Bacterial ß-carbonic anhydrases.

ß-Carbonic anhydrases (ß-CA; EC 4.2.1.1) are widespread zinc metalloenzymes which catalyze the interconversion of carbon dioxide and bicarbonate. They have been isolated in many pathogenic and non-pathogenic bacteria where they are involved in multiple roles, often related to their growth and survival. ß-CAs are structurally distant from the CAs of other classes. In the active site, located at the interface of a fundamental dimer, the zinc ion is coordinated to two cysteines and one histidine. ß-CAs have been divided in two subgroups depending on the nature of the fourth ligand on the zinc ion: class I have a zinc open configuration with a hydroxide ion completing the metal coordination, which is the catalytically active species in the mechanism proposed for the ß-CAs similar to the well-known of α-CAs, while in class II an Asp residue substitute the hydroxide. This latter active site configuration has been showed to be typical of an inactive form at pH below 8. An Asp-Arg dyad is thought to play a key role in the pH-induced catalytic switch regulating the opening and closing of the active site in class II ß-CAs, by displacing the zinc-bound solvent molecule. An allosteric site well-suited for bicarbonate stabilizes the inactive form. This bicarbonate binding site is composed by a triad of well conserved residues, strictly connected to the coordination state of the zinc ion. Moreover, the escort site is a promiscuous site for a variety of ligands, including bicarbonate, at the dimer interface, which may be the route for bicarbonate to the allosteric site.

Staphylococcus aureus Stress Response to Bicarbonate Depletion.

Bicarbonate and CO2 are essential substrates for carboxylation reactions in bacterial central metabolism. In Staphylococcus aureus, the bicarbonate transporter, MpsABC (membrane potential-generating system) is the only carbon concentrating system. An mpsABC deletion mutant can hardly grow in ambient air. In this study, we investigated the changes that occur in S. aureus when it suffers from CO2/bicarbonate deficiency. Electron microscopy revealed that ΔmpsABC has a twofold thicker cell wall thickness compared to the parent strain. The mutant was also substantially inert to cell lysis induced by lysostaphin and the non-ionic surfactant Triton X-100. Mass spectrometry analysis of muropeptides revealed the incorporation of alanine into the pentaglycine interpeptide bridge, which explains the mutant's lysostaphin resistance. Flow cytometry analysis of wall teichoic acid (WTA) glycosylation patterns revealed a significantly lower α-glycosylated and higher ß-glycosylated WTA, explaining the mutant's increased resistance towards Triton X-100. Comparative transcriptome analysis showed altered gene expression profiles. Autolysin-encoding genes such as sceD, a lytic transglycosylase encoding gene, were upregulated, like in vancomycin-intermediate S. aureus mutants (VISA). Genes related to cell wall-anchored proteins, secreted proteins, transporters, and toxins were downregulated. Overall, we demonstrate that bicarbonate deficiency is a stress response that causes changes in cell wall composition and global gene expression resulting in increased resilience to cell wall lytic enzymes and detergents.

The release of GLP-1 from gut L cells is inhibited by low extracellular pH.

OBJECTIVE: The intestinal luminal pH profile varies from stomach to rectum and becomes disrupted in diseases. However, little is known about the pH dependence of incretin hormone secretion, with most in vitro studies having failed to consider this modulatory factor or having used nonphysiological buffer systems. Here, we report the extracellular pH (pHe) dependence of glucagon-like peptide-1 (GLP-1) exocytosis from L cells. METHODS: The pHe dependence of GLP-1 release from GLUTag cells and murine ex vivo primary gut cultures was detected by ELISA. GLP-1 release was measured over a range of pHe under a physiological (CO2/HCO3 -) buffering regime and in its absence (HEPES buffer). The relationship between intracellular pH (pHi) and pHe was mapped given that at least some component of pH sensitivity is likely to be intracellular. RESULTS: GLP-1 secretion from L cells was pHe-dependent and stimulated under alkaline conditions. In the absence of glucose or extracellular calcium, secretion remained at a pHe-insensitive baseline. pHi followed changes in pHe, but the relationship was offset to more alkaline levels in the absence of CO2/HCO3 - buffer and became shallower if [Cl-] changes that normally accompany [HCO3 -] changes were compensated iso-osmotically with gluconate. CONCLUSIONS: GLP-1 secretion is sensitive to pHe and the buffer present. Exploiting this mechanism therapeutically may benefit patients with obesity.

Cryo-EM structures of the human band 3 transporter indicate a transport mechanism involving the coupled movement of chloride and bicarbonate ions.

