Glycolate production by a Chlamydomonas reinhardtii mutant lacking carbon-concentrating mechanism.

The green alga Chlamydomonas reinhardtii serves as a model organism for plant and photosynthesis research due to many commonalities in metabolism and to the fast growth rate of C. reinhardtii which accelerates experimental turnaround time. In addition, C. reinhardtii is a focus of research efforts in metabolic engineering and synthetic biology for the potential production of biofuels and value-added chemicals. Here, we report that the C. reinhardtii cia5 mutant, which lacks a functional carbon-concentrating mechanism (CCM), can produce substantial amounts of glycolate, a high-value cosmetic ingredient, when the mutant is cultured under ambient air conditions. In order to reveal the metabolic basis of glycolate accumulation by the cia5 mutant, we investigated the metabolomes of the cia5 mutant and a wild type strain CC-125 (WT) through the global metabolic profiling of intracellular and extracellular fractions using gas chromatography and mass spectrometry. We observed the intracellular and extracellular metabolic profiles of the WT and the cia5 mutant were similar during the mixotrophic phase at 30 h. However, when the cells entered the photoautotrophic phase (i.e., 96 h and 120 h), both the intracellular and extracellular metabolic profiles of cia5 mutant differed significantly when compared to WT. In the cia5 mutant strain, a group of photorespiration pathway intermediates including glycolate, glyoxylate, glycine, and serine accumulated to significantly higher levels compared to WT. In the photorespiration pathway, glycolate is metabolized to glyoxylate and glycine leading to NH3 and CO2 generation during the mitochondrial conversion of glycine to serine. This result provides further evidence that the CIA5 mutation increased the photorespiration rate. Because the cia5 mutant lacks a CCM, and C. reinhardtii might harbor an inefficient or incomplete photorespiration pathway, glycolate may accumulate when the CCM is not functional. We envision that investigating photorespiration controls in C. reinhardtii provides tools for producers to use the cia5 mutant to produce glycolate as well as platform to engineer alternative pathways for glycolate metabolism.