Ivan Alberto Sandoval Salazar¹, Leonardo Murilo², Felipe Stolf¹, Ana Paula Duarte Ramin¹, Flavia Vischi Winck¹

Introduction: The induction of neutral lipids (TAGs) accumulation in microalgae in response to nitrogen deprivation reduces cell growth, limiting biomass productivity. We investigate the role of arginine-ornithine biosynthesis pathway in the regulation of TAGs accumulation by inhibiting Ornithine decarboxylase (ODC) enzyme with difluoromethylornithine (DFMO) and transiently silencing Arginine decarboxylase (ADC) enzyme with antisense DNA. A proteomic analysis was performed to elucidate the impact of these treatments on TAGs´s metabolism, seeking to optimize lipid accumulation without compromising cell growth.

Materials and methods: C. reinhardtii CC503 was treated with 1 mM DFMO to chemically modulate the polyamines pathway and samples were collected at 0 h (control) and 5 h. In addition, C. reinhardtii CC5325 mutant for N-acetylglutamate synthase (NAGS) was transiently silenced photothermally for ADC gene via gold nanoparticle-carried antisense DNA released at 39 °C. Samples were collected 4 h post silencing induction (0 h control). Neutral lipids were quantified relatively using Nile red fluorescence. For proteomics, 2 × 10⁶ cells were collected from each treatment at the specified time points. Total protein extracts were obtained with urea-thiourea buffer and sonication. Proteins (10 µg) were reduced, alkylated, and trypsin digested. Peptides were desalted using C18 StageTips and eluted in 0.1% formic acid. LC-Mass spectrometry was performed with an Orbitrap Fusion Lumos. Protein identification/quantification employed MaxQuant (1% FDR), and data analysis was performed using Perseus with LFQ intensities. Functional annotation used ClueGO and String (FDR 0.05%, P<0.05). 

Results and Discussion: Proteomic analysis of DFMO treatment identified 12 differentially expressed proteins at 5 h treatment, coinciding with lipid fluorescence data. Downregulated proteins included chloroplast starch synthase, Fe-hydrogenases, flagellar apparatus components, SET domain-containing protein, and Nudix hydrolase. Upregulated proteins comprised proliferating cell nuclear antigen (PCNA), SAP domain-containing protein, and a lysine-induced co-expression cluster. These changes indicate DFMO-mediated ODC inhibition redirects carbon from starch synthesis toward lipid accumulation, likely through redox imbalance and transcriptional remodeling under polyamine depletion. On the other hand, proteomic analysis at 4 h after transient silencing of the ADC gene in the NAGS mutant resulted 515 differentially expressed proteins, coinciding with increased lipid´s accumulation. This dual interference in polyamine biosynthesis (reduced ornithine and blocked arginine decarboxylation) led to an enrichment of cellular amide metabolism (GO:0043603), amino acid metabolism (GO:0006520), and oxidoreductase activity (GO:0016491), suggesting altered nitrogen and redox balance. Pathways related to mitochondrial energy metabolism (tricarboxylic acid cycle and electron transport; Reactome: MAP-1428517) and carbon metabolism (KEGG: cre01200) were also affected (p<0.01). Therefore, the disruption of the polyamine pathway triggered metabolic remodeling associated with lipid accumulation.

Conclusion: Polyamine biosynthesis inhibition (DFMO or ADC silencing in the NAGS mutant) alters metabolism in Chlamydomonas, reducing starch proteins, chromatin regulation, and stress factors, while promoting lipid accumulation via redox-sensitive shifts in carbon/nitrogen fluxes.

Agradecimentos: CNPq, CAPES, FAPESP (2016/06601-4, 2023/02511-4). We thank Professors Paolo Di Mascio and Graziella E. Ronsein for the Mass spectrometry analyses performed at the Redox Proteomics Core of the Mass Spectrometry Resource at Chemistry Institute, University of Sao Paulo (FAPESP grant numbers 2016/06601-4, 2012/12663-1, 2023/00995-4, 2023/02511-4, CEPID Redoxoma 2013/07937-8), and Dr. Mariana P. Massafera for her technical assistance. This study utilized equipment funded by the FAPESP Multi-User Equipment Program (Grant No. 2019/27255-5).