The band 3 transporter is a critical integral membrane protein of the red blood cell (RBC), as it is responsible for catalyzing the exchange of bicarbonate and chloride anions across the plasma membrane. To elucidate the structural mechanism of the band 3 transporter, detergent solubilized human ghost membrane reconstituted in nanodiscs was applied to a cryo-EM holey carbon grid to define its composition. With this approach, we identified and determined structural information of the human band 3 transporter. Here, we present 5 different cryo-EM structures of the transmembrane domain of dimeric band 3, either alone or bound with chloride or bicarbonate. Interestingly, we observed that human band 3 can form both symmetric and asymmetric dimers with a different combination of outward-facing (OF) and inward-facing (IF) states. These structures also allow us to obtain the first model of a human band 3 molecule at the IF conformation. Based on the structural data of these dimers, we propose a model of ion transport that is in favor of the elevator-type mechanism.

Metabolic alkalosis in cystic fibrosis: from vascular volume depletion to impaired bicarbonate excretion.

Cystic fibrosis (CF) is the most common life-threatening genetic disease in the United States and among people of European descent. Despite the widespread distribution of the cystic fibrosis transmembrane conductance regulator (CFTR) along kidney tubules, specific renal phenotypes attributable to CF have not been well documented. Recent studies have demonstrated the downregulation of the apical Cl-/HCO3 - exchanger pendrin (Slc26a4) in kidney B-intercalated cells of CF mouse models. These studies have shown that kidneys of both mice and humans with CF have an impaired ability to excrete excess HCO3 -, thus developing metabolic alkalosis when subjected to excess HCO3 - intake. The purpose of this minireview is to discuss the latest advances on the role of pendrin as a molecule with dual critical roles in acid base regulation and systemic vascular volume homeostasis, specifically in CF. Given the immense prevalence of vascular volume depletion, which is primarily precipitated via enhanced chloride loss through perspiration, we suggest that the dominant presentation of metabolic alkalosis in CF is due to the impaired function of pendrin, which plays a critical role in systemic vascular volume and acid base homeostasis.

Functional plasticity of HCO3- uptake and CO2 fixation in Cupriavidus necator H16.

Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of ß-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO2 similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3- uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing.

Ciliary Motility Decreased by a CO2/HCO3--Free Solution in Ciliated Human Nasal Epithelial Cells Having a pH Elevated by Carbonic Anhydrase IV.

An application of CO2/HCO3--free solution (Zero-CO2) did not increase intracellular pH (pHi) in ciliated human nasal epithelial cells (c-hNECs), leading to no increase in frequency (CBF) or amplitude (CBA) of the ciliary beating. This study demonstrated that the pHi of c-hNECs expressing carbonic anhydrase IV (CAIV) is high (7.64), while the pHi of ciliated human bronchial epithelial cells (c-hBECs) expressing no CAIV is low (7.10). An extremely high pHi of c-hNECs caused pHi, CBF and CBA to decrease upon Zero-CO2 application, while a low pHi of c-hBECs caused them to increase. An extremely high pHi was generated by a high rate of HCO3- influx via interactions between CAIV and Na+/HCO3- cotransport (NBC) in c-hNECs. An NBC inhibitor (S0859) decreased pHi, CBF and CBA and increased CBF and CBA in c-hNECs upon Zero-CO2 application. In conclusion, the interactions of CAIV and NBC maximize HCO3- influx to increase pHi in c-hNECs. This novel mechanism causes pHi to decrease, leading to no increase in CBF and CBA in c-hNECs upon Zero-CO2 application, and appears to play a crucial role in maintaining pHi, CBF and CBA in c-hNECs periodically exposed to air (0.04% CO2) with respiration.

In search of the pH limit of growth in halo-alkaliphilic cyanobacteria.

Cyanobacteria have many biotechnological applications. Increasing their cultivation pH can assist in capturing carbon dioxide and avoiding invasion by other organisms. However, alkaline media may have adverse effects on cyanobacteria, such as reducing the Carbon-Concentrating Mechanism's efficiency. Here, we cultivated two halo-alkaliphilic cyanobacteria consortia in chemostats at pH 10.2-11.4. One consortium was dominated by Ca. Sodalinema alkaliphilum, the other by a species of Nodosilinea. These two cyanobacteria dominate natural communities in Canadian and Asian alkaline soda lakes. We show that increasing the pH decreased biomass yield. This decrease was caused, in part, by a dramatic increase in carbon transfer to heterotrophs. At pH 11.4, cyanobacterial growth became limited by bicarbonate uptake, which was mainly ATP dependent. In parallel, the higher the pH, the more sensitive cyanobacteria became to light, resulting in photoinhibition and upregulation of DNA repair systems.

