Regulatory network rewiring drives strain‐specific lipid accumulation response in <i>Chlorella sorokiniana</i> under nutrient starvation

Microalgae modulate lipid metabolism in response to nutrient stress, offering a promising avenue for sustainable biofuel production. However, a mechanistic understanding of the transcriptional programs driving triacylglycerol (TAG) accumulation remains limited, particularly in non-model species. Here, we employ a systems-level approach to dissect the regulatory basis of TAG biosynthesis in two Chlorella sorokiniana strains exhibiting contrasting lipid accumulation phenotypes under nitrogen (N) and phosphorus (P) deprivation. Through physiological, metabolic, and transcriptomic analyses, we confirmed C. sorokiniana DOE1412 (CsDOE1412) as a high TAG-accumulator and C. sorokiniana UTEX1228 (Cs1228) as a low TAG-accumulator, providing a comparative framework for inferring transcriptional regulatory networks (TRNs). Both stressors induced rapid TAG accumulation within 6 h, with CsDOE1412 reaching 40% TAG content by 48 h under N conditions. While N deprivation primarily promoted TAG accumulation, P starvation favored diacylglyceryl trimethylhomoserine biosynthesis, reaching up to 21 and 30% of the lipid composition in Cs1228 and CsDOE1412, respectively. TRNs analysis revealed a distinct regulatory logic between strains: CsDOE1412 exhibited a stress-specific, narrowly focused transcriptional response, with five transcription factors (TFs) identified as leading regulators based on centrality measures, whereas Cs1228 mounted a broader, overlapping response, with 30 key TFs across conditions. A detailed analysis of the inferred TRNs identified 15 and 14 candidate TFs in CsDOE1412 and Cs1228, respectively, with predicted interactions involving key steps in carbon metabolism and lipid biosynthesis, suggesting their involvement in metabolic rewiring during nutrient stress. Among them, we found two CH3-type ortholog pairs, Cs1228_21g10473/CsDOE1412_2079g07848 and Cs1228_02g00899/CsDOE1412_2296g01133, showing upregulation in TAG-accumulating conditions; and one AP2-type ortholog pair, Cs1228_04g03113/CsDOE1412_2160g02163, with contrasting transcription profiles, pointing to transcriptional regulatory pathways with shared and unique regulators between strains. These findings expand the repertoire of regulatory components associated with algal lipid metabolism and highlight C. sorokiniana as a robust model for elucidating complex transcriptional responses to environmental cues. Furthermore, this study provides candidate TFs for engineering enhanced lipid productivity in microalgae.