Australian Institute for Bioengineering and Nanotechnology (AIBN), the University of Queensland, St. Lucia QLD 4072, Australia.
Australian Institute for Bioengineering and Nanotechnology (AIBN), the University of Queensland, St. Lucia QLD 4072, Australia; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet, 2800 Kgs Lyngby, Denmark.
Metab Eng. 2018 May;47:83-93. doi: 10.1016/j.ymben.2018.02.005. Epub 2018 Feb 19.
Monoterpene production in Saccharomyces cerevisae requires the introduction of heterologous monoterpene synthases (MTSs). The endogenous farnesyl pyrosphosphate synthase (FPPS; Erg20p) competes with MTSs for the precursor geranyl pyrophosphate (GPP), which limits the production of monoterpenes. ERG20 is an essential gene that cannot be deleted and transcriptional down-regulation of ERG20 has failed to improve monoterpene production. Here, we investigated an N-degron-dependent protein degradation strategy to down-regulate Erg20p activity. Degron tagging decreased GFP protein half-life drastically to 1 h (degron K3K15) or 15 min (degrons KN113 and KN119). Degron tagging of ERG20 was therefore paired with a sterol responsive promoter to ensure sufficient metabolic flux to essential downstream sterols despite the severe destabilisation effect of degron tagging. A dual monoterpene/sesquiterpene (linalool/nerolidol) synthase, AcNES1, was used as a reporter of intracellular GPP and FPP production. Transcription of the synthetic pathway was controlled by either constitutive or diauxie-inducible promoters. A combination of degron K3K15 and the ERG1 promoter increased linalool titre by 27-fold to 11 mg L in the strain with constitutive promoter constructs, and by 17-fold to 18 mg L in the strain with diauxie-inducible promoter constructs. The sesquiterpene nerolidol remained the major product in both strains. The same strategies were applied to construct a limonene-producing strain, which produced 76 mg L in batch cultivation. The FPPS regulation method developed here successfully redirected metabolic flux toward monoterpene production. Examination of growth defects in various strains suggested that the intracellular FPP concentration had a significant effect on growth rate. Further strategies are required to balance intracellular production of FPP and GPP so as to maximise monoterpene production without impacting on cellular growth.
酵母中单萜的生产需要引入异源单萜合酶（MTS）。内源性法呢基焦磷酸合酶（FPPS； Erg20p）与 MTS 竞争前体香叶基焦磷酸（GPP），这限制了单萜的产生。ERG20 是一个必需基因，不能被删除，而 ERG20 的转录下调未能提高单萜的产量。在这里，我们研究了一种基于 N-降解结构域的蛋白质降解策略来下调 Erg20p 的活性。降解结构域标记使 GFP 蛋白的半衰期大大缩短至 1 小时（降解结构域 K3K15）或 15 分钟（降解结构域 KN113 和 KN119）。因此，ERG20 的降解结构域标记与固醇响应启动子配对，以确保尽管降解结构域标记有严重的不稳定化作用，但仍有足够的代谢通量流向必需的下游固醇。使用双单萜/倍半萜（芳樟醇/橙花叔醇）合酶 AcNES1 作为细胞内 GPP 和 FPP 产生的报告基因。合成途径的转录由组成型或双重诱导启动子控制。降解结构域 K3K15 和 ERG1 启动子的组合使具有组成型启动子构建体的菌株中的芳樟醇产量增加了 27 倍，达到 11mg/L，具有双重诱导启动子构建体的菌株中的产量增加了 17 倍，达到 18mg/L。在这两种菌株中，倍半萜橙花叔醇仍然是主要产物。同样的策略也被应用于构建柠檬烯生产菌株，该菌株在分批培养中产生了 76mg/L 的柠檬烯。这里开发的 FPPS 调节方法成功地将代谢通量重新定向到单萜生产。对各种菌株生长缺陷的检查表明，细胞内 FPP 浓度对生长速率有显著影响。需要进一步的策略来平衡细胞内 FPP 和 GPP 的产生，以最大限度地提高单萜的产量，而不影响细胞生长。
