Results

Module 1: Bioleaching

Overview

In the bioleaching module, we first need to prove that succinic acid has the ability to leach rare earth ions from rare earth ores and measure the efficiency of metal leaching. Since the key content of our project is not the metabolic engineering of Issatchenkia orientalis strain in the bioleaching module to overproduce succinic acid, we only did the concept verification experiment.

1.1 Acid leaching of rare earth ore

Monazite is a phosphate rare earth mineral containing cerium and lanthanum, a nd its chemical composition is close to (Ce, Y, La, Th) PO₄. Here, monazite was leached with succinic acid and rare earth ion concentration was determined by the Arsenazo III-based assay. We first directly purchased succinic acid to prepare a solution, and conducted a bioleaching experiment on monazite. 0.82 g monazite powder was weighed and added with 0.67 M succinic acid saturated solution, then leached in a 50 mL centrifuge tube. We shook it once a day, 4 days later, got monazite biological leaching solution.

Due to the high cost of the commonly used elemental analysis techniques ICP-MS or ICP-OES, we used an assay based on Arsenazo III dye, which can be used to roughly determine the content of rare earth ions in the solution. See our Protocols page for details. The absorbance value at 650 nm can reflect the content of rare earth ions in a certain range.

The mother liquor of 10 mM TbCl₃ standard sample was prepared with high purity TbCl₃ as standard sample. Firstly, we carried out the pre-experiment to explore the experimental conditions, and finally 25, 50, 75, 100, 125, 150 ,175, 200 μM Tb(Ⅲ) was determined as the original concentration gradient, and the sample without Tb(Ⅲ) was used as the blank control. The absorbance of Arsenazo III-REE complex at 650 nm in each group of standard samples was determined by Arsenazo III method under spectrophotometer, and we draw the Ln(Ⅲ)-A₆₅₀ standard curve (Figure 1-1). Due to the high concentration of rare earth ions in the monazite leaching solution, according to the pre-experimental results, the A₆₅₀ value of the monazite leaching s olution was determined by the same method after being diluted 6.45 times.

Figure 1-1. A-D. The leaching concentration of rare earth ions in monazite was determined by Arsenazo III method. E. The standard curve was plotted by Arsenazo III method.

Results:

The standard curve of Ln(Ⅲ)-A₆₅₀ shown in Figure 1-2A was drawn with the sample without Tb(Ⅲ) as the blank control group. The results of monazite leaching solution are shown in Figure 1-2B. In the 40 mL succinic acid leaching system, the final leaching concentration of rare earth ions obtained from 0.82 g monazite sample is 420.17 μM, and the leaching ratio is 20.50 μmol Ln³⁺/g Monazite. The feasibility of leaching rare earth elements from ore by succinic acid was preliminarily proved.

Figure 1-2. A. Ln(Ⅲ)-A₆₅₀ standard curve drawn by Arsenazo III method. B. Succinate acid leaching results of monazite.

1.2 Determination of the ability of Issatchenkia orientalis to produce succinic acid

Our Primary PI Dr. Jiazhang Lian has previously constructed the Issatchenkia orientalis strain overexpressing the succinic acid synthesis pathway gene, genotype: Io-FUMR-FRDg-PCKa-spMAE1-MDH3. We originally planned to conduct direct fermentation experiments on this strain to determine its succinic acid production capacity and use it to evaluate its potential for bioleaching. However, for some reasons, we failed to carry out this work. Anyway, the succinic acid leaching experiment in Section 1.1 has preliminarily proved the feasibility of our bioleaching module design through conceptual verification.

Module 2: Protein Engineering

Overview

In this module, we carried out protein engineering modification of lanthanide binding protein TFD-S (BBa_K5261001), constructed mutant protein TFD-M (BBa_K5261003) from the perspective of enhanced rare earth binding and detection, conducted molecular dynamics simulation of protein and rare earth coordination, and characterized its rare earth binding ability through experiments.

