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

Abstract

Background: 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. 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.

Results: By 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.

Conclusions: The 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.

Figure 1

GFP expressions under different promoters in iron-limitation (A), under P_fhuA in variant iron concentrations (B). A.E. coli Top10 transformants harboring p_tP_XG were cultured in LB medium to OD₆₀₀ = 0.8 and induced by 200 μM 2,2’-dipyridyl. Samples were taken at 20 h after induction for GFP assay; B. LB medium with 40 μM FeSO₄ as iron-rich condition, LB medium with 200 μM 2,2’-dipyridyl as iron-limiting condition. The error bars represent the standard deviation (SD) for one independent experiment, performed in triplicate, the experiment was repeated for three times.

Figure 2

P_fhuA modified strategies used in this study. Continuous line frame, -35 or −10 region; dash line frame, Fur binding box; P_fhuA, fhuA promoter; TT, transcriptional terminator rrnBT1T2 derived from the rrnB rRNA operon of E. coli.

Figure 3

P_fhuA1-controlled GFP expression induced by 2,2’-dipyridyl. A. GFP expression levels of E. coli Top10/p_tP_fhuA1 under the induction of different 2,2’-dipyridyl concentrations. OD₆₀₀ in the table represented the OD₆₀₀ value of cultures at 20 h post-induction. B. Time course analysis of GFP expression in E. coli Top10/p_tP_fhuA1 after induction with 200 μM 2,2’-dipyridyl. 2,2’-dipyridyl was added at 0 h. The error bars represent the standard deviation (SD) for one independent experiment, performed in triplicate, the experiment was repeated for three times.

Figure 4

E. coli growth with toxic gene expression under the control of P_fhuA1. A. Growth curves to demonstrate the tightly controllable expression of lysis gene E in p_tP_fhuA1E, p_bP_fhuA1E and p_aP_fhuA1E under repression (+Fe, 40 μM FeSO₄), or induction (+DP, 200 μM 2,2’dipyridyl). At −3 h, 40 μM FeSO₄ was added; at 0 h, 200 μM 2,2’dipyridyl was added. Cultures were in duplicate. Bacterial growth was measured at OD₆₀₀ nm. B. Colony forming units of Top10/p_atP_fhuA1gef and Top10/p_atP_fhuA1mazF after induction. At 0 h, 200 μM 2,2’dipyridyl was added. The error bars represent the standard deviation (SD) for three independent experiments, performed in triplicate.

Figure 5

A. A schematic diagram of the mechanism of tight Fur-dependent iron-limitation regulation switch developed for E. coli stains. The mechanism is described in Results and Discussion. B. Physical map of the p_YP_fhuA1 expression vector. Y, different replicate origins; MCS, multi-cloning sites; rrnBT1T2, ribosomal terminators T1 and T2.