Lipid droplets (LDs) are specialized organelles that store neutral lipids to reduce the negative effects of lipotoxicity on cells. However, many neutral lipids are precursors for the synthesis of sterols and complex terpenoids, and this sequestration often greatly limits the efficient biosynthesis of sterols and complex terpenoids. In this study, taking 7-dehydrocholesterol (7-DHC) synthesis in Saccharomyces cerevisiae as an example, we revealed the blocking mechanism of LD sequestration on the efficient synthesis of metabolic products and found that LDs can sequester a significant amount of squalene, the precursor of 7-DHC, effectively preventing it from being directed toward the post-squalene pathway. Based on this, a post-squalene pathway was reconstructed on LDs, which resulted in a 28.7% increase in the 7-DHC titer, reaching 684.1 mg/L, whereas the squalene titer was reduced by approximately 97%. Subsequently, the triacylglycerol degradation pathway was weakened to release the storage space in LDs, and the esterification pathway was concurrently strengthened to guide 7-DHC storage within LDs, which further increased 7-DHC production, reaching 792.9 mg/L. Finally, by reducing the NADH/NAD + ratio to alleviate the redox imbalance, the 7-DHC titer reached 867.6 mg/L in shake flask and 5.1 g/L in a 3-L bioreactor, which is the highest reported titer to date. In summary, this study provides new insights into the important role of LDs in sterol synthesis and offers a novel strategy for constructing cell factories for the efficient synthesis of sterol compounds.

中文翻译：

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通过重新连接酿酒酵母脂滴中的角鲨烯后模块，过度产生 7-脱氢胆固醇


脂滴 （LD） 是储存中性脂质以减少脂毒性对细胞的负面影响的专用细胞器。然而，许多中性脂质是合成甾醇和复杂萜类化合物的前体，这种螯合通常极大地限制了甾醇和复杂萜类化合物的有效生物合成。在本研究中，以酿酒酵母中 7-脱氢胆固醇 （7-DHC） 合成为例，我们揭示了 LD 隔离对代谢产物有效合成的阻断机制，发现 LDs 可以隔离大量 7-DHC 的前体角鲨烯，有效阻止其定向到后角鲨烯途径。基于此，在 LDs 上重建了角鲨烯后途径，导致 7-DHC 滴度增加了 28.7%，达到 684.1 mg/L，而角鲨烯滴度降低了约 97%。随后，三酰基甘油降解途径减弱以释放 LDs 中的储存空间，同时加强酯化途径以指导 LDs 内的 7-DHC 储存，进一步提高 7-DHC 的产量，达到 792.9 mg/L。最后，通过降低 NADH/NAD + 比率以缓解氧化还原失衡，摇瓶中的 7-DHC 滴度达到 867.6 mg/L，在 3 L 生物反应器中达到 5.1 g/L，这是迄今为止报道的最高滴度。综上所述，本研究为 LDs 在甾醇合成中的重要作用提供了新的见解，并为构建细胞工厂以高效合成甾醇化合物提供了一种新的策略。