For years, the ability to take up carbon dioxide (CO₂) and convert it into sugar has been naturally restricted to plants, algae, and some bacteria. However, researchers have now incorporated this function into a type of yeast used in ethanol production to reduce CO₂ emissions of this process while also increasing ethanol production for biofuels.

The use of ethanol as a fuel dates back to 1900 with the invention of the first car. However, it had low adoption in those years because petroleum-based fuels were cheaper and more readily available.

In 1970, amid the energy crisis and environmental concerns, there was a renewed interest in ethanol as an alternative to fossil fuels as it has a cleaner combustion, releasing less CO₂ and other pollutants into the atmosphere. Today, ethanol is used in some countries blended with gasoline in different proportions to power flexible fuel cars.

Currently, ethanol is produced on large scales using yeast, specifically Saccharomyces Cerevisiae (S. Cerevisiae), through the fermentation of sugars sourced from sugarcane, wood, straw, and algae. But here’s the hitch: Fermentation not only yields ethanol but releases CO₂, throwing a wrench into the eco-friendly use of ethanol as a biofuel.

“While emission from bioprocesses is generally lower than that from fossil fuel-based processes, emission is inevitable when utilizing S. cerevisiae,” explained Sujeong Park, a doctorate student at the School of Food Science and Biotechnology at Kyungpook National University and first author of the study.

Greenhouse gases, like CO₂, act like a blanket that prevents heat escaping from the atmosphere. Moderate levels create optimal temperature conditions for life on Earth, but the excess causes abnormal temperature increases that make living conditions more challenging.

“Global warming seriously threatens human survival and ecosystem[s], triggering problems such as natural disasters and food shortages. Therefore, diverse efforts to reduce CO₂ emissions should be made,” said Park.

Recycling CO₂ and boosting bioethanol

To tackle this issue, the team genetically engineered an enzyme called Rubisco into S. Cerevisiae yeast. Rubisco is a key enzyme that allows for different plants, some bacteria, algae, and protists to fix CO₂, meaning to capture atmospheric CO₂ and incorporate it into sugars used for energy.

“Native S. cerevisiae does not naturally contain Rubisco,” said Park. “However, it has been shown that when Rubisco from other microorganisms is introduced into S. cerevisiae, it can work.

“We tried to reduce the CO₂ emissions from bioethanol production into the atmosphere by introducing Rubisco in yeast to reutilize the CO₂ released in fermentation.”  To do this, the researchers used CRISPR-gene editing technology to precisely modify the yeast’s genome, inserting the gene for the production of Rubisco.

With this strategy, the researchers created S. Cerevisiae SJ03, a genetically modified yeast strain with the ability to fix CO₂. However, instead of using the atmospheric gas as photosynthetic organisms do, this yeast make use of the CO₂ released during fermentation and recycles it.

To test the performance of the new strain in producing ethanol and minimizing CO₂ production, the team began by fermenting the sugar, xylose.

When they measured CO₂ emission and ethanol yield in the cultures, they found that the new strain produced 0.657g of CO₂ for every gram of ethanol and 0.36g of ethanol for each gram of xylose consumed. This means nearly 10% less CO₂ release and 17% more ethanol yield than a control S. Cerevisiae that lacked the CO₂ fixing pathway.

But where does the boost in ethanol production come from? “In SJ03, after fixation by Rubisco, CO₂ is converted to 3-phosphoglycerate, a key intermediate in ethanol production,” explained Park.

The trapped CO₂ feeds the yeast of this intermediary molecule, which is then converted during the normal ethanol synthesis, producing more ethanol and removing more CO₂ from the reaction.

Although the percentages obtained are moderate, this is the highest efficiency of conversion of recycled CO₂ into ethanol reported for CO₂-fixing yeasts.

According to Park, one of the hurdles to overcome that could enhance the efficiency of CO₂ fixation and ethanol yield is the low specificity of Rubisco for CO₂. This enzyme not only fixes CO₂ but also oxygen; for every three CO₂ molecules fixed, it fixes one of oxygen. “Rubisco is […] an inefficient enzyme,” explained Park. “In environments with oxygen, Rubisco reacts with oxygen instead of utilizing CO₂, consuming energy.

“If research is conducted to enhance rubisco affinity for CO₂ by reducing its sensitivity to oxygen, it could significantly increase dioxide carbon fixation,” she concluded. 

Reference: Sujeong Park, Bo-Ram Park, et al., Functional expression of RuBisCO reduces CO2 emission during fermentation by engineered Saccharomyces cerevisiae, Process Biochemistry (2023). DOI: doi.org/10.1016/j.procbio.2023.10.013

Feature image credit: Kai Dahms on Unsplash