The Liquid Core of Photosynthesis

Researchers at the Max Planck Society have gained fundamental insights into the pyrenoid, a key enhancer of CO₂ fixation

Green algae boost CO₂ fixation efficiency with the help of the pyrenoid—a liquid droplet within the chloroplast that concentrates CO₂ in the direct vicinity of the photosynthetic enzyme Rubisco. Researchers at the Max Planck Institute for Terrestrial Microbiology and the University of Marburg reduced this organelle to its simplest form and unlocked its evolutionary history by reconstructing ancient proteins. The findings could help improve photosynthesis in crops, thereby promoting food security and climate protection.

To the point:

• The pyrenoid, a concentrated droplet within the chloroplast of green algae, increases the local CO₂ concentration via the protein EPYC1 and enhances the efficiency of Rubisco.

• Reconstruction of ancient EPYC1 versions showed that the ability to form pyrenoid condensates is an ancient, conserved trait.

• Condensates do not modify Rubisco but rather create an environment in which CO₂ is enriched solely through phase separation.

• The formation of minimal pyrenoids holds the potential to enhance photosynthesis in crops and thereby contribute to food security and climate protection.

A Tiny droplet with a big task

Rubisco is the most important and abundant enzyme on Earth; it catalyzes CO₂ fixation during photosynthesis. However, the enzyme is imperfect: it operates relatively slowly and frequently binds oxygen (O₂) instead of CO₂. This results in photorespiration, an inefficient process that leads to the loss of valuable energy.  

Green algae, such as the model organism Chlamydomonas reinhardtii, face the additional challenge of capturing CO₂ from aquatic environments where its concentration is very low. To overcome these limitations, they utilize condensates—membraneless organelles known as pyrenoids. These employ a linker protein named EPYC1 to bind Rubisco and segregate it into a concentrated droplet through phase separation. CO₂ is enriched in this condensate, enhancing Rubisco’s carboxylation reaction and suppressing photorespiration. 

Can EPYC1 alone achieve carbon concentration? Does it directly influence Rubisco’s catalytic activity, or is the improved CO₂ fixation solely due to locally elevated substrate concentrations?  Dr. Andreas Küffner, currently research group leader for Carbon Capture at the Max Planck Institute for Multidisciplinary Sciences in Göttingen, investigated these questions during his post-doctoral research within the team of Prof. Dr. Tobias Erb at the Max Planck Institute for Terrestrial Microbiology in Marburg. The pyrenoid condensate system serves as a unique model to study the interplay between biocatalysis and physical conditions. However, modern pyrenoids are complex structures featuring elaborate cellular architectures and numerous components, complicating the isolated study of EPYC1 function. 

“In synthetic biology, we aim to understand biological functions by building them from the bottom up”, explains Tobias Erb. “Reconstructing a minimal pyrenoid from scratch reveals which components are essential  - and sufficient -, providing insights into function as it evolved”, adds Andreas Küffner.  

Rebuilding Pyrenoid Evolution in the Lab

To understand how pyrenoids developed from simpler condensates, the team, together with Prof. Dr. Georg Hochberg from the University of Marburg, reconstructed the gene sequences of the evolutionary ancestors of today’s EPYC1 (ancestral sequence reconstruction). “By rebuilding the earliest protein forms in the laboratory, we studied their mechanisms and capabilities for condensate formation in vitro. This enabled us to propose an evolutionary path from minimal pyrenoids to the more complex structures observed in green algae today,” says Küffner. 

Almost all ancestral versions of EPYC1 formed condensates, providing evidence that the ability to organize Rubisco into a concentrated droplet is an ancient trait conserved throughout evolution. Structural analyses using cryo-electron microscopy showed that Rubisco inside the condensates retains its natural shape and active site configuration. Thus, the condensates do not alter Rubisco itself. Instead, they likely create a local environment in which CO₂ is enriched relative to oxygen solely through the physical chemistry of phase separation, as demonstrated by kinetic and biophysical experiments.

From Evolutionary Insights to Synthetic Applications

The principle that EPYC1 sequences provide carbon-concentrating functions through pure phase separation, rather than by improving Rubisco itself, can be utilized by both natural and synthetic pyrenoids. Using simple phase-separating proteins to build minimal carbon-concentrating structures offers a simpler strategy than complex pyrenoid reconstructions or other approaches to improving carbon fixation and opens new possibilities for future applications in synthetic biology. Understanding how nature’s simplest CO₂ concentrators operate could ultimately assist researchers in developing more efficient photosynthesis in crops, contributing to food security and climate change mitigation.