Antimony sensing whole-cell bioreporters derived from ArsR genetic engineering.

Despite the known hazardous effects of antimony (Sb) on human health, Sb monitoring biosensors have not been as actively investigated as arsenic (As) biosensors. Whole-cell bioreporters (WCBs) employing an arsenic-responsive operon and a regulatory protein (ArsR) are reportedly capable of monitoring arsenite, arsenate, and antimonite. However, the potential of WCBs as Sb biosensors has been largely ignored. Here, the metal-binding site of ArsR (sequenced as ELCVCDLCTA from amino acid number 30 to 39) was modified via genetic engineering to enhance Sb specificity. By relocating cysteine residues and introducing point mutations, nine ArsR mutants were generated and tested for metal(loid) ion specificity. The Sb specificity of WCBs was enhanced by the C37S/A39C and L36C/C37S mutations on the As binding site of ArsR. Additionally, WCBs with other ArsR mutants exhibited new target sensing capabilities toward Cd and Pb. Although further research is required to enhance the specificity and sensitivity of WCBs and to broaden their practical applications, our proposed strategy based on genetic engineering of regulatory proteins provides a valuable basis to generate WCBs to monitor novel targets.

Shifting the Specificity of E. coli Biosensor from Inorganic Arsenic to Phenylarsine Oxide through Genetic Engineering.

It has recently been discovered that organic and inorganic arsenics could be detrimental to human health. Although organic arsenic is less toxic than inorganic arsenic, it could form inorganic arsenic through chemical and biological processes in environmental systems. In this regard, the availability of tools for detecting organic arsenic species would be beneficial. Because As-sensing biosensors employing arsenic responsive genetic systems are regulated by ArsR which detects arsenics, the target selectivity of biosensors could be obtained by modulating the selectivity of ArsR. In this study, we demonstrated a shift in the specificity of E. coli cell-based biosensors from the detection of inorganic arsenic to that of organic arsenic, specifically phenylarsine oxide (PAO), through the genetic engineering of ArsR. By modulating the number and location of cysteines forming coordinate covalent bonds with arsenic species, an E. coli cell-based biosensor that was specific to PAO was obtained. Despite its restriction to PAO at the moment, it offers invaluable evidence of the potential to generate new biosensors for sensing organic arsenic species through the genetic engineering of ArsR.

Heavy metal(loid) biosensor based on split-enhanced green fluorescent protein: development and characterization.

Heavy metal(loid)s such as Cd and Hg adversely affect human health and are therefore strictly regulated and monitored; however, their quantitation in the environment is usually performed by expensive and time-consuming instrumental analysis techniques, which necessitates the search for more practical alternatives. Herein, we prepare enhanced green fluorescent protein (eGFP)-based biomolecules for metal(loid) sensing by insertion of metal-binding loops (MBLs) into a loop region of eGFP to render this protein inactive and show that the binding of metal ions to MBLs induces a conformational change and restores the original activity. Specifically, eGFP with an MBL sequenced as CTTCGCG regains fluorescence upon exposure to Cd and Hg, which allows the above metals to be quantified in the concentration range of 0-5 µM. For practical applicability verification, the developed sensing platform is used to quantify Cd in artificially amended soil and water samples. Although the obtained results imply that sensor performance needs to be significantly improved, the presented design concept is believed to be of high value to researchers in the field of heavy metal sensing and facilitate the development of new biosensors.

A Biosensor Platform for Metal Detection Based on Enhanced Green Fluorescent Protein.

Microbial cell-based biosensors, which mostly rely on stress-responsive operons, have been widely developed to monitor environmental pollutants. Biosensors are usually more convenient and inexpensive than traditional instrumental analyses of environmental pollutants. However, the targets of biosensors are restricted by the limited number of genetic operon systems available. In this study, we demonstrated a novel strategy to overcome this limitation by engineering an enhanced green fluorescent protein (eGFP). It has been reported that combining two fragments of split-eGFP can form a native structure. Thus, we engineered new biosensors by inserting metal-binding loops (MBLs) between ß-strands 9 and 10 of the eGFP, which then undergoes conformational changes upon interaction between the MBLs and targets, thereby emitting fluorescence. The two designed MLBs based on our previous study were employed as linkers between two fragments of eGFP. As a result, an Escherichia coli biosensor exhibited a fluorescent signal only when interacting with cadmium ions, revealing the prospect of a new biosensor for cadmium detection. Although this study is a starting stage for further developing biosensors, we believe that the proposed strategy can serve as basis to develop new biosensors to target various environmental pollutants.

