Development of a Fur-dependent and tightly regulated expression system in Escherichia colifor toxic protein synthesis

Lingyu Guan,Qin Liu,Chao Li,Yuanxing Zhang

doi:10.1186/1472-6750-13-25

Abstract

BackgroundThere is a continuous demanding for tightly regulated prokaryotic expression systems, which allow functional synthesis of toxic proteins in Escherichia coli for bioscience or biotechnology application. However, most of the current promoter options either are tightly repressed only with low protein production levels, or produce substantial protein but lacking of the necessary repression to avoid mutations initiated by leaky expression in the absence of inducer. The aim of this study was to develop a tightly regulated, relatively high-efficient expression vector in E. coli based on the principle of iron uptake system.ResultsBy using GFP as reporter, PfhuA with the highest relative fluorescence units, but leaky expression, was screened from 23 iron-regulated promoter candidates. PfhuA was repressed by ferric uptake regulator (Fur)-Fe2+ complex binding to Fur box locating at the promoter sequence. Otherwise, PfhuA was activated without Fur-Fe2+ binding in the absence of iron. In order to improve the tightness of PfhuA regulation for toxic gene expression, Fur box in promoter sequence and fur expression were refined through five different approaches. Eventually, through substituting E. coli consensus Fur box for original one of PfhuA, the induction ratio of modified PfhuA (named PfhuA1) was improved from 3 to 101. Under the control of PfhuA1, strong toxic gene E was successfully expressed in high, middle, low copy-number vectors, and other two toxic proteins, Gef and MazF were functionally synthesized without E. coli death before induction.ConclusionsThe features of easy control, tight regulation and relatively high efficiency were combined in the newly engineered PfhuA1. Under this promoter, the toxic genes E, gef and mazF were functionally expressed in E. coli induced by iron chelator in a tightly controllable way. This study provides a tightly regulated expression system that might enable the stable cloning, and functional synthesis of toxic proteins for their function study, bacterial programmed cell death in biological containment system and bacterial vector vaccine development.

Highlights

There is a continuous demanding for tightly regulated prokaryotic expression systems, which allow functional synthesis of toxic proteins in Escherichia coli for bioscience or biotechnology application
Preliminary screening for iron-regulated promoters As described in the previous study [18], 23 promoter candidates were selected from Vibrio parahaemolyticus, Vibrio cholerae, E. coli and Vibrio anguillarum, and their transcription abilities were investigated with pUTtG as screening vector and iron chelator 2,2’-dipyridyl as inducer
To evaluate the regulation performance of PfhuA, the GFP synthesis was detected when Top10/ptPfhuAG growing in lysogeny broth (LB) medium supplemented with repressor (40 μM FeSO4) or inducer (200 μM 2,2’-dipyridyl)

Summary

Introduction

There is a continuous demanding for tightly regulated prokaryotic expression systems, which allow functional synthesis of toxic proteins in Escherichia coli for bioscience or biotechnology application. With the advent of the post-genomic era coming, the need is boosting to express a growing number of genes originating from different organisms [1]. Many of these foreign genes severely interfere with the survival of Escherichia coli cells, which could lead to bacteria death or cause significant defects in bacteria growth. What’s more, following the development of biological technology, many genetically engineered E. coli have been constructed and developed for different purposes, such as bioremediation, biomedicine and bioenergy [2] Their practical applications in the field are still restricted because engineered bacteria may cause new environmental contaminations. In order to activate the killer gene expression only at expected condition, usually a specific environment, the promoter that controls bacteria programmed cell death needs to be controllable, tightly regulated and environment-responsive

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