Metlock: A Biomining Library

Abstract

Global demand for zinc is rising, but conventional extraction remains energy-intensive, waste-generating, and geopolitically vulnerable. We propose to develop a modular synthetic-biology platform that engineers Escherichia coli for selective, tunable sequestration of zinc from low-grade ores and industrial effluents. The system will:

1. Utilize engineered intracellular metal-binding proteins that lock the captured zinc within the cytoplasm[5][9].
2. Integrate a LuxR/3OC6-HSL-inducible ZnuABC high-affinity transporter to amplify uptake in response to user-defined cues[2][4].

A standardized genetic toolkit comprising interchangeable promoters, ribosome-binding sites, regulatory elements, and metal-specific cassettes will be compiled with detailed protocols for media composition, induction scheduling, and bioreactor operation to ensure reproducibility and portability. Phase-I milestones include achieving a ≥5-fold increase in zinc capture from ≤0.5 mM solutions and validating scalability from benchtop to pilot-scale reactors[3] [4]. Success will deliver an open-source biomining library that enables low-carbon, decentralized recovery of zinc and lays the groundwork for future expansion to lead, cadmium, and rare-earth elements, strengthening critical-mineral supply resilience.

Figure 1: Synthetic operon design illustrating zinc ion sequestration and LuxR/3OC6-HSL regulation of ZnuABC gene to express zinc transport proteins. Zinc ions are held within the cytoplasm by endogenous zinc finger proteins.

Methods & Toolkit

• Modular genetic toolkit: interchangeable promoters, RBS, regulators
• LuxR/3OC6-HSL inducible ZnuABC cassette
• Engineered cytoplasmic binding proteins
• Standard assembly & media/induction workflow

Figure 2: Information flow of ZnuABC mirroring central dogma of biology.

Figure 3: Arranged crystal structures of ZnuA[7], ZnuB[1], and ZnuC[8] with zinc binding sites on ZnuA highlighted.

Figure 4: Crystal structure of bacterioferretin[6], as an analog to the desired finalized design of an engineered cytosolic zinc sequestering protein.

Benchmarks & Scale-Up: Target Outputs

• ≥5-fold zinc uptake vs wild-type capture from ≤0.5 mM solutions
• Consistent capture in triplicate batches
• Lab-scale (100 mL) → Pilot-scale (10 L)

Modeled Data Displays

Figure 5: Zinc uptake over time in E. coli expressing iZnuABC under varying induction levels. If zinc uptake (μM per OD600) is measured over 60 minutes, it may show that the high induction group demonstrates the greatest zinc uptake, followed by low induction and no induction conditions[9]. The knockout strain (ΔznuB) exhibits minimal zinc uptake, which would confirm the essential role of the ZnuB (and therefore ZnuABC) in zinc transport. These results hypothetically show that synthetic operon iZnuABC expression enhances zinc uptake in an induction-modulated manner.

Figure 6: Modeled total zinc capture in wild-type vs. iZnuABC E. coli. Engineered E. coli expressing inducible ZnuABC transport system theoretically captures approximately 6-times more zinc under controlled conditions. This demonstrates the enhanced zinc uptake capacity mediated by iZnuABC expression, highlighting its potential applications in bioremediation.

Figure 7: Heat map of zinc uptake across a 96-well plate. Each cell corresponds to a well (rows A-H, columns 1-12) and is shaded by the measured zinc capture (µM), with warmer colors indicating higher uptake; this visualization would highlight top-performing wells for downstream scale-up.

Future Directions & IP

• Expansion to Pb²⁺, Cd²⁺, and other rare-earth metals
• Automated promoter/RBS tuning for selectivity
• High-throughput mutagenesis of binding proteins
• Release of open-source metadata and sequences

Figure 8: Proposed progressive deployment of system from strain identification to environmental deployment for diverse industrial applications ranging from bioremediation to critical mineral processing.

Sources

[1] AlphaFold Protein Structure Database. ZnuB protein (Escherichia coli), UniProt ID: P39832. Available from: https://alphafold.ebi.ac.uk/entry/P39832. Accessed 2025 Aug 4.
[2] Bernstein DA, Zittel MC, Keck JL. High-resolution structure of the E.coli RecQ helicase catalytic core. EMBO J. 2003 Oct 1;22(19):4910-21. doi: 10.1093/emboj/cdg500. PMID: 14517231; PMCID: PMC204483.
[3] Bird AJ, McCall K, Kramer M, Blankman E, Winge DR, Eide DJ. Zinc fingers can act as Zn2+ sensors to regulate transcriptional activation domain function. EMBO J. 2003 Oct 1;22(19):5137-46. doi: 10.1093/emboj/cdg484. PMID: 14517251; PMCID: PMC204467.
[4] Collins CH, Arnold FH, Leadbetter JR. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Mol Microbiol. 2005 Feb;55(3):712-23. doi: 10.1111/j.1365-2958.2004.04437.x. PMID: 15660998.
[5] Graham AI, Hunt S, Stokes SL, Bramall N, Bunch J, Cox AG, McLeod CW, Poole RK. Severe zinc depletion of Escherichia coli: roles for high affinity zinc binding by ZinT, zinc transport and zinc-independent proteins. J Biol Chem. 2009 Jul 3;284(27):18377-89. doi: 10.1074/jbc.M109.001503. Epub 2009 Apr 19. PMID: 19377097; PMCID: PMC2709383.
[6] Rivera M. Bacterioferritin: Structure, Dynamics, and Protein-Protein Interactions at Play in Iron Storage and Mobilization. Acc Chem Res. 2017 Feb 21;50(2):331-340. doi: 10.1021/acs.accounts.6b00514. Epub 2017 Feb 8. PMID: 28177216; PMCID: PMC5358871.
[7] SWISS-MODEL Repository. ZnuA protein (Escherichia coli), UniProt ID: A1AC19. Available from: https://swissmodel.expasy.org/repository/uniprot/A1AC19. Accessed 2025 Aug 4.
[8] SWISS-MODEL Repository. ZnuC protein (Escherichia coli), Model MD5: 0eb68fb20fe9ce365ac15a74498d040a. Available from: https://swissmodel.expasy.org/repository/md5/0eb68fb20fe9ce365ac15a74498d040a. Accessed 2025 Aug 4.
[9] Patzer SI, Hantke K. The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli. Mol Microbiol. 1998 Jun;28(6):1199-210. doi: 10.1046/j.1365-2958.1998.00883.x. PMID: 9680209.