Climate warming is progressively issuing a severe warning to humanity through its multifaceted and immense threats.

We plan to design an efficient algal-bacterial co-culture system, utilizing three engineered bacteria, Vibrio natriegens, Shewanella oneidensis MR-1, and Synechococcus elongatus PCC 7942, to sustainably and efficiently convert carbon dioxide into calcium carbonate, achieving carbon sequestration for millennia.

Shewanella oneidensis MR-1, as an electrochemically active microorganism (EAM), is one of the most widely studied model microorganisms in the field of bioelectrochemistry. In addition to its application in microbial fuel cells (MFCs) through extracellular electron transfer (EET), it also plays a significant role in microbial electrosynthesis (MES) via extracellular electron uptake (EEU). In EEU, electrons transfer from an electrode to S. oneidensis MR-1, where they are utilized in terminal electron receptor reductases, such as formate dehydrogenases (FDHs), enabling the biocatalytic conversion of CO₂ to formate.

Synechococcus elongatus PCC 7942, a well-characterized cyanobacterium, is vital in cyanobacterial molecular genetics. As an obligate photoautotroph, it relies on light for energy and CO₂for carbon. When cultured in high salinity, S. elongatus PCC 7942 synthesizes sucrose as an osmolyte. Following our engineering efforts, it effectively converts CO₂ to sucrose and secrete it to culture media, which is subsequently utilized by V. natriegens .

Vibrio natriegens ATCC 14048 is recognized for its rapid generation time and high ribosomal content, capable of doubling in under 10 minutes under optimal conditions. This halophilic bacterium thrives in environments with approximately 2% NaCl, aligning with artificial seawater conditions. It metabolizes various carbon sources, including formate and with sucrose being the most efficient. Its rapid growth and ability to utilize inexpensive carbon sources make it a candidate for biotechnological applications, including recombinant protein production.

The biocatalytical conversion of CO₂ to formate can be produced through the hydrogenation of CO₂ by formate hydrogen lyase or the direct reduction of CO₂ catalyzed by formate dehydrogenases (FDHs) with the cofactor NADH or electrons generated from electrodes as reducing power ^[1] . Microorganisms harboring powerful electron transfer systems as well as formate dehydrogenases would be anticipated to perform efficient syntheses of formic acid through the electro-biocatalytical reduction of CO₂. Shewanella oneidensis MR-1, a facultative aerobic gram-negative bacterium, has been well-known for its electron transfer system (e.g., cytochromes, reductases, iron-sulfur proteins, and quinones) ^[1] .

In extracellular electron uptake (EEU), electrons first be taken up from electrode to OmcA and MtrC, directly or through electron mediators like riboflavin ^[2] . Then electrons go from MtrABC system to inner membrane cytochrome, CymA, mainly through shutters like the small tetrahaem cytochrome CctA (STC) ^[3] . And finally some electrons reach formate dehydrogenases (FDHs) and reduce CO₂ to formate. The following figure shows the pathway of electrosynthesis of formic acid.

To enhance the produce of formate, we consider to optimize the extracellular electron uptake (EEU) pathway.

3.1 Overexpression cctA Gene

First, CctA, as a mainly used electron shutter in periplasmic, has been proposed to be the bottleneck of EEU efficiency. ^[4] Overexpression of the periplasmic electron shutter protein CctA can enhance electrosynthesis efficiency and promotes formic acid production.

We designed a plasmid that achieves the high expression of CctA. pGEX-4T-1 is chosen as it has the promoter required and high popularity in Gram-negative bacteria engineering. Under the addition of IPTG, the promoter will activate the expression of ccta. According to literature ^[5] , Shewanella is resistant to ampicillin, leading to a substitution of ampicillin resistant gene for Kanamycin resistant gene.

