From lab anomaly to climate solution: Decoding the reductive glycine pathway

As the impacts of climate change become increasingly pressing, so does the search for alternatives to fossil fuels and strategies to mitigate greenhouse gas emissions. Biological CO₂ fixation pathways have been an important part of this effort for some time, but the discovery of a brand-new, highly energy-efficient pathway has sparked fresh interest.

Biological CO₂ fixation pathways convert inorganic CO₂ in the atmosphere into organic compounds. They consume CO₂, an important greenhouse gases driving climate change, and use them to produce valuable products we need. While six of these pathways were identified between 1950 and 2008, a groundbreaking seventh CO₂ fixation pathway called the reductive glycine pathway was discovered recently. Spearheaded by IE University microbiologist Dr. Irene Sánchez-Andrea, the research was carried out in collaboration with Wageningen University & Research in The Netherlands and the Max Planck Institute in Germany.

Here, we trace this research from the biological anomaly that sparked it to the pioneering results it delivered with a deep dive into Dr. Sánchez-Andrea’s work on CO₂ fixation pathways.

Digging into Desulfovibrio desulfuricans

Dr. Sánchez-Andrea’s work began with a detailed examination of Desulfovibrio desulfuricans, a well-known and widely studied sulfate-reducing bacterium.

Desulfovibrio is common in soil, water, animal stools, aquatic environments with organic material, water-logged soils, brackish water, sewage and freshwater sediments. Previously, extensive research had always suggested that it needs organic carbon sources like acetate to grow. In the early phases of her research, however, Dr. Sánchez-Andrea and her team found in literature that D. desulfuricans thrived for extended periods without any organic matter at all. This felt like an impossibility—at the very least, an error. How could this bacterium appear and flourish in environments that lacked its essential nutrients?

Dr. Sánchez-Andrea’s early results were slow. When she first attempted to grow the bacterium on hydrogen and CO₂ alone, the changes were so slight that they felt unreliable. “Typically, bacterial cultures become cloudy as cells multiply, but the cloudiness—or turbidity—I was seeing was so subtle that for a long time I wasn’t sure if it was growth or just contamination, which is always a risk,” she explains. After the first transfer to the new environment, she only saw growth after a lag phase longer than six days and with an optical density of less than 0.12—in other words, a very low yield.

However, her results started to change after several transfers, when she found that the growth characteristics in autotrophic conditions improved and reached those observed in heterotrophic environments. To test whether this improvement came from laboratory evolution, she took bacterial populations that had been grown heterotrophically for three years with no previous exposure to autotrophic conditions and transferred them to CO₂-only growth. While they initially showed the same long lag phases, after just one additional autotrophic transfer, they achieved the same rapid growth as cultures that had been transferred autotrophically for years. 

This ruled out laboratory evolution. The bacterium didn’t need to evolve or adapt over time to grow on CO₂. Instead, the capability was already encoded in its genetics. And with that, the real detective work began. 

Uncovering the seventh pathway

The next step was to sequence the bacterium’s complete genome, and in the process, search for the molecular structures of CO₂ fixation pathways. “Because I knew that it was fixing CO₂, I checked the genome for the six known pathways,” elaborates Dr. Sánchez-Andrea. “It was obvious that it was fixing  CO₂. as it was growing on it, but when I checked for the known routes, none were complete.”

The genome lacked crucial enzymes for every existing route. There was no ribulose-1,5-bisphosphate carboxylase, the key enzyme of photosynthesis; no acetyl-CoA synthase, which is central to another major pathway; and no malate dehydrogenase or succinyl-CoA synthetase, essential for reductive TCA cycle variants. With these discoveries, Dr. Sánchez-Andrea came to a startling realization. As she says in her final paper, “The observed autotrophic growth of D. desulfuricans indicates the presence of an unknown CO₂ fixation pathway in this microorganism.”

To unpack these initial observations further, she analyzed not just what genes the bacterium possessed, but which ones it used under a variety of conditions. This comparative multiomics strategy examined the transcriptome (which genes were active), proteome (which proteins were produced) and metabolome (which chemical compounds were present). 

Her results revealed specific genes and proteins that became more active during autotrophic growth, bringing several key players to light: the glycine cleavage system, glycine reductase complex, formate-tetrahydrofolate ligase and acetyl-CoA synthetase. Together, these components formed the molecular machinery of what would be recognized as the seventh CO₂ fixation pathway: the reductive glycine pathway. 

And with it, a breakthrough. From an initial observation of unusual, even impossible bacterial behavior, Dr. Sánchez-Andrea had discovered an entirely new biological mechanism for capturing atmospheric CO₂.

A breakthrough in efficiency

What sets the seventh CO₂ fixation pathway apart from its predecessors is its remarkable energy efficiency—a factor that could have far-reaching implications for both research and application.

In cellular metabolism, energy is currency, and every biological process requires it. Typically, this energy comes in the form of adenosine triphosphate, or ATP. When bacteria fix CO₂, they have to invest energy to convert waste gas into valuable products. But not all fixation pathways require the same amount of energy.

