saline (PBS, pH 7.4) three times and fixing with 4% paraformaldehyde. After blocking using 1% bovine serum albumin for 1 h, the cells were incubated with Alexa Fluor® 647 Anti-6X His tag® antibody (Abeam) overnight, then washed 3 times with PBS. Control samples were obtained by processing the P. thermoglucosidasius strain with the vector backbone in the same manner. The treated cells were visualized on a Leica Microsystems Confocal Microscope (Leica, SP8, LMI-005), with nuclei counterstained by prior incubation with 2 drops / mL DAPI (NucBlue Fixed Cell ReadyProbes Reagent, Invitrogen) for 15 minutes at room temperature. 1.6 Protease activity assay

[0150] The hydrolyzing activity of the protease was determined using both an independent substrate like casein and a specific substrate such as commercial bovine CA and purified SazCA from the inventors' lab. Each strain was cultured, normalized to OD6oo=l with SPY medium, and homogenized using a Precellys 24 touch Homogenizer (Bertin Technologies) to obtain the supernatant, referred to as the protease solution. SPY medium only served as the control.

[0151] When using casein as the substrate, the protease activity assay was conducted following Sigma's universal protocol with a modification in the incubation temperature set at 60°C. Initially, 5 mL of 0.65% casein, pre-equilibrated at 60°C, was added to each protease solution. After a 10-minute incubation at 60°C, the reaction was stopped by adding 5 mL of 110 mM TCA and incubating for an additional 30 minutes. Subsequently, the reaction mix was supplemented with 0.5 M sodium carbonate and 1 mL of Folin's reagent. Colour development was quantified using a spectrophotometer, measuring absorbance at 660 nm. One unit of enzyme activity was defined as the volume of enzyme required to induce a 1-unit absorbance increase per minute.

[0152] When employing BCA and SazCA as the substrates, the protease activity was based on the residual CO2 hydration activity of digested SazCA or BCA. Specifically, 1 mL of protease solution was combined with either 0.5 mL of 5 mg / mL BCA or 1.5 mg / mL SazCA, and the mixture was incubated for 12 hours. Subsequently, the reaction mixture was then diluted, and the CO2 hydration activity assay was conducted. The protease activity was calculated by the following formula.

[0153] Protease activity are the CO2 hydration activity in the control and experimental group, respectively.

[0154] 1.7 CO mineralization

[0155] CaCO3production yield

[0156] The precipitation method used in this study was largely based on the protocol previously described by Koo et al. (Robust Biocatalysts Displayed on Crystalline Protein-Layered Cells for Efficient and Sustainable Hydration of Carbon Dioxide-Supplementary materials. Advanced Functional Materials 31, (2021)). In particular, CO2 was bubbled through 50 mL of 1 M Tris-HCI (pH 9) solution containing the whole-cell biocatalyst (OD6oo=0.3) for 30 seconds. Subsequently, the mixture was filled with a solution of CaCl2 to achieve a final concentration of 0.5 g / mL. The mixture was then incubated at 60°C for 1 hour, after which the precipitate was separated from the solution by passing through qualitative filter paper (Whatman, gradel) utilizing a glass vacuum distiller. After being dried in the oven at 60°C overnight, the mass of the precipitate was determined by weighing and normalizing with the mass of the empty filter paper and the added cells. Control was also established in the Tris- HCI solution containing CaCh, but without the whole-cell biocatalyst.

[0157] CaCO3formation rates

[0158] The whole-cell biocatalysts were diluted to OD6oo=0.3 in 1 M Tris-HCI (pH 9). In a 24-well plate (Greiner Bio-One), 1 mL of this suspension was mixed with 1 mL of a 3 M CaCh solution in IM Tris- HCI (pH 9) (Jo, Byong H. et al. "Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration", Appl Environ Microbiol. 2013 Nov;79(21):6697-705). Then the plate was placed in the ELx808TM microplate reader (BioTek) with shaking. After the addition of 1 mL of CO2-saturated water to the wells, the absorbance at 600 nm was monitored for lh. The resulting curves were then fitted to the Hill equation (see, e.g., Weiss, James N. "The Hill equation revisited: uses and misuses." The FASEB Journal 11.11 (1997): 835-841), the slope for the exponential stage was defined as "CaCOs formation rates".

[0159] Crystal characterization

[0160] The CaCOs precipitates, along with the associated bacteria described in section 2.6, were collected and subsequently fixed using glutaraldehyde for lh. Following this, the specimens underwent dehydration through a gradient concentration of ethanol. The crystal morphology was verified and imaged using Scanning Electron Microscopy (FEI Quanta FEG 250) with energy-dispersive X-ray spectroscopy (EDS), operated at a 10 kV acceleration voltage. The identification and polymorphs of dried precipitates was carried out through X-ray powder diffraction (XRD) techniques with a Malvern Panalytical Empyrean diffractometer, equipped with a IDer detector, using Cu Ko (A = 1.5406 A) radiation in reflection mode at a voltage of 45 kV and a current of 40 mA. Bulk samples were measured at room temperature on a reflection / transmission spinner between 20 = 4-90° with a step size of 0.008° at a scan speed of 0.08° / s. And the thermal properties were analyzed by Thermal Gravimetrical Analysis (TGA, Mettler, Columbus, OH, USA) with a heating rate of 10°C / min. Samples were heated from 35°C to 950°C in a stream of N2 with a flow rate of 20 mL / min as following established procedures (Clara Saracho, A. et al. Controlling the calcium carbonate microstructure of engineered living building materials. J. Mater. Chem. A 9, 24438-24451 (2021)).