2.1 pET-28a (+)-TFD-S plasmid construction

The original TFD-EE(BBa_K5261000) is a homologous protein with C2 symmetry. When expressed separately in E. coli, the two monomers self-assemble in solution to form a complete TFD-EE. However, when applying TFD to yeast surface display, the surface display system may limit the self-assembly of protein monomers. Therefore, we first fused the homologous dimer protein gene into a single monomer protein gene (Figure 2-1. B), named TFD-S, through a head-to-tail junction.

We sent the TFD-S gene sequence to Azenta for gene synthesis and cloned it into the pET-28a (+) plasmid vector. We received E. coli containing the pET-28a (+)-TFD-S plasmid (Figure 2-1. A). After sequencing verification, the successful insertion of the TFD-S gene was confirmed by sequence comparison (Figure 2-1. C).

Figure 2-1. A. pET-28a (+)-TFD-S plasmid B. TFD-S gene sequence. C. The sequencing results showed that the TFD-S gene was successfully inserted into the pET-28a (+).

Subsequently, we expressed and purified TFD-S protein in Escherichia coli BL21 (DE3), which was verified by SDS-PAGE, indicating that the TFD-S protein was successfully obtained (theoretical band 37.49 kDa) (Figure 2-2).

Figure 2-2. SDS-PAGE and Coomassie bright blue staining of TFD-S

(Note: TFD-S-1 and TFD-S-2 are two parallel samples. Lane 1: Initial supernatant, Lane 2: precipitation after centrifugation, Lane 3: buffer elute, Lane 4: Dilute imidazole elute, Lane 5: concentrated imidazole elute, Lane 6: protein concentrate obtained after ultrafiltration)

2.2 Design of mutant protein TFD-M

To further enhance the rare earth binding and rare earth detection performance of TFD-S, we first introduced site-specific mutations of amino acids into the protein internal cavity through visual inspection based on the good C2 symmetry structure of TFD-S.

We use PyMol to modify the amino acid sequence. First, to introduce new lanthanide metal coordination residues, we introduce I6E/I52E/I175E/I221E mutations into TFD-S to obtain TIM-FD-Change (TFD-C). Secondly, to detect the binding of rare earth ions to proteins, we continued to introduce Q154W/Q323W mutations into TFD-C, and finally obtained the mutant TIM-FD-Mutation (TFD-M).

2.3 Computer modeling and molecular dynamics simulation

To predict the binding ability of mutant proteins TFD-C and TFD-M to rare earth elements, we submitted amino acid sequences to AlphaFold3 for prediction, and screened the spatial conformation with the highest prediction score index. Molecular dynamics simulation is then used to simulate the binding of proteins and rare earth ions (in the case of Tb³⁺). See the Model page for more details.

Results：

The results showed that among the two active binding sites of TFD-M, the average distance between the 8 active oxygen atoms and terbium ions was 2.64 Å and 2.84 Å, respectively (Figure 2-3). In the coordination structure, the distance between the ligand and the central atom can show the size of the binding force, but if the distance is too far, it is difficult to effectively bind. The average distance between the ligands at the two active sites of TFD-M and the central atom is close to 2.41 Å of TFD-S, so we believe that TFD-M has the potential to bind lanthanides. The MD simulation results of TFD-M combined with Tb³⁺ are shown in the following Figure 2-4.

Figure 2-3. Schematic diagram of binding of eight metal coordination residues to terbium ions in TFD-M. The Tb(III) ions are shown as green spheres, and coordination bonds are shown as dashed lines. The numbers near the coordination bonds represent the distances between the atoms, with the unit of Å.

Figure 2-4. The MD process, and kinetic curve of TFD-M. The ordinate of the RMSD curve is the root-mean-square deviation, indicating the distance the system moves.

2.4 Circular PCR site-specific mutation

Through several rounds of circular PCR in the TFD-S sequence within the pET-28a (+)-TFD-S plasmid, introducing I6E/I52E/I175E/I221E/Q154W/Q323W mutations, and building the E. coli expression plasmid pET-28a (+)-TFD-M (Figure 2-5. A). After the positive monoclone of transformed E. coli was obtained, it was sent for sequencing. Sequence alignment results confirmed that TFD-M gene was successfully obtained (Figure 2-5. C).

Figure 2-5. A. pET-28a (+)-TFD-M plasmid. B. TFD-M genetic sequence. C. The sequencing results showed that the TFD-M gene was successfully obtained in the pET-28a (+) plasmid

Subsequently, we expressed and purified TFD-M protein in Escherichia coli BL21 (DE3), and verified by SDS-PAGE, the mutant protein TFD-M was successfully obtained (theoretical band 37.67 kDa) (Figure 2-6).

Figure 2-6. SDS-PAGE and Coomassie bright blue staining of TFD-M

(Note: TFD-M-1 and TFD-M-2 are two parallel samples. Lane 1: Initial supernatant, Lane 2: precipitation after centrifugation, Lane 3: buffer elute, Lane 4: Dilute imidazole elute, Lane 5: concentrated imidazole elute, Lane 6: protein concentrate obtained after ultrafiltration)

2.5 Binding ability of TFD-S to different REEs

We then characterized the binding ability of TFD to different rare earth ions. Firstly, the binding of TFD-S and Tb(III) was characterized by the terbium luminescence mechanism sensitized by the antenna effect. The 10 μM TbCl₃ and 5 μM TFD proteins were pre-incubated in Tris-HCl buffer pH 7.5 for 1 hour, then added into 96-well plates. The excitation wavelength was set at 280 nm, the detection emission wavelength ranged from 520 nm to 570 nm, and the step length was 5 nm using a time-resolved fluorescence mode (TRF) and detect the terbium emission enhanced by tryptophan.

Subsequently, to detect the adsorption capacity of proteins for different rare earth ions, Tb(III) in the incubated proteins was replaced with other lanthanide ions, and the luminescence intensity was detected every 30nmin, and the decay curve of the Tb(III) fluorescence value enhanced by tryptophan was measured over time (Figure 2-7).

Results：

Taking Pr(III), Nd(III), Sm(III), and Eu(III) as examples, the experimental results show that TFD-S has the binding ability to all the above rare earth metal ions. As the reaction time went by, the emission intensity of terbium showed a decreasing trend, indicating that TFD-S-bound Tb (III) was gradually replaced.

Figure 2-7. The binding of other lanthanides, such as Pr(III), Nd(III), Sm (III), and Eu(III), was measured in displacement titrations using Tb(III)-bound TFD-S. The left figure showed the attenuation curve of the characteristic luminescence intensity of Tb³⁺ with time, and the right figure shows the fitting curve of the replacement ion concentration and the equilibrium fluorescence intensity.

Additionally, the equilibrium dissociation constant K_D can usually reflect the affinity of protein and ligand, and the two are inversely proportional. Therefore, in the fitting curve of the replacement ion concentration and equilibrium fluorescence intensity, c_1/2(X³⁺) at half attenuation of A₅₄₅ was extracted, and \(\frac{K_D(Tb^{3+})} {K_D(X^{3+})}\) was obtained. Shane J. Caldwell et al. titrated the pre-incubated TbCl₃ and EDTA mixture with metal-free TFD protein, and obtained an equilibrium dissociation constant K_D(Tb³⁺/EDTA) = 1.6×10⁻¹⁸ M [1], which can be used as a reference. The final results are shown in the following table.

Table 1. Fitting Results

Among them, the binding ability of Pr(III), Nd(III), Sm(III), and Eu(III) to TFD-S protein decreases in order. It is speculated that the possible reason is that the ion radius size Pr(III) < Nd(III) < Sm(III) < Eu(III), and with the increase of the rare earth metal ion radius, the acceptance ability of TFD-S to the rare earth ions decreases, which leads to a decrease in the adsorption capacity of TFD-S to the rare earth metal ions.

