Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR

Abstract

The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5-and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd 2؉ or Zn 2؉ accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.Arsenic (As) is an extremely toxic metalloid that adversely affects human health. Both highly toxic trivalent arsenite [As(III)] and less-toxic pentavalent arsenate [As(V)] have been associated with increased risk of skin, kidney, lung, and bladder cancer (12). The toxicity of arsenic is attributed to the substitution of As(V) for phosphate, affinity of As(III) for protein thiol groups, and protein-DNA and DNA-DNA crosslinking (20). Arsenic enters the water supply primarily from geochemical sources, such as the mining of arsenopyrite gold ores (16), which constitute about one-third of world gold reserves. Additional contaminations arise from anthropomorphic sources such as arsenical-containing herbicides, pesticides, and the widely used wood preservative chromated copper arsenic (17, 21). Untreated, highly toxic arsenic effluent has been disposed of in rivers and ended up in groundwater. An estimated 20 million Americans consume water containing arsenic at levels presenting a potentially fatal cancer risk (9). Worldwide, 57 million people have been exposed to arsenic through contaminated wells in Bangladesh (18). These incidents again serve as a reminder of the toxic consequences of arsenic mobilization and the needs for efficient removal of arsenic in aquatic systems (19).Because of the toxicity, the regulatory limit on arsenic in the United States is currently set at 10 ppb. There are a variety of methods currently available for removal of arsenic from contaminated water. Conventional technologies, such as coagulation, do not discriminate between arsenic and other elements and involve alteration of the water chemistry and addition of other chemicals (7). Current technologies, such as activated alumina sorption, polymeric anion exchange, and polymeric ligand exchange (6), are more effective for As(V) than As(III), and most commonly used methods require pri...

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

“…The level of enhancement is consistent with the observed increase in As(III) uptake due to GlpF overexpression (Table 2), reflecting the additive effect on accumulation of coexpression of fMT and GlpF. The final level of 8.1 mol/g (dry cell weight [DCW]) is three times higher than levels recently reported for other engineered E. coli strains (16,30,32).…”

“…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.…”

“…To investigate the engineered cells' ability to accumulate As, whole-cell binding experiments were conducted as described previously (16,36). Cells expressing fMT accumulated levels of As(III) 30-fold higher than those accumulated by the control ( Table 2), indicating that the fMT fusions retain their As(III)-binding functionality in vivo.…”

Peptide-based biopolymers in biomedicine and biotechnology

“…It is possible to isolate glyco-polypeptides using the interaction between the capture molecule and the sugar group [86] (Figure 8). It has also been shown that ELPs fused to an appropriate fusion partner can bind to heavy metals such as mercury [87], arsenic [88] or cadmium [89,90], and facilitate the rapid isolation of these toxic metal contaminants.…”

Affinity purification of plasmid DNA by temperature-triggered precipitation

“…To recycle the ELP-MerR fusion protein for reuse, resuspend ELP aggregates with ice-cold TB7.4M, preferably in the same volume added in Step 20. 24 are also used (see Fig. 2b).…”