[0161] 1.8 Stability characterization

[0162] The residual catalytic activity was measured using the Wilbur-Anderson assay as detailed in section 1.3. All data were obtained by the mean of three independent determinations. Prior to conducting any stability characterization experiments, the bacteria were cultured overnight at 60°C and collected as whole-cell biocatalysts, then diluted and normalized to ODeoo = 1 with SPY medium. In the thermal stability assay, the bacteria were subjected to various heat treatments, ranging from 60°C to 90°C, within sealed containers to prevent buffer evaporation. The exposure duration ranged from 1 to 20 days.

[0163] In the pH stability assay, the bacteria were mixed with pH working buffers covering a pH range of 4 to 11, in a 1: 3 ratio. These samples were then stored in sealed containers at 4°C for periods ranging from 1 to 24 hours. Tris-HCI and glycine-NaOH were used as pH buffers for the pH ranges of 4 to 8 and 9 to 12, respectively.

[0164] In the calcium tolerance assay, the bacteria were mixed with calcium chloride solutions to achieve final calcium concentrations of 10, 50, 100, 200, 500, and 1000 mM. Subsequently, they were incubated at 4°C for 24 hours within sealed containers.

[0165] 1.9 Statistical analysis

[0166] All the graphs, calculations, and statistical analyses were performed using GraphPad Prism software version 9.0 (GraphPad Software, San Diego, CA, USA). Each analytical result is presented as the mean of triplicate sample measurements. The comparison of means between different groups of numerical variables was performed using one-way ANOVA followed by Dunnett's multiple comparisons test, and a p-value less than 0.05 (p < 0.05) was considered statistically significant.

[0167] Results

[0168] 2.1 Selection of optimal carbonic anhydrase candidates

[0169] First, the goal was to identify a suitable CA, which ideally possess high catalytic efficiency and robustness under harsh conditions, including elevated temperature, pH extremes and the presence of metal ions, as well as Cl-, SO42-, NOs- commonly found in flue gases. For this purpose, the present inventors evaluated six thoroughly characterized CA candidates, cf. Fig. 2A (MICA, CA from Micrococcus luteus; hCAII, human; SazCA, Sulfurihydrogenibium azorense; TaCA, Thermovibrio ammonificans; ApCA, Aerbaciiius paiiidus,- and Cab, Methanobacterium thermoautotrophicum). MICA served as the baseline in this study.

[0170] To express and secrete the CAs in the host cell, P. thermoglucosidasius DSM2542, codon-optimized genes that encoded a translational fusion between a signal peptide and the respective CAs, were placed under the control of a strong constitutive promoter.

[0171] To evaluate the transformed strains, the inventors conducted a CO2 hydration activity assay. Both supernatant containing secreted proteins and cell pellets were evaluated. The results demonstrated high CA activity in the samples from strains expressing SazCA or TaCA. Most of the activity was observed in the supernatant fraction. For the remaining four strains, CA activity was detected but was substantially lower compared to that of SazCA and TaCA (Fig. 2A). Moreover, both SazCA and TaCA showed a relatively high temperature stability. After incubation for one day at 60°C or 70°C, SazCA retained 58.2% and 39.5% activity, respectively (Fig. 2C). Even higher stability was observed for TaCA that retained 85.9% and 75.0% activity after incubation at 60°C or 70°C, respectively (Fig. 2D). Alkaline conditions are essential for the formation of CCh2’ and subsequently generation of CaCOs crystals. SazCA exhibited a high pH stability, maintaining after 1 hour of incubation at pH 9, 9.5 or 10 an activity of 100%, 70.9%, and 53.1%, respectively. In contrast, TaCA retained only 49.6%, 42.8%, and 18.5% activity at pH 9, 9.5, and 10, respectively (Fig. 2B).

[0172] 2.2 Screening of functional cell surface anchoring motif candidate

[0173] In order to express SazCA on the cell surface, the inventors fused it with five different anchoring motifs, binding either to the peptidoglycan or S-layer. This led to the following fusion proteins: LytE- SazCA (51 kDa), LysM-SazCA (46 kDa), PG-SazCA (39 kDa), SazCA-LPxTG (36 kDa), and SLH- SazCA (50 kDa). In choosing between N- or C-terminal fusion, the inventors were guided by the organization in the native proteins from which the anchoring motifs were derived. The fusion proteins additionally contained a signal peptide and a FLAG-tag at the N-terminus, and a His6 tag at the C- terminus. For all strains, the growth-rates and final cell densities were similar. The supernatant, cell wall, and protoplast subcellular fractions were carefully separated, and the 5 different CA fusion proteins were analyzed by Western Blot using anti-FLAG® and anti-6X His tag® antibodies. Fig. 3 shows the Western Blots for all five fusion proteins. As expected, the control strain that lacked any anchoring motif primarily secreted SazCA into the medium (Fig 3, top panel, Pt-SazCA_Sec (S)). In contrast, cells expressing LysM-SazCA retained the CA in the cell wall fraction as evidenced by the presence of a band at 46 kDa (Fig 3, top panel, Pt-LysM-SazCA (CW)). The LysM-SazCA fusion protein appeared to undergo partial proteolysis that would cause its release from the cell surface. This was indicated by a weak band observed in the supernatant fraction at ~26 kDa that presumably corresponds to a SazCA degradation fragment (Fig 3, top panel, Pt-LysM-SazCA (S)).

