Highly Selective and Rapid Arsenic Removal by Metabolically Engineered Escherichia coli Cells Expressing Fucus vesiculosus Metallothionein

Abstract

An arsenic-chelating metallothionein (fMT) from the arsenic-tolerant marine alga Fucus vesiculosus was expressed in Escherichia coli, resulting in 30-and 26-fold-higher As(III) and As(V) binding, respectively. Coexpression of the As(III)-specific transporter GlpF with fMT further improved arsenic accumulation and offered high selectivity toward As. Resting E. coli cells coexpressing fMT and GlpF completely removed trace amounts (35 ppb) of As(III) within 20 min, providing a promising technology for compliance with the As limit of 10 ppb newly recommended by the U.S. EPA.Arsenic (As), a metalloid, is a known human carcinogen affecting millions of people worldwide (25,33). Arsenic exists in two forms: trivalent arsenite [As(III)] and pentavalent arsenate [As(V)]. Exposure to As can result in increased risks of hypertension (5, 6), skin, lung, and bladder cancers (14), and hyperkeratosis (4), due to inhibition of oxidative phosphorylation (11), interference with cell signaling by binding to hormone receptors (12), or generation of reactive oxygen species (19).Conventional techniques for As treatment are mostly ineffective for the uncharged form, As(III) (9, 37), or at low arsenic concentrations. Recently, bioremediation has been gaining momentum as an environmentally friendly and effective alternative for removal of heavy metals (6,7,15,18,22,26). Although metal-chelating peptides such as metallothionein (MT) have been overexpressed in microorganisms for enhanced accumulation of Cd and Cu, almost all such peptides lack specificity for As (1,2,20,29,31,34,35). Specific arsenic accumulation has been reported by utilizing the metalloregulatory protein ArsR (16) or phytochelatins (13,21,32). However, enzymatic synthesis and the availability of precursors such as glutathione and ␥-glutamylcysteine require actively growing cells and limit the utility of the metal-chelating ArsR and phytochelatins.Recently, a newly identified MT from an arsenic-tolerant marine alga, Fucus vesiculosus (fMT), has been cloned and stably expressed as a fusion protein (24) in Escherichia coli and has been shown to bind arsenite with high affinity in vitro (23). However, the utility of E. coli cells expressing fMT for As removal has not been reported. Here we report the overexpression of fMT in E. coli for enhanced accumulation of both As(V) and the uncharged form, As(III). To remove the bottleneck in As(III) uptake, the As(III) transporter GlpF was coexpressed with fMT, resulting not only in further improvement in As(III) accumulation but also in selectivity for As(III). Even resting cells could remove trace amounts of As(III) within 20 min.Expression of recombinant fMT and its effect on arsenic accumulation.

Systematic engineering of phytochelatin synthesis and arsenic transport for enhanced arsenic accumulation in E. coli

“…4). This level of As accumulation is 10-fold higher than those observed with cells expressing either AtPCS or ArsR (Kostal et al, 2004;Sauge-Merle et al, 2003) and is twofold higher than the fMT-expressing cells (Singh et al, 2008b).…”

“…We have previously reported (Singh et al, 2008b) that over-expression of an arsenic transporter GlpF improves the arsenic uptake by E. coli. Therefore, a similar approach was exploited to investigate if this could enable more As complexation by the intracellular PCs.…”

“…For constructing pQE-SpPCS-GshI Ã -GlpF, the GlpF gene was amplified from pMT-GlpF (Singh et al, 2008b) with the forward primer GCCGGGATCC AAGGAGATATACAT-CAGAAGGAGATATACATATGAGTCA and the reverse primer TAGGATCC GTCGACTCTAGAGCTCTTCAAG-TTAATGGTGATGGT GATGGT. The PCR product was digested with BamHI and ligated into BamHI-digested pQE-SpPCSGshI Ã yielding pQE-SpPCS-GshI Ã -GlpF.…”

Protective effect of resveratrol on arsenic trioxide-induced nephrotoxicity in rats

“…Toxicity due to trivalent arsenic might be attributed to direct binding with the -SH group [3]. In addition, zinc and selenium are essential for the activity of many enzymes that defend biological systems against damage caused by activated oxygen [10,11].…”

“…Arsenic is known to be a potent carcinogen that can induce the formation of various types of solid tumors, including lung, prostate, bladder, renal, and skin cancers, as well as other malignancies via inhibition of oxidative phosphorylation, interference with cell signaling by binding to hormone receptors, or generation of reactive oxygen species (ROS) [3,4]. Conversely, arsenic has been used for the treatment of various diseases since 400 BC, and it is currently employed as an effective chemotherapeutic agent for the treatment of certain human cancers, such as acute promyelocytic leukemia (APL), non-APL acutemyeloid leukemia cells, chronic myeloid leukemia cells, and some solid tumor cells.…”