Modulating the sensing properties of Escherichia coli-based bioreporters for cadmium and mercury.

Despite the large number of bioreporters developed to date, the ability to detect heavy metal(loid)s with bioreporters has thus far been limited owing to the lack of appropriate genetic systems. We here present a novel approach to modulate the selectivity and sensitivity of microbial whole-cell bioreporters (WCBs) for sensing metal(loid)s via the znt-operon from Escherichia coli, which were applied to quantify the bioavailability of these contaminants in environmental samples. The WCB harboring the fusion gene zntAp::egfp was used as a microbial metal(loid) sensor, which was turned on by the interaction between ZntR and metal(loid) ions. This design makes it possible to modulate the selectivity and sensitivity to metal(loid)s simply by changing the metal-binding property of ZntR and by disrupting the metal efflux system of E. coli, respectively. In fact, the E. coli cell-based bioreporter harboring zntAp::egfp showed multi-target responses to Cd(II), Hg(II), and Zn(II). However, the WCBs showed responses toward only Cd(II) and Hg(II) when the amino acid sequence of the metal-binding loop of ZntR was changed to CNHEPGTVCPIC and CPGDDSADC, respectively. Moreover, the sensitivity toward both Cd(II) and Hg(II) was enhanced when copA, which is known to export copper and silver, was deleted. Thus, our findings provide a strong foundation for expanding the target of WCBs from the currently limited number of genetic systems available.

Enhancing the copper-sensing capability of Escherichia coli-based whole-cell bioreporters by genetic engineering.

Metals are essential to all organisms; accordingly, cells employ numerous genes to maintain metal homeostasis as high levels can be toxic. In the present study, the gene operons responsive to metal(loid)s were employed to generate bacterial cell-based biosensors to detect target metal(loid)s. The cluster of genes related to copper transport known as the cop-operon is regulated by the interaction between the copA promoter region (copAp) and CueR, turning on and off gene expression upon copper ion binding. Therefore, the detection of copper ions could be achieved by inserting a plasmid harboring the fusion of copAp and reporter genes, such as enzymes and fluorescent genes. However, copAp is not as strong a promoter as other metal-inducible promoters, such as znt-, mer-, and ars-operons; thereby, its sensitivity toward copper ions was not sufficient for quantification. To overcome this problem, we engineered Escherichia coli with a deletion of copA to interfere with copper export from cells. The engineered E. coli whole-cell bioreporter was able to detect copper ions at 0 to 10 µM in an aqueous solution. Most importantly, it was specific to copper among several tested heavy metal(loid)s. Therefore, it will likely be useful to detect copper in diverse environmental systems. Although additional improvements are still required to optimize the E. coli-based copper-sensing whole-cell bioreporters presented in this study, our results suggest that there is huge potential to generate whole-cell bioreporters for additional targets by molecular engineering.

Modulating the Properties of Metal-Sensing Whole-Cell Bioreporters by Interfering with Escherichia coli Metal Homeostasis.

In Escherichia coli, the transcription of genes related to metal homeostasis is activated by the presence of target metals. The promoter regions of those genes can be fused with reporter genes to generate whole-cell bioreporters (WCBs); these organisms sense the presence of target metals through reporter gene expression. However, the limited number of available promoters for sensing domains restricts the number of WCB targets. In this study, we have demonstrated an alternative method to generate novel WCBs, based on the notion that since the sensing mechanisms of WCBs are related to metal transportation systems, their properties can be modulated by disrupting metal homeostasis. Mutant E. coli strains were generated by deleting the znt-operon genes zntA, which encodes a zinc-export protein, and zntR, which encodes a znt-operon regulatory protein, to investigate the effects on the metal-sensing properties of WCBs. Deletion of zntA increased the sensitivity but abolished the selectivity of cadmium-sensing WCBs, whereas arsenic-sensing WCBs gained sensitivity toward cadmium. When zntR was deleted, cadmium-sensing WCBs lost the ability to detect cadmium, and this was recovered by introducing exogenous zntR. In addition, the metal-binding site of ZntR was genetically engineered to modulate metal selectivity. This study provides a valuable platform for the development of novel E. coli-based WCBs.