Nannochloropsis oceanica occupies a special place among photosynthetic chassis organisms. It is a haploid alga whose nuclear genome has been fully sequenced and extensively annotated, and it naturally produces high levels of triacylglycerol rich in eicosapentaenoic acid, a high-value omega-3 long-chain polyunsaturated fatty acid increasingly sought after as a food resource for human consumption. Genetic tools for gene overexpression, suppression, subcellular localization and genome editing have been developed for the species over the past decade, and transgene expression has proven stable. Yet most published engineering efforts have introduced no more than three transgenes at a time, and the plasmid construction behind them remained laborious. That bottleneck is precisely what a MoClo system is designed to eliminate.

MoClo is built on the Golden Gate cloning technique, which exploits Type IIS restriction enzymes that recognize asymmetric DNA sequences and cut outside their recognition sites, generating defined overhangs. Because these overhangs can be assigned to specific positions in a genetic construct, multiple DNA fragments can be ligated together seamlessly in a single reaction tube, with no scar sequences left behind. The approach, first formalized in 2011, has since spawned tailored toolkits for bacteria such as Escherichia coli, the yeast Saccharomyces cerevisiae, the land plant Nicotiana benthamiana, and microalgae including Synechocystis, Synechococcus elongatus and Chlamydomonas reinhardtii. The new work extends this lineage to the stramenopile algae, adopting the syntax proposed by the plant synthetic biology community so that parts can, in principle, be exchanged across platforms.

The architecture of the Nannochloropsis toolkit follows the familiar three-level hierarchy of MoClo systems. Level 0 vectors carry individual genetic parts flanked by eight distinct fusion sites; these parts can be shuttled into level 1 vectors to build single transcriptional modules, and those modules can then be stacked into level 2 vectors carrying multiple expression cassettes. The 91 parts represent 39 unique genetic elements distributed across the standard framework and fall into clear functional categories: 12 promoters placed at three different positions, five terminators plus one polygenic terminator, five selectable markers, six signal peptides with two position options each, four reporter genes with five position options, five affinity tags, and one 2A peptide for expressing fused proteins from a single open reading frame.

Functional validation began with the selectable markers, which determine how efficiently transformed cells can be recovered. The team assembled four vectors pairing either the zeocin-resistance gene Ble from Streptoalloteichus hindustanus or the hygromycin B-resistance gene Hyg from Streptomyces hygroscopicus with two different promoter-terminator combinations: the RIBI promoter paired with the HS terminator, both of Nannochloropsis origin, and the VCP2 promoter paired with the FCPA terminator from the diatom Phaeodactylum tricornutum. Counting antibiotic-resistant colonies after electroporation revealed that the RIBI-HS combination outperformed VCP2-FCPA regardless of which marker was used, and reverse transcription quantitative PCR confirmed that the winning pair also drove higher transcription of the marker genes. The lesson, the authors note, is that promoter activity in this alga depends strongly on the terminator it is paired with, a phenomenon also documented in Chlamydomonas.

To quantify expression more precisely, the researchers turned to NanoLuciferase, a small and highly sensitive reporter. They built six level 2 vectors in which the Nluc gene was driven by three promoters, RIBI, VCP2 and the beta-tubulin promoter TUB, each paired with either the HS or FCPA terminator. Luminescence from transformed cultures correlated linearly with cell density, allowing quantitative comparison, and the spread between constructs was striking: otherwise identical constructs differing only in terminator produced dramatically different light output. The best-performing combination, pairing the TUB promoter with the FCPA terminator, yielded the highest luminescence, reinforcing the conclusion that promoter-terminator compatibility must be evaluated as a unit rather than assuming promoter strength is an intrinsic property.