The unique allosteric property of crocodilian haemoglobin elucidated by cryo-EM.

The principal effect controlling the oxygen affinity of vertebrate haemoglobins (Hbs) is the allosteric switch between R and T forms with relatively high and low oxygen affinity respectively. Uniquely among jawed vertebrates, crocodilians possess Hb that shows a profound drop in oxygen affinity in the presence of bicarbonate ions. This allows them to stay underwater for extended periods by consuming almost all the oxygen present in the blood-stream, as metabolism releases carbon dioxide, whose conversion to bicarbonate and hydrogen ions is catalysed by carbonic anhydrase. Despite the apparent universal utility of bicarbonate as an allosteric regulator of Hb, this property evolved only in crocodilians. We report here the molecular structures of both human and a crocodilian Hb in the deoxy and liganded states, solved by cryo-electron microscopy. We reveal the precise interactions between two bicarbonate ions and the crocodilian protein at symmetry-related sites found only in the T state. No other known effector of vertebrate Hbs binds anywhere near these sites.

Bicarbonate concentration influences carbon utilization rates and biochemical profiles of freshwater and marine microalgae.

Selecting the optimal microalgal strain for carbon capture and biomass production is crucial for ensuring the commercial viability of microalgae-based biorefinery processes. This study aimed to evaluate the impact of varying bicarbonate concentrations on the growth rates, inorganic carbon (IC) utilization, and biochemical composition of three freshwater and two marine microalgal species. Parachlorella kessleri, Vischeria cf. stellata, and Porphyridium purpureum achieved the highest carbon removal efficiency (>85%) and biomass production at 6 g L-1 sodium bicarbonate (NaHCO3), while Phaeodactylum tricornutum showed optimal performance at 1 g L-1 NaHCO3. The growth and carbon removal rate of Scenedesmus quadricauda increased with increasing NaHCO3 concentrations, although its highest carbon removal efficiency (∼70%) was lower than the other species. Varying NaHCO3 levels significantly impacted the biochemical composition of P. kessleri, S. quadricauda, and P. purpureum but did not affect the composition of the remaining species. The fatty acid profiles of the microalgae were dominated by C16 and C18 fatty acids, with P. purpureum and P. tricornutum yielding relatively high polyunsaturated fatty acid content ranging between 14% and 30%. Furthermore, bicarbonate concentration had a species-specific effect on the fatty acid and chlorophyll-a content. This study demonstrates the potential of bicarbonate as an effective IC source for microalgal cultivation, highlighting its ability to select microalgal species for various applications based on their carbon capture efficiency and biochemical composition.

Bicarbonate is a key regulator but not a substrate for O2 evolution in Photosystem II.

Photosystem II (PSII) uses light energy to oxidize water and to reduce plastoquinone in the photosynthetic electron transport chain. O2 is produced as a byproduct. While most members of the PSII research community agree that O2 originates from water molecules, alternative hypotheses involving bicarbonate persist in the literature. In this perspective, we provide an overview of the important roles of bicarbonate in regulating PSII activity and assembly. Further, we emphasize that biochemistry, spectroscopy, and structural biology experiments have all failed to detect bicarbonate near the active site of O2 evolution. While thermodynamic arguments for oxygen-centered bicarbonate oxidation are valid, the claim that bicarbonate is a substrate for photosynthetic O2 evolution is challenged.

Optimizing wheat productivity through integrated management of irrigation, nutrition, and organic amendments.

Enhancing wheat productivity by implementing a comprehensive approach that combines irrigation, nutrition, and organic amendments shows potential for collectively enhancing crop performance. This study examined the individual and combined effects of using irrigation systems (IS), foliar potassium bicarbonate (PBR) application, and compost application methods (CM) on nine traits related to the growth, physiology, and yield of the Giza-171 wheat cultivar. Analysis of variance revealed significant (P ≤ 0.05) main effects of IS, PBR, and CM on wheat growth, physiology, and yield traits over the two growing seasons of the study. Drip irrigation resulted in a 16% increase in plant height, leaf area index, crop growth rate, yield components, and grain yield compared to spray irrigation. Additionally, the application of foliar PBR at a concentration of 0.08 g/L boosted these parameters by up to 22% compared to the control. Furthermore, the application of compost using the role method resulted in enhanced wheat performance compared to the treatment including mix application. Importantly, the combined analysis revealed that the three-way interaction between the three factors had a significant effect (P ≤ 0.05) on all the studied traits, with drip irrigation at 0.08 g PBR rate and role compost application method (referred as Drip_0.08g_Role) resulting in the best performance across all traits, while sprinkle irrigation without PBR and conventional mixed compost method (referred as sprinkle_CK_Mix) produced the poorest results. This highlights the potential to synergistically improve wheat performance through optimized agronomic inputs.

