Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor.
ACS Synth Biol
; 13(7): 2177-2187, 2024 Jul 19.
Article
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| MEDLINE
| ID: mdl-38968698
ABSTRACT
Transcription factor (TF)-based biosensors have arisen as powerful tools in the advancement of metabolic engineering. However, with the emergence of numerous bioproduction targets, the variety of applicable TF-based biosensors remains severely limited. In this study, we investigated and engineered an 1,2-propanediol (1,2-PD)-responsive transcription activator, PocR, from Salmonella typhimurium to enrich the current biosensor repertoire. Heterologous characterization of PocR in E. coli revealed a significantly limited operational range and dynamic range, primarily attributed to the leaky binding between PocR and its corresponding promoters in the absence of the 1,2-PD inducer. Promiscuity characterization uncovered the minor responsiveness of PocR toward glycerol and 1,2-butanediol (1,2-BD). Using AlphaFold-predicted structure and protein mutagenesis, we preliminarily explored the underlying mechanism of PocR. Based on the investigated mechanism, we engineered a PcoR-F46R/G105D variant with an altered inducer specificity to glycerol, as well as a PocR-ARE (Q107A/S192R/A203E) variant with nearly a 4-fold higher dynamic range (6.7-fold activation) and a 20-fold wider operational range (0-20 mM 1,2-PD). Finally, we successfully converted PocR to a repressor through promoter engineering. Integrating the activation and repression functions established a versatile 1,2-PD-induced bifunctional regulation system based on PocR-ARE. Our work showcases the exploration and exploitation of an underexplored type of transcriptional activator capable of recruiting RNA polymerase. It also expands the biosensor toolbox by providing a 1,2-PD-responsive bifunctional regulator and glycerol-responsive activator.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Salmonella typhimurium
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Factores de Transcripción
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Técnicas Biosensibles
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Propilenglicol
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Escherichia coli
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Ingeniería Metabólica
Idioma:
En
Revista:
ACS Synth Biol
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos