Overview
We introduce synthetic biology into the metallurgical industry to develop a new biomining process for rare earth elements, CaptuREE, which differs from traditional pyrometallurgy or hydrometallurgy extraction methods. The biomining process we constructed is divided into three main modules: Bioleaching, Biosorption, and Biosafety (Figure 1).
Module 1: Bioleaching
The bioleaching module uses the Issatchenkia orientalis strain to overproduce succinic acid as a bioleaching agent for acid leaching to dissolve metallic elements in rare earth ores.
Module 2: Biosorption
The core of the biosorption module is to construct a whole-cell bio-adsorbent to adsorb,
enrich, and recover rare earth metal ions in the upstream bioleaching solution. The biosorption module is our key content,
which is subdivided into three parts: Protein Engineering, Yeast Surface Display, and Biofilm Adsorption.
Part 1: Protein Engineering
We choose a de novo designed lanthanide binding protein, TFD, as the starting point and first enhance TFD's rare earth binding ability through protein engineering modification.
Part 2: Yeast Surface Display
We choose a de novo designed lanthanide binding protein, TFD, as the starting point and first enhance TFD's rare earth binding ability through protein engineering modification.
Part 3: Biofilm Adsorption
To achieve continuous large-scale processing in the industry, we choose the membrane bioreactor as the hardware carrier and construct yeast biofilm in the reactor to ensure more adequate adsorption of rare earth elements.
Module 3: Biosafety
To prevent cell escape and gene leakage, we design a biosafety protection mechanism based on copper ion concentration response to restrict engineered yeast in the treatment environment of ore wastewater.
In our design, the strains of the bioleaching module are relatively independent. The chassis strain of choice for the Biosorption and Biosafety module remains Issatchenkia orientalis.
Because mine wastewater is usually acidic, Issatchenkia orientalis is known for its excellent tolerance to highly acidic conditions [1],
which will give it better performance on biological adsorption.
However, in actual experiments, due to the difficulty of genetic modification of the non-model strain Issatchenkia orientalis and possible biosafety issues,
we later carried out experiments on the GRAS strain Saccharomyces cerevisiae.
Figure 1. Project design of CaptuREE
Module 1: Bioleaching
Bioleaching is the process of extracting the target metal by the oxidation and dissolution of rare earth metal elements in ores into trivalent cations (REE³+)
released into solution through the acidolysis and complexation of organic acids using microorganisms to produce leaching agents such as acids.
Therefore, a low pH environment is more conducive to the leaching of rare earth elements [2]. Compared with traditional physical and chemical metallurgical methods,
bioleaching can effectively reduce environmental pollution and is a low-cost, cost-effective, and sustainable way.
Therefore, our task is to find a suitable bioleaching strain. Considering the great biological potential
of Issatchenkia orientalis to be able to perform fermentation at low pH, we ultimately select this strain
to produce succinic acid on a large scale as a bioleaching agent for extracting lanthanides from rare earth
ores. Overexpression of the succinic acid synthesis pathway gene in Issatchenkia orientalis can be
achieved through metabolic engineering modification, and related studies have been reported [1] [3].
Module 2: Biosorption
Part 1: Protein Engineering
We select an artificially designed lanthanide binding protein, TIM-Ferredoxin (TFD),
(BBa K5261000)
for biosorption, which is a C2-symmetric dimer protein that incorporates TIM barrel fold and
ferredoxin fold and has a large protein cavity [4]. The de novo designed TFD reported a much
higher affinity for lanthanides than the currently common natural lanthanide binding protein,
lanmodulin (LanM).
The perfect symmetrical structure and stable protein cavity of TFD give us the possibility to
further modify the rare earth binding pocket of TFD to further enhance its rare earth binding
performance. To facilitate the subsequent transformation of the yeast surface display module,
we first fused the homologous dimer protein TFD-EE into a monomer, called TFD-S
( BBa K5261001)
.
Subsequently, this module mainly includes two tasks, which are: (1) Strengthening rare earth
bonding, and (2) Strengthening rare earth detection.