GPR30 is a potential player between islet cells and ductal HCO3- secretion.

The primary role of pancreatic ductal HCO3- secretion is to prevent premature activation of digestive enzymes and to provide a vehicle for the delivery of enzymes to the duodenum. In addition, HCO3-is responsible for the neutralization of gastric juice and protect against the formation of protein plugs and viscous mucus. Due to this multifaceted role of HCO3- in the pancreas, its altered functioning can greatly contribute to the development of various exocrine diseases. It is well known that the exocrine and endocrine pancreas interact lively with each other, but not all details of this relationship are known. An interesting finding of a recent study by Jo-Watanabe et al. is that the G protein-coupled oestrogen receptor, GPR30, which is expressed in the endocrine pancreas, can be also activated by HCO3-. This raises the possibility that ductal cells play a key role not only in the exocrine pancreas, but presumably also in endocrine function through HCO3- secretion.

DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function.

Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Protocol for producing hyperpolarized 13C-bicarbonate for clinical MRI of extracellular pH in aggressive tumors.

Tumor acidosis is one of the hallmarks indicating the initiation and progression of various cancers. Here, we present a protocol for preparing a hyperpolarized (HP) 13C-bicarbonate tissue pH MRI imaging contrast agent to detect aggressive tumors. We describe the steps for the formulation and polarization of a precursor molecule 13C-glycerol carbonate (13C-GLC), the post-dissolution reaction, and converting HP 13C-GLC to an injectable HP 13C-bicarbonate solution. We then detail procedures for MRI data acquisition to generate tumor pH maps for assessing tumor aggressiveness. For complete details on the use and execution of this protocol, please refer to Mu et al.1.

Impact of AKI on metabolic compensation for respiratory acidosis in ICU patients with AECOPD.

PURPOSE: Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) can result in severe respiratory acidosis. Metabolic compensation is primarily achieved by renal retention of bicarbonate. The extent to which acute kidney injury (AKI) impairs the kidney's capacity to compensate for respiratory acidosis remains unclear. MATERIALS AND METHODS: This retrospective analysis covers clinical data between January 2009 and December 2021 for 498 ICU patients with AECOPD and need for respiratory support. RESULTS: 278 patients (55.8%) presented with or developed AKI. Patients with AKI exhibited higher 30-day-mortality rates (14.5% vs. 4.5% p = 0.001), longer duration of mechanical ventilation (median 90 h vs. 14 h; p = 0.001) and more severe hypercapnic acidosis (pH 7.23 vs. 7.28; pCO2 68.5 mmHg vs. 61.8 mmHg). Patients with higher AKI stages exhibited lower HCO3-/pCO2 ratios and did not reach expected HCO3- levels. In a mixed model analysis with random intercept per patient we analyzed the association of pCO2 (independent) and HCO3- (dependent variable). Lower estimates for averaged change in HCO3- were observed in patients with more severe AKI. CONCLUSION: AKI leads to poor outcomes and compromises metabolic compensation of respiratory acidosis in ICU patients with AECOPD. While buffering agents may aid compensation for severe AKI, their use should be approached with caution.

Production of Peroxymonocarbonate by Steady-State Micromolar H2O2 and Activated Macrophages in the Presence of CO2/HCO3- Evidenced by Boronate Probes.

Peroxymonocarbonate (HCO4-/HOOCO2-) is produced by the reversible reaction of CO2/HCO3- with H2O2 (K = 0.33 M-1, pH 7.0). Although produced in low yields at physiological pHs and H2O2 and CO2/HCO3- concentrations, HCO4- oxidizes most nucleophiles with rate constants 10 to 100 times higher than those of H2O2. Boronate probes are known examples because HCO4- reacts with coumarin-7-boronic acid pinacolate ester (CBE) with a rate constant that is approximately 100 times higher than that of H2O2 and the same holds for fluorescein-boronate (Fl-B) as reported here. Therefore, we tested whether boronate probes could provide evidence for HCO4- formation under biologically relevant conditions. Glucose/glucose oxidase/catalase were adjusted to produce low steady-state H2O2 concentrations (2-18 µM) in Pi buffer at pH 7.4 and 37 °C. Then, CBE (100 µM) was added and fluorescence increase was monitored with time. The results showed that each steady-state H2O2 concentration reacted more rapidly (∼30%) in the presence of CO2/HCO3- (25 mM) than in its absence, and the data permitted the calculation of consistent rate constants. Also, RAW 264.7 macrophages were activated with phorbol 12-myristate 13-acetate (PMA) (1 µg/mL) at pH 7.4 and 37 °C to produce a time-dependent H2O2 concentration (8.0 ± 2.5 µM after 60 min). The media contained 0, 21.6, or 42.2 mM HCO3- equilibrated with 0, 5, or 10% CO2, respectively. In the presence of CBE or Fl-B (30 µM), a time-dependent increase in the fluorescence of the bulk solution was observed, which was higher in the presence of CO2/HCO3- in a concentration-dependent manner. The Fl-B samples were also examined by fluorescence microscopy. Our results demonstrated that mammalian cells produce HCO4- and boronate probes can evidence and distinguish it from H2O2 under biologically relevant concentrations of H2O2 and CO2/HCO3-.

