Heavy metal pollution has become a global environmental problem that seriously endangers human health and ecological safety (Adimalla, 2020; Zhu et al., 2022). Some heavy metals, including lead (Pb), mercury (Hg), arsenic (As), chromium (Cr), and cadmium (Cd), can cause significant damage to multiple organs such as the liver, kidneys, and brain by disrupting the body’s metabolic functions, and lead to different types of cancer, neurological disorders, and other endocrine abnormalities (Bridges and Zalups, 2017; Klotz and Goen, 2017; Rehman et al., 2018). As one of the critical heavy metal pollutants, Pb can quietly threaten human life through various hidden pathways (Mulhern et al., 2022). Due to its high toxicity and non-biodegradability (Yuvaraja et al., 2019), Pb can accumulate in living organisms through the food chain and threaten their bioactivities (Naikoo et al., 2019). Pb is toxic to almost all human organs (Charkiewicz and Backstrand, 2020; Hemmaphan and Bordeerat, 2022). Thus, the biological exposure limit of blood lead is constantly being lowered from 60 μg/dL in 1960 to 3.5 μg/dL in 2016 (Rocha and Trujillo, 2019).

Implementation of rational control policies and continuous environmental monitoring are prerequisites for avoiding increased Pb pollution (Pohl et al., 2017). Accurate detection of bioavailable Pb in soil and water is necessary to predict its ecological risk (Roux and Marra, 2007; Kumar et al., 2020). From available studies, most methods for detecting Pb rely on expensive instruments that are not portable and limited to specific analytical systems (Klotz and Goen, 2017; Tudosie et al., 2021; Manousi et al., 2022). Although the mainstream instrumental methods are sensitive and accurate, the analytical results mainly reflect the total amount of elemental Pb. The bioavailable Pb that is genuinely hazardous to organisms was not exactly reflected using traditional instruments, including atomic absorption spectroscopy and inductively coupled plasma-mass spectrometry. The specification analysis of toxic Pb largely depends on complex pre-treatment (Manousi et al., 2022). As an alternative to instrumental assays, the novel microbial cell-based approach showed potential in the practical determination of bioavailable heavy metals (Cui et al., 2018; Hui et al., 2020). These biological methods also have the advantage of solid operability, low cost, and ease of use (Kannappan and Ramisetty, 2022). Despite the lack of rigorous selectivity and background noise, microbial cell-based biosensors can be continuously improved by evolving genetic techniques to meet on-site testing requirements (Wang and Buck, 2012).

Bioaccessible Pb(II) is critical to the transcription initiation of Pb resistance genes, mediated by the metalloregulator PbrR (Borremans et al., 2001). The Pb(II)-resistant pbr operon, a member of the mer-like operons, was commonly engineered to develop several microbial cell-based biosensors toward Pb (Fang and Zhang, 2022). With the rapid advances in synthetic biology, it is now possible to artificially reconstruct heavy metal-resistant operons, including optimization of the sensory modules (Levin-Karp et al., 2013; Jia et al., 2020) and development of novel reporter modules (Jiang et al., 2008). When bioavailable metals enter the biosensing cell, the transcription and translation of downstream reporter genes will be induced, and the resultant biosensing signals can be emitted. Various biosensing reporters are employed to develop genetic devices responsive to heavy metal pollutants. Versatile engineered microbial cells became bioluminescent biosensors (Viviani et al., 2022), fluorescent biosensors (Guo et al., 2021b; Hui et al., 2021b), and colorimetric biosensors (Hui et al., 2021a) toward toxic metals.

Bioluminescent and fluorescent reporters as primary actuators depend highly on expensive instruments to determine biosensing signals (Hui et al., 2021b; Viviani et al., 2022). These instruments, such as photometers, multi-wavelength microplate readers, and fluorescence spectrometers, are not conducive to rapidly detecting Pb contamination in the field. Recently, several natural pigments as easy-to-read biosensing reporters were successfully designed to develop whole-cell biosensors for the detection of environmental Hg(II) (Hui et al., 2021a), Cd(II) (Hui et al., 2022a) and Pb(II) (Hui et al., 2022b). These novel pigment-based biosensors have great potential in developing rapid colorimetric methods for in situ detection of contaminant metals, with low-cost, mini-equipment, and high-throughput advantages.

