Reķēna Alīna, Pals Kristjan, Gavrilović Srðan, Lahtvee Petri-Jaan
Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia.
Appl Microbiol Biotechnol. 2025 Mar 29;109(1):77. doi: 10.1007/s00253-025-13454-w.
Rhodotorula toruloides 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 R. toruloides 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 R. toruloides 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 R. toruloides. KEY POINTS: • ACL is crucial for the fatty acid synthesis and growth in R. toruloides IFO0880. • Lipid production and cell growth is are unchanged as a result of PK knockout. • Cytosolic malic enzyme does not play a significant role in lipogenesis.
红酵母是一种产油酵母，因其具有较强的适应性和能够合成高含量的中性脂质而闻名。早期的生化研究表明，ATP柠檬酸裂解酶（ACL）、磷酸酮醇酶（PK）和胞质苹果酸酶（cMAE）与从头合成脂质有关。在本研究中，我们发现，通过CRISPR/Cas9介导敲除ACL基因后，在葡萄糖培养基中，红酵母IFO0880的脂质含量从其干细胞重量（DCW）的50%降至9%，并导致严重的生长缺陷（比生长速率降低、细胞形态改变）。在木糖培养基中，脂质含量从DCW的43%降至38%。然而，当以乙酸盐作为唯一碳源生长时，脂质含量从DCW的45%降至20%。在所有底物上均观察到由于敲除ACL而导致的显著生长缺陷。相比之下，敲除PK对生长或脂质合成没有影响。敲除cMAE基因导致DCW脂质增加2.9%，在葡萄糖上的比生长速率增加23%。在木糖或乙酸盐培养基中，未观察到由于敲除cMAE基因而导致的脂质产量变化。这些结果表明，ACL在红酵母IFO0880的脂质合成中起关键作用，与PK途径或cMAE不同，PK途径或cMAE在某些条件下甚至不利于脂质产生。这些结果为红酵母未来的代谢工程提供了有价值的信息。要点：• ACL对红酵母IFO0880的脂肪酸合成和生长至关重要。• 敲除PK后脂质产量和细胞生长不变。• 胞质苹果酸酶在脂肪生成中不发挥重要作用。