[0174] Among the five different anchoring motif fusion proteins examined, it was observed that LysM-SazCA exhibited the most intensive band in the cell wall fraction. The LysM anchoring motif was therefore chosen for the surface display of SazCA.

[0175] 2.3 Optimization of anchor motif positioning

[0176] Compared to the secreted SazCA, the CO2 hydration of LysM-SazCA decreased from 222.8 to 24.6 U / mL / ODeoo, which revealed the discernible impact of the anchor motif on the enzyme activity (Fig. 4B). We therefore entertained the idea that optimization of the fusion system might improve the performance. This was done by positioning SazCA either N-terminally or C-terminally relative to the LysM motif and by either incorporating, or not incorporating, the long and flexible FnBPB linker that was previously used successfully in B. subtilis (Fig. 4A). It was observed that the C-terminal fusion with LysM (SazCA-LysM) resulted in a 1.8-fold increase in activity (44.1 U / mL / ODeoo). The introduction of the flexible linker in the C-terminal fusion (SazCA-linker-LysM) led to a modest but significant improvement in activity. Overall, the best variant (SazCA-linker-LysM) exhibited a CO2 hydration activity of 63.8 U / mL / ODeoo representing a 2.9-fold increase compared to our initial system (Fig. 4B).

[0177] The inventors observed a reduced CA activity for the surface-display system compared to secreted CA. This could obviously be due to constraints related to enzyme loading on the surface. To elucidate whether structural alterations could also played a role, the inventors performed molecular dynamics (MD) simulations on the modeled structures of SazCA and its fusion complexes. In summary, the MD analyses indicated that the differences in activity among the different SazCA variants could be attributed to structural factors such as dimerization (SazCA is a dimer, whereas the fusion complexes are monomeric due to the interference of LysM with the dimerization interface), interface interactions, active site conformation, and the presence of a flexible linker, which influenced stability, substrate binding, and penetration efficiency through the cell wall.

[0178] The bacterial cell wall has a significantly smaller surface area or volume in contrast to liquids with virtually limitless spatial dimensions, and it acts as a physical barrier, thereby restricting the number of enzymes that can be displayed on the surface. The inventors estimated that the maximum number of surface-displayed enzymes is about 3-4 orders of magnitude lower than the number of secreted enzymes theoretically. It is also worth noting that the surface-displayed enzymes may also compete with other cell wall associated proteins or affect each other's conformation and function. Despite these limitations, the activity measured by the present inventors was only 4.25-fold lower, highlighting the activity of the surface-display strategy and the importance of optimization efforts.

[0179] 2.4 Protease knockout strategy implementation

[0180] The above analysis of different anchor proteins indicated some degree of proteolysis of the fusion protein which in turn might negatively affect the activity of displayed CA. In B. subtilis, strains devoid of up to eight proteases have been shown to lead to reduced degradation of secreted, heterologous proteins. A similar approach was therefore attempted.

[0181] Through sequence homology analysis with B. subtilis serine proteases, six candidates (Table 2) were identified. Two were predicted to encode extracellular proteases (AOT13_01245 and AOT13_01525), two intracellular proteases (AOT13__01905 and AOT13_10345), one putative "quality control" (AOT13__03220), and one other type of protease (AOT13_06160). Employing a marker-less approach that avoids the introduction of antibiotic resistance determinants, those six genes were inactivated individually or in combination. These deletions had little or no impact on cell biomass compared with the wild-type strain. The assessment of protease activity, detected by tyrosine release through Sigma's nonspecific assay using casein as the substrate, revealed no significant differences between any protease-deficient strains and the wild-type strain. However, upon varying the substrate to purified SazCA or commercial bovine CA, clear differences in protease activity emerged. High-copy plasmids were introduced into these protease-deficient strains to express cell surface- displayed SazCA, and their catalytic activity was evaluated (Fig. 5). The deletion of AOT13_01245 or AOT13_01525 resulted in the most significant increase in catalytic activity, confirming that these two may be the dominant proteases. The deletion of other genes had little or no effect on the catalytic activity. By deleting all six proteases, a 2.7-fold increase in CO2 hydration activity was achieved.

[0182] Table 2. Protease prediction and identification

[0183] 2.5 CaCOs formation rate

[0184] The Wilbur-Anderson assay served as a reliable indicator of CO2 hydration activity of CA, but it does not directly reflect the ability of cells to precipitate CaCCh. This is because the assay measures the protons while CO2 sequestration requires the CCh2-ions, which are produced from HCCh’ generated by CO2 hydration. Therefore, it is not valid to assume a single linear relationship between protons and CO32-. Besides, the Wilbur-Anderson assay is performed at low temperature, where the cellbased biocatalyst according to the present invention is envisioned to work at conditions such as high temperature, high pH levels and in the presence of Ca2+. Therefore, the time-dependent solution turbidity test was adopted as a proxy method to determine the precipitation rate. Specifically, the engineered strain was combined with CaCh and CO2 at pH 9, and the resulting change in Abseoo by CaCO3precipitation was monitored, and the initial precipitation rate (the slope of the curve in the linear part) was calculated.