Pigments with striking colors could be used to develop visual bacterial biosensors toward pollutants. Several studies have clarified that the branched violacein biosynthetic pathway can produce four-color metabolites, including violacein (V), deoxyviolacein (DV), proviolacein (PV), and prodeoxyviolacein (PDV) (Yang et al., 2021). Differential colored signals contribute to the design of multiple functional biosensors. Whole-cell biosensors based on navy V (Hui et al., 2020) and purple DV (Hui et al., 2022b) showing differential responsive properties have been successfully developed. The PV-based colored signal responsive to Pb(II) was previously obtained by employing a vioABDE gene cluster as a reporter module fused downstream of the Pb(II) sensory element. Due to the high reducibility of the chromogenic compounds, the color rendering of PV is unstable. Thus its feasibility as a biosensing reporter has yet to be verified in previous studies. In the current study, The PV biosynthesis was demonstrated to be selectively activated by soluble Pb(II) in a time- and dose–response manner. Due to the extreme reducibility of PV, green and brown PV derivatives were generated under oxidizing and non-oxidizing conditions, and the dual-color signals could be quantified by visible light colorimetry. A high-throughput colorimetric method based on a 96-well plate was developed to detect environmental Pb pollutants. It has the potential to become a complementary tool to instrumental means.

Microorganisms are inexpensive weapons for defending against heavy metal pollution, as they can adapt to high concentrations of heavy metal exposure through metal ions sequestration, transport, and export (Dave et al., 2020). The primary heavy metal pollutants in environmental water include Pb(II), Hg(II), Cd(II), As(III), and Cr(III) (Kiran Marella et al., 2020). Recently, biological assays have been extensively investigated to detect residual heavy metals in surface water or soil using genetically modified bacteria. Compared with non-cellular devices such as protein-based biosensors, DNA-based biosensors, and electrochemical sensors, whole-cell biosensors can potentially monitor cytotoxic, bioavailable, and bioaccessible metals in a low-cost, mini-equipment, and high-throughput manner. Various whole-cell biosensors have been successfully developed with fluorescent (Hui et al., 2021b; Kolosova et al., 2022), and colorimetric (Hui et al., 2021a) signal outputs.

Recent studies showed that natural pigment biosynthesis based on metabolic engineering made designed bacteria become colorimetric biosensors toward pollutants (Watstein and Styczynski, 2017; Hui et al., 2020, 2021a, 2022c; Guo et al., 2021a). Redesign of the metabolic pathway facilitated the biosynthesis of interested colorants (Hui et al., 2022b). The colored PV could be selectively accumulated by interrupting the branched violacein biosynthetic pathway (Tong et al., 2021; Hui et al., 2022b). The PV biosynthetic gene cluster was employed as a pigment-based reporter and transcriptionally fused to the well-characterized Pb(II) sensing element. The resultant engineered bacterium was expected to become a PV-based Pb(II) biosensor. As an intermediate of the violacein biosynthetic pathway, PV is chemically active (Zhao et al., 2019). Its extreme reducibility was first demonstrated in the present study. Bimodal PV derivatives with two peaks at about 480 nm and 652 nm were gradually generated, and stable dual-color signals could be captured at about 6 h after extraction into butanol (Figures 1C,D). Our findings shew that natural oxidation led to the accumulation of brown-colored PV derivatives in the n-butanol phase. Unexpectedly, more stable green-colored PV derivatives were predominantly generated due to the rapid and intense oxidation mediated by hydrogen peroxide. Although background signal noise is difficult to eliminate in whole-cell biosensors employing natural metalloregulators as sensory elements (Bereza-Malcolm et al., 2016; Hui et al., 2021a), the results showed that the intense oxidant treatments facilitated stabling and decreasing the background in the control group and reducing signal fluctuations in parallel groups (Figure 2).

Due to the stable green signal, the PV-based biosensor had a low LOD of 0.183 nM after an extra oxidation treatment (Figure 3B). However, the LOD was increased to 2.93 nM when PV suffered from natural oxidation (Figure 3A), which was consistent with previously developed V- and DV-based bacterial biosensors toward Pb(II) (Hui et al., 2022b). Considering the antibacterial activity of V, the signal intensities of both non-cytotoxic PV and DV were significantly more robust than that of V under the same concentration of lead exposure (Hui et al., 2022b). As expected, the LOD of the PV-based biosensor is significantly lower than that of fluorescent biosensors resulting from the amplification effect of continuous pigment biosynthesis. Furthermore, no linear relationship was observed between the fluorescent signal and Pb(II) concentration (Wei et al., 2014). A good regression relationship was found in this study. The quantifiable Pb(II) concentration reached 3,000 nM, which was similar to the DV-based lead biosensor but significantly more comprehensive than previously developed cytotoxic V-based biosensors (Hui et al., 2022b), non-cellular biosensors (Zhang et al., 2019; Yu et al., 2022) and cellular fluorescent biosensors (Bereza-Malcolm et al., 2016). In contrast to fluorescent or bioluminescent biosensors, the naked eye could directly distinguish gradient changes of dual-color signals (Figure 3G). The accumulation of visible colorants helps direct the reading of high Pb(II) exposure with the naked eye and the development of visible light colorimetric methods.

