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<p>Genetically-encoded sensors are used to control protein and metabolite production in bacterial fermentations. However, these sensors are generally optimized for exponential growth rather than stationary phase where production occurs. Here, we find that our previously engineered <i>E. coli</i> green light sensor CcaSR, which functions robustly in exponential phase, fails in stationary phase due to spontaneous loss of an engineered chromophore biosynthetic pathway and accumulation of CcaS and CcaR. We optimize the genetic context and expression determinants of each component, resulting in a stable system named CcaSR<sub>stat</sub> that imposes little metabolic burden, exhibits low leakiness and an 80-fold green light response, and functions exclusively in stationary phase. We combine CcaSR<sub>stat</sub>-driven enzyme expression with varied static and periodic illumination patterns to achieve high titers of the industrially-relevant phenylpropanoid <i>p</i>-Coumaric acid and demonstrate that these optimizations scale to benchtop bioreactor conditions. Finally, we use CcaSR<sub>stat</sub> to optimize the expression level of a co-transcribed multi-enzyme metabolic pathway encoding production of plant-derived betaxanthin family pigments. Stationary phase-optimized bacterial sensors should enhance fermentation productivity by enabling rapid interrogation of the impact of enzyme expression level and induction dynamics.</p>

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A stationary phase-specific bacterial green light sensor for enhancing metabolite production

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月24日

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  • John T. Lazar,
  • Daniel J. Haller,
  • Abbas Ghaddar,
  • Jae J. Kim,
  • Kevin Yang,
  • Sebastián M. Castillo-Hair,
  • Andrew R. Gilmour,
  • Ross Thyer,
  • Jeffrey J. Tabor

摘要

Genetically-encoded sensors are used to control protein and metabolite production in bacterial fermentations. However, these sensors are generally optimized for exponential growth rather than stationary phase where production occurs. Here, we find that our previously engineered E. coli green light sensor CcaSR, which functions robustly in exponential phase, fails in stationary phase due to spontaneous loss of an engineered chromophore biosynthetic pathway and accumulation of CcaS and CcaR. We optimize the genetic context and expression determinants of each component, resulting in a stable system named CcaSRstat that imposes little metabolic burden, exhibits low leakiness and an 80-fold green light response, and functions exclusively in stationary phase. We combine CcaSRstat-driven enzyme expression with varied static and periodic illumination patterns to achieve high titers of the industrially-relevant phenylpropanoid p-Coumaric acid and demonstrate that these optimizations scale to benchtop bioreactor conditions. Finally, we use CcaSRstat to optimize the expression level of a co-transcribed multi-enzyme metabolic pathway encoding production of plant-derived betaxanthin family pigments. Stationary phase-optimized bacterial sensors should enhance fermentation productivity by enabling rapid interrogation of the impact of enzyme expression level and induction dynamics.