Monoterpenes and monoterpenoids such as (S)-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. Saccharomyces cerevisiae has proven to be an effective host to produce various terpenes and terpenoids. (S)-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, Erg20K197G (Erg20G) and Erg20F96W, N127W (Erg20WW), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20WWG mutant with over four times higher GPP accumulating capability than Erg20WW, as observed through geraniol production levels. The Erg20WWG mutant was fused to the LS from Mentha spicata or the GES from Catharanthus roseus for efficient conversion of GPP to (S)-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20WWG-fused enzymes in peroxisomes, while simultaneously downregulating the essential ERG20 gene using the glucose-sensing HXT1 promoter. In the case of (S)-limonene production, additional Erg20WWG-LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of (S)-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20WWG mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids.

中文翻译：

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通过使用具有增强 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，如 （S）-柠檬烯和香叶醇，是具有广泛应用的有价值的化学品，包括化妆品、药品和生物燃料。酿酒酵母已被证明是产生各种萜烯和萜类化合物的有效宿主。（S）-柠檬烯和香叶醇分别通过柠檬烯合酶 （LS） 和香叶醇合酶 （GES） 的酶促作用由香叶酰焦磷酸酯 （GPP） 产生。然而，它们生产中的一个主要障碍是 Erg20 的双重功能，Erg20 是一种负责产生 GPP 的法呢基焦磷酸 （FPP） 合酶。Erg20 不仅通过将焦磷酸异戊烯酯 （IPP） 与焦磷酸二甲基烯丙酯缩合来合成 GPP，而且还催化 IPP 与 GPP 进一步缩合以产生 FPP。在这项研究中，我们通过利用先前开发的 Erg20 突变体 Erg20K197G （Erg20G） 和 Erg20F96W、N127W （Erg20WW） 来解决这个问题，它们增强了 GPP 积累。通过这些突变体的组合，我们生成了一种新的 Erg20WWG 突变体，其 GPP 积累能力是 Erg20WW 的四倍以上，从香叶醇生产水平中观察到。将 Erg20WWG 突变体与薄荷的 LS 或 Catharanthus roseus 的 GES 融合，以将 GPP 分别高效转化为 （S）-柠檬烯和香叶醇。通过在过氧化物酶体中定位整个甲羟戊酸通路和 Erg20WWG 融合酶，同时使用葡萄糖感应 HXT1 启动子下调必需的 ERG20 基因，实现了进一步的改进。在 （S）-柠檬烯产生的情况下，额外的 Erg20WWG-LS 在胞质溶胶中表达。 结果，最终菌株通过乙醇进料分批补料双相发酵产生了 1063 mg/L 的 （S）-柠檬烯和 1234 mg/L 的香叶醇。新发现的 Erg20WWG 突变体为高效生产各种其他 GPP 衍生化学品（包括单萜衍生物和大麻素）打开了大门。