The greenhouse effect constitutes a critical anthropogenic perturbation of the Earth's climate system, with cascading ramifications across the biosphere, lithosphere, and hydrosphere^[1]. Carbon capture, utilization, and storage technologies are therefore pivotal for mitigating these impacts. Autotrophic microorganisms provide a promising pathway for carbon dioxide (CO₂) reduction, capable of fixing CO₂ into biomass and valuable products in industrial settings using either light (photoautotrophy) or inorganic electron donors (chemolithoautotrophy). However, biological photosynthesis suffers from low energy efficiency—typically 1–2% for plants^[2] and up to around 3% for microalgae^[3]—which limits its practical application. In contrast, photovoltaic solar panels achieve much higher solar-to-electricity efficiencies, currently around 18%, with emerging technologies potentially exceeding 40%^[4,5].

This efficiency advantage has spurred the development of microbial electrosynthesis^[4,6], a process that uses electricity generated from solar or other renewable sources to drive CO₂ fixation by chemolithoautotrophic microorganisms. By integrating electrocatalysis with microbial metabolism, this approach effectively couples solar power with CO₂ conversion into chemicals and biofuels, transforming electrical energy into storable chemical energy. Not only does microbial electrosynthesis enable efficient CO₂ fixation, but it also offers an innovative strategy for storing electricity—addressing the critical challenge of intermittency and grid-scale storage associated with renewables like photovoltaics and wind power. Thus, it represents a dual solution to both greenhouse gas accumulation and renewable energy storage, holding significant potential to mitigate climate change and energy crises.

To this end, CUG-China 2025 aims to engineer the chemolithoautotrophic bacteriumAcidithiobacillus ferrooxidans to establish a novel pathway for electricity-driven carbon fixation and chemical synthesis. This inherent trait offers a key advantage as a chassis organism, effectively minimizing contamination risks. Our engineered strain integrates three synthetic biology modules: (1) a cyclic di-GMP synthase module to enhance extracellular electron uptake; (2) a glycerol synthesis and transport module for converting fixed carbon into glycerol; and (3) a phosphoribulokinase (PRK) module designed to improve CO₂ fixation efficiency within the Calvin–Benson–Bassham (CBB) cycle. By incorporating the engineered A. ferrooxidans into a microbial electrochemical system, we achieve efficient electricity-driven carbon fixation, enabling sustainable production of valuable chemicals.

A. ferrooxidans is an extremely acidophilic, chemolithoautotrophic, Gram-negative bacterium that thrives in highly acidic environments (pH 1.5–2.5). This inherent trait offers a key advantage as a chassis organism, effectively minimizing contamination risks. It derives energy from the oxidation of ferrous iron (Fe²⁺) or reduced sulfur compounds—such as sulfides, elemental sulfur, and thiosulfates—while utilizing CO₂ as its sole carbon source for autotrophic growth^[7]. Notably, A. ferrooxidans can also grow using electricity as its only energy source, employing soluble iron as an electron shuttle^[8]. This unique capability enables its application in microbial electrochemical synthesis systems, facilitating the efficient conversion of electrical energy into storable chemical energy.

Figure 1 Microbial electrochemical synthesis system of A. ferrooxidans ^[9]

Based on its extracellular electron transfer mechanism, A. ferrooxidans serves as a key biocatalyst in the microbial electrochemical synthesis system we designed. Like many electroactive microorganisms, A. ferrooxidans takes up electrons from the extracellular environment such as ferrous iron (Fe²⁺). This inward electron flow enables the organism to use electrical energy—supplied via a cathode—to drive intracellular metabolic processes. In this system (Figure 1), electrons provided from the cathode reduce extracellular ferric iron (Fe³⁺) to ferrous iron (Fe²⁺). A. ferrooxidans , acting as the cathode biocatalyst, then re-oxidizes Fe²⁺ back to Fe³⁺, capturing the electrons in the process. These electrons are utilized to support carbon dioxide fixation and biomass synthesis, effectively converting electrical energy into storable chemical energy. Through this cyclic iron-mediated electron transfer, the system integrates CO₂ reduction and bio-production in a single electrochemical platform.

Figure 2 Three-electrode reactor system used in our project for biocathode development. Here, WE: working electrode (i.e. cathode); CE, counter electrode (auxiliary); RE, reference electrode. All the electrodes were immersed in the electrolyte.

c-di-GMP (bis-(3',5')-cyclic dimeric guanosine monophosphate) is a ubiquitous bacterial second messenger that plays a central role in regulating the transition between motile and biofilm lifestyles^[10,11]. Generally, elevated intracellular levels of c-di-GMP promote the production of biofilm matrix components (e.g., exopolysaccharides and adhesins) and suppress bacterial motility, facilitating surface colonization and biofilm formation^[12]. It is synthesized from two GTP molecules by diguanylate cyclase enzymes (DGCs), which contain GGDEF domains[13]. In our iGEM project in 2024, we promoted biofilm formation of A. ferrooxidans by elevating its c-di-GMP concentrations through overexpression of its DGCs (Table 1)^[14], thereby enhancing the ability of microorganisms to leach metals from mineral matrices.

