Periodic Reporting for period 1 - ECOFix (Engineering Catalytic Membraneless Organelles for CO2 Fixation)
Reporting period: 2023-11-01 to 2025-04-30
The accelerating climate crisis urges humanity to understand the global carbon cycle and the biosphere’s capacity to capture and fix atmospheric carbon dioxide. Despite a dramatic rise in CO2 levels from 280 to 420 ppm since the industrial revolution, the effective CO2 concentration in the atmosphere remains low. This presents a fundamental challenge for carbon fixation and carbon capture. To grow at these low concentrations, aquatic phototrophs have evolved so-called carbon-concentrating mechanisms (CCMs) that locally increase CO2 levels in close proximity to Rubisco (Ribulose-1,5-bisphosphate carboxylase oxygenase) for enhancing carbon fixation. Algae, which account for an estimated 30–50% of global carbon fixation, have independently developed two distinct CCMs: cyanobacteria utilize carboxysomes - highly ordered protein microcompartments - while green algae employ membraneless organelles known as pyrenoids. Because Rubisco’s catalytic efficiency is inherently limited by a Pareto optimum between its activity and selectivity for CO2 over O2, with the latter leading to energetically costly photorespiration, CCMs such as the pyrenoid help overcome this limitation by creating microenvironments with elevated CO2, suppressing the wasteful side reaction with O2. Pyrenoids are particularly intriguing because it is crucially formed by just one intrinsically disordered protein (IDP) called EPYC1. In this project we aimed at engineering bottom-up artificial pyrenoids fusion proteins of Rubisco and IDPs that drive liquid-liquid phase separation. We set out with three main objectives: (1) generating artificial pyrenoid-like condensates in vitro and characterizing their structural and catalytic properties, (2) transplanting these artificial organelles into cyanobacteria to replace native carboxysomes and studying their physiological impact, and (3) applying adaptive laboratory evolution under CO2-limiting conditions to optimize these synthetic CCMs for enhanced carbon fixation. This project aims not only to unravel the principles governing pyrenoid assembly and function but also to explore new strategies for improving photosynthetic efficiency in diverse organisms with implications for climate change mitigation and synthetic biology.
The project achieved a series of crucial advances in understanding the pyrenoid, a membraneless organelle critical for carbon concentration in photosynthetic organisms. By employing a bottom-up synthetic biology approach, we demonstrated that modern EPYC1 proteins - intrinsically disordered proteins (IDPs) from green algae - are uniquely capable of inducing liquid-liquid phase separation of Rubisco, resulting in the formation of minimal, functional pyrenoid-like condensates in vitro. Unlike other IDPs, only EPYC1 and its homologs were found to cause the assembly of these condensates, highlighting the evolutionary importance of EPYC1 sequences.
To dive into the evolution of EPYCs we used ancestral sequence reconstruction for EPYC1 and compared ancestral and extant EPYC1-Rubisco fusion proteins, revealing that the ability to enhance carboxylation rates through condensate formation is a deeply conserved and ancestral trait within the green algae. Notably, these synthetic pyrenoids increase CO2 fixation rates up to fivefold compared to non-phase-separated Rubisco, demonstrating that phase separation alone can provide a minimal carbon concentrating mechanism (CCM). We further investigated whether the primary mechanism behind this enhancement is substrate partitioning and mass action kinetics or if the condensate allosterically modulates Rubisco. Using dedicated kinetics assays, physico-chemical assays and structural analyses confirmed that the condensates act like a multi-phase reaction system and Rubisco retains its native conformation within condensates.
Additionally, we established the first transgenic Synechococcus elongatus with EPYC1 and EPYC1 fusion proteins inside showing early signs of growth enhancement.
To dive into the evolution of EPYCs we used ancestral sequence reconstruction for EPYC1 and compared ancestral and extant EPYC1-Rubisco fusion proteins, revealing that the ability to enhance carboxylation rates through condensate formation is a deeply conserved and ancestral trait within the green algae. Notably, these synthetic pyrenoids increase CO2 fixation rates up to fivefold compared to non-phase-separated Rubisco, demonstrating that phase separation alone can provide a minimal carbon concentrating mechanism (CCM). We further investigated whether the primary mechanism behind this enhancement is substrate partitioning and mass action kinetics or if the condensate allosterically modulates Rubisco. Using dedicated kinetics assays, physico-chemical assays and structural analyses confirmed that the condensates act like a multi-phase reaction system and Rubisco retains its native conformation within condensates.
Additionally, we established the first transgenic Synechococcus elongatus with EPYC1 and EPYC1 fusion proteins inside showing early signs of growth enhancement.
The main achievements include the successful in vitro generation of minimal and functional pyrenoid-like condensates using a singular IDP. Additionally we demonstrated that also IDPs are driven by evolutionary selection based on metabolic pressures despite the high mutation rates in IDPs. The work culminated in the first transgenic Synechococcus elongatus strains expressing EPYC1 and EPYC1 fusion proteins, which showed indications of a growth benefit brought about by a singular IDP. The potential impacts of this work are substantial: it provides a blueprint for engineering efficient but minimal CCMs in diverse organisms, with applications in crop improvement, carbon capture, and climate change mitigation. Nonetheless, further research, particularly on the transgenic strains, has to be conducted to confirm a potential growth benefit and the actual creation of an organelle in vivo. The research remains in an early stage and is not yet ready for commercialization. While our work underlines the importance of synthetic biology for society and economy, the regulatory frameworks are not yet adapted towards application of transgenic organisms for carbon capture and utilization.