Leandro Luis Lavandosque¹, Richard Law², Junyoung Park², Clissia Barboza Mastrangelo¹, Flavia Vischi Winck¹

Introduction: Chlamydomonas reinhardtii is a well-established model microalga with great potential for biotechnological production of triacylglycerols (TAG), particularly for sustainable biofuel development. Despite significant advancements, efficient and non-stressful strategies to enhance lipid accumulation remain a major challenge. The nitrogen deprivation effectively induce TAG synthesis but often trigger physiological stress responses, including growth arrest and programmed cell death, compromising industrial scalability. Recent results pointed to the regulatory role of the arginine metabolism in the TAG biosynthesis. Then, we investigated the effects of L-ornithine on lipid accumulation in C. reinhardtii. We explored the role of amino acid metabolism as a regulatory axis for lipid biosynthesis under non-cellular stress conditions.

Methodology: C. reinhardtii strain CC503 cw92mt+ was cultured in Tris-Acetate-Phosphate medium under controlled mixotrophic conditions (100uE, 25ºC, 100 rpm). Cultures were supplemented with 10 mM L-ornithine, and samples were collected at 0 h, 10 min, 30 min, 1 h, 3 h, and 5 h for downstream analyses. Neutral lipid bodies were visualized via Nile Red staining under confocal fluorescence microscopy and relatively quantified by fluorimetry. A complementary experimental setup used isotopically labeled L-arginine (¹³C₆,¹⁵N₄) to monitor metabolic fluxes in the presence or absence of L-ornithine. High-throughput molecular phenotyping approaches included shotgun label-free proteomics (nanoLC-MS/MS), time-resolved targeted metabolomics (HPLC-MS), multispectral imaging, and qPCR analysis of key genes from the arginine catabolism and associated nitrogen stress response pathways.

Results/Discussion: L-ornithine supplementation led to a 45% increase in neutral lipid accumulation compared to control conditions, while avoiding the stress-related responses typically observed under nitrogen deprivation (used as a positive control). Multispectral imaging and gene expression data confirmed the absence of stress markers, including those from the nitrogen scavenging/salvaging machinery. Confocal microscopy and fluorometric quantification indicated enhanced lipid body formation. Metabolomic analysis of L-ornithine-treated cells revealed elevated levels of 2-oxoglutarate, linking carbon and nitrogen metabolism, suggesting activation of the glyoxylate cycle. Isotopic tracing indicated altered arginine-to-citrulline fluxes upon L-ornithine addition, implying potential feedback regulation involving ornithine carbamoyltransferase. Proteomic profiling highlighted modulation of upregulating redox and downregulating malic acid-related pathways, while transcriptional analysis identified compensatory upregulating of Argininosuccinate synthase 1 gene expression. Then, omics data suggest the involvement of a novel signaling pathway, as a link between ornithine metabolism and TAG biosynthesis.

Conclusion: Our findings demonstrate that L-ornithine supplementation can enhance lipid accumulation in C. reinhardtii without compromising photosynthetic activity or inducing stress-related responses. This highlights amino acid metabolism as a promising regulatory mechanism for lipid biosynthesis in microalgae. The data uncover novel interactions between ornithine/arginine metabolism and neutral lipid accumulation, offering new perspectives for metabolic engineering and industrial biofuel production.

Agradecimentos: The authors thank the University of São Paulo (USP); and The São Paulo Research Foundation (FAPESP), Grants #2024/02042-7, #2022/15431-6, #2016/06601-4.