Visualization tools received equally systematic treatment. The toolkit’s fluorescent protein set includes enhanced green fluorescent protein, enhanced yellow fluorescent protein and StayGold, a highly photostable and bright GFP variant that had never before been evaluated in Nannochloropsis. All three produced strong cytosolic fluorescence when expressed alone. The team then fused transit peptides to direct the reporters to specific compartments: chloroplast transit peptides from the glycine cleavage system L protein and from the violaxanthin/chlorophyll a-binding protein steered eGFP into the chloroplast, while the simian virus 40 nuclear localization sequence confined eYFP to a single nuclear spot. Crucially, dual-cassette constructs allowed two reporters to be targeted simultaneously, one to the chloroplast and one to the nucleus, demonstrating that the system supports co-localization studies within a single transformant. Five epitope tags, HA, 6xHis, Flag, Myc and StrepII, were also validated by immunoblotting, with the HA tag producing the strongest signal and four of the five tags being demonstrated in this organism for the first time.

The demonstration that ties the toolkit together is metabolic: the modular assembly of keto-carotenoid biosynthetic pathways. Canthaxanthin and astaxanthin are red-pigmented carotenoids with powerful antioxidant properties and established markets in food, feed and nutraceuticals. The researchers assembled a beta-carotene ketolase from Chlamydomonas, targeted to the chloroplast, to produce canthaxanthin, and a two-enzyme module combining that ketolase with a beta-carotene hydroxylase from Haematococcus lacustris to produce astaxanthin. Transformants turned visibly orange, accumulating 4.5 milligrams of canthaxanthin or 2.8 milligrams of astaxanthin per gram of dry weight, comparable to yields previously achieved with traditional cloning. Interestingly, the engineered strains accumulated more keto-carotenoid under normal light than under high light, the opposite of the stress-induced production seen in natural producers like Haematococcus, likely because high light depletes the beta-carotene precursor pool in Nannochloropsis. Canthaxanthin was even detected in isolated thylakoid membranes, at 4.4 moles per 100 moles of chlorophyll a, and transcriptomic analysis showed that the engineered accumulation perturbed endogenous carotenoid gene expression only mildly, with fold changes below 1.5.

The practical implications extend well beyond pigments. Because assembly is modular, the authors estimate that researchers can now design and construct complex constructs carrying up to six distinct genes in a single level 2 vector within days rather than weeks, and the availability of multiple selectable markers means more than one level 2 vector can be introduced at once, theoretically allowing over ten genes to be stacked. The toolkit also proved portable: introducing the canthaxanthin module into Nannochloropsis gaditana, a close industrial relative, likewise yielded substantial canthaxanthin. Combined with existing CRISPR-Cas genome editing systems and high-throughput microfluidic screening, the toolkit positions Nannochloropsis as a genuine sunlight-driven chassis for carbon-neutral biomanufacturing, in which captured CO2 becomes feedstock for lipids, carotenoids and other high-value biochemicals grown in seawater rather than on arable land.

There remain honest limits. Astaxanthin yields in the engineered Nannochloropsis still fall far short of the roughly four percent of cell dry weight that stressed Haematococcus cultures can reach, and the authors point to strengthening the plastidial MEP pathway and beta-carotene supply as the logical next engineering targets. But the structural obstacle, the absence of a fast, standardized, expandable assembly system, has now been removed. With 91 validated parts, a shared syntax compatible with the broader plant and algal synthetic biology community, and demonstrated performance across two Nannochloropsis species, the toolkit converts what was once a craft-based cloning exercise into an engineering discipline, opening the door to rapid design-build-test cycles for anyone hoping to turn these tiny marine cells into photosynthetic factories.

Subject of Research: Development of a modular cloning toolkit for metabolic engineering of the microalga Nannochloropsis

Article Title: A modular synthetic biology toolkit unlocks metabolic engineering of the industrially relevant alga Nannochloropsis

Article References: A modular synthetic biology toolkit unlocks metabolic engineering of the industrially relevant alga Nannochloropsis. (n.d.). https://doi.org/10.1007/s44307-026-00096-w

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00096-w

Keywords: synthetic biology, Nannochloropsis, MoClo, Golden Gate cloning, metabolic engineering, microalgae, canthaxanthin, astaxanthin, carotenoid biosynthesis, CO2 valorization, genetic toolkit, algal biotechnology

