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<p>Heavy metal toxicity is a severe global threat, adversely affecting environmental health, food safety, and human well-being. Microbial biosorbents are a renowned effective solution for environmental pollution; however, their success depends on the adsorption capacity of the microbes. Metallothionein (MT) proteins are high in cysteine content and have a strong affinity to bind divalent metal ions. Thus, deliberately increasing cysteine content in bacterial MTs might hold promise in enhancing their metal-binding capacity. However, expressing these MTs in bacterial hosts is challenging due to many genetic constraints. In this study, we applied sequence-based protein design to generate three novel synthetic MT proteins (M_MT01, M_MT02, and M_MT03) with 32–38% cysteine content and enhanced metal ion binding potential. When expressed in <i>Escherichia coli</i> (<i>E. coli</i>) cytosol and on the cell surface, these MTs exhibited enhanced Cd/Cu tolerance and bioaccumulation capabilities. Notably, cytosolic expression of M_MT02 removed 153.6% more Cu and grew faster than the control, while M_MT03 enhanced Cd removal by 214.5%. However, cytotoxicity affected the growth rate. Moreover, the MT recombinant cells demonstrated antioxidant activity up to 81.7% higher than the control. To overcome intracellular metal toxicity, we further anchored M_MT02 and M_MT03 to the bacterial surface using the Lpp-OmpA (lipoprotein–outer membrane protein A) system to develop bacterial cell surface expression proteins that we called Lp-M_MT02 and Lp-M_MT03, accordingly. The Scanning Electron Microscopy–Energy Dispersive X-ray Spectroscopy (SEM–EDS) assay verified the expected cell surface expression and metal binding. Recombinant <i>E. coli</i> expressed Lp-M_MT02 and Lp-M_MT03 on their cell surface exhibited faster growth in Cd- and Cu-containing media than cells with only cytosolic expression. In fact, Lp-M_MT03 expression removed 46.4% more Cd from the media compared to M_MT03 expression. This study demonstrates the potential of de novo MT design technology and opens the door to utilizing the developed MTs for strain engineering to advance bio-absorption technologies.</p>

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Design and functional assessment of novel metallothionein in bacteria for cadmium and copper bioremediation

  • 期刊论文

  • 发表日期：

    2026年3月16日

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  • Md Mominul Islam,
  • Xin Zheng,
  • Roy Njoroge Kimotho,
  • Md Moniruzzaman,
  • Xiaofang Li

摘要

Heavy metal toxicity is a severe global threat, adversely affecting environmental health, food safety, and human well-being. Microbial biosorbents are a renowned effective solution for environmental pollution; however, their success depends on the adsorption capacity of the microbes. Metallothionein (MT) proteins are high in cysteine content and have a strong affinity to bind divalent metal ions. Thus, deliberately increasing cysteine content in bacterial MTs might hold promise in enhancing their metal-binding capacity. However, expressing these MTs in bacterial hosts is challenging due to many genetic constraints. In this study, we applied sequence-based protein design to generate three novel synthetic MT proteins (M_MT01, M_MT02, and M_MT03) with 32–38% cysteine content and enhanced metal ion binding potential. When expressed in Escherichia coli (E. coli) cytosol and on the cell surface, these MTs exhibited enhanced Cd/Cu tolerance and bioaccumulation capabilities. Notably, cytosolic expression of M_MT02 removed 153.6% more Cu and grew faster than the control, while M_MT03 enhanced Cd removal by 214.5%. However, cytotoxicity affected the growth rate. Moreover, the MT recombinant cells demonstrated antioxidant activity up to 81.7% higher than the control. To overcome intracellular metal toxicity, we further anchored M_MT02 and M_MT03 to the bacterial surface using the Lpp-OmpA (lipoprotein–outer membrane protein A) system to develop bacterial cell surface expression proteins that we called Lp-M_MT02 and Lp-M_MT03, accordingly. The Scanning Electron Microscopy–Energy Dispersive X-ray Spectroscopy (SEM–EDS) assay verified the expected cell surface expression and metal binding. Recombinant E. coli expressed Lp-M_MT02 and Lp-M_MT03 on their cell surface exhibited faster growth in Cd- and Cu-containing media than cells with only cytosolic expression. In fact, Lp-M_MT03 expression removed 46.4% more Cd from the media compared to M_MT03 expression. This study demonstrates the potential of de novo MT design technology and opens the door to utilizing the developed MTs for strain engineering to advance bio-absorption technologies.