Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30°C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. ‘Turbo’ mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80p-supressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37–33°C) to a low temperature (≤30°C). Overall, we present a synthetic biology procedure that leverages ‘leaky’ biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

中文翻译：

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LowTempGAL：酿酒酵母中高度响应的低温诱导 GAL 系统


温度是生物制剂生物制造精准发酵的重要控制因素。在这里，我们在模型酵母酿酒酵母中探索了高度响应的低温诱导遗传系统（LowTempGAL）。两个温度生物传感器，一个热诱导降解决定子和一个热诱导蛋白聚集域，被用来调节 GAL 激活剂 Gal4p，从而呈现泄漏的 LowTempGAL 系统。布尔型诱导是通过对 GAL 阻遏基因 GAL80 进行低温介导的抑制来实现第二层控制，但对低温触发的反应延迟，并且在 30°C 时反应较弱。验证了蛋白质和小分子生产的应用潜力。蛋白质组学分析表明残留的 Gal80p 和 Gal4p 不足导致诱导效果欠佳。 “Turbo”机制是通过整合基础 Gal4p 表达和不依赖半乳糖的 Gal80p 抑制 Gal3p 突变体 (Gal3Cp) 来设计的。通过改变 Gal3Cp 配置，我们部署了 LowTempGAL 系统，能够在从高温 (37–33°C) 转变到低温 (≤30°C) 时进行快速、严格的高水平感应。总的来说，我们提出了一种合成生物学程序，利用“泄漏”生物传感器来部署高度响应的布尔型遗传电路。关键在于多因素系统错综复杂布局的优化。 LowTempGAL 系统可适用于精密生物制造的非传统酵母平台。