Growth of CRISPRi library of Synechocystis at 4% and 30% CO2 gas feed

Optimal cultivation of photosynthetic microbes is often performed under high CO2 conditions to promote growth, but excessively high CO2 levels inhibit growth through mechanisms that are not fully understood. This study investigates the physiological responses of the cyanobacterium Synechocystis sp. PCC 6803 to very high CO2 concentrations under conditions where pH is maintained at around 7.5. The growth rate of the wild type (WT) at a gas phase content of 30% CO2 was 2.6-fold lower compared to 4% CO2. Using a CRISPR interference knockdown mutant library, we identified mutations that either enhanced or impaired growth under these conditions. The results showed that the knockdown of phycocyanin biosynthesis genes (cpc) promoted growth in 30% CO2 suggesting that the cells were under high light stress under very high CO2. Conversely, knockdown of key regulators of CO2 fixation (cp12 and rbcR) and carbon utilization (pmgA) resulted in increased growth inhibition at 30% CO2, suggesting that adaptation to very high CO2 conditions requires alterations in carbon fixation and utilization pathways. Further experiments confirmed that WT cells under 30% CO2 experienced light stress at lower light intensities than WT cells under 4% CO2. In addition, experiments with knockout mutants lacking cpcG1 showed better growth than WT at 30% CO2, while pmgA and cp12 mutants performed worse. These findings suggest that enhanced fitness under very high CO2 involves modifications in light harvest, CO2 fixation, and carbon management pathways. The genetic profiling in this study provides potential targets for engineering cyanobacteria with improved photosynthetic efficiency and stress resilience, which could be valuable for biotechnological applications.