Single Regulatory Protein Supercharges Hydrogen Production in Deep-Sea Archaeon

Deep beneath the ocean’s surface, in hydrothermal vents where scalding fluid rich in carbon monoxide and formate streams into oxygen-free seawater, microorganisms have evolved remarkable ways to eke out a living. Among them is Thermococcus onnurineus NA1, a hyperthermophilic archaeon isolated from a vent field near Papua New Guinea that can grow by oxidizing carbon monoxide or formate and releasing hydrogen gas as a byproduct. That metabolic trick has made the organism a favorite among researchers hunting for biological routes to clean hydrogen fuel. Now, a team at the Korea Institute of Ocean Science and Technology has revealed how a single regulatory protein, called EnfR, exerts a surprisingly broad influence over this formate-driven metabolism, and their findings suggest new ways to engineer microbes for more efficient hydrogen production.

The new study, published in Applied Microbiology and Biotechnology, builds on earlier work showing that EnfR acts as a crucial regulator of carboxydotrophic metabolism, the suite of reactions that lets T. onnurineus NA1 thrive on carbon monoxide. EnfR controls the expression of the codh-mch-mnh3 gene cluster, which encodes the carbon monoxide dehydrogenase and associated membrane complexes that funnel electrons from CO oxidation into energy conservation. But carbon monoxide is only half of the story. The archaeon also grows vigorously on formate, a one-carbon compound that is increasingly viewed as a promising liquid carrier for hydrogen and as a key intermediate in synthetic carbon-fixation pathways. Whether EnfR also governed formate metabolism was an open question.

To answer it, the researchers took a two-pronged approach: they deleted the enfR gene entirely and, in a separate strain, cranked up its expression well beyond normal levels. The results were striking in both directions. When enfR was deleted, cells grown on formate suffered a clear growth defect, confirming that the regulator matters even for this non-CO substrate. When enfR was overexpressed, the opposite occurred. In pH-stat batch cultures, a controlled setup in which pH is held constant by automated addition of base as acids accumulate, the overexpression strain grew significantly faster than the parental strain and produced markedly more hydrogen gas. A single transcriptional tweak, in other words, was enough to boost one of the organism’s most industrially attractive traits.

The obvious hypothesis would be that EnfR simply switches on the genes that handle formate directly. Formate-dependent growth in T. onnurineus NA1 relies on a gene cluster called fdh2-mfh2-mnh2, which encodes formate dehydrogenase, the enzyme that splits formate into carbon dioxide and electrons, together with a membrane-bound hydrogenase complex that uses those electrons to make hydrogen while pumping ions to generate energy. But when the team measured transcript and protein levels for fdh2 in the overexpression strain, they found no significant differences from the parental strain. The formate-splitting machinery itself was not being induced. Something else was responsible for the growth advantage.

To find out what, the researchers turned to transcriptomics, sequencing the messenger RNA of both strains to build a genome-wide picture of gene activity. The comparison revealed 219 genes with differential expression between the EnfR-overexpression strain and the control. That is a substantial rewiring for a single regulatory protein, and the affected genes clustered into several functionally coherent groups. Among the upregulated players were genes encoding a ferredoxin-dependent membrane-bound hydrogenase, a family of enzymes that oxidize reduced ferredoxin, a small iron-sulfur protein that serves as a cellular electron shuttle, and channel the electrons into hydrogen production at the membrane.

Also among the differentially expressed genes were 2-ketoacid ferredoxin oxidoreductases, enzymes central to the archaeon’s central carbon metabolism. These enzymes catalyze the oxidation of 2-ketoacids, intermediates in amino acid and sugar breakdown, and in doing so they load electrons onto ferredoxin. Together with the ferredoxin-dependent hydrogenase, they form a pipeline: central metabolism generates reduced ferredoxin, and the hydrogenase drains those electrons off to produce hydrogen. By adjusting the expression of both ends of this pipeline, EnfR overexpression appears to change how much reducing power flows through the cell and how efficiently it is converted into hydrogen gas.

A third notable group of affected genes encoded flagellins, the protein subunits that assemble into the rotating filaments archaea use for motility. Changes in flagellin expression hint that the overexpression strain may alter its swimming behavior or surface properties as part of its overall physiological response, though the study’s central finding lies elsewhere. The authors propose that the collective effect of the 219 differentially expressed genes is to modulate the levels of reduced electron carriers, particularly ferredoxin, inside the cell. In this view, the growth and hydrogen-production gains do not come from making more formate dehydrogenase, but from reshaping the redox landscape so that the existing formate-oxidizing machinery operates under more favorable conditions.

This redox-balancing interpretation carries real weight for biotechnology. Microbial hydrogen production is often limited not by the abundance of the key enzymes but by the traffic of electrons between them. When reduced electron carriers accumulate faster than they can be consumed, metabolism stalls; when they are scarce, energy conservation suffers. Engineering a regulator that tunes the expression of ferredoxin-linked oxidoreductases and hydrogenases in concert offers a way to keep that electron traffic flowing smoothly. The fact that EnfR overexpression achieved this without touching fdh2 transcription suggests that indirect, systems-level tuning can be as powerful as direct induction of the canonical pathway genes, a lesson that may generalize to other hydrogen-producing microbes.

The work also deepens understanding of how archaea, the third domain of life, orchestrate their energy metabolism. T. onnurineus NA1 occupies a niche where formate and carbon monoxide are abundant, and its regulatory network must decide how to allocate resources between these alternative fuels. EnfR’s dual role in carboxydotrophic and formate-linked metabolism suggests it functions as a hub connecting carbon and electron flow across substrates. Because the organism grows at high temperatures and thrives in conditions mimicking hydrothermal vents, it is also a robust chassis for industrial processes that would challenge many conventional microbes, and regulators like EnfR provide natural control points for optimization.

The research, funded by the Korea Institute of Ocean Science and Technology in-house program, was led by Bo Gyoung Choi and Sungjin Pyo, who contributed equally, together with Sung-Mok Lee, Ji-in Yang, Seong Hyuk Lee, Myeong-Eun Jegal, Hyun Sook Lee and Sung Gyun Kang, with correspondence from Hyun Sook Lee and Sung Gyun Kang. As hydrogen economies gather momentum worldwide, studies like this one show that some of the most useful engineering blueprints may already be written into the genomes of organisms living at the bottom of the sea. The next step will be to pin down exactly which of the 219 regulated genes deliver the biggest gains, and whether the same redox-tuning strategy can be transplanted into other production strains.

Subject of Research: Regulation of formate-dependent hydrogen production by the EnfR transcriptional regulator in the archaeon Thermococcus onnurineus NA1

Article Title: Physiological and transcriptomic changes by enfR overexpression in a formate- utilizing archaeon

Article References: Choi, B. G., Pyo, S., Lee, S.-M., Yang, J.-I., Lee, S. H., Jegal, M.-E., Lee, H. S., & Kang, S. G. (2026). Physiological and transcriptomic changes by enfR overexpression in a formate- utilizing archaeon. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-026-14047-x

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14047-x

Keywords: Thermococcus onnurineus NA1, EnfR, archaea, formate metabolism, hydrogen production, formate dehydrogenase, ferredoxin, transcriptomics, gene regulation, redox balance, hydrothermal vents, biohydrogen