Molecular engineering of the salicylate-inducible transcription factor Sal7AR for orthogonal and high gene expression in Escherichia coli.

ABSTRACT: I have previously identified a metagenomic fragment (~4 kb) containing the salicylate (2-hydroxybenzoate)-responsive transcriptional regulator Sal7AR. Taking advantage of the inert nature of salicylate to common genetic switches used in Escherichia coli, here I developed a salicylate-inducible high expression system in E. coli. I first applied a deletion analysis to the metagenomic fragment to identify the core region (~1 kb) necessary for the salicylate-dependent expression. Sal7AR was subjected to an error-prone PCR, and a library was screened for an enhanced expression of a reporter green fluorescent protein (GFP) gene in the presence of 1 mM salicylate, where virtually no growth inhibition was observed. Three beneficial amino acid substitutions were identified (N282K, Q292R, and V295G)

SUBMITTER: Miyazaki K 

PROVIDER: S-EPMC5894983 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature