Abstract <p><i>Rhodotorula toruloides</i> is an oleaginous yeast recognized for its robustness and the production of high content of neutral lipids. Early biochemical studies have linked ATP citrate lyase (ACL), phosphoketolase (PK), and cytosolic malic enzyme (cMAE) with de novo lipid synthesis. In this study, we discovered that upon a CRISPR/Cas9-mediated knockout of the ACL gene, lipid content in <i>R. toruloides</i> IFO0880 decreased from 50 to 9% of its dry cell weight (DCW) in glucose medium and caused severe growth defects (reduced specific growth rate, changes in cell morphology). In xylose medium, the lipid content decreased from 43 to 38% of DCW. However, when grown on acetate as the sole carbon source, the lipid content decreased from 45 to 20% of DCW. Significant growth defects as a result of ACL knockout were observed on all substrates. In contrast, PK knockout resulted in no change in growth or lipid synthesis. Knocking out cMAE gene resulted in lipid increase of 2.9% of DCW and 23% increase in specific growth rate on glucose. In xylose or acetate medium, no change in lipid production as a result of cMAE gene knockout was observed. These results demonstrated that ACL plays a crucial role in lipid synthesis in <i>R. toruloides</i> IFO0880, as opposed to PK pathway or cMAE, whose presence in some conditions even disfavors lipid production. These results provided valuable information for future metabolic engineering of <i>R. toruloides</i>.</p> Graphical Abstract <p></p> Key points <p>• <i>ACL is crucial for the fatty acid synthesis and growth in R. toruloides IFO0880.</i></p> <p>• <i>Lipid production and cell growth is are unchanged as a result of PK knockout.</i></p> <p>• <i>Cytosolic malic enzyme does not play a significant role in lipogenesis.</i></p>