Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells
Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells
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DOI：
10.1126/sciadv.abf5851
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发表时间：
2021-06-01
期刊：
影响因子：
13.6
通讯作者：
Church, George M.
中科院分区：
文献类型：
--
作者：
Kunjapur, Aditya M.;Napolitano, Michael G.;Church, George M.
了解生物遏制技术的进化稳定性和可能的背景依赖性是至关重要的，因为工程微生物越来越多地被考虑用于生物制造以外的应用。虽然合成的营养缺陷型以前阻止大肠杆菌表现出可检测的逃避分批培养，其长期有效性是未知的。在这里，我们报告自动化的连续进化的合成营养缺陷型，同时提供浓度降低的必需联苯丙氨酸（BPA）。经过100天的进化，一式三份的群体没有表现出可观察到的逃逸，并表现出正常的生长速率比祖先的合成营养缺陷型低10倍的BPA浓度。等位基因重建揭示了三个基因的贡献，以增加健身在低BPA浓度。基于其进化稳定性，我们将祖菌株直接引入哺乳动物细胞培养物中，并观察到细菌的遏制，而不会对HEK293T细胞产生有害影响。总的来说，我们的研究结果表明，合成营养缺陷型是有效的时间尺度和背景下，使不同的应用程序。
Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented Escherichia coli from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.