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), 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.

Metal-ion-induced expression of gene fragments from subseafloor micro-organisms in the Kumano forearc basin, Nankai Trough

“…Therefore, the use of host microbes other than E. coli needs to be carefully considered with regard to the size of the genomic library for future studies. Another advantage of using E. coli as the genetic transformation host is that the identified genetic fragment in E. coli transformants can be directly transferred via genetic engineering for further enhancement of its ability for biotechnological applications (Miyazaki ).…”

“…Additionally, Uchiyama and Miyazaki () utilized the SIGEX method to discover a novel transcriptional regulator induced by salicylate, which is non‐metabolizable in standard laboratory strains of Escherichia coli . Moreover, Miyazaki () recently modified the induction sensitivity of the salicylate‐inducible promoter by a directed‐evolution approach and constructed a gene‐expression system orthogonal to the lactose‐based expression system in order to allow regulation of multiple promoters in one cell and demonstrating the applicability of novel promoters for future biotechnological applications.…”

Transcriptional factor engineering in microbes for industrial biotechnology

“…Based on the core region of the 2-hydroxybenzoate-responsive TF Sal7AR identified from a metagenomic fragment, an inducible protein expression system was developed by Miyazaki. 53 This inducible protein expression system offered a solution to situations in which an additional inducible protein expression orthogonal to the lacbased system was needed. To enrich the choice for constructing metabolite-responsive biosensors utilizable in metabolic engineering or synthetic biology, Younger et al developed a general method termed as biosensor engineering by random domain insertion (or BERDI), which is capable of converting a metabolite-binding protein into a TF responsive to the metabolites.…”

“…The development of more expression systems that are responsive to novel specific stimuli will enrich the toolbox of synthetic biology and simplify the selection of desired modules. Based on the core region of the 2‐hydroxybenzoate‐responsive TF Sal7AR identified from a metagenomic fragment, an inducible protein expression system was developed by Miyazaki 53 . This inducible protein expression system offered a solution to situations in which an additional inducible protein expression orthogonal to the lac ‐based system was needed.…”

Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology

“…Several small-molecule inducible/repressible systems have been characterised in E. coli [169,170,171]. However, only a small number have been characterised in cyanobacteria, and thus far, only in model species [31,37,164,172,173,174].…”