Analysis of Sensitive CO2 Pathways and Genes Related to Carbon Uptake and Accumulation in Chlamydomonas reinhardtii through Genomic Scale Modeling and Experimental Validation

Flavia V. Winck, David O. Páez Melo, Diego M. Riaño-Pachón, Marina C. M. Martins, Camila Caldana, Andrés F. González Barrios

The development of microalgae sustainable applications needs better understanding of microalgae biology. Moreover, how cells coordinate their metabolism toward biomass accumulation is not fully understood. In this present study, flux balance analysis (FBA) was performed to identify sensitive metabolic pathways of Chlamydomonas reinhardtii under varied CO2 inputs. The metabolic network model of Chlamydomonas was updated based on the genome annotation data and sensitivity analysis revealed CO2 sensitive reactions. Biological experiments were performed with cells cultivated at 0.04% (air), 2.5, 5, 8, and 10% CO2 concentration under controlled conditions and cell growth profiles and biomass content were measured. Pigments, lipids, proteins, and starch were further quantified for the reference low (0.04%) and high (10%) CO2 conditions. The expression level of candidate genes of sensitive reactions was measured and validated by quantitative real time PCR. The sensitive analysis revealed mitochondrial compartment as the major affected by changes on the CO2 concentrations and glycolysis/gluconeogenesis, glyoxylate, and dicarboxylate metabolism among the affected metabolic pathways. Genes coding for glycerate kinase (GLYK), glycine cleavage system, H-protein (GCSH), NAD-dependent malate dehydrogenase (MDH3), low-CO2 inducible protein A (LCIA), carbonic anhydrase 5 (CAH5), E1 component, alpha subunit (PDC3), dual function alcohol dehydrogenase/acetaldehyde dehydrogenase (ADH1), and phosphoglucomutase (GPM2), were defined, among other genes, as sensitive nodes in the metabolic network simulations. These genes were experimentally responsive to the changes in the carbon fluxes in the system. We performed metabolomics analysis using mass spectrometry validating the modulation of carbon dioxide responsive pathways and metabolites. The changes on CO2 levels mostly affected the metabolism of amino acids found in the photorespiration pathway. Our updated metabolic network was compared to previous model and it showed more consistent results once considering the experimental data. Possible roles of the sensitive pathways in the biomass metabolism are discussed.