[0185] The detailed analysis results are presented in Table 3. It was evident that the deletion of all 6 proteases combined significantly enhanced the rate of CaCO3formation. Compared to expression of CA in the wild type, the precipitation rate (slope) for strains expressing cellsurface displayed and secreted SazCA in the protease knockout strain increased by 61.7% and 90.9%, respectively. Furthermore, in both wild type and protease-deficient hosts, cell surface display led to a precipitous slope, which means higher CaCO3formation rate. This observation aligns with the expectations that the bacterial surface display system can accelerate the generation of CaCO3by promoting its nucleation. This is grounded in the understanding that the CA enzyme, crucial for carbonate ion production, is localized on the cell wall surface. Given this localization, it is reasonable to anticipate the highest concentration of carbonate ions near the cell surface. Besides, microbial cell surfaces, with their negatively charged properties, act as scavengers for cations, particularly Ca2+ions. These Ca2+ions are not likely utilized by metabolic processes but accumulate outside the cell. It has previously been demonstrated that a Ca2+gradient exists between the intracellular and extracellular compartment, where extracellular free Ca2+concentrations can be as much as 100-1000 times higher than their intracellular counterparts. This disparity effectively places a higher concentration of Ca2+in an area of heightened carbonate ion supersaturation adjacent to the cell surface. Consequently, this effectively minimizes transport distances for reactants, optimizing spatial allocation and ultimately enhancing the overall rate of calcium carbonate formation.

[0186] Table 3. CaCO3formation rates 2.6 Surface expression of calcium binding proteins

[0187] To further pursue the hypothesis described above that the physical proximity of CA to the Ca2+ions bound to the negatively charged bacterial envelope creates a local environment where biomineralization can occur in a confined space, the P. thermoglucosidasius bacteria were further modified to express calcium binding proteins (CaBPs) on their surface, aiming to boost calcium ion accumulation around the displayed CAs. Opting for a CaBP compatible with the host system at elevated temperatures, dockerins from Acetivibrio thermocellus were employed. Dockerin Docl24A (structure PDB code 4DH2) can bind two Ca2+, while CtCell24 (structure PDB code 2XQO) can bind one Ca2+. As shown in Table 4, surface display of Docl24A increased the CaCOs formation rate of the whole-cell biocatalyst, while expression of CtCell24 resulted in no significant improvement.

[0188] Table 4. CO? hydration activity and CaCO? formation rates for different P. thermoglucosidasius strains as indicated. Pt = P. thermoglucosidasius , Sec = secreted. Dis = displayed, Pko = protease knock-out (knock-out of all 6 proteases described above).

[0189] 2.7 CaCO? crystallography

[0190] The present inventors investigated the morphology of CaCCh precipitation across three distinct sample sets: i) CaCh only, iii) engineered bacteria only, and iii) a combination of engineered bacteria and CaCh. These components were introduced into a solution with a pH of 9.5, enriched with saturated CO?. After 1 day of incubation at 60°C, the samples were analyzed for crystallization by Scanning Electron Microscopy (SEM) with Energy-Dispersive X-ray Spectroscopy (EDS), X-ray Diffraction (XRD), and Thermogravimetric Analysis (TGA).

[0191] SEM analysis indicated that in the bacteria-only group, cell aggregation was observed, yet no formation of CaCO?. Conversely, SEM observations revealed the presence of particles in groups i) and iii), which, according to EDS analysis, contained calcium (Ca), carbon (C), and oxygen (O) elements, confirming their identity as calcium carbonate minerals. Specifically, CaCI? as a source of Ca2+in a CO?-saturated solution at high pH resulted in the formation of rhombohedral cubic crystals. XRD analysis confirmed that these crystals were entirely composed of calcite. With the engineered bacteria, more crystals were generated, and these crystals looked more spotted / deformed and slightly larger compared to the uniform calcite crystals in the CaCh group. XRD patterns revealed the presence both of calcite and vaterite phases in the engineered bacteria group. The ratio between the peaks of 29.3° (calcite) and 27.0° (vaterite) was 1:0.36, indicating that these CaCCh crystals comprised 89 wt.% calcite and 11 wt.% vaterite. Remarkably, the morphology of these crystals closely resembled what was previously documented in SEM micrographs, which are spherical and rhomboidal spherulites. The formation of vaterite was attributed to the mediated calcium carbonate precipitation by the engineered bacteria since only the peaks of calcite were found in the CaCh group. The inventors hypothesized that the bacterial surface proteins, particularly cell surface-displayed SazCA, influenced the polymorphs by affecting the nucleation and growth kinetics of CaCOs. This discovery provides a compelling rationale for the activity of the strategy according to the present invention, facilitating the biomineralization process. TGA analysis supported SEM and XRD findings on CaCOs crystallization and polymorphism, revealing thermal decomposition behaviour from 35°C to 950°C. Continuous organic decomposition extending to 800°C was observed in the bacteria-only group, without notable weight loss peaks. CO2 removal from CaCOs to CaO between 600°C and 800°C occurred both in the CaCh-only and the CaCh mixed with bacteria group. However, in the CaCh mixed with bacteria group, there was an additional derivative weight curve around 465°C, indicating crystal water elimination and organic decomposition in calcite and vaterite. These TGA analyses confirmed the generation of vaterites by the biocatalyst and the opportunities for more versatile applications through its transformation into calcite.

