Siu Yik, Fenno Jesse, Lindle Jessica M, Dunlop Mary J
School of Engineering, University of Vermont , Burlington, Vermont 05405, United States.
Biomedical Engineering, Boston University , Boston, Massachusetts 02215, United States.
ACS Synth Biol. 2018 Jan 19;7(1):16-23. doi: 10.1021/acssynbio.7b00260. Epub 2017 Oct 12.
Feedback control allows cells to dynamically sense and respond to environmental changes. However, synthetic controller designs can be challenging because of implementation issues, such as determining optimal expression levels for circuit components within a feedback loop. Here, we addressed this by coupling rational design with selection to engineer a synthetic feedback circuit to optimize tolerance of Escherichia coli to the biojet fuel pinene. E. coli can be engineered to produce pinene, but it is toxic to cells. Efflux pumps, such as the AcrAB-TolC pump, can improve tolerance, but pump expression impacts growth. To address this, we used feedback to dynamically regulate pump expression in response to stress. We developed a library with thousands of synthetic circuit variants and subjected it to three types of pinene treatment (none, constant, and varying pinene). We were able to select for strains that were biofuel tolerant without a significant growth cost in the absence of biofuel. Using next-generation sequencing, we found common characteristics in the designs and identified controllers that dramatically improved biofuel tolerance.
反馈控制使细胞能够动态感知并响应环境变化。然而，由于实施问题，如确定反馈回路中电路组件的最佳表达水平，合成控制器的设计可能具有挑战性。在这里，我们通过将合理设计与筛选相结合来解决这个问题，构建一个合成反馈回路，以优化大肠杆菌对生物喷气燃料蒎烯的耐受性。大肠杆菌可以被改造以产生蒎烯，但蒎烯对细胞有毒性。外排泵，如AcrAB-TolC泵，可以提高耐受性，但泵的表达会影响生长。为了解决这个问题，我们利用反馈来动态调节泵的表达以应对压力。我们开发了一个包含数千个合成回路变体的文库，并对其进行三种类型的蒎烯处理（无、恒定和变化的蒎烯）。我们能够筛选出在没有生物燃料时具有生物燃料耐受性且生长成本没有显著增加的菌株。通过下一代测序，我们发现了设计中的共同特征，并鉴定出显著提高生物燃料耐受性的控制器。
