A synthetic biology platform for engineering pyrenoid matrix-traversing membranes to enhance photosynthetic CO2 concentration

Background

Photosynthesis is the fundamental process by which plants, algae, and some bacteria convert light energy into chemical energy, sustaining life on Earth. In land plants, the efficiency of photosynthesis is limited by the enzyme Rubisco, which not only fixes carbon dioxide (CO2) but also reacts with oxygen, leading to energy loss through photorespiration. Algae have evolved specialized organelles called pyrenoids that concentrate CO2 around Rubisco, dramatically increasing its efficiency and reducing photorespiration. This CO2 concentrating mechanism (CCM) is a major reason why many algae outperform land plants in carbon fixation, especially under low CO2 conditions. There is a pressing need to engineer similar CCMs into crop plants to boost yields, improve resource use efficiency, and enhance resilience to climate change, as global food demand rises and arable land becomes increasingly limited.

Despite advances in synthetic biology and the partial reconstruction of algal CCM components in land plants, a major structural and functional bottleneck has persisted: the inability to engineer the specialized thylakoid membranes that traverse the pyrenoid matrix and are essential for efficient CO2 delivery to Rubisco. Current approaches have successfully created synthetic pyrenoid matrices in plants, but these lack the traversing membranes required to localize key enzymes and deliver concentrated CO2 directly to Rubisco. Without these membranes, the engineered CCMs fail to replicate the high-efficiency carbon fixation seen in algae, resulting in incomplete or non-functional systems. This limitation has prevented the realization of the full potential of CCM engineering in crops, underscoring the need for new strategies to reconstruct these critical membrane structures and achieve robust, high-yield photosynthesis in agricultural plants.

Technology

This technology enables the engineering of pyrenoid matrix-traversing thylakoid membranes into land plants, a key step toward reconstructing the algal CO₂ concentrating mechanism (CCM) in crops. The core innovation lies in the heterologous expression of two proteins, SAGA1 and MITH1, which are both necessary and sufficient to induce thylakoid membranes to penetrate and extend through the synthetic pyrenoid matrix already formed by EPYC1 and compatible Rubisco small subunits. SAGA1 initiates membrane contact with the Rubisco matrix by binding both membranes and Rubisco, while MITH1 binds SAGA1 and membranes to strengthen this adhesion and facilitate membrane extension through the matrix. When co-expressed in engineered plants, these proteins induce the formation of traversing membranes, confirmed by advanced microscopy techniques. These membranes are essential for delivering concentrated CO₂ to Rubisco, maintaining pyrenoid integrity, and localizing carbonic anhydrase for efficient CO₂ conversion, thereby addressing a major structural bottleneck in engineering functional CCMs into crops.

This solution is differentiated by its precise identification and functional characterization of the minimal protein machinery required for pyrenoid matrix-traversing membrane formation, overcoming a longstanding barrier in synthetic biology. Unlike previous approaches that could only reconstitute the pyrenoid matrix, this technology successfully reconstructs the critical membrane architecture necessary for efficient CO₂ delivery and fixation in land plants. The method is validated by genetic, biochemical, and imaging evidence, demonstrating that SAGA1 and MITH1 are both necessary and sufficient for this process. This breakthrough not only enables the next steps in engineering fully functional CCMs in crops—potentially increasing yields and resource efficiency—but also provides a foundational blueprint for studying and manipulating membrane-condensate interactions in diverse biological systems. The ability to reconstitute these structures in heterologous systems marks a significant advance toward transforming agricultural productivity and sustainability.

Benefits

• Enables formation of pyrenoid matrix-traversing thylakoid membranes in land plants, overcoming a major structural bottleneck in engineering functional CO2 concentrating mechanisms (CCMs).
• Enhances CO2 delivery to Rubisco by localizing carbonic anhydrase and maintaining pyrenoid integrity, improving photosynthetic efficiency and carbon fixation.
• Increases crop biomass and yield by 30-60%, contributing to improved agricultural productivity and sustainability.
• Reduces water and nitrogen fertilizer requirements, promoting resource-efficient crop growth.
• Improves plant tolerance to heat and drought stress, enhancing climate resilience.
• Provides a molecular blueprint for synthetic biology approaches to enhance photosynthesis in diverse crops.
• Facilitates global food security by addressing CO2 availability limitations in major C3 crops such as rice, wheat, soybean, and cassava.

Applications

• High-yield transgenic crop development
• Climate-resilient agriculture solutions
• Water-efficient farming systems
• Low-fertilizer input crop varieties

Additional info

This technology enables engineering pyrenoid matrix-traversing thylakoid membranes into land plants. It utilizes heterologous expression of SAGA1 and MITH1 proteins. SAGA1 initiates membrane contact with the Rubisco matrix, while MITH1 strengthens adhesion and facilitates membrane extension through it. These induced membranes deliver concentrated CO2 to Rubisco, reconstructing a critical component of the algal CO2 concentrating mechanism for enhanced photosynthesis.