The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering

Abstract

SummaryThe XylS/Pm regulator/promoter system originating from the Pseudomonas putida TOL plasmid pWW0 is widely used for regulated low‐ and high‐level recombinant expression of genes and gene clusters in Escherichia coli and other bacteria. Induction of this system can be graded by using different cheap benzoic acid derivatives, which enter cells by passive diffusion, operate in a dose‐dependent manner and are typically not metabolized by the host cells. Combinatorial mutagenesis and selection using the bla gene encoding β‐lactamase as a reporter have demonstrated that the Pm promoter, the DNA sequence corresponding to the 5′ untranslated end of its cognate mRNA and the xylS coding region can be modified and improved relative to various types of applications. By combining such mutant genetic elements, altered and extended expression profiles were achieved. Due to their unique properties, obtained systems serve as a genetic toolbox valuable for heterologous protein production and metabolic engineering, as well as for basic studies aiming at understanding fundamental parameters affecting bacterial gene expression. The approaches used to modify XylS/Pm should be adaptable for similar improvements also of other microbial expression systems. In this review, we summarize constructions, characteristics, refinements and applications of expression tools using the XylS/Pm system.

Physical decoupling of XylS/Pm regulatory elements and conditional proteolysis enable precise control of gene expression in Pseudomonas putida

“…The XylS/ Pm regulatory system originates from the catabolic megaplasmid pWW0 of P. putida mt‐2, where it controls the transcription of genes in the lower ( meta ) cleavage pathway for degradation of aromatic compounds (Ramos et al ., ). Expression systems based on the XylS/ Pm pair have been extensively adopted for both fundamental studies and metabolic engineering of Pseudomonas , because of its low basal expression, relatively high fold‐change induction, and the use of low‐cost inducers such as 3‐methylbenzoate (3‐ m Bz) and derivatives thereof (Gawin et al ., ). In its native configuration, the XylS protein binds as a dimer to two operator sites (distal and proximal) closely upstream to the −35 motif of the Pm promoter, where it recruits the RNA polymerase with the σ 32 or σ 38 factors (Marqués et al ., ; González‐Pérez et al ., ).…”

Tailor-made sRNAs: a toolbox to control metabolic targets

“…In this plasmid, gene expression is driven from the inducible P. putida native promoter XylS/ Pm . This regulator/promoter system has been widely used for regulated gene expression and it is suitable for use in metabolic engineering (recently reviewed in (Gawin et al 2017). MicC scaffold was cloned exactly in the +1 position relative to the Pm promoter, so that addition of a target specific sequence (seed sequence) immediately upstream of MicC scaffold will allow the expression of a target specific synthetic sRNA after induction (Fig.…”

“…For the construction of this genetic tool we have used an inducible plasmid from the SEVA collection, in which the expression of the synthetic sRNA is driven from the well-known P. putida promoter XylS/ Pm . Induction of this promoter can be graded by using different, non-expensive, benzoic acid derivatives, which enter cells by passive diffusion and operate in a dose-dependent manner (reviewed by (Gawin et al 2017)). It combines a high expression level with a low fraction of non-induced cells (Calero et al 2016).…”

pTRA – A reporter system for monitoring the intracellular dynamics of gene expression

“…In order to show the potential of pTRA, we constructed pTRA-51hd ( Fig 1D ). As promoter the well-characterised XylS/Pm promoter was used [ 9 , 10 ]. As ribosome binding site (RBS) we chose the one that is used for the reporter genes of the SEVA database [ 4 ], and as the proxy of any gene of interest (GOI) the sequence encoding the fluorescent mCherry protein.…”