Figure 2. De novo designed lanthanide binding protein, TFD
(1) Strengthening rare earth binding: introducing new lanthanide
coordination residues to increase the binding sites of TFD and rare earth ions
The protein internal cavity of TFD itself introduced four glutamate residues to provide lanthanide
ion coordination, and TFD-EE protein was obtained. We are concerned about TFD-EE's high affinity
for lanthanide ions (binding constant KD ≈ 10-18 M) and high tolerance to thermal and chemical
cosolvents, so we hope to introduce it into iGEM for efficient lanthanide recovery from rare earth
ore bioleaching liquid.
However, at present, each unit of TFD-EE protein can only bind one unit of rare earth ions,
and we hope to improve this. Inspired by the lanthanide coordination on the EF hands of the
natural lanthanide binding protein, lanmodulin, we noted that oxygen atoms on glutamic acid,
aspartic acid, and asparagine residues can provide a suitable coordination environment for
lanthanides, an oxygen-philic, large trivalent cation [5]. Therefore, we try to increase the
rare earth ion binding number of unit protein by introducing new lanthanide coordination
residues by continuing site-directed mutations near the symmetry axis of TFD.
(2) Strengthening rare earth detection: introducing the tryptophan-enhanced
"antenna effect" luminescence mechanism to quantitatively detect lanthanide binding
To facilitate the quantitative detection of lanthanide binding, we further introduce the "antenna effect"
luminescence mechanism [6]. Different lanthanide metals themselves have different fluorescence emission
wavelengths (Figure 3), but due to the low extinction coefficient of lanthanide metals themselves,
they cannot be directly excited, and the surrounding "antenna" needs to absorb external energy and
pass it to lanthanide metal ions to be excited to produce fluorescence. The indole ring on tryptophan
can function as an "antenna".
Figure 3. Emission spectra of trivalent lanthanide ions [7].
The central rare earth binding site of TFD-EE itself is equipped with tryptophan residues for fluorescence
excitation of terbium ions, but the energy transfer of the "antenna" is limited by distance. Therefore,
we need to continue to install tryptophan near the newly introduced rare earth ion binding site on TFD-EE
to quantitatively detect lanthanide binding of unit TFD proteins.
Figure 4. Enhancing the rare earth binding properties of TFD-EE protein through protein
engineering modification
Part 2: Yeast Surface Display
Cell surface display is to fuse foreign protein and anchor protein genes into host
cells to achieve the expression of foreign proteins or peptides on the cell surface.
Cell surface display for biosorption of heavy metals in the environment is a promising
application area. Therefore, if our modified lanthanide binding protein TFD with a
high affinity for lanthanide rare earth is displayed on the surface of yeast cells,
yeast can be directly cultured as a whole-cell bio-adsorbent. Compared with the direct
use of free proteins as adsorbents, whole-cell bio-sorbents can enhance the stability
of proteins to changes in organic solvents, temperature, and pH in the environment,
and greatly simplify a series of operations such as cell fragmentation, protein purification,
and loading.
Here, we choose a yeast surface display system based on a-lectin. The a-agglutinin receptor
consists of two subunits encoded by AGA1 and AGA2 genes. Aga1p is secreted extracellular and
covalently binds to β-glucan on the cell wall surface. Aga2p binds to Aga1p on the N-terminus
through two disulfide bonds and thus also attaches to the yeast cell surface [8], and its
C-terminus is fused with lanthanide binding protein gene TFD. Thus, it is jointly displayed
on the surface of yeast cells under the guidance of signal peptides [9]. At the same time,
short peptide sequences (Xpress™, V5, 6×His) are also present on both sides of the TFD
insertion site as epitope labels, facilitating subsequent detection and characterization.
Figure 5. Schematic diagram of yeast surface display system based on a-lectin
Part 3: Biofilm Adsorption
(1) Coupling MBBR and MABR for rare earth biosorption
After constructing the yeast whole-cell bio-adsorbent, we design the MBBR-MABR series reflux
membrane bioreactor as the hardware carrier for the biosorption process. For details, see our
Hardware page. Yeast cells will form biofilms on the surface of the hydrophobic carrier
fillers of MBBR and MABR, and achieve a fuller and more continuous lanthanide rare earth
adsorption recovery process during continuous aeration and reflux in the reactor (Figure 6).
