Genome-Wide Identification of Novel Genes Involved with Photosynthesis and the Carbon Concentrating Mechanism of the Green Alga Chlamydomonas reinhardtii

Satisfying the world’s food and fuel demands in the coming decades will require innovative and sustainable solutions. Both food and biofuel require photosynthesis to absorb abundant solar energy and store it in biomass via carbon fixation. Photosynthesis is often limited by the availability of carbon. Interestingly, the alga Chlamydomonas reinhardtii enhances photosynthetic efficiency with a genetically encoded Carbon Concentrating Mechanism (CCM) that actively accumulates inorganic carbon (CO₂ and HCO₃^-) inside the cell, circumventing most of the energy required by photorespiration. The specific objective of this research was to comprehensively identify the genes required for photosynthesis and a subset of genes constituting the CCM. We generated 120,048 independent mutant strains via insertional mutagenesis, representing knockouts for an estimated >85% of all protein coding genes. We screened these mutants via a unique high-throughput quantitative growth phenotyping pipeline and isolated 3,601 mutant strains with statistically significant deficient photoautotrophic growth. From these mutants, 336 candidate CCM mutants were dissected by further growth phenotyping in air (0.04% CO₂), where a functional CCM is required for growth, verses in high carbon dioxide (3% CO₂), where a CCM is unnecessary. A high-throughput deep-sequencing based approach was used to determine the mutation loci of these strains which identified numerous novel genes involved in photosynthesis and establishing essential genes for the CCM. This comprehensive identification of photosynthesis and CCM genes expands our understanding of these processes and paves ways to engineer more efficient crops for increased food and biofuel production.