The answer that MES offers is deceptively simple in concept. A typical system consists of two chambers separated by an ion-exchange membrane. In the anodic compartment, water or organic substrates are oxidized to release electrons. In the cathodic compartment, electroactive microorganisms intercept those electrons either directly from the cathode surface, where they form biofilms, or indirectly via hydrogen gas and redox mediators generated at the electrode. Once inside the cell, the electrons enter microbial metabolism and serve as reducing power for fixing carbon dioxide. Because the microbial catalysts are alive, they replicate themselves, operate under mild near-ambient conditions, and tolerate feedstock variability in a way that expensive metal catalysts cannot. And because microbial metabolism is enormously diverse, MES can in principle reach C3 and longer-chain molecules that remain stubbornly out of reach for conventional electrochemistry.

The contrast with existing CO2 conversion technologies is stark. Thermocatalytic routes such as the Sabatier reaction and reverse water-gas shift chemistry require high temperatures, high-purity hydrogen as a reductant, and durable metal catalysts that suffer from carbon deposition and poisoning. Electrochemical CO2 reduction on copper and other catalysts can make carbon monoxide, formate, and C2 products under ambient conditions, but selectivity for C3 and more complex molecules remains poor, catalysts deactivate, and long-term stability is inadequate. Meanwhile, green hydrogen produced by water electrolysis currently costs between 4.5 and 6.0 US dollars per kilogram, meaning that simply reducing CO2 with hydrogen often yields chemicals worth less than the hydrogen consumed. Microbial catalysts sidestep many of these constraints, using self-assembled enzymatic pathways such as the Wood-Ljungdahl route to weave carbon dioxide into multi-carbon products with remarkable specificity.

The performance numbers reported in recent studies are striking. Acetate, the workhorse product of MES, is routinely produced with coulombic efficiencies exceeding 90 percent, meaning that more than nine out of every ten electrons supplied by the circuit end up stored in the target molecule. Most impressively, a continuous thermophilic hydrogen-mediated system using the acetogenic bacterium Thermoanaerobacter kivui has achieved acetate concentrations of up to 29.4 grams per liter, roughly 490 millimolar, from carbon dioxide. That is an order of magnitude beyond typical laboratory titers and begins to approach concentrations relevant to industrial separation. For context, commercial acetic acid is produced today by methanol carbonylation in plants rated at 200,000 to 650,000 tonnes per year, so MES still has far to travel in scale, current density, and process intensification, but the trajectory of improvement is unmistakable.

Beyond acetate, the product spectrum widens considerably. When acetate and ethanol accumulate in the reactor, chain-elongating microbes take over, running reverse beta-oxidation pathways that stitch short-chain intermediates into C4 through C8 medium-chain fatty acids such as butyrate and caproate, which command far higher market prices as feed and chemical precursors. Solventogenic metabolism can be triggered by tuning reactor operating conditions, reducing accumulated organic acids to ethanol, butanol, and 2,3-butanediol. Reductive branches of the tricarboxylic acid cycle yield lactate and succinate. Photo-bioelectrochemical systems using the purple bacterium Rhodobacter sphaeroides go further still, simultaneously converting CO2 into biomass and hydrogen gas, while MES-integrated setups direct CO2-derived carbon into intracellular polyhydroxybutyrate, a biodegradable plastic. In each case, the electron source is the electrode rather than sugar, decoupling production from agricultural feedstocks.

One of the most commercially mature applications is biogas upgrading. Anaerobic digestion plants in Germany, Denmark, and the Netherlands already supply 10 to 20 percent of renewable power in parts of the European Union, but raw biogas contains only 50 to 70 percent methane, with the remainder mostly CO2 plus troublesome impurities such as siloxanes and sulfur compounds. Conventional pressure swing adsorption can polish biogas to roughly 97 percent methane for pipeline injection, but contaminants foul the adsorbents and raise costs. MES offers an elegant pre-treatment: raw biogas is sparged into the cathode chamber, where methanogenic archaea electrochemically reduce the CO2 fraction to additional methane. Recent work with biogas from an operating anaerobic digestion plant achieved 95 percent methane in the upgraded gas at a methane production rate of 8.8 liters of CH4 per square meter of catalyst per day. Because gaseous impurities dissolve into the liquid phase during this step, the downstream PSA unit faces a lighter, cleaner load, and the captured CO2 is not merely discarded but converted into fuel.

