RESUMO
The existence of crosstalk between quorum sensing systems limits their application in a complex environment. In this study, two completely orthogonal quorum sensing systems with self-produced autoinducers were built in one cell to enable the systems to be signal orthogonal and promoter orthogonal to each other. The systems were designed on the basis of the las system from Pseudomonas aeruginosa and the tra system from Agrobacterium tumefaciens. Both were optimized with respect to the orthogonality of signals and promoters by using a series of synthetic biology strategies and high-throughput screening. The systems were applied intracellularly, and an automatic delayed cascade circuit was successfully demonstrated, which can realize sequential gene expression without exogenous inducer. This circuit provides a new tool for biotechnological applications, such as metabolic regulation, that require sequential gene control. This cascade model expands the toolkit of synthetic biology research and indicates a high application potential of quorum sensing systems that are orthogonal to each other.
Assuntos
4-Butirolactona/análogos & derivados , Homosserina/análogos & derivados , Lactonas/metabolismo , Percepção de Quorum/genética , 4-Butirolactona/metabolismo , 4-Butirolactona/farmacologia , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Homosserina/metabolismo , Homosserina/farmacologia , Lactonas/farmacologia , Mutagênese , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/metabolismo , Transativadores/genéticaRESUMO
Metabolic engineering aims to achieve high yields of desired products. The most common strategies focus on optimization of metabolic flux distributions. The dynamic activation or inhibition of gene expression through quorum sensing (QS) has been applied to metabolic engineering. In this study, we designed and constructed a series of QS-based bifunctional dynamic switches (QS switches) capable of synchronizing the up-regulation and down-regulation of genes at different times and intervals. The bifunctional QS switches were based on the Esa QS system, because EsaR regulatory proteins can act as transcriptional activator and repressor. The QS switches' effectiveness and feasibility were verified through fluorescence characterization. Finally, the QS switches were applied to the production of 5-aminolevulinic acid (ALA) and poly-ß-hydroxybutyrate (PHB) to solve two key metabolic engineering problems: necessary gene knockout and redirection of metabolic flux. The production of PHB and ALA was increased 6- and 12-fold in Escherichia coli, respectively.