[0192] 2.8 Stability test

[0193] The engineered whole-cell biocatalyst, utilizing SazCA displayed on the cell surface in a proteasedeficient strain, has been designed to enhance stability for application in demanding industrial conditions and environments conducive to CaCOs formation (>60°C, pH 8.7-9.5, Ca2+concentration 25-250 mM). To evaluate its performance, the inventors assessed the activity of the enzyme following exposure to various temperatures (60-90°C) for 20 days, pH treatments (pH 3-12) for 1 and 24 hours and CaCh treatments (25-250 mM) for 24 hours.

[0194] No discernible differences were observed between the performance of displayed SazCA in the wildtype and protease-deficient strains. Following a 5-day incubation at 60°C, the whole-cell biocatalyst displaying SazCA on the cell surface retained 46.3% of its CO2 hydration activity. Remarkably, even after exposure to 80°C, the biocatalyst maintained 33.6% of its initial activity (Fig. 6A). Upon 10 days of incubation at 60°C, the whole-cell biocatalyst still preserved 34.3% of its CA activity. However, this activity drastically declined to 9.7% at 80°C. At 90°C, the whole-cell biocatalyst exhibited complete inactivity even after 1 day of incubation.

[0195] In the case of incubation in various pH buffers for 1 hour, the whole-cell biocatalyst consistently remained fully active (even exhibited increased activity) within the pH range of 8-9.5 (Fig. 6B). However, its activity diminished to 43% when subjected to a pH of 10. Notably, even in more alkaline conditions (pH 11), relatively high activity levels were maintained with 40% activity (Fig. 6B). Extending the incubation time to 24 hours resulted in the preservation of 40%-60% activity at pH 8-9.5, which corresponds to the predominant pH range during CaCOs formation.

[0196] The selection of the ideal calcium source and concentration is crucial for CaCCh precipitation. Our whole-cell biocatalyst exhibited almost full activity (>92.5%) within the range of 0-200 mM CaCh, with optimal activity observed at 50-100 mM (Fig. 6C). When the concentration of CaCh increased to 500 mM, the activity mildly decreased to 76.9% and further decreased to 62.1% when the concentration of CaCh increased to 1000 mM. This calcium ion tolerance is sufficient for CO2 mineralization and CaCOs precipitation, as the ideal CaCh concentrations for calcite precipitation have been reported to be 50-250 mM.

[0197] EXAMPLE 2

[0198] Rational engineering of carbonic anhydrase from Persephonella hydrogeniphila

[0199] Materials and methods

[0200] 1.1 Sequence identification and retrieval

[0201] The sequence for P. hydrogeniphila a-CA (PhyCA, NCBI accession number: WP 096999253.1) was acquired from NCBI and submitted to NCBI BLASTp to search for other a-CAs from the genus Persephonella. From this analysis, two proteins originating from the bacteria Persephonella sp. KM09-Lau-8, (PlauCA, Accession number WP 231475403.1) and Persephonella atlantica (PaCA, Accession number: WP 200672902.1) were selected. Sequence alignments were performed using T-COFFEE for retrieved CAs PhyCA, PaCA and PlauCA (the amino acid sequences of which are listed in Table 5) as well as sequences from Persephonella marina, PmCAl (PDB ID: 6IM3) and PmCA2 (PDB ID: 6EKI). TaCA (PDB ID:43CT) from Thermovibrio ammonificans, which is in the same order as Persephonella, as well SazCA (PDB ID:4X5S), one of the most efficient CAs currently and from Sulfurihydrogenibium azorense, were also included in the sequence alignment for comparison.

[0202] 1.2 DNA manipulations and strain construction

[0203] Genes encoding the mature part of PaCA, PlauCA, PhyCA and SazCA were codon-optimised for Escherichia coll and synthesised by Integrated DNA Technologies (IDT). The recombinant plasmids pETMlO-PhyCA-His, pETMlO-PaCA-His, pETmlO-PlauCA-His and pETMlO-SazCA-His, were constructed using the Gibson Assembly method. The vector backbone was PCR-amplified using relevant primers and pETMIO vector as template. All oligonucleotide primers were procured from IDT and are listed in Table 5.

[0204] E. coli strains NM522 and BL21(DE3) were used for cloning and expression, respectively. The strains containing the plasmid were grown overnight in LB medium (1% (w / v) peptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCI) supplemented with 50 pg / ml kanamycin.

