PDF(Pubmed)
Abstract：
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 (<~10-9) 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.
摘要：
细胞活力取决于非天然氨基酸（unAA）的存在的合成营养缺陷型提供了一种强大的策略来限制转基因生物（GMO）在开放环境中的不需要的繁殖，并可能防止工业间谍活动。这里,我们描述了一种依赖于unAA的出芽酵母的强大生物抑制的通用方法。通过了解逃生机制，我们通过在琥珀抑制tRNA的产生中引入设计的“免疫”并开发基于转录和翻译的生物抑制开关来特别优化我们的策略。我们进一步开发了一种面向适应性的筛选方法，以轻松获得多重保护菌株，该菌株在固体培养基上表现出14天的强劲生长和无法检测到的逃逸频率（<〜10-9）。最后,我们表明，采用我们的多重保障体系可以限制在真实的发酵场景中感兴趣的菌株的增殖，突出了我们的酵母生物防护策略的巨大潜力，以保护工业专有菌株。