Table 1 Identification of proteins involved in the c-di-GMP pathway of Acidithiobacillus ferrooxidans ^[14]

In addition to its role in regulating biofilm formation, c-di-GMP has also been shown to modulate the expression of genes associated with extracellular electron transfer (EET). For instance, elevated levels of c-di-GMP upregulate EET-related genes in Geobacter^[15] and Shewanella species^[16]. Based on our 2024 project and transcriptomic analysis, overexpression of the AFE_0053 gene encoding DGC in A. ferrooxidans was found to elevate intracellular cyclic di-GMP levels and upregulate electron transport-related genes. Building on this finding, we aim to enhance electron transfer efficiency in A. ferrooxidans through targeted overexpression of AFE_0053, with the goal of optimizing its performance in microbial electrochemical devices.

During carbon sequestration, the chemical energy generated is ultimately stored in organic matter^[17]. This form of energy storage is particularly significant for this project, especially since microbial electrosynthesis offers a distinct advantage by minimizing energy losses compared to conventional electrical energy storage methods. Through electrocatalysis, microorganisms can effectively integrate solar energy with CO₂ fixation and fuel production processes. By utilizing electricity, they convert CO₂ into organic compounds, thereby achieving efficient transformation of electrical energy into chemical energy.

In industrial contexts, glycerol (also known as glycerine) serves as a fundamental chemical feedstock, often referred to as the 'industrial monosodium glutamate' due to its widespread applications. Its value stems from properties such as high hygroscopicity, humectancy, non-toxicity, and mildness, making it a key moisturizing agent in cosmetics and toothpaste. In the food industry, glycerol functions as both a sweetener and humectant, helping to maintain soft texture in products like candies and baked goods. Moreover, glycerol is extensively used in manufacturing explosives (e.g., nitroglycerin), synthetic resins, lubricants, and antifreeze ^[18]. Given the broad industrial utility of glycerol and the relative maturity of microbial metabolic engineering for its large-scale production, this project proposes the introduction of an exogenous glycerol synthesis pathway to redirect carbon flux toward glycerol biosynthesis.

Figure 3 Metabolic pathways associated with glycerol production in A. ferrooxidans

To achieve glycerol production, we introduced a glycerol synthesis pathway from Saccharomyces cerevisiae, where the gpd1 gene encodes glycerol-3-phosphate dehydrogenase, which catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, and the gpp2 gene encodes glycerol-3-phosphatase, responsible for dephosphorylating glycerol-3-phosphate to yield glycerol^[19] (Figure 2). To improve the catalytic efficiency of these enzymes, we designed fusion proteins by linking Gpd1 and Gpp2 via a flexible peptide linker (Figure 3), creating a spatially coordinated enzyme complex to facilitate substrate channeling and enhance glycerol synthesis. Furthermore, to improve glycerol recovery, we introduced the fps1 gene, which encodes a glycerol facilitator protein (aquaglyceroporin) in the S. cerevisiae plasma membrane. This protein enables efficient passive transport of glycerol from the intracellular space to the extracellular environment, thereby streamlining downstream separation and purification processes.

Figure 4 Gene circuit of the Glycerol Synthesis and Exocytosis Module

In cells, chemical reactions are primarily catalyzed by enzymes. A key enzyme, also known as a rate-limiting enzyme, plays a decisive role in regulating metabolic pathways. It typically catalyzes the slowest and irreversible step in a reaction, acting as a 'bottleneck' in an assembly line. Its activity is precisely regulated by various intracellular factors, such as substrate concentration, hormonal signals, or feedback inhibition. Therefore, by controlling their own reaction rates, key enzymes can effectively regulate the direction and speed of entire metabolic pathways, serving as the central hub for maintaining metabolic homeostasis and energy stability in cells ^[20].

A. ferrooxidans primarily relies on the Calvin–Benson–Bassham (CBB) cycle for CO₂ fixation ^[21]. Phosphoribulokinase (PRK) is one of the key enzymes in this process We selected PRK enzymes from ten different organisms, obtained their structural files directly from the UniProt database or predicted their structures using AlphaFold, performed molecular docking with the substrate Ru5P using AutoDock Vina, and screened two PRK enzymes based on binding energy values. In addition, based on our 2024 project experience, we included the native PRK enzyme of A. ferrooxidans (encoded by the AFE_0053 gene). For instance, although the yedQ gene from E. coli exhibits high efficiency in other organisms, its expression is impaired in extremophiles like A. ferrooxidans, resulting in lower diguanylate cyclase (DGC) activity compared to the native PRK.

PRK catalyzes the phosphorylation of ribulose-5-phosphate (Ru5P) to produce ribulose-1,5-bisphosphate (RuBP). This ATP-dependent reaction is a critical rate-limiting step in the carbon fixation pathway, directly influencing the flux of CO₂ assimilation^[22]. To experimentally verify the activities of the three selected PRK enzymes in A. ferrooxidans, we codon-optimized the corresponding PRK genes to enhance their expression in this host. We anticipate that overexpression of PRK will enhance CO₂ fixation efficiency, thereby accelerating the accumulation of carbonaceous organic matter and promoting cell growth.