Autonomous Control of Gene Expression Using Endogenous Transcription Signals

Autonomous cell-based control of heterologous gene expression can simplify batch-culture bioprocessing by eliminating external monitoring and extrinsic control of culture conditions. Existing approaches use auto-induction media, synthetic cell-cell communication systems, or application-specific biosensors. A simpler, resource-efficient, and general-purpose expression control system responsive to common changes during batch culture would be highly valuable. We used native E. coli promoters, including PhdeA, PdpS, PfumA, PrpoA, PrpoS, PgadA, and PyiaG, and recombinase-based switches to repurpose endogenous transcription signals for control of heterologous gene expression. Specifically, natural changes, covering 1-2 orders of magnitude across growth phases, in transcription from endogenous promoters result in recombinase expression at the exponential-to-stationary phase transition. So-expressed recombinases invert a constitutive promoter regulating expression of arbitrary heterologous genes amplifying the endogenous transcriptional input signal by more than 10-fold. We realized reversible and single-use switching with reduced static and dynamic cell-to-cell variation and overall expression amplification. We used "off-the-shelf" genetic parts and abstraction-based composition frameworks to realize reliable forward engineering of our synthetic genetic systems. We engineered autonomous control systems for regulating heterologous gene expression. Our system uses generic endogenous promoters to sense and control heterologous expression during growth-phase transitions. Our system does not require specialized auto-induction media, production or activation of quorum sensing, or the development of application-specific biosensors. Cells programmed to control themselves could simplify existing bioprocess operations and enable the development of more powerful synthetic genetic systems. Modern biotechnology uses engineered microbes to manufacture molecules that are incorporated into medicines, flavors, fuels, and other materials. Making molecules inside cells requires enzymes, whose expression levels and timing need to be optimized to maximize final product yields. Many engineered microbes use enzymes that should only be expressed during the later stages of cell growth to avoid overconsumption of resources or accumulation of toxic intermediates. Here, we develop simple genetic devices that enable engineered cells to control themselves, automatically switching on or off enzyme expression during batch cell culture. Unlike existing approaches, our devices do not require specialized growth media or wasteful synthesis of cell-cell signaling molecule.