Lupeol exhibits novel physiological and pharmacological activities, such as anticancer and immunity-enhancing activities. However, cytotoxicity remains a challenge for triterpenoid overproduction in microbial cell factories. As lipophilic and relatively small molecular compounds, triterpenes are generally secreted into the extracellular space. The effect of increasing triterpene efflux on the synthesis capacity remains unknown. In this study, we developed a strategy to enhance triterpene efflux through manipulation of lipid components in Y. lipolytica by overexpressing the enzyme Δ9-fatty acid desaturase (OLE1) and disturbing phosphatidic acid phosphatase (PAH1) and diacylglycerol kinase (DGK1). By this strategy combined with two-phase fermentation, the highest lupeol production reported to date was achieved, where the titer in the organic phase reached 381.67 mg/L and the total production was 411.72 mg/L in shake flasks, exhibiting a 33.20-fold improvement over the initial strain. Lipid manipulation led to a twofold increase in the unsaturated fatty acid (UFA) content, up to 61–73%, and an exceptionally elongated cell morphology, which might have been caused by enhanced membrane phospholipid biosynthesis flux. Both phenotypes accelerated the export of toxic products to the extracellular space and ultimately stimulated the capacity for triterpenoid synthesis, which was proven by the 5.11-fold higher ratio of extra/intracellular lupeol concentrations, 2.79-fold higher biomass accumulation and 2.56-fold higher lupeol productivity per unit OD in the modified strains. This strategy was also highly efficient for the biosynthesis of other triterpenes and sesquiterpenes, including α-amyrin, β-amyrin, longifolene, longipinene and longicyclene. In conclusion, we successfully created a high-yield lupeol-producing strain via lipid manipulation. We demonstrated that the enhancement of lupeol efflux and synthesis capacity was induced by the increased UFA content and elongated cell morphology. Our study provides a novel strategy to promote the biosynthesis of valuable but toxic products in microbial cell factories.

中文翻译：

---

通过操纵脂质成分在解脂耶氏酵母中高产三萜类化合物。

羽扇豆具有新的生理和药理活性，例如抗癌和增强免疫活性。然而，细胞毒性仍然是微生物细胞工厂过度生产三萜类化合物的挑战。作为亲脂性和相对较小的分子化合物，三萜类化合物通常被分泌到细胞外空间中。增加三萜流出量对合成能力的影响仍然未知。在本研究中，我们开发了一种策略，通过过度表达酶 Δ9-脂肪酸去饱和酶 (OLE1) 和干扰磷脂酸磷酸酶 (PAH1) 和二酰基甘油激酶 (DGK1) 来操纵解脂耶氏酵母中的脂质成分来增强三萜外流。通过这种结合两阶段发酵的策略，实现了迄今为止报道的最高的羽扇豆醇产量，其中有机相中的滴度达到 381.67 mg/L，在摇瓶中总产量为 411.72 mg/L，比初始菌株提高了 33.20 倍。脂质操作导致不饱和脂肪酸 (UFA) 含量增加两倍，高达 61-73%，并且细胞形态异常延长，这可能是由于膜磷脂生物合成通量增强所致。两种表型都加速了有毒产物向细胞外空间的输出，并最终刺激了三萜类化合物的合成能力，这通过 5.11 倍的细胞外/细胞内羽扇豆醇浓度比、2.79 倍的生物量积累和 2.56 倍的羽扇豆醇得到证明改良菌株中每单位 OD 的生产力。该策略对于其他三萜和倍半萜的生物合成也非常有效，包括α-香脂素、β-香脂素、longifolene、longipinene 和 longicyclene。总之，我们通过脂质操作成功地创建了一种高产羽扇豆醇生产菌株。我们证明了羽扇豆醇流出和合成能力的增强是由增加的 UFA 含量和拉长的细胞形态引起的。我们的研究提供了一种新的策略来促进微生物细胞工厂中有价值但有毒的产品的生物合成。我们证明了羽扇豆醇流出和合成能力的增强是由增加的 UFA 含量和拉长的细胞形态引起的。我们的研究提供了一种新的策略来促进微生物细胞工厂中有价值但有毒的产品的生物合成。我们证明了羽扇豆醇流出和合成能力的增强是由增加的 UFA 含量和拉长的细胞形态引起的。我们的研究提供了一种新的策略来促进微生物细胞工厂中有价值但有毒的产品的生物合成。