3.2 Addition of Exogenous Electron Mediators

Riboflavin (RF) and 2-hydroxy-1,4-naphthoquinone (2-HNQ) are effective exogenous electron mediators (EMs) that enhance extracellular electron uptake (EEU) through electron shuttling ^[6] . RF, known for its role in S. oneidensis MR-1, has an optimal redox potential for this process. Recent research shows that RF binds to OmcA as a cofactor, lowering OmcA's redox potential by about 100 mV and increasing the EEU rate ^[2] . Thus, incorporating EMs like RF and 2-HNQ can boost electrosynthesis efficiency and promote formic acid production.

3.3 Form Self-Assembled Electroactive Reduced-Graphene-Oxide-Hybridized Biofilm

The self-assembly of graphene oxide (GO) with S. oneidensis MR-1 creates an electroactive, reduced-graphene-oxide-hybridized, three-dimensional macroporous biofilm. This structure allows efficient bidirectional electron transfer between S. oneidensis MR-1 and electrodes due to high biomass and enhanced direct contact. GO nanosheets capture bacterial cells through a “fishing” process, acting as nets. Upon reduction to rGO, they form a 3D macroporous network, incorporating many bacteria and creating conductive pathways that facilitate extracellular electron uptake (EEU) ^[7] . Thus, adding graphene oxide (GO) can enhance electrosynthesis efficiency and promotes formic acid production.

4.1 Import and expression of the cscB gene.

When S. elongatus PCC7942 are cultured in an environment with high salt concentration, they can synthesize sucrose as an osmolyte. However, S. elongatus do not actively release sucrose outside the cell. On this basis, S. elongatus were designed to express sucrose permease(CscB), a sucrose/proton symporter, which could enable S. elongatus to secrete sucrose into the growth medium, providing an auxiliary carbon source for Vibrio natriegens and promoting their growth and metabolism.

The plasmid introduced into the cyanobacteria was obtained from the ShanghaiTech_China 2022 team. They constructed a plasmid containing the Sucrose permease (CscB) gene, which can integrate into the cyanobacterial genome. We expressed this plasmid in S. elongatus . For more information, please check the ShanghaiTech_China 2022 website at https://2022.igem.wiki/shanghaitech-china/engineering.

5.1 Overexpression of Carbonic Anhydrase (CA) Gene

Carbonic anhydrase is an enzyme that catalyzes the reversible reaction between carbon dioxide (CO₂) and water (H₂O) to form carbonic acid (H₂CO₃).

An improved part of carbonic anhydrase (csoS3) from Halothiobacillus neapolitanus is introduced to Vibrio natriegens , which converts the incoming bicarbonate into carbon dioxide in the carboxysome, a step that is essential for CO₂ fixation.

5.2 Expression of Coral Acid-rich Proteins (CARPs)

Coral Acid-rich Proteins (CARPs) are characterized by their high content of acidic amino acids, such as aspartic and glutamic acids. These proteins are primarily found in corals and play crucial roles in biomineralization, the process through which corals form their calcium carbonate skeletons.

CARPs regulate calcium carbonate precipitation by controlling the nucleation and growth of calcium carbonate crystals.

We selected three coral acid-rich proteins (CARP1-3, GenBank accession numbers KC148537–KC148539) based on the work of Tali Mass et al. They identified, cloned, sequenced, and characterized four novel coral acid-rich proteins (CARP1-4) derived from the stony coral Stylophora pistillata ^[8] . Each of these proteins can spontaneously catalyze calcium carbonate precipitation in vitro. Their findings demonstrate that CARPs bind Ca²⁺ stoichiometrically and precipitate aragonite in vitro in seawater at pH levels of 8.2 and 7.6, through electrostatic interactions with protons on bicarbonate anions ^[8] . The selected CARP1-3, unlike CARP4, contain a secretory signal peptide, indicating they are secreted from cells ^[8] .

We integrated the gene sequences of CARP1-3 into plasmids and introduced them into Vibrio natriegens , aiming for their expression to promote calcium carbonate precipitation for carbon fixation.

