Halvorsen Tiffany M, Ricci Dante P, Park Dan M, Jiao Yongqin, Yung Mimi C
Lawrence Livermore National Laboratory, Biosciences and Biotechnology Division, Livermore, California 94550, United States.
ACS Synth Biol. 2022 Nov 18;11(11):3785-3796. doi: 10.1021/acssynbio.2c00386. Epub 2022 Nov 8.
Kill switches provide a biocontainment strategy in which unwanted growth of an engineered microorganism is prevented by expression of a toxin gene. A major challenge in kill switch engineering is balancing evolutionary stability with robust cell killing activity in application relevant host strains. Understanding host-specific containment dynamics and modes of failure helps to develop potent yet stable kill switches. To guide the design of robust kill switches in the agriculturally relevant strain SBW25, we present a comparison of lethality, stability, and genetic escape of eight different toxic effectors in the presence of their cognate inactivators (i.e., toxin-antitoxin modules, polymorphic exotoxin-immunity systems, restriction endonuclease-methyltransferase pair). We find that cell killing capacity and evolutionary stability are inversely correlated and dependent on the level of protection provided by the inactivator gene. Decreasing the proteolytic stability of the inactivator protein can increase cell killing capacity, but at the cost of long-term circuit stability. By comparing toxins within the same genetic context, we determine that modes of genetic escape increase with circuit complexity and are driven by toxin activity, the protective capacity of the inactivator, and the presence of mutation-prone sequences within the circuit. Collectively, the results of our study reveal that circuit complexity, toxin choice, inactivator stability, and DNA sequence design are powerful drivers of kill switch stability and valuable targets for optimization of biocontainment systems.
杀伤开关提供了一种生物遏制策略，即通过毒素基因的表达来防止工程微生物的不必要生长。杀伤开关工程中的一个主要挑战是在与应用相关的宿主菌株中，平衡进化稳定性与强大的细胞杀伤活性。了解宿主特异性的遏制动态和失效模式有助于开发高效且稳定的杀伤开关。为了指导在农业相关菌株SBW25中设计强大的杀伤开关，我们比较了八种不同毒性效应物在其同源失活剂（即毒素-抗毒素模块、多态性外毒素-免疫系统、限制内切酶-甲基转移酶对）存在下的致死性、稳定性和遗传逃逸情况。我们发现细胞杀伤能力和进化稳定性呈负相关，且取决于失活剂基因提供的保护水平。降低失活剂蛋白的蛋白水解稳定性可提高细胞杀伤能力，但会以长期电路稳定性为代价。通过在相同遗传背景下比较毒素，我们确定遗传逃逸模式会随着电路复杂性的增加而增加，并且由毒素活性、失活剂的保护能力以及电路中易发生突变的序列的存在所驱动。总体而言，我们的研究结果表明，电路复杂性、毒素选择、失活剂稳定性和DNA序列设计是杀伤开关稳定性的强大驱动因素，也是生物遏制系统优化的有价值目标。
