Monoterpenes and monoterpenoids such as ()-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. has proven to be an effective host to produce various terpenes and terpenoids. ()-limonene and geraniol are produced from geranyl pyrophosphate (GPP) through the enzymatic actions of limonene synthase (LS) and geraniol synthase (GES), respectively. However, a major hurdle in their production arises from the dual functionality of the Erg20, a farnesyl pyrophosphate (FPP) synthase, responsible for generating GPP. Erg20 not only synthesizes GPP by condensing isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate but also catalyzes further condensation of IPP with GPP to produce FPP. In this study, we have tackled this issue by harnessing previously developed Erg20 mutants, Erg20 (Erg20) and Erg20 (Erg20), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20 mutant with over four times higher GPP accumulating capability than Erg20, as observed through geraniol production levels. The Erg20 mutant was fused to the LS from or the GES from for efficient conversion of GPP to ()-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20-fused enzymes in peroxisomes, while simultaneously downregulating the essential gene using the glucose-sensing promoter. In the case of ()-limonene production, additional Erg20-LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of ()-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20 mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids.

中文翻译：

---

通过利用具有增强的 GPP 积累能力的 Erg20 突变体在酿酒酵母中高效生产 (S)-柠檬烯和香叶醇


单萜和单萜类化合物，例如()-柠檬烯和香叶醇，是有价值的化学品，具有广泛的应用，包括化妆品、药品和生物燃料。已被证明是生产各种萜烯和萜类化合物的有效宿主。 ()-柠檬烯和香叶醇分别由香叶基焦磷酸 (GPP) 通过柠檬烯合酶 (LS) 和香叶醇合酶 (GES) 的酶促作用产生。然而，其生产的一个主要障碍来自于 Erg20 的双重功能，Erg20 是一种法尼基焦磷酸 (FPP) 合酶，负责生成 GPP。 Erg20不仅可以通过异戊烯基焦磷酸（IPP）与二甲基烯丙基焦磷酸缩合合成GPP，还可以催化IPP与GPP进一步缩合生成FPP。在这项研究中，我们通过利用先前开发的 Erg20 突变体 Erg20 (Erg20) 和 Erg20 (Erg20) 来解决这个问题，它们可以增强 GPP 的积累。通过这些突变体的组合，我们产生了一种新的 Erg20 突变体，其 GPP 积累能力比 Erg20 高四倍以上，如通过香叶醇生产水平观察到的。将 Erg20 突变体与 LS from 或 GES from 融合，分别将 GPP 有效转化为 ()-柠檬烯和香叶醇。通过将整个甲羟戊酸途径和 Erg20 融合酶定位在过氧化物酶体中，同时使用葡萄糖感应启动子下调必需基因，实现了进一步的改进。在 ()-柠檬烯生产的情况下，额外的 Erg20-LS 在细胞质中表达。结果，通过添加乙醇的分批补料双相发酵，最终菌株产生了 1063 mg/L 的 ()-柠檬烯和 1234 mg/L 香叶醇。 新发现的 Erg20 突变体为有效生产各种其他 GPP 衍生化学物质（包括单萜衍生物和大麻素）打开了大门。