The sesquiterpene α-humulene is an important plant natural product, which has been used in the pharmaceutical industry due to its anti-inflammatory and anticancer activities. Although phytoextraction and chemical synthesis have previously been applied in α-humulene production, the low efficiency and high costs limit the development. In this study, Yarrowia lipolytica was engineered as the robust cell factory for sustainable α-humulene production. First, a chassis with high α-humulene output in the cytoplasm was constructed by integrating α-humulene synthases with high catalytic activity, optimizing the flux of mevalonate and acetyl-CoA pathways. Subsequently, the strategy of dual cytoplasmic-peroxisomal engineering was adopted in Y. lipolytica; the best strain GQ3006 generated by introducing 31 copies of 12 different genes could produce 2280.3± 38.2 mg/l (98.7 ± 4.2 mg/g dry cell weight) α-humulene, a 100-fold improvement relative to the baseline strain. To further improve the titer, a novel strategy for downregulation of squalene biosynthesis based on Cu²⁺-repressible promoters was firstly established, which significantly improved the α-humulene titer by 54.2% to 3516.6 ± 34.3 mg/l. Finally, the engineered strain could produce 21.7 g/l α-humulene in a 5-L bioreactor, 6.8-fold higher than the highest α-humulene titer reported before this study. Overall, system metabolic engineering strategies used in this study provide a valuable reference for the highly sustainable production of terpenoids in Y. lipolytica.

中文翻译：

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双胞质-过氧化物酶体工程在解脂耶氏酵母中高产高产倍半萜烯α-葎草烯

倍半萜烯α-葎草烯是一种重要的植物天然产物，由于其抗炎和抗癌活性，已被用于制药工业。虽然植物提取和化学合成先前已应用于α-葎草烯的生产，但效率低和成本高限制了发展。在这项研究中，解脂耶氏酵母被设计为用于可持续生产 α-葎草烯的强大细胞工厂。首先，通过整合具有高催化活性的α-葎草烯合酶，优化甲羟戊酸和乙酰辅酶A途径的通量，构建了在细胞质中具有高α-葎草烯输出的底盘。随后，解脂耶氏酵母采用胞质-过氧化物酶体双重工程策略。; 通过引入 12 个不同基因的 31 个拷贝产生的最佳菌株 GQ3006 可产生 2280.3± 38.2 mg/l（98.7 ± 4.2 mg/g 细胞干重）α-humulene，相对于基线菌株提高了 100 倍。为了进一步提高效价，首先建立了一种基于Cu ²⁺阻抑型启动子下调角鲨烯生物合成的新策略，将α-葎草烯效价显着提高了54.2%，达到3516.6 ± 34.3 mg/l。最后，工程菌株可以在 5-L 生物反应器中产生 21.7 g/l α-humulene，比本研究之前报道的最高 α-humulene 滴度高 6.8 倍。总体而言，本研究中使用的系统代谢工程策略为解脂耶氏酵母中萜类化合物的高度可持续生产提供了有价值的参考。