Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA.
Nucleic Acids Res. 2023 Jul 21;51(13):7094-7108. doi: 10.1093/nar/gkad484.
The development of synthetic biological circuits that maintain functionality over application-relevant time scales remains a significant challenge. Here, we employed synthetic overlapping sequences in which one gene is encoded or 'entangled' entirely within an alternative reading frame of another gene. In this design, the toxin-encoding relE was entangled within ilvA, which encodes threonine deaminase, an enzyme essential for isoleucine biosynthesis. A functional entanglement construct was obtained upon modification of the ribosome-binding site of the internal relE gene. Using this optimized design, we found that the selection pressure to maintain functional IlvA stabilized the production of burdensome RelE for >130 generations, which compares favorably with the most stable kill-switch circuits developed to date. This stabilizing effect was achieved through a complete alteration of the allowable landscape of mutations such that mutations inactivating the entangled genes were disfavored. Instead, the majority of lineages accumulated mutations within the regulatory region of ilvA. By reducing baseline relE expression, these more 'benign' mutations lowered circuit burden, which suppressed the accumulation of relE-inactivating mutations, thereby prolonging kill-switch function. Overall, this work demonstrates the utility of sequence entanglement paired with an adaptive laboratory evolution campaign to increase the evolutionary stability of burdensome synthetic circuits.
在应用相关时间范围内保持功能的合成生物电路的发展仍然是一个重大挑战。在这里，我们采用了合成重叠序列，其中一个基因完全编码或“纠缠”在另一个基因的替代阅读框中。在这种设计中，编码毒素的 relE 被纠缠在编码苏氨酸脱氨酶的 ilvA 中，苏氨酸脱氨酶是异亮氨酸生物合成所必需的酶。通过修饰内部 relE 基因的核糖体结合位点，获得了具有功能的纠缠构建体。使用这种优化设计，我们发现维持 IlvA 功能的选择压力稳定了累赘 RelE 的产生超过 130 代，这与迄今为止开发的最稳定的杀伤开关电路相比具有优势。这种稳定作用是通过完全改变允许的突变景观来实现的，使得失活纠缠基因的突变不受青睐。相反，大多数谱系在 ilvA 的调控区域积累了突变。通过降低基线 relE 表达，这些更“良性”的突变降低了电路负担，从而抑制了 relE 失活突变的积累，从而延长了杀伤开关功能。总的来说，这项工作证明了序列纠缠与适应性实验室进化相结合的实用性，可提高繁琐的合成电路的进化稳定性。
