Jadhav Prajakta, Roy Sudeshna, Butzin Xuan Yi, Butzin Nicholas C
Department of Biology and Microbiology, South Dakota State University, Brookings, South Dakota 57007, United States.
ACS Synth Biol. 2025 Apr 18;14(4):1062-1071. doi: 10.1021/acssynbio.4c00612. Epub 2025 Mar 19.
The ATP-dependent ClpXP-SspB protease complex is responsible for the degradation of intracellular proteins and is maintained at low levels in to avoid nonspecific degradation. The rate-limiting step in the protease complex leads to proteolytic queueing, where the proteins form waiting lines, and their overall degradation rate is slowed. Synthetic biologists have leveraged proteolytic queueing to design robust synthetic circuits by tagging proteins with the SsrA tag, an 11-amino acid sequence recognized by the complex. Previous work has demonstrated the binding site of each component of the ClpXP-SspB complex to the SsrA tag. However, the precise component responsible for queueing was unknown. To identify the bottleneck in the complex, we designed different SsrA tag variants depending on the chaperone binding sequences. We further overexpressed each protein in the ClpXP-SspB complex to determine how an increased amount of each component affects the tagged protein levels. Based on the degradation of the SsrA variants, upon overexpression of each component of the ClpXP-SspB system, evidence supports that ClpX (the ATP-dependent chaperone) is responsible for queueing but not ClpP (the protease) or SspB (the adapter, ATP-independent chaperone). In the process, we identified LAA-LAA, a 6-amino acid ClpX-dependent tag that degraded faster than the original SsrA tag, AANDENYALAA. We speculated that this high degradation tag could be useful in a dynamic-synthetic circuit, so we modified the well-characterized dual-feedback oscillator by replacing its original SsrA tag with the LAA-LAA tag to form the LAA-LAA-Osc oscillator. Both population and single-cell level experiments show that the new and old oscillators have distinct frequencies. Like the original oscillator, thousands of cells containing the new oscillator could be synchronized by entrainment using an external signal. Thus, the new LAA-LAA-Osc oscillator retains the original oscillator's best characteristics (robustness to fluctuations, a steady oscillation period, and entrainment across 1000s of cells to an external signal) but oscillates at a different frequency.
依赖ATP的ClpXP - SspB蛋白酶复合体负责细胞内蛋白质的降解，并维持在低水平以避免非特异性降解。蛋白酶复合体中的限速步骤导致蛋白水解排队，即蛋白质形成等待队列，其整体降解速率减慢。合成生物学家利用蛋白水解排队，通过用SsrA标签（一种被该复合体识别的11个氨基酸序列）标记蛋白质来设计稳健的合成电路。先前的工作已经证明了ClpXP - SspB复合体各组分与SsrA标签的结合位点。然而，负责排队的确切组分尚不清楚。为了确定复合体中的瓶颈，我们根据伴侣蛋白结合序列设计了不同的SsrA标签变体。我们进一步在ClpXP - SspB复合体中过表达每种蛋白质，以确定每种组分数量的增加如何影响被标记蛋白质的水平。基于SsrA变体的降解情况，在ClpXP - SspB系统各组分过表达后，有证据支持ClpX（依赖ATP的伴侣蛋白）负责排队，而不是ClpP（蛋白酶）或SspB（衔接蛋白，不依赖ATP的伴侣蛋白）。在此过程中，我们鉴定出LAA - LAA，一种6个氨基酸的依赖ClpX的标签，其降解速度比原始的SsrA标签AANDENYALAA更快。我们推测这种高降解标签可能在动态合成电路中有用，因此我们通过用LAA - LAA标签替换其原始的SsrA标签来修饰特征明确的双反馈振荡器，形成LAA - LAA - Osc振荡器。群体水平和单细胞水平实验均表明，新旧振荡器具有不同的频率。与原始振荡器一样，数千个含有新振荡器的细胞可以通过外部信号的诱导实现同步。因此，新的LAA - LAA - Osc振荡器保留了原始振荡器的最佳特性（对波动的稳健性、稳定的振荡周期以及数千个细胞对外部信号的诱导同步），但振荡频率不同。
