Indigoidine biosynthesis triggered by the heavy metal-responsive transcription regulator: a visual whole-cell biosensor

Abstract

During the last few decades, whole-cell biosensors have attracted increasing attention for their enormous potential in monitoring bioavailable heavy metal contaminations in the ecosystem. Visual and measurable output signals by employing natural pigments have been demonstrated to offer another potential choice to indicate the existence of bioavailable heavy metals in recent years. The biosynthesis of the blue pigment indigoidine has been achieved in E. coli following heterologous expression of both BpsA (a single-module non-ribosomal peptide synthetase) and PcpS (a PPTase to activate apo-BpsA). Moreover, we demonstrated herein the development of the indigoidine-based whole-cell biosensors to detect bioavailable Hg(II) and Pb(II) in water samples by employing metal-responsive transcriptional regulator MerR and PbrR as the sensory elements, and the indigoidine biosynthesis gene cluster as a reporter element. The resulting indigoidine-based biosensors presented a good selectivity and high sensitivity to target metal ions. High concentration of target metal exposure could be clearly recognized by the naked eye due to the color change by the secretion of indigoidine, and quantified by measuring the absorbance of the culture supernatants at 600 nm. Doseresponse relationships existed between the exposure concentrations of target heavy metals and the production of indigoidine. Although fairly good linear relationships were obtained in a relatively limited concentration range of the concentrations of heavy metal ions, these findings suggest that genetically controlled indigoidine biosynthesis triggered by the MerR family transcriptional regulator can enable a sensitive, visual, and qualitative whole-cell biosensor for bioindicating the presence of bioaccessible heavy metal in environmental water samples. Key points• Biosynthesis pathway of indigoidine reconstructed in a high copy number plasmid in E. coli.• Visual and colorimetric detection of Hg(II) and Pb(II) by manipulation of indigoidine biosynthesis through MerR family metalloregulator. •Enhanced detection sensitivity toward Hg(II) and Pb(II) achieved using novel pigment-based whole-cell biosensors.

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

“…The whole dose-response of TOP10/pPb-CACD toward Pb(II) was shown in Figure 4C, which was similar to that of the previously constructed PbrR-based bacterial biosensor with the blue pigment indigoidine as the output signal (Hui et al, 2021a). The CACD-derived signal was significantly increased from 0 to 12.5 μM and gradually stabilized from 25 to 200 μM (Figure 4C).…”

Detection of environmental pollutant cadmium in water using a visual bacterial biosensor

“…The detection limit could be defined as the lowest metal concentration that evokes a significant increase in biosensing signal (background + 3 × SD) 7 , 11 . The detection limit using bacterial biosensor in the present investigation was about 0.049 μM (Fig.…”

“…To validate the capability of the biosensor to detect bioavailable Cd(II) in environmental water samples, engineered TOP10/pPcad-vio was cultured in LB medium prepared with purified water, tap water, and two kinds of surface water as described previously 11 . The early exponential-phase culture were spiked with 0, 0.39, 0.78, 1.56, 3.125, 6.25, and 12.5 μM Cd(II) using a double dilution method.…”

Teaching an old ‘doc’ new tricks for algal biotechnology: Strategic filter use enables multi-scale fluorescent protein signal detection

“…In-gel fluorescent protein detection can be of value if a non-precise molecular mass determination, yet confirmation of difference in size from control is needed. The ability to use fluorescence for plate-level rapid colony screening and target recombinant protein visualization in gel allowed determination of the expression of two new transgene targets after integration into the algal nuclear genome: the nonribosomal peptide synthase (NRPS) A from Streptomyces lavendulae (BpsA, NCBI: AB240063.1, (Hui et al, 2021)) and the 4′phosphopantetheinyl transferase (4'-PPTase) from Pseudomonas aeruginosa (PaPcpS, Protein Data Bank ID: 7BCZ_A, (Carivenc et al, 2021)). BpsA expression was chosen for several reasons: it is the first example of heterologous NRPS expression in a eukaryotic algal host, it is a validation of C-terminal fusion to mVenus using the current iteration of intron spreading, and the fusion protein is ~170 kDa making it one of the larger heterologous proteins to be expressed from the nuclear genome of this alga.…”