College of Life Sciences, University of Chinese Academy of Sciences, 100049, Beijing, China.
BGI Research, Shenzhen, 518083, China.
Nat Commun. 2023 Oct 14;14(1):6487. doi: 10.1038/s41467-023-42358-4.
Synthetic auxotrophy in which cell viability depends on the presence of an unnatural amino acid (unAA) provides a powerful strategy to restrict unwanted propagation of genetically modified organisms (GMOs) in open environments and potentially prevent industrial espionage. Here, we describe a generic approach for robust biocontainment of budding yeast dependent on unAA. By understanding escape mechanisms, we specifically optimize our strategies by introducing designed "immunity" to the generation of amber-suppressor tRNAs and developing the transcriptional- and translational-based biocontainment switch. We further develop a fitness-oriented screening method to easily obtain multiplex safeguard strains that exhibit robust growth and undetectable escape frequency (<~10) on solid media for 14 days. Finally, we show that employing our multiplex safeguard system could restrict the proliferation of strains of interest in a real fermentation scenario, highlighting the great potential of our yeast biocontainment strategy to protect the industrial proprietary strains.
合成营养缺陷型，其中细胞活力依赖于非天然氨基酸（unAA）的存在，为限制遗传修饰生物体（GMO）在开放环境中的不受控制的繁殖提供了一种强大的策略，并有可能防止工业间谍活动。在这里，我们描述了一种依赖 unAA 的通用方法，用于稳健的芽殖酵母生物控制。通过了解逃逸机制，我们通过引入设计的“免疫”琥珀抑制 tRNA 的产生并开发基于转录和翻译的生物控制开关来专门优化我们的策略。我们进一步开发了一种基于适应性的筛选方法，以方便地获得多重保护菌株，这些菌株在固体培养基上表现出稳健的生长，并且在 14 天内逃逸频率（<~10）无法检测到。最后，我们表明，采用我们的多重保护系统可以限制感兴趣的菌株在实际发酵情况下的增殖，突出了我们的酵母生物控制策略保护工业专有菌株的巨大潜力。
