Engineering stringent genetic biocontainment of yeast with a protein stability switch
Engineering stringent genetic biocontainment of yeast with a protein stability switch
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DOI：
10.1101/2022.11.24.517818
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发表时间：
2023-02
影响因子：
16.6
通讯作者：
Stefan A. Hoffmann;Yizhi Cai
中科院分区：
文献类型：
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作者：
Stefan A. Hoffmann;Yizhi Cai
合成生物学在解决我们面临的关键问题方面有着巨大的希望，例如在资源利用、环境健康和人类健康方面。然而，在开放环境应用中部署基因工程微生物需要全面的安全措施。在这里，我们描述了一个遗传生物遏制系统的基础上的条件稳定的必需蛋白质。我们使用酵母适应的去稳定结构域降解决定子，其可以通过雌二醇添加（ERdd）来稳定。利用酵母GFP收集和实验室自动化平台，我们ERdd标记了775个必需基因，并筛选了具有雌二醇依赖性生长的菌株。发现SPC 110、DIS 3和RRP 46三个基因是特别合适的靶标。各菌株在雌二醇的存在下没有表现出生长缺陷，而在其不存在下表现出强烈的生长抑制。SPC 110-ERdd提供了最严格的遏制，逃逸频率为7.0×10-8，在100 nM雌二醇下完全恢复生长。通过系统分析安全壳逃逸突变，我们确定SPC 110的非必需C-末端区域为逃逸突变的靶点。它的去除使单个ERdd标签的逃逸频率进一步降低至4.3×10-9。将SPC 110-ERdd与DIS 3或RRP 46上的第二个ERdd标签组合导致逃逸频率低于所用测定的检测限（<2×10-10）。基于有条件的蛋白质稳定性，这种方法是机械正交先前报道的内在生物遏制系统。因此，它可以容易地与其他系统，例如基于转录或翻译控制必需基因表达的系统组合，以实现对工程生物体存活的多重、极其严格的控制。合成生物学具有解决人类面临的关键问题的巨大潜力，例如可以彻底改变农业，生物修复或医疗保健。在每一种情况下，都必须防止工程生物在自然环境中不受限制地扩散。这对于开放环境中的工程微生物的使用情况尤其成问题。内在的、遗传编码的生物遏制系统可以解决这个问题，该系统根据环境线索控制细胞存活。我们已经开发了这样一种遗传生物遏制系统，作用于必需蛋白质的稳定性，利用可切换的降解决定子。通过大规模筛选合适的必需靶基因，我们能够创建严格依赖雌二醇的酵母菌株。提供这种小分子，工程细胞保持高适应性，并像未修饰的菌株一样健壮地生长。
Synthetic biology holds immense promise to tackle key problems we are facing, for instance in resource use, environmental health, and human health care. However, comprehensive safety measures are needed to deploy genetically engineered microorganisms in open-environment applications. Here, we describe a genetic biocontainment system based on conditional stability of essential proteins. We used a yeast-adapted destabilizing domain degron, which can be stabilized by estradiol addition (ERdd). Leveraging the yeast GFP collection and lab automation platforms, we ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of 7.0×10-8, and full growth restoration at 100 nM estradiol. By systematically analyzing the containment escapees, we identified the non-essential C-terminal region of SPC110 as target for escape mutations. Its removal decreased the escape frequency with a single ERdd tag further to 4.3×10-9. Combining SPC110-ERdd with a second ERdd tag on either DIS3 or RRP46 resulted in escape frequencies below the detection limit of the used assay (<2×10-10). Being based on conditional protein stability, this approach is mechanistically orthogonal to previously reported intrinsic biocontainment systems. It thus can be readily combined with other systems, for instance ones based on transcriptional or translational control of essential gene expression, to achieve multiplexed, extremely stringent control over the survival of engineered organisms. Significance Synthetic biology holds enormous potential to tackle key issues humanity is facing and can for instance revolutionize agriculture, bioremediation or health care. In each case, the unchecked spread of engineered organisms in natural environments must be prevented. This is particularly problematic with use cases of engineered microbes in open environments. Intrinsic, genetically encoded biocontainment systems, which control cell survival based on environmental cues, can solve this issue. We have developed such a genetic biocontainment system acting on the stability of essential proteins, leveraging a switchable degron. Through a large-scale screening for suitable essential target genes, we were able to create yeast strains that are strictly dependent on estradiol. Supplied with this small molecule, the engineered cells maintain high fitness and grow as robustly as the unmodified strains.