One major challenge in synthetic biology is the deleterious impacts of cellular stress caused by expression of heterologous pathways, sensors, and circuits. Feedback control and dynamic regulation are broadly proposed strategies to mitigate this cellular stress by optimizing gene expression levels temporally and in response to biological cues. While a variety of approaches for feedback implementation exist, they are often complex and cannot be easily manipulated. Here, we report a strategy that uses RNA transcriptional regulators to integrate additional layers of control over the output of natural and engineered feedback responsive circuits. Called riboregulated switchable feedback promoters (rSFPs), these gene expression cassettes can be modularly activated using multiple mechanisms, from manual induction to autonomous quorum sensing, allowing control over the timing, magnitude, and autonomy of expression. We develop rSFPs in Escherichia coli to regulate multiple feedback networks and apply them to control the output of two metabolic pathways. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, biological therapeutic production, and many other applications.
Dynamic pathway regulation has emerged as a promising strategy in metabolic engineering for improved system productivity and yield, and continues to grow in sophistication. Bacterial stress-response promoters allow dynamic gene regulation using the host's natural transcriptional networks, but lack the flexibility to control the expression timing and overall magnitude of pathway genes. Here, we report a strategy that uses RNA transcriptional regulators to introduce another layer of control over the output of natural stress-response promoters. This new class of gene expression cassette, called a riboregulated switchable feedback promoter (rSFP), can be modularly activated using a variety of mechanisms, from manual induction to quorum sensing. We develop and apply rSFPs to regulate a toxic cytochrome P450 enzyme in the context of a Taxol precursor biosynthesis pathway and show this leads to 2.4x fold higher titers than from the best reported strain. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, protein and biologic production, and many other applications.
Techniques by which to genetically manipulate members of the microbiota enable both the evaluation of host-microbe interactions and an avenue by which to monitor and modulate human physiology. Genetic engineering applications have traditionally focused on model gut residents, such as
Escherichia coli
and lactic acid bacteria.
The human microbiome has been inextricably linked to multiple facets of human physiology. From an engineering standpoint, the ability to precisely control the composition and activity of the microbiome holds great promise for furthering our understanding of disease etiology and for new avenues of therapeutic and diagnostic agents. While the field of microbiome research is still in its infancy, growing engineering efforts are emerging to enable new studies in the microbiome and to rapidly translate these findings to microbiome-based interventions. At the 3rd International Conference on Microbiome Engineering, leading experts in the field presented state-ofthe-art work in microbiome engineering, discussing probiotics, prebiotics, engineered microbes, microbially derived biomolecules, and bacteriophage.
Lifestyle-induced changes to the diversity of the commensal microbiota have been causally linked to the increasing prevalence of food allergies and other non-communicable chronic diseases. We have shown that bacteria from the Clostridia class prevent an allergic response to food by eliciting an IL-22 dependent barrier protective response that limits allergen access to the systemic circulation. We have now examined the mechanisms by which commensal Clostridia induce this allergy-protective effect. We identified taxa in a consortium of Clostridia that possess flagella and produce indole, which are ligands for TLR5 and AhR, respectively. Lysates and flagella isolated from this consortium induced IL-22 in mouse intestinal explants. IL-22 was not induced in explants from mice in which TLR5 or MyD88 was knocked out globally or conditionally in CD11c+ cells. Treatment with the commensal flagellar isolate also reduced detection of intragastrically administered FITC dextran in the serum of antibiotic-treated mice. Similarly, indole exposure induced IL-22 in intestinal explants and reduced intestinal permeability to FITC dextran. Importantly, AhR signaling in RORγt+ cells was necessary for IL-22 induction by flagella. These results suggest that flagella and indole act synergistically to prevent an allergic response. Finally, we have isolated and characterized two Clostridial taxa which bear flagella and produce indole. We hypothesize that germ-free mice colonized with these two taxa will exhibit improved IL-22 dependent barrier function and be protected against an allergic response. Our work reveals novel features of Clostridia key to their allergy-protective capability which may be further exploited to develop therapeutics.
Supported by NIH AI106302 and the Bunning Professorship Endowment Fund
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