The review also highlights an emerging application with obvious public appeal: power-to-protein. In these schemes, renewable electricity splits water to generate hydrogen, formate, or methanol, which feed bioreactors cultivating protein-rich microorganisms for food and feed. Single-cell protein is not new; companies such as Unibio and Calysta have commercialized fermentation-based production, but their processes traditionally rely on sugar substrates that compete with food supply. A techno-economic assessment of solar-driven microbial protein production found that photovoltaic-powered systems could achieve protein yields per unit of land up to an order of magnitude higher than conventional agriculture, and that estimate assumed conservative solar-to-electricity and power-to-chemical conversion efficiencies of 5 percent or less. Because electricity delivers reducing power to microbes far more efficiently than photosynthesis delivers it to crops, the land-use arithmetic of protein production could be transformed, freeing farmland while feeding a growing population.

None of this means MES is ready for prime time, and the authors are candid about the obstacles. Most laboratory studies still rely on small H-type reactors whose distant electrodes and ion-exchange membranes impose severe ohmic resistance and overpotentials, often pushing cell voltages above 3 volts and crushing energy efficiency. The oxygen evolution reaction at the anode is kinetically sluggish, particularly on carbon-based electrodes, and acts as a bottleneck for the entire system. Mixed microbial consortia, while robust, tend to foul membranes and default to acetate rather than more valuable products, whereas pure cultures of Shewanella, Sporomusa, Geobacter, or Rhodobacter offer precision but demand sterility. The field is responding with nanostructured and conductive-polymer-coated cathodes such as polyaniline-deposited graphite felt, synthetic-biology strains with engineered electron-transfer and metabolic pathways, artificial redox mediators like neutral red, and scalable reactor geometries including bubble columns and 3D-printed electrodes designed to improve hydrogen delivery.

The most pragmatic near-term strategy may be integration rather than replacement. Because MES alone is unlikely to reach commercial viability at current productivities, the authors describe two-stage processes in which MES first converts CO2 to acetate, which is then recovered and fed to a second, optimized bioprocess that converts it into long-chain alkyl esters or high-value isoprenoids. Such hybrid configurations sidestep the selectivity limits of mixed-culture MES while still exploiting its unique ability to fix carbon with electricity. Coupled with direct air capture and low-carbon power, an integrated MES platform could even operate as a carbon-negative factory, drawing down atmospheric CO2 while selling chemicals, fuel, and protein. The remaining challenges, from current density to product recovery costs, are formidable but increasingly quantified, and for the first time the road from laboratory biofilm to industrial biorefinery looks less like a leap of faith and more like an engineering schedule.

Subject of Research: Microbial electrosynthesis using renewable electricity to convert CO2 into value-added chemicals, biogas, and protein

Article Title: Renewable electricity–driven microbial electrosynthesis for high-value CO2 valorization: recent trends

Article References: Lee, C. H., Kim, M., Kong, D. S., Son, H., & Kim, J. R. (2026). Renewable electricity–driven microbial electrosynthesis for high-value CO2 valorization: recent trends. Advances in Industrial and Engineering Chemistry, 2(1), Article 4. https://doi.org/10.1007/s44405-026-00044-1

Image Credits: AI Generated

DOI: 10.1007/s44405-026-00044-1

Keywords: microbial electrosynthesis, CO2 valorization, renewable electricity, acetate production, biogas upgrading, single-cell protein, polyhydroxybutyrate, bioelectrochemical systems, Wood-Ljungdahl pathway, power-to-protein, carbon capture and utilization, electroactive bacteria