[0205] For protein synthesis, the E. coli BL21(DE3) with relevant plasmids were cultured in LB medium to an ODeoo of 0.5-0.6, at which point IPTG (final concentration 1 mM) was added to induce protein expression. Cultures were grown for a further three hours after which cells were harvested by centrifugation at 6000 x g for 10 minutes. The cell pellet was resuspended in lysis buffer containing 2 pl DNAse and 0.5 mg / mL lysozyme, prior to sonication using a probe sonicator (2 min, 40% amplitude). The lysed cells were centrifuged at 6000 x g for 15 minutes, and the supernatant was collected. This was loaded onto an Ni-NTA column equilibrated with binding buffer (20 mM Tris, 0.5 M NaCI, pH 7.5), washed with washing buffer (20 mM Tris, 0.5 M NaCI, 10 mM imidazole, pH 7.5) and eluted with elution buffer (20 mM Tris, 0.5 M NaCI, 250 mM imidazole, pH 7.5). Protein concentration was measured using the Bradford's assay using bovine serum albumin (BSA) as a standard.

[0206] Table 5: Sequences of proteins and primers used in this Example

[0207] 1.3 CO? hydration assay

[0208] CO? hydration activity was measured by the Wilbur-Anderson method. CO2 was bubbled into deionized water to attain a CO2-saturated solution as the substrate. Tris-CI buffer (50 mM, pH 8.4) was mixed with bromothymol blue indicator to record the change in pH from 8.4 (blue) to 6.4 (yellow). All reactions were performed on ice. To start the reaction, 3 ml CCh-saturated solution was added to 2 ml of coloured buffer and 50 pl enzyme solution (50 pl Tris-CI buffer for the uncatalyzed reaction). Time for the uncatalyzed reaction was noted as To and the enzyme-driven reaction was T. Activity was calculated in Wilbur-Anderson units (WAU) using the formula (To - T) / T. Specific activity (WAU / mg) was determined by dividing WAU with protein concentration.

[0209] 1.4 Thermo- and pH-stability assays

[0210] Thermostability was investigated by incubating samples at individual temperatures from 30°C to 90°C in 10°C increments for one hour. Samples were cooled on ice for an hour and then assayed on ice as described in Section 1.3 above. pH stability was explored for the pH range 4 to 10. For the acidic pH values 4, 5 and 6, citrate-phosphate buffer (25 mM) was used while Tris-CI buffer (25 mM) was used at pH values of 7-10. Enzyme solutions were incubated in each buffer (volume ratio of 1:4) for an hour. Residual activity was evaluated using the CO2 hydration assay.

[0211] 1.5 Molecular dynamics simulations

[0212] Dimeric structures for PhyCA and its mutants were modelled using SWISS-MODEL using PmCA2 (PDB ID: 6EKI) as a template. Following validation using z-DOPE (Discrete Optimized Protein Energy) and VERIFY3D, the structures were protonated using H++. AMBERTOOLS22 was applied for the inference of metal parameters to Zn2+coordinating residues and those immediately surrounding them. MD simulations were performed at 363 K using GROMACS v2020.6 following minimization, canonical ensemble and isothermal-isobaric equilibration consecutively at 363 K. GROMACS was used to calculate root mean square deviation (RMSD) to observe changes in conformations, RMSF to investigate individual residue fluctuations, and radius of gyration (Rg) to understand changes in protein compactness over the course of the simulation. Visual molecular dynamics was used to measure atomic distances between proton transfer residues and Zn2+metal ion in the active site and their evolution throughout the trajectory, and PyMOL was used to view structures.

[0213] Results and discussion

[0214] 2.1 Analysis of PhyCA, PaCA and PlauCA

[0215] Persephonella sp. are Gram-negative bacteria that are often associated with hydrothermal vent systems, and they grow at high temperatures (60-90°C). Using the PhyCA sequence as query in BLAST p analysis led to identification of the CAs from Persephonella sp. KM09-Lau-8 (PlauCA) and Persephonella atlantica (PaCA) with sequence identities of 80% and 72%, respectively. Next, these enzymes were compared to other CAs from the genus Persephonella as well as the well- characterised enzymes from Thermovibrio ammonificans (TaCA) and Sulfurihydrogenibium azorense (SazCA).

[0216] The sequence alignment revealed high sequence similarity amongst the CA genes from Persephonella, with each pair exhibiting over 70% sequence identity. PhyCA and PaCA were most similar to PmCA2, which was isolated from the Logatchev hydrothermal fields, with 89% and 84% sequence identities, respectively. With regards to the proton shuttling residue K88, it is noted that the Lys is conserved in all the enzymes except in the SazCA enzyme where a Gin (Q) is employed. Note that the amino acid numbering follows that of PhyCA (i.e., SEQ ID NO: 1) unless explicitly stated otherwise.

[0217] To perform an experimental characterisation of the proteins, the present inventors synthesised 6xHis-tagged variants in E. coll and purified the proteins. SDS-PAGE revealed the sizes of the proteins to be approximately 26 kDa (PhyCA), 30 kDa (PlauCA) and 34 kDa (PaCA) (not shown).

[0218] To evaluate the thermostability, the proteins were incubated at various temperatures for 1 h and the residual activity was tested, see Fig. 7. Further, Fig. 8 shows the specific activity and residual activity of Persephonella hydrogeniphila PhyCA, its mutants K88Q, K88A, K88H and K88Y, as well as Sulfurihydrogenibium azorense SazCA, at 30°C to 90°C in 10°C increments.

