Pb(II)-inducible proviolacein biosynthesis enables a dual-color biosensor toward environmental lead.

With the rapid development of synthetic biology, various whole-cell biosensors have been designed as valuable biological devices for the selective and sensitive detection of toxic heavy metals in environmental water. However, most proposed biosensors are based on fluorescent and bioluminescent signals invisible to the naked eye. The development of visible pigment-based biosensors can address this issue. The pbr operon from Klebsiella pneumoniae is selectively induced by bioavailable Pb(II). In the present study, the proviolacein biosynthetic gene cluster was transcriptionally fused to the pbr Pb(II) responsive element and introduced into Escherichia coli. The resultant biosensor responded to Pb(II) in a time- and dose-dependent manner. After a 5-h incubation with Pb(II), the brown pigment was produced, which could be extracted into n-butanol. Extra hydrogen peroxide treatment during n-butanol extract resulted in the generation of a stable green pigment. An increased brown signal was observed upon exposure to lead concentrations above 2.93 nM, and a linear regression was fitted from 2.93 to 3,000 nM. Extra oxidation significantly decreased the difference between parallel groups. The green signal responded to as low as 0.183 nM Pb(II), and a non-linear regression was fitted in a wide concentration range from 0.183 to 3,000 nM. The specific response toward Pb(II) was not interfered with by various metals except for Cd(II) and Hg(II). The PV-based biosensor was validated in monitoring bioaccessible Pb(II) spiked into environmental water. The complex matrices did not influence the regression relationship between spiked Pb(II) and the dual-color signals. Direct reading with the naked eye and colorimetric quantification enable the PV-based biosensor to be a dual-color and low-cost bioindicator for pollutant heavy metal.

Anthocyanin biosynthetic pathway switched by metalloregulator PbrR to enable a biosensor for the detection of lead toxicity.

Environmental lead pollution mainly caused by previous anthropogenic activities continuously threatens human health. The determination of bioavailable lead is of great significance to predict its ecological risk. Bacterial biosensors using visual pigments as output signals have been demonstrated to have great potential in developing minimal-equipment biosensors for environmental pollutant detection. In this study, the biosynthesis pathway of anthocyanin was heterogeneously reconstructed under the control of the PbrR-based Pb(II) sensory element in Escherichia coli. The resultant metabolic engineered biosensor with colored anthocyanin derivatives as the visual signal selectively responded to concentrations as low as 0.012 µM Pb(II), which is lower than the detection limit of traditional fluorescent protein-based biosensors. A good linear dose-response pattern in a wide Pb(II) concentration range (0.012-3.125 µM) was observed. The color deepening of culture was recognized to the naked eye in Pb(II) concentrations ranging from 0 to 200 µM. Importantly, the response of metabolic engineered biosensors toward Pb(II) was not significantly interfered with by organic and inorganic ingredients in environmental water samples. Our findings show that the metabolic engineering of natural colorants has great potential in developing visual, sensitive, and low-cost bacterial biosensors for the detection and determination of pollutant heavy metals.

Metabolic engineering of the violacein biosynthetic pathway toward a low-cost, minimal-equipment lead biosensor.

Metabolic engineered bacteria have been successfully employed to produce various natural colorants, which are expected to be used as the visually recognizable signals to develop mini-equipment biological devices for monitoring toxic heavy metals. The violacein biosynthetic pathway has been reconstructed in Escherichia coli (E. coli). Here the successful production of four violacein derivatives was achieved by integrating metabolic engineering and synthetic biology. Lead binding to the metalloregulator enables whole-cell colorimetric biosensors capable of assessing bioavailable lead. Deoxyviolacein-derived signal showed the most satisfied biosensing properties among prodeoxyviolacein (green), proviolacein (blue), deoxyviolacein (purple), and violacein (navy). The limit of detection (LOD) of pigment-based biosensors was 2.93 nM Pb(II), which is lower than that of graphite furnace atomic absorption spectrometry. Importantly, a good linear dose-response model in a wide dose range (2.93-6000 nM) was obtained in a non-cytotoxic deoxyviolacein-based biosensor, which was significantly better than cytotoxic violacein-based biosensor (2.93-750 nM). Among ten metal ions, only Cd(II) and Hg(II) exerted a slight influence on the response of the deoxyviolacein-based biosensor toward Pb(II). The deoxyviolacein-based biosensor was validated in detecting bioaccessible Pb(II) in environmental samples. Factors such as low cost and minimal-equipment requirement make this biosensor a suitable biological device for monitoring toxic lead in the environment.