Figure 6. Yeast biofilm attached to MBBR-MABR
(2) Overexpression of FLO11 to enhance bioadhesion and form yeast biofilm
However, Saccharomyces Cerevisiae BY4741 cannot form biofilms on its own. To enable yeast
cells to stably adhere to hydrophobic carrier fillers to form biofilms for rare-earth e
lement adsorption, we overexpress the internal FLO11 (BBa_K5261015) gene of Saccharomyces Cerevisiae to
achieve bioadhesion.
Flo11p is a member of the Flo family of special yeast adhesion surface glycoproteins,
which is involved in the surface adhesion of cells to hydrophobic substrates and the
formation of bacterial biofilm, and can enable yeast cells to adhere to the surfaces
of agar and plastics [10]. The FLO11 gene encodes a serine - and threonine-rich GPI-anchored
glycoprotein that enhances cell-to-cell and cell-to-substrate carrier interactions in
the presence of Ca(Ⅱ) in solution. However, in Saccharomyces Cerevisiae BY4741, the
promoter region of FLO11 is inhibited by signal regulation caused by nonsense mutation
of transcription regulator Flo8p, and the FLO11 gene is silenced, thus losing the surface
adhesion phenotype [10]. Therefore, the phenotype of biofilm can be formed by overexpression
of FLO11 (BBa_K5261017).
Figure 7. Overexpressing FLO11 with the PTEF1 promoter
Module 3: Biosafety
Our ultimate goal is to apply engineered yeast to the rare earth industry,
so special controls on gene leakage and biosafety are also required. The working environment
of engineered yeast is wastewater rich in a large number of metal ions such as rare earth
ore leach liquid and mine wastewater in MBR, which allows us to select a metal ion as a
"signal" to control the suicide switch of engineered yeast.
We find that the vast majority of mine or industrial wastewater contains a certain concentration
of copper ions, which is related to the wide abundance of copper in crustal ores [11].
Taking conventional industrial wastewater as an example, the Ministry of Ecology and
Environment, PRC limits the discharge standard of total copper in conventional industrial
wastewater pollutants to 1.0 mg/L (on the order of μM) [12], while the average concentration
of copper ions in ordinary fresh water in the natural environment is only about 3 μg/L
(on the order of nM), and it is even lower in seawater [13]. The large difference between
the two allows us to design a yeast suicide mechanism based on copper ion concentration
sensing, so that engineered yeast can spontaneously die after accidental leakage from the
wastewater environment.
Therefore, in this module, we will introduce a copper ion-inhibited promoter CTR3 (BBa_K5261020) and a
toxin-antitoxin system RelE/RelB (BBa_K185047 / BBa_K185048) to control yeast cell
death and ensure biosafety.
(1) RelE/RelB toxin-antitoxin system
Here, we use the RelE/RelB toxin-antitoxin system to construct the kill
switch. The RelE/RelB system is derived from Escherichia coli, but in yeast,
expression of the RelE toxin gene causes serious growth defects, while the concomitant
expression of the RelB antitoxin gene partially restored growth [14].
(2) Design of suicide switch in response to copper ion concentration
Copper ion-inhibited promoter PCTR3, a regulatory element from Saccharomyces cerevisiae,
can be inhibited in the presence of μM concentration of copper ions [15], which is used
to control the expression of toxin gene RelE. The high concentration of copper ions in
the ore waste liquid inhibited the expression of the toxin gene RelE driven by the CTR3
promoter, and the engineered yeast worked normally. If the engineered yeast leaks into
the outside of the MBR, the concentration of copper ions in the environment decreases,
the suicide mechanism of the response of low concentration copper ions is activated,
and the expressed RelE toxin can kill the yeast cells.
In addition, we note that the CTR3 promoter will also have certain leakage expression
in the absence of copper ions [15], so we select a composition of weak promoter PCYC1
with similar relative expression intensity to activate the expression of antitoxin gene
RelB to avoid accidental cell death caused by the leakage expression of CTR3 promoter [16].
Figure 8. Biosafety module design drawing
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