[0219] PlauCA exhibited low thermostability compared to PhyCA and PaCA, with only 12% residual activity at 60°C and losing all activity by 70°C. Its activity at 40°C, however, was comparable to that of SazCA. A comparable thermostability profile was observed for PhyCA and PaCA, but the former had lower specific activity at temperatures 30°C-50°C. Thereafter, PhyCA maintained its activity until 70°C, whereas a decline had already started for PaCA from 60°C onwards. At 90°C, PhyCA had just above 5% of its initial activity. However, unlike the rest of the enzymes, PaCA showed almost 25% residual activity at 90°C, showing great potential for enzyme engineering. Both PaCA and PhyCA were interesting candidates for further mutation studies.

[0220] 2.2 PhyCA rational engineering

[0221] PhyCA was used as a case study for rational mutagenesis to investigate if activity could be increased to match or surpass that of SazCA, the most active bacterial CA to date. To do this, the present inventors targeted the proton transfer residue K88. Since SazCA, which has higher activity compared to PhyCA, has a Gin in the position of K88, the first mutation consideration was K88Q. Then, K88 was further mutated to His and Tyr, which are also possible proton shuttling residues. Mutation to non-reactive Ala was to investigate the effect of the absence of this proton shuttle. Since there is still H85 in the catalytic site, it is possible that the absence of a proton transfer residue in position 88 would not render the enzyme completely inactive. Conformational changes of the active sites in the presence of the mutations were an important part of the analysis. RMSD was therefore calculated for active sites to investigate the evolution of the pockets throughout the simulation. Of all the mutants, K88Y showed quite a contrasting pattern compared to the WT and the other mutants and was thus investigated further, as described in Section 2.2.2. All mutant residues considered for PhyCA, except Tyr, were smaller than the original Lys, thus a change in the active site opening was expected upon mutation.

[0222] 2.2.1 K88A and K88H

[0223] Results for mutant K88A were quite unexpected. Not only was this mutant very active but actually more active than wildtype (WT) PhyCA. Specific activity for this mutant was 3-fold higher than the WT at 90°C, which was a very welcome improvement. A temperature activation was detected, noted by the increase in activity at 70°C. Ala is a small non-reactive residue which is not capable of taking up the role of proton donor and / or acceptor exhibited by the other mutant residues as well as K88. The mere presence of activity in this mutant is evidence that the other proton shuttle present in a- CAs (H85) is more than capable of carrying out CO2 hydration and, like in human CAs, is likely the main proton shuttle. K88 may thus not be as important a proton transfer residue as H85 and the positive impact of Ala in its place is assumed to be structural.

[0224] Accordingly, the root mean square fluctuation (RMSF) was investigated for the structure K88A, which interestingly revealed that most residues attained lower fluctuations, inferring structural stability brought about by the mutation. A closer look revealed a decrease in RMSF of mutated residue A88 in both chain A and B, possibly bringing about a degree of stability to the catalytic pocket.

[0225] The size of mutant residue A88 also allowed a visibly larger opening of the active site, possibly explaining the increase in activity compared to the WT.

[0226] Thermostability of K88H was comparable to that of the WT, maintaining a similar activity after incubation at temperatures from 30°C up to 70°C, followed by a decline at 80°C and a sharp decrease at 90°C. Specific activity for this mutant, however, was high, similarly to that of K88Q and significantly higher than for the WT.

[0227] 2.2.2 K88Q and K88Y

[0228] Compared to Lys, Gin is a relatively medium sized and uncharged residue. For mutant K88Q, a similar thermostability trend was observed as compared to the WT. Incubation of the enzyme variant K88Q at temperatures up to 80°C did not affect residual activity, whereas incubation at 90°C resulted in a drastic drop in activity. This variant interestingly had more than 3-fold higher activity than the WT at 30°C. To verify that Lys is indeed insignificant as a proton shuttle in the position 88, the corresponding residue in SazCA (Q74) was mutated to a Lys and its activity as well as its thermostability were tested. Results of these experiments substantiated this hypothesis as the inventors saw the activity of SazCA Q74K drop below half the activity of SazCA and its thermostability decrease as well. PhyCA K88Q had comparable activity to SazCA at higher temperatures, from 60°C to 80°C, but both are still surpassed by PhyCA K88Y at 90°C.

[0229] Mutant K88Y was less active than the other mutants, showing a similar specific activity to the WT, but exhibiting a surprisingly higher thermal stability than all three other variants as well as the WT. Similar to K88A, a temperature activation at 70°C was observed. In both chains A and B, mutant residue Y88 fluctuated twice as much as in the WT, and H85 also had higher RMSF values. One would assume this is an indication of increased proton transfer, directly inferring higher activity, but perhaps something else was deterring this increase. Tyr is quite a large residue and appeared as though it was slightly restricting the active site, which might explain the lower activity. However, its ability to preserve activity at 90°C clearly portrays that this mutation was good for maintenance of the active site at high temperatures.