5.3 Design and Expression of Engineered Protein Cabp-Chbd

In nearshore marine environments, particularly on the seabed, the presence of biological remains results in relatively high chitin content. Inspired by the work of Thiloththama H. K. Nawarathna and colleagues, we explored the potential of an artificial fusion protein to enhance calcium carbonate mineralization on insoluble polysaccharides for effective biocementation.

The fusion protein, CaBP-ChBD, was engineered by integrating a calcite-binding peptide (CaBP) into a chitin-binding domain (ChBD). This protein significantly increased CaCO₃ formation and facilitated its deposition on chitin particles. Their work revealed that CaBP-ChBD improved sand solidification efficiently and sustainably, enhancing the biocementation process ^[9] .

This inspired us to introduce the fusion protein (CaBP-ChBD) in chitin-rich nearshore seabeds to enhance calcium carbonate precipitation and stabilization. Chitin was chosen as the organic matrix due to its abundance and association with CaCO₃ biomineralization ^[9] . The chitin-binding domain (ChBD) from chitinase, with high chitin affinity, was used as the binding site, and a short calcite-binding peptide (CaBP) served as the calcite-binding site ^[9] . This fusion protein's dual binding sites facilitated efficient CaCO₃ precipitation on the chitin matrix ^[9] .

Based on the work of Thiloththama H. K. Nawarathna et al., the synthetic gene for the chitin-binding domain (ChBD) was derived from chitinase A1 of Bacillus circulans WL-12 (GenBank: AAA81528.1), and the calcite-binding peptide (CaBP: DVFSSFNLKHMR) was obtained from a phage-display system. We introduced these gene sequences into Vibrio natriegens via plasmids to enhance calcium carbonate precipitation and stability in chitin-rich coastal seabeds, thereby promoting carbon fixation.

6.1 Design and Construction of the Co-culture System Device

In the co-culture system, engineered Shewanella oneidensis MR-1 will be cultured at the cathode region (where electrons flow in, and voltage and current are generated and maintained by solar panels). Here, it can perform microbial electrosynthesis (MES) and complete the extracellular electron uptake (EEU) process, facilitating the biocatalytic conversion of CO₂ to formate. Due to the presence of a mildly alkaline environment (pH = 7-8), the chemical equation is as follows: CO₂ + H₂O == HCOO^- + OH^-

Formate and hydroxide ions, under the influence of an electric field, will pass through the anion exchange membrane (AEM) and enter the chamber containing Vibrio natriegens.

The chamber containing Synechococcus elongatus PCC 7942 is placed parallel to the other chambers. Engineered S. elongatus PCC 7942 can convert CO₂ into sucrose through photosynthesis and secrete it extracellularly. Due to the sucrose concentration gradient, sucrose can diffuse into the chamber containing Vibrio natriegens.

To maintain the stability of the electrolytic cell, an anion exchange membrane (AEM), CaCl₂solution, and cation exchange membrane (CEM) are used between the anode and cathode chambers. This setup allows the solution to form a relatively stable internal circuit under the influence of an electric field, which, together with the external circuit (connected by wires), forms a complete electrolytic cell. During operation, Ca²⁺ ions in the CaCl₂ solution will pass through the CEM into the chamber containing Vibrio natriegens under the influence of the electric field.

Finally, in the chamber containing Vibrio natriegens , the engineered Vibrio natriegens can utilize formate and sucrose for growth and express Carbonic Anhydrase (CA), Coral Acid-rich Proteins (CARPs), and the engineered protein Cabp-Chbd. CA catalyzes the conversion of CO₂ to HCO₃^⁻, which then reacts with OH^- and Ca²⁺ to form calcium carbonate precipitates. CARPs significantly accelerate this process and increase the size of the precipitate particles. Additionally, in the presence of chitin, the engineered protein Cabp-Chbd promotes the binding of chitin with calcium carbonate precipitates, achieving more robust and stable carbon fixation.