Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University, Chiba, Japan.
Nat Commun. 2021 Mar 23;12(1):1846. doi: 10.1038/s41467-021-22134-y.
A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system's utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.
一个广泛的遗传开关库加速了原核合成生物学的发展，而真核合成生物学在模式生物酿酒酵母中却落后了。真核遗传开关比原核遗传开关更大、更复杂，这使得它们的合理设计和进化变得复杂。在这里，我们提出了一个用于酵母遗传开关的创建和进化的稳健工作流程。选择器系统的设计使得遗传开关的 ON 和 OFF 状态的选择都仅通过液体处理完成，并且它能够在不同的选择条件下并行筛选/选择不同的基序。由于 ON 和 OFF 状态选择的选择阈值都可以灵活地调节，因此无需事先了解文库群体，就可以快速确定每个定向进化实验的所需选择条件。该系统的实用性通过 20 个独立的定向进化实验得到了证明，产生了具有更高诱导剂敏感性、反转开关行为、感官功能和提高信噪比（>100 倍诱导）的遗传开关。所得的酵母遗传开关可以以即插即用的方式轻松集成到 AND 门控类胡萝卜素生物合成途径中。
