Synergistic transcriptional activation by one regulatory protein in response to two metabolites

Bundy, Becky M.; Collier, Lauren S.; Hoover, Timothy R.; Neidle, Ellen L.

doi:10.1073/pnas.102605799

Cited by 84 publications

(139 citation statements)

References 37 publications

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“…1). Finally, this protein does not require any binding to regulatory subunits apart from its cognate inducers, which should ensure its orthogonality in nonnative chassis 26 .…”

Section: A Prokaryote Transcription Activator In Yeastmentioning

confidence: 99%

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

et al. 2016

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TitleEngineering prokaryotic transcriptional activators as metabolite biosensors in yeast io-based production of chemicals and fuels is an attractive avenue to reduce dependence on petroleum. For bio-based production, biocatalysts must often be genetically modified to increase production. However, the current efficiency of genomeengineering methods and parts prospecting allows for unprecedented genotype diversity that vastly outstrips our ability to screen for best cell performance 1,2 .To meet current demand, bioengineers have started to develop genetically encoded devices and systems that enable screening and selection of better-performing biocatalysts in higher throughput. Genetic devices including oscillators, amplifiers and recorders, which have been developed based on fine-tuned relationships between input and output signals, are promising tools for programming and controlling gene expression in living cells [3][4][5] . These devices sense extracellular or intracellular perturbations and actuate cellular decision-making processes akin to logic gates in electrical circuits. Hence, from a diverse set of inputs, molecular gating components such as RNA aptamers and allosterically regulated transcription factors have been engineered to control outputs for applications such as high-throughput screening, actuation on cellular metabolism and evolution-based selection of optimal cell performance [6][7][8] .A key component in many of the reported devices is a ligandinducible transcriptional regulator. Transcriptional regulators are straightforward and powerful components, with many uses in genetic designs. Owing to their modular structure, transcriptional regulators have proven to be versatile platforms for genetically encoded Boolean logic functions 9,10 . In particular, gene switches based on ligand-binding transcriptional repressors bind to genomic targets in the absence of their cognate ligand and thereby repress gene expression of the downstream gene(s), whereas binding between ligand and repressor causes the release of the repressor from the DNA and thereby a derepression 11 . In such 'NOT' gates, simple steric hindrance of RNA polymerase progression, as in the case of the tetracycline-responsive gene switch TetR, have for decades been used for conditional control of gene expression in both prokaryotic and eukaryotic chassis 12,13 . Transcriptional repressors and other artificial transcriptional regulators can be further engineered, for example, via the addition of nuclear localization signals, destabilization domains and transcriptional activation regions, to repurpose conditional repressors into activators [13][14][15] . Though conceptually intriguing and practically relevant, the repurposing of logic gates can suffer from the inherent need for extensive engineering 9,16,17 .Though most ligand-inducible genetic devices adopted for eukaryotes historically have been founded on transcriptional repressors, a hitherto untapped resource for use in genetic designs is ligand-inducible transcriptional activators. Bac...

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“…1). Finally, this protein does not require any binding to regulatory subunits apart from its cognate inducers, which should ensure its orthogonality in nonnative chassis 26 .…”

Section: A Prokaryote Transcription Activator In Yeastmentioning

confidence: 99%

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

et al. 2016

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“…1). Furthermore, benzoate and muconate together have a synergistic effect on transcriptional activation (1). Thus, it seems likely that both compounds bind simultaneously to a BenM tetramer.…”

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confidence: 99%

“…Thus, it seems likely that both compounds bind simultaneously to a BenM tetramer. Effectordependent transcription and DNA-binding properties have been characterized for several muconate-responsive proteins (1,3,14,15,18). However, there have been no direct studies of their interactions with effectors.…”

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confidence: 99%

“…Two amino acids, leucine and glutamate, were also added between the native C terminus and the histidine tag. The methods used to generate and purify this protein were similar to those used for the histidine-tagged full-length BenM protein (1,4). BenM-EBD was obtained at greater than 95% purity, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.…”

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confidence: 99%

“…BenM-EBD was obtained at greater than 95% purity, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration and liquid chromatography-mass spectrometry methods indicated that BenM-EBD is a dimer in solution (5) while the full-length protein is a tetramer in solution (1).…”

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Benzoate Decreases the Binding of cis , cis -Muconate to the BenM Regulator despite the Synergistic Effect of Both Compounds on Transcriptional Activation

Clark¹,

Phillips²,

Bundy³

et al. 2004

J Bacteriol

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Fluorescence emission spectroscopy was used to investigate interactions between two effectors and BenM, a transcriptional regulator of benzoate catabolism. BenM had a higher affinity for cis,cis-muconate than for benzoate as the sole effector. However, the presence of benzoate increased the apparent dissociation constant (reduced the affinity) of the protein for cis,cis-muconate. Similar results were obtained with truncated BenM lacking the DNA-binding domain. High-level transcriptional activation may require that some monomers within a BenM tetramer bind benzoate and others bind cis,cis-muconate.

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Design, Engineering, and Characterization of Prokaryotic Ligand-Binding Transcriptional Activators as Biosensors in Yeast

Ambri

Snoek

Skjoedt

et al. 2018

Methods in Molecular Biology

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In cell factory development, screening procedures, often relying on low-throughput analytical methods, are lagging far behind diversity generation methods. This renders the identification and selection of the best cell factory designs tiresome and costly, conclusively hindering the manufacturing process. In the yeast Saccharomyces cerevisiae, implementation of allosterically regulated transcription factors from prokaryotes as metabolite biosensors has proven a valuable strategy to alleviate this screening bottleneck. Here, we present a protocol to select and incorporate prokaryotic transcriptional activators as metabolite biosensors in S. cerevisiae. As an example, we outline the engineering and characterization of the LysR-type transcriptional regulator (LTTR) family member BenM from Acetinobacter sp. ADP1 for monitoring accumulation of cis,cis-muconic acid, a bioplast precursor, in yeast by means of flow cytometry.

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Synergistic transcriptional activation by one regulatory protein in response to two metabolites

Cited by 84 publications

References 37 publications

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

Benzoate Decreases the Binding of cis , cis -Muconate to the BenM Regulator despite the Synergistic Effect of Both Compounds on Transcriptional Activation

Design, Engineering, and Characterization of Prokaryotic Ligand-Binding Transcriptional Activators as Biosensors in Yeast

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