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

Kentaro Miyazaki,Seon-Woo Lee

doi:10.1371/journal.pone.0194090

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), each of which improved the expression of GFP relative to the wildtype by several-fold. The three sites were then completely randomized by saturation mutagenesis either individually or combinatorially to identify three variants carrying a single point mutation, N282L, V295F, or V295S; no further improvements were observed by combining these mutations. Salicylate-dependent expression of these mutants was highly repressed in its absence and escalated in response to ~10 μM salicylate, and gradually increased up to 1 mM salicylate; the induction rate was approximately 15 times greater than that achieved with a lactose promoter. Orthogonality to the lactose-based expression system was also confirmed. This salicylate-based expression system should thus be advantageously used for high-level production of recombinant proteins in combination with common lactose-dependent induction systems.

Highlights

The most common inducible promoters are T7, T5, lactose, and tac, all of which rely on a LacI-negative transcription regulator and the expression is commonly induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), a non-metabolizable analogue of lactose
Apart from the lactose-based induction system, there are a couple of sugar-based expression systems; PBAD-based expression is activated by L-arabinose and highly repressed in its absence
One drawback in the PBAD system is that L-arabinose is readily metabolized in E. coli, and non-metabolized analogues are not available to date; for continuous induction, a special mutant strain, which lacks the ability to assimilate L-arabinose, is required as a host

Summary

Introduction

Salicylate-inducible expression system in E. coli that respond to a specific compound as an inducer. Error-prone PCR and subsequent saturation mutagenesis [12] identified variants with a ~20-fold enhanced induction rate relative to the original pSAL7A, which was ~15-fold that achieved by the lactose promoter. Competent E. coli JM109 cells were transformed with the mixture and grown on LB/Amp agar plates at 37 ̊C.

Results

Conclusion