Temporal Transcriptomic Profiling Reveals Dynamic Changes in Gene Expression of Giant Freshwater Prawn upon Acute Saline-Alkaline Stresses.

Bicarbonate and sulfate are among two primary ion constituents of saline-alkaline water, with excessive levels potentially causing metabolic disorders in crustaceans, affecting their molting and interrupting development. As an economically important crustacean species, the molecular adaptive mechanism of giant freshwater prawn Macrobrachium rosenbergii in response to the stress of bicarbonate and sulfate remains unexplored. To investigate the mechanism underlying NaHCO3, Na2SO4, and mixed NaHCO3, Na2SO4 stresses, M. rosenbergii larvae were exposed to the above three stress conditions, followed by total RNA extraction and high-throughput sequencing at eight distinct time points (0, 4, 8, 12, 24, 48, 72, and 96 h). Subsequent analysis revealed 13, 16, and 13 consistently identified differentially expressed genes (DEGs) across eight time points under three stress conditions. These consistently identified DEGs were significantly involved in the Gene Ontology (GO) terms of chitin-based cuticle development, protein-carbohydrate complex, structural constituent of cuticle, carnitine biosynthetic process, extracellular matrix, and polysaccharide catabolic process, indicating that alkaline stresses might potentially impact the energy metabolism, growth, and molting of M. rosenbergii larvae. Particularly, the transcriptome data revealed that DEGs associated with energy metabolism, immunity, and amino acid metabolism were enriched across multiple time points under three stress conditions. These DEGs are linked to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including glycolysis/glucogenesis, amino sugar and nucleotide sugar metabolism, and lysine degradation. Consistent enrichment findings across the three stress conditions support conclusions above. Together, these insights are instrumental in enhancing our understanding of the molecular mechanisms underlying the alkaline response in M. rosenbergii larvae. Additionally, they offer valuable perspectives on the regulatory mechanisms of freshwater crustaceans amid saline-alkaline water development.

Mechanisms of whole body, respiratory, acid-base buffering: a first computer-model test of three physicochemical, acid-base theories.

Acid-base disorders are currently analyzed and treated using a bicarbonate-centered approach derived from blood studies prior to the advent of digital computers, which could solve computer models capable of quantifying the complex physicochemical nature governing distribution of water and ions between fluid compartments. An alternative is the Stewart approach, which can predict the pH of a simple mixture of ions and electrically charged proteins; hence, the role of extravascular fluids has been largely ignored. The present study uses a new, comprehensive computer model of four major fluid compartments, based on a recent blood model, which included ion binding to proteins, electroneutrality constraints, and other essential physicochemical laws. The present model predicts quantitative respiratory acid-base buffering behavior in the whole body, as well as determining roles of each compartment and their species, particularly compartmental electrically charged proteins, largely responsible for buffering. The model tested an early theory that H+ was conserved in the body fluids; hence, when changing Pco2 states, intracellular buffering could be predicted by net changes in bicarbonate and protein electrical charge in the remaining fluids. Even though H+ is not conserved in the model, the theory held in simulated respiratory disorders. Model results also agreed with a second part of the theory, that ion movements between cells and interstitial fluid were linked with H+ buffering, but by electroneutrality constraints, not necessarily by some membrane-related mechanisms, and that the strong ion difference (SID), an amalgamation of ionic electrical charges, was approximately conserved when going between equilibrium states caused by Pco2 changes in the body-fluid system.NEW & NOTEWORTHY For the first time, a physicochemically based, whole body, four-compartment, computer model was used to study respiratory whole body acid-base buffering. An improved approach to quantify acid-base buffering, previously used by this author, was able to determine contributions of the various compartmental fluids to whole body buffering. The model was used to test, for the first time, three fundamental theories of whole body acid-base homeostasis, namely, H+-conservation, its linkage to ion transport, and strong ion difference conservation.

Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.