The biosensing selectivity largely depends on the metal sensory element. Previous studies showed that group 12 metals exerted some influence on the response of biosensors employing metalloregulator PbrR as a Pb(II) sensory component (Hui et al., 2022b). The developed biosensor comprised a PbrR-based sensory element and a vioABDE reporter gene cluster. As expected, excellent biosensing selectivity was demonstrated when PV-based biosensors were exposed to metal ions alone (Figures 4A,B). Only Cd(II) and Hg(II) exerted some adverse effects on the Pb(II) induced signal output (Figures 4C,D). The electrochemical property is similar among Pb, Cd, and Hg (Chen et al., 2005). The Cd(II) and Hg(II) may compete with Pb(II) to occupy the metal binding domains of dimeric PbrR. However, the association with non-target metal cannot efficiently trigger the downstream reporter gene’s transcription (Hui et al., 2022b). The molecular evolution of metalloregulator was always thought to be a promising solution to improve the metal selectivity of biosensors (Jia et al., 2020; Cai et al., 2022).

Soluble inorganic and organic components in natural aquatic systems can change metal morphology, resulting in its differential bioavailability and ecotoxicity (Ryan et al., 2009). It is necessary to validate a novel biosensor in monitoring environmental pollutant metals (Gavrilas et al., 2022). This study developed a simple and three-step detection process to detect residual Pb(II) in the environmental water (Figure 5A). Artificially contaminated environmental water samples were prepared and mixed into the biosensing culture system. Engineered biosensor cells were simulated to be exposed to increased Pb(II) concentrations in four water samples. Although the whole dose–response curves of environmental water are similar to that under experimental conditions, environmental water matrices exerted a specific influence on the generation of pigment signals, particularly on the heterogeneous brown biosensing signal toward Pb(II), which might lead to a differential LOD (Figure 5B) and a narrow quantifiable concentration range (Figure 5F) in detecting various environmental samples. The naked eye quickly identified the gradient changes in color deepening (Figures 5H,I).

Interestingly, a significantly decreased relative standard deviation was observed in the oxidation treatment group compared to the non-oxidation treatment group (Supplementary Tables S3, S4). It can be well explained that PV may be oxidized into homogeneous derivatives under extreme oxidizing conditions. However, the autoxidation of PV in the n-butanol tends to produce more ambiguous products. The above findings suggest that PV-based biosensors, especially employing green signals after oxidation treatment, become a semi-quantitative device powerful in warning high dose Pb(II) pollution through direct reading with the naked eye.

Novel visible pigment-based bacterial biosensors share the same advantages over luminescent and fluorescent bacterial biosensors (Liao et al., 2006; Hui et al., 2021b). It differs from widely used enzyme-based biosensors because no extra substrate is necessary. The pigment-based colorimetric method is also superior to traditional colorimetric methods. The critical devices involved in these biosensors are a cheap incubator, a low-speed centrifuge, and a microplate reader with a visible detection range. Regarding the initial investment in equipment, its cost is far lower than traditional sensors. The biosensing plasmid and host cells are stably stored at low temperatures. Fresh biosensor cells may be transformed regularly to avoid genetic variation. However, the costs associated with bacterial culture, plasmid preservation, and transformation can be ignored. All these properties make metabolically engineered bacteria potentially become low-cost and mini-equipment biosensors (Naik and Dubey, 2013; Guo et al., 2021a).

In conclusion, this study transformed an intermediate of the branched violacein biosynthetic pathway into a dual-color biosensing reporter. The PV-derived dual-color signals had excellent biosensing properties and showed great potential in developing whole-cell biosensors toward pollutants such as toxic Pb(II). Colored signals read directly by the naked eye enabled a PV-based biosensor, a potential on-site detection method. The sensitivity and selectivity of bacterial biosensors are expected to be improved by optimization of genetic circuits and molecular evolution of metalloregulator PbrR in future studies.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

C-yH, N-xZ, and HL designed the experimental protocol. D-lZ and C-yH drafted the manuscript. D-lZ, YG, B-cM, M-qL, C-xG, Y-qL, and H-jW carried out most of the study. D-lZ, YG, and B-cM analyzed the data. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (82073517), the Natural Science Foundation of Guangdong Province (2021A1515012472 and 2023A1515011184), the Science and Technology Program of Shenzhen (KCXFZ20201221173602007), Shenzhen Key Medical Discipline Construction Fund (SZXK068), and Shenzhen Fund for Guangdong Provincial High-level Clinical Key Specialties (SZGSP015).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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