RESUMO
In recent years, small-molecule biosensors have become increasingly important in synthetic biology and biochemistry, with numerous new applications continuing to be developed throughout the field. For many biosensors, however, their utility is hindered by poor functionality. Here, we review the known types of mechanisms of biosensors within bacterial cells, and the types of approaches for optimizing different biosensor functional parameters. Discussed approaches for improving biosensor functionality include methods of directly engineering biosensor genes, considerations for choosing genetic reporters, approaches for tuning gene expression, and strategies for incorporating additional genetic modules.
Assuntos
Técnicas Biossensoriais , Bactérias , Biologia SintéticaRESUMO
Pyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we also develop Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl-containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl-proteins in E. coli.
Assuntos
Vias Biossintéticas/genética , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Lisina/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Colífagos/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lisina/biossíntese , Microrganismos Geneticamente Modificados , Mutação , Óperon , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Developing and optimizing small-molecule biosensors is a central goal of synthetic biology. Here we incorporate additional cellular components to improve biosensor sensitivity by preventing target molecules from diffusing out of cells. We demonstrate that trapping erythromycin within Escherichia coli through phosphorylation increases the sensitivity of its biosensor (MphR) by approximately 10-fold. When combined with prior engineering efforts, our optimized biosensor can detect erythromycin concentrations as low as 13 nM. We show that this strategy works with a range of macrolide substrates, enabling the potential usage of our optimized system for drug development and metabolic engineering. This strategy can be extended in future studies to improve the sensitivity of other biosensors. Our findings further suggest that many naturally evolved genes involved in resistance to multiple classes of antibiotics may increase intracellular drug concentrations to modulate their own expression, acting as a form of regulatory feedback.
Assuntos
Antibacterianos/metabolismo , Técnicas Biossensoriais/métodos , Eritromicina/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Fatores de Transcrição/genética , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Fosforilação , Biologia Sintética/métodos , Fatores de Transcrição/metabolismoRESUMO
Ligand-inducible genetic systems are the mainstay of synthetic biology, allowing gene expression to be controlled by the presence of a small molecule. However, 'leaky' gene expression in the absence of inducer remains a persistent problem. We developed a leak dampener tool that drastically reduces the leak of inducible genetic systems while retaining signal in Escherichia coli. Our system relies on a coherent feedforward loop featuring a suppressor tRNA that enables conditional readthrough of silent non-sense mutations in a regulated gene, and this approach can be applied to any ligand-inducible transcription factor. We demonstrate proof-of-principle of our system with the lactate biosensor LldR and the arabinose biosensor AraC, which displayed a 70-fold and 630-fold change in output after induction of a fluorescence reporter, respectively, without any background subtraction. Application of the tool to an arabinose-inducible mutagenesis plasmid led to a 540-fold change in its output after induction, with leak decreasing to the level of background mutagenesis. This study provides a modular tool for reducing leak and improving the fold-induction within genetic circuits, demonstrated here using two types of biosensors relevant to cancer detection and genetic engineering.
Assuntos
Regulação Bacteriana da Expressão Gênica , RNA de Transferência/metabolismo , Fator de Transcrição AraC/metabolismo , Arabinose/metabolismo , Códon de Terminação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácido Láctico/metabolismo , Mutagênese , Plasmídeos/genética , Biossíntese de Proteínas , RNA Catalítico , RNA de Transferência/química , Fatores de Transcrição/metabolismoRESUMO
Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans-a phenomenon that provides a valuable source of data for antimicrobial drug development.