Characterization and Diversification of AraC/XylS Family Regulators Guided by Transposon Sequencing

ACS Synth Biol. 2024 Jan 19;13(1):206-219. doi: 10.1021/acssynbio.3c00441. Epub 2023 Dec 19.

Abstract

In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For AraC family regulators (AFRs) represented in these data, we posited AFR/promoter/inducer groupings. Twelve promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in Escherichia coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled the further diversification of these inducible plasmids.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • AraC Transcription Factor / genetics
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins* / genetics
  • Gene Expression Regulation, Bacterial / genetics
  • Plasmids / genetics
  • Promoter Regions, Genetic / genetics
  • Pseudomonas putida* / genetics
  • Pseudomonas putida* / metabolism

Substances

  • Bacterial Proteins
  • AraC protein, E coli
  • Escherichia coli Proteins
  • AraC Transcription Factor