We present a synthetic gene circuit for decoupling cell growth from metabolite production through autonomous regulation of enzymatic pathways by integrated modules that sense nutrient and substrate. The two-layer circuit allows Escherichia coli to selectively utilize target substrates in a mixed pool; channel metabolic resources to growth by delaying enzymatic conversion until nutrient depletion; and activate, terminate, and re-activate conversion upon substrate availability. We developed two versions of controller, both of which have glucose nutrient sensors but differ in their substrate-sensing modules. One controller is specific for hydroxycinnamic acid and the other for oleic acid. Our hydroxycinnamic acid controller lowered metabolic stress 2-fold and increased the growth rate 2-fold and productivity 5-fold, whereas our oleic acid controller lowered metabolic stress 2-fold and increased the growth rate 1.3-fold and productivity 2.4-fold. These results demonstrate the potential for engineering strategies that decouple growth and production to make bio-based production more economical and sustainable.
Environmental pH is a fundamental signal continuously directing the metabolism and behavior of living cells. Programming the precise cellular response toward environmental pH is, therefore, crucial for engineering cells for increasingly sophisticated functions. Herein, we engineer a set of riboswitch-based pH-sensing genetic devices to enable the control of gene expression according to differential environmental pH. We next develop a digital pH-sensing system to utilize the analogue-sensing behavior of these devices for high-resolution recording of host cell exposure to discrete external pH levels. The application of this digital pH-sensing system is demonstrated in a genetic program that autonomously regulated the evolutionary engineering of host cells for improved tolerance to a broad spectrum of organic acids, a valuable phenotype for metabolic engineering and bioremediation applications.
Fatty acid derivatives have ideal properties for use as drop‐in biofuels. An effective strategy in engineering microbial cells to maximize productivity and yield involves dynamic control of protein production in response to concentrations of key intermediates. In Saccharomyces cerevisiae, the activities of the native transcription factors responsive to fatty acids are repressed in the presence of a glucose carbon source. In order to develop a modular fatty acid regulation system in S. cerevisiae, we constructed fatty acid/fatty acyl‐CoA biosensors in S. cerevisiae using bacterial FadR transcriptional repressors and yeast synthetic promoters containing DNA‐binding operators. We demonstrated the functionality of FadR repressors in S. cerevisiae, and tuned the sensing system by varying the promoter strength upstream to the FadR‐coding sequence by varying the number of operator sites in the synthetic promoter and by using FadR from two bacterial sources (Escherichia coli and Vibrio cholerae) with different ligand sensitivities. We envision that our fatty acid/fatty acyl‐CoA biosensors can be used for regulation of protein expression based on the availability of fatty acid intermediates, which will assist in balancing of cellular metabolism during fatty acid derivatives production in yeast.
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