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.

绿藻莱茵衣藻可作为植物和光合作用研究的模式生物，因为它具有许多新陈代谢的共性以及莱茵衣藻的快速生长速度，从而加快了实验周转时间。此外，莱茵衣藻是代谢工程和合成生物学研究的重点，用于潜在生产生物燃料和增值化学品。在这里，我们报告了缺乏功能性碳浓缩机制 (CCM) 的莱茵衣藻 cia5突变体，当该突变体在环境空气条件下培养时，可以产生大量乙醇酸，这是一种高价值的化妆品成分。为了揭示乙醇酸积累的代谢基础对于 cia5突变体，我们通过使用气相色谱和质谱法对细胞内和细胞外部分的整体代谢分析，研究了cia5突变体和野生型菌株 CC-125 (WT) 的代谢组。我们观察到 WT 和cia5突变体的细胞内和细胞外代谢谱在 30 小时的混合营养阶段相似。然而，当细胞进入光合自养阶段（即 96 小时和 120 小时）时，与 WT 相比，cia5突变体的细胞内和细胞外代谢谱显着不同。在cia5在突变菌株中，一组光呼吸途径中间体，包括乙醇酸、乙醛酸、甘氨酸和丝氨酸，与 WT 相比积累到显着更高的水平。在光呼吸途径中，乙醇酸被代谢为乙醛酸和甘氨酸，从而在甘氨酸向丝氨酸的线粒体转化过程中产生NH ₃和 CO ₂。这一结果提供了进一步的证据，证明CIA5突变增加了光呼吸率。由于cia5突变体缺乏 CCM，并且莱茵衣藻可能具有低效或不完整的光呼吸途径，因此当 CCM 不起作用时，乙醇酸可能会积聚。我们设想研究光呼吸控制C. reinhardtii为生产者提供了使用cia5突变体生产乙醇酸的工具以及设计乙醇酸代谢替代途径的平台。