Framework for the TIMO genetic circuit-assisted strategy for remodeling and synergistic regulating IPP metabolism. (IMAGE)
Caption
(a) Liquid chromatography–mass spectrometry (LC-MS) was used to detect the content of key intracellular metabolites, including MVA, MEP, pyruvate, and acetyl-CoA. Thus, the intracellular metabolic flux distribution was analyzed. (b) According to the analysis results, the corresponding regulatory system based on the genetic circuit cascade was designed to facilitate the flow of metabolic fluxes into the IPP supply module. Pyr: pyruvate; Aco: acetyl-CoA; Mco: malonyl-CoA. Pink arrow: pyruvate activated genetic circuit; blue blunt-end arrow: malonyl-CoA-inhibited genetic circuit; switch: AND/OR gate in response to pyruvate and malonyl-CoA. (c) The genetic circuits involved in the regulatory system were constructed separately, including one-input genetic circuits and two-input genetic circuits. eGFP: enhanced green fluorescence protein; FapR: malonyl-CoA-responsive transcriptional factor; PdhR: pyruvate-responsive transcriptional factor. (d) The performance and logic behavior of these genetic circuits were characterized and optimized, including the dynamic range, responsive threshold, and specificity. r: dynamic range; a: the asymmetry of the gate with respect to its two inducers; l: the logical behavior of the promoter; asym: asymmetry. (e) The optimized genetic circuits were introduced into the host to build a regulatory system for regulating IPP metabolism. (f) The product yield and intracellular metabolite content of the recombinant strain in a shake flask and bioreactor were detected.
Credit
Xianhao Xu et al.
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