[0230] Conclusion

[0231] In this Example, the present inventors analysed three CAs from Persephonella bacteria, namely P. hydrogeniphila (PhyCA), Persephonella sp. KM09-Lau-8, (PlauCA) and P. atlantica (PaCA). PhyCA showed good thermostability potential, thus further mutations were carried out in this CA to enhance both its catalytic activity and thermostability. The proton transfer residue, K88, was targeted and mutated into three alternative proton shuttles (His, Gin and Tyr) and Ala as a non-reactive residue. It is worth noting that all mutants showed higher stability compared to the WT PhyCA at 90°C, with mutant K88Y showing a 10-fold higher residual activity after incubation at this temperature. The specific activity of PhyCA was successfully improved in the mutants K88Q and K88H almost 3-fold. The reduced catalytic activity with Lys in position 88 was confirmed by introducing it into SazCA which originally has a Gin in the corresponding position, and this mutant (Saz_Q74K) consequently showed a decrease both in activity and thermostability. The PhyCA mutant K88A surprisingly had a higher activity and stability than the WT, with residue fluctuations of most of the structure, including interface residues, decreasing with this mutation. Overall, the aim to engineer the CA from P. hydrogeniphila was a success, resulting in the discovery of mutants, including K88Y, that are suitable for CO2 sequestration at high temperatures.

Claims

CLAIMS1. A thermophilic bacterium comprising a carbonic anhydrase, wherein the carbonic anhydrase is attached to and / or expressed on the surface of the thermophilic bacterium.

2. The thermophilic bacterium according to claim 1, wherein the thermophilic bacterium has an optimal growth temperature of at least 50°C, such as in a range between 50°C and 64°C, in a range between 65°C and 79°C, or such as at least 80°C; preferably it is in a range between 50°C and 64°C, such as around 60°C.

3. The thermophilic bacterium according to claim 1 or 2, wherein the thermophilic bacterium belongs to a Parageobacillus species, preferably Parageobacillus thermoglucosidasius.

4. The thermophilic bacterium according to any one of the preceding claims, wherein the carbonic anhydrase is obtained or derived from a second thermophilic bacterium selected from: Sulfurihydrogenibium azorense and a Persephonella species, wherein the Persephonella species is optionally selected from any one of: Persephonella hydrogeniphila, Persephonella atlantica, Persephonella marina, and Persephonella sp. KM09-Lau-8, preferably from Persephonella hydrogeniphila, or is a variant or fragment thereof, optionally wherein the carbonic anhydrase comprises or consists of a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-5 or a polypeptide having at least 60% sequence identity with any one of these sequences.

5. The thermophilic bacterium according to any one of the preceding claims, wherein the carbonic anhydrase is attached to the surface of the thermophilic bacterium as a part of a fusion protein comprising at least one lysine motif (LysM) domain.

6. The thermophilic bacterium according to claim 5, wherein the at least one LysM domain is located C-terminally to the carbonic anhydrase in the fusion protein.

7. The thermophilic bacterium according to claim 5 or 6, wherein the fusion protein comprises a linker, such as a Fibronectin-binding protein B (FnBPB) linker, between the carbonic anhydrase and the at least one LysM domain.

8. The thermophilic bacterium according to any one of the preceding claims, wherein the thermophilic bacterium has been genetically modified to reduce or abolish the expression and / or activity of at least one serine protease in the bacterium, as compared to an unmodified parent strain, optionally wherein the at least one serine protease comprises the protease with NCBI reference AOT13_01245, the protease with NCBI reference AOT13_01525, or a combination thereof, such as a combination comprising AOT13_01245,AOT13_01525 and the proteases with NCBI references AOT13_01905, AOT13_10345, AOT13_03220, and AOT13_06160.

9. The thermophilic bacterium according to any one of the preceding claims, wherein the thermophilic bacterium has been genetically modified to overexpress and anchor at least one calcium binding protein on the surface of the bacterium, such as the dockerin Docl24A, and / or wherein the thermophilic bacterium has been genetically modified to overexpress at least one calcium exchanger-encoding gene, wherein the overexpression is compared to an unmodified parent strain.

10. A carbonic anhydrase having at least 72% sequence identity with SEQ ID NO: 1 and having an amino acid selected from A, H, Y, and Q in the position corresponding to position 88 of SEQ ID NO: 1.

11. The carbonic anhydrase according to claim 10, wherein the sequence identity is at least73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least97%, such as at least 98%, and such as at least 99%.

12. The thermophilic bacterium according to any one of claims 1-9 or the carbonic anhydrase according to claim 10 or 11, wherein the carbonic anhydrase maintains at least 90% of its specific CO2 hydration activity at 70°C as compared to at 50°C, as measured by a Wilbur- Anderson assay after 1 hour of incubation.

13. The thermophilic bacterium according to any one of claims 1-9 and 12, wherein the carbonic anhydrase is the carbonic anhydrase according to any one of claims 10-12.

14. Use of the thermophilic bacterium according to any one of claims 1-9 and 12-13 or the carbonic anhydrase according to any one of claims 10-12 for capturing carbon dioxide from a gaseous mixture, such as flue gas.

15. A method of capturing carbon dioxide and forming an alkaline earth metal carbonate, the method comprising a step of:- contacting carbon dioxide with the thermophilic bacterium according to any one of claims 1-9 and 12-13 or the carbonic anhydrase according to any one of claims 10-12 and alkaline earth metal ions, so as to form one or more alkaline earth metal carbonate(s), and- optionally, extracting the alkaline earth metal carbonate(s).

16. The method according to claim 15, wherein the alkaline earth metal carbonate(s) comprise(s) calcium carbonate and / or magnesium carbonate, and the alkaline earth metal ions comprise calcium and / or magnesium ions.

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