The reductive glycine pathway, Dr. Sánchez-Andrea´s team found, consumes just one to two ATP molecules to produce one molecule of pyruvate. Pyruvate is a fundamental building block that cells use to construct everything from amino acids to complex organic compounds. At this rate, the reductive glycine pathway quickly becomes one of the most energy-efficient pathways available, comparable to the reductive acetyl-CoA pathway, which was previously considered the gold standard of energy-efficient CO₂ fixation.

This efficiency stems from the pathway’s elegant design. Instead of complex, multi-step processes that burn through ATP, its approach is more streamlined. CO₂ is first reduced to formate by formate dehydrogenase, then converted through a series of reactions involving tetrahydrofolate derivatives to produce glycine, and subsequently reduced to acetyl-phosphate and converted to useful compounds.

While we’re on formate dehydrogenase, it’s important to note that this key enzyme helps the pathway tolerate oxygen well, too. This once again sets the reductive glycine pathway apart from other pathways, which are sometimes destroyed by oxygen exposure.

Why are these characteristics important?

Because they directly affect how commercially viable the pathway is. “If its ATP requirements are lower, bacteria can grow faster, produce more product per unit of input and operate economically at scale,” explains Dr. Sánchez-Andrea. Better oxygen tolerance helps to lower industrial implementation costs and expand the number of potential applications. This makes the pathway more likely to get out of the lab and into industrial environments, where it can have a material impact. 

Ammonia matters

In addition to its energy efficiency and oxygen tolerance, one of the most surprising discoveries about the reductive glycine pathway was its relationship with ammonia. Unlike other CO₂ fixation routes, the research found that the pathway’s efficiency depended directly on the availability of nitrogen. 

This emerged when the researchers noticed that genes associated with ammonia limitation were strongly upregulated during autotrophic growth, even though the bacteria were provided with the same ammonia concentrations used in heterotrophic conditions. The upregulated genes included ammonia transporters and nitrogen-processing enzymes, suggesting the bacteria needed higher internal ammonia concentrations specifically for CO₂ fixation.

A few experimental tests confirmed this. While heterotrophic growth was unaffected by changes in ammonia levels, autotrophic growth rates increased dramatically with higher ammonia concentrations.

Importantly, the final biomass yield stayed constant, indicating that excess ammonia wasn’t being incorporated into cellular structures but rather used temporarily and then released. This suggested that it was playing a catalytic role in the CO₂ fixation process. This ammonia dependency stems from the glycine cleavage system, and higher ammonia concentrations help drive this thermodynamically challenging reaction in the desired direction, making the pathway more efficient.

This is important because it affects how the pathway might be scaled. But it means industrial applications will need to manage the availability of nitrogen as an important cofactor. 

The ammonia dependency distinguishes the reductive glycine pathway from other CO₂ fixation mechanisms and highlights the interconnected nature of cellular metabolism. Instead of viewing carbon and nitrogen cycles as separate processes, this pathway demonstrates how they can be intimately linked in unexpected ways.

The seventh pathway in the real world

Within the lab, researchers have already shown how the pathway’s components can be transplanted across diverse biological systems. The serine variant, for example, has been successfully implemented in Escherichia coli, Cupriavidus necator and even yeast. This transferability means that engineers aren’t limited to working with the original sulfate-reducing bacterium, and that they can insert this CO₂-fixing machinery into established industrial microorganisms that are easier to cultivate and scale.

But what happens when we go beyond the lab? Does the seventh pathway have the potential to effect real-world change? Can it help us find truly sustainable alternatives to fossil fuels? 

Dr. Sánchez-Andrea is quick to emphasize that there is no one-size-fits-all solution to the fossil fuel crisis. “It can be detrimental to put all our hopes on one technology that we think will solve all of our problems for us,” she explains. “To have a net positive effect, CO₂ removal technologies will have to be deployed at scale all over the world—and we’re going to need as many as possible to effect meaningful change.”

That said, the seventh pathway should form an integral part of any future approach.

Even in its unmodified state, D. desulfuricans produces an impressive array of important compounds, including biomass, amino acids, acetate and formate. These products have implications for alternative food, as well as flavor and feed additives. And formate is increasingly being investigated as a safer, easier-to-transport alternative to hydrogen in green economy applications.

Add its synthetic applications to this list, and the pathway’s usage broadens exponentially. By inserting specific genes, scientists can turn engineered microorganisms into biological factories that consume CO₂ and produce desired products for industries that include the chemicals, pharmaceutical, cosmetics and metals sectors. It’s here that the seventh pathway’s excellent energy efficiency comes into play, making it an attractive option.

Challenges ahead

Ultimately, there are several factors that will determine the seventh pathway’s industrial success. Its need for ammonia as a cofactor adds operational complexity—industrial systems will have to maintain optimal ammonia concentrations. And while the pathway shows oxygen tolerance, further research needs to be done in terms of optimizing these conditions in commercial environments.

These challenges are certainly worth investigating and overcoming, however. Rising atmospheric CO₂ levels demand innovative and scalable solutions, and every advance that supports the transition away from fossil fuels contributes to the broader effort to mitigate climate change. The reductive glycine pathway, with its exceptional energy efficiency, offers a promising addition to the portfolio of carbon fixation strategies. By converting the primary driver of climate change into raw material for sustainable production, it offers not only a scientific breakthrough but also a potential pathway toward practical climate solutions.