More data can be found on the registry page of TFD-S (BBa_K5261001).

2.6 Measurement of binding capacity of REEs by TFD-S and TFD-M under different pH conditions

Rare earth ore bioleaching liquid is usually acidic, so we need to further examine the difference in the binding ability of TFD protein to lanthanide metals under acidic conditions. We selected Tb (III) ions for adsorption and detected tryptophan-enhanced terbium luminescence in the buffer solution at pH 2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, and 8.5 with a microplate reader at a time interval of 15 min. The curves of the adsorption reaction of TFD-S and TFD-M with Tb (III) at different pH were obtained.

Results：

For TFD-S (Figure 2-8. A), obvious terbium luminescence was detected in the pH range of 5.0 ~ 8.5, indicating that TFD-S had good adsorption capacity for Tb (III) at different pH. The most suitable pH is 6.5, when terbium has the highest luminous intensity, indicating the strongest adsorption capacity for Tb (III).

For TFD-M (Figure 2-8. B), when pH < 6.0 or pH > 7.0, terbium luminous intensity is at a low level, indicating that TFD-M has low adsorption capacity for Tb (III). When the pH is 6.5, the terbium luminescence intensity also reaches the maximum, but it is far less than that produced by the binging of TFD-S and Tb (III) in the same pH, which indicates that our modified mutant TFD-M cannot achieve the purpose of increasing rare earth binding sites in the protein.

Through visual examination of the rare earth binding site of TFD-M, we speculated that the possible reason was that the internal cavity of TFD-M protein could not accommodate two rare earth ions in the barrel, and two Tb (III) ions led to unstable binding under the action of repulsion.

More data can be found on the registry page of TFD-S (BBa_K5261001) and TFD-M (BBa_K5261003).

Figure 2-8. A. Adsorption of Tb (III) by TFD-S in the pH range of 5.0-8.5

B. Adsorption of Tb (III) by TFD-M in the pH range of 5.0-8.5

Conclusion

In the end, we successfully obtained a monomeric protein TFD-S that is more suitable for yeast surface display purposes, and through site-directed mutagenesis, we generated a mutant protein TFD-M with two rare earth ion binding sites. Characterization of TFD-S and TFD-M confirmed their binding capabilities to rare earth ions, with binding ability correlated to the ionic radius of the rare earth ions and the environmental pH. However, the mutant protein TFD-M did not exhibit stronger rare earth binding capacity compared to TFD-S, possibly due to its smaller size limiting the accommodation of two rare earth ions within the barrel structure, which makes it more difficult for the ions to be adsorbed by the protein under repulsive forces.

According to the analysis of the protein structure, increasing the length of the TIM barrel requires extending the α-helices and β-sheets that compose the walls of the TIM barrel. Therefore, our next step will focus on increasing the length of the α-helices on the barrel walls to separate the two rare earth binding sites and reduce the electrostatic interactions between the rare earth ions. We will also explore additional modification strategies for the rare earth binding sites, including adding or removing metal coordination residues within the redesigned TIM barrel. Based on these, we will proceed with further protein modifications and validations in the future.

Module 3: Yeast Surface Display

Overview

In this section, we introduced the AGA2-TFD fusion gene (BBa_K5261010) driven by GAL1 promoter into Saccharomyces cerevisiae EBY100 strain, induced TFD protein expression with galactose, detected the TFD protein on the cell surface by antigen-antibody reaction and flow cell technology, and verified the adsorption capacity of the surface display strain Saccharomyces cerevisiae EBY100 (pYD1-TFD).

Saccharomyces cerevisiae EBY100 is a commonly used strain for cell surface display, Genotype: MATa AGA1:: GAL1AGA1:: URA3 ura3-52 trp1 leu2200 his3200 pep 4:: HIS3 prbd1.6Rcan1 GAL. Due to time constraints, we only used the Saccharomyces cerevisiae EBY100 as the chassis strain for proof-of-concept in our experiments in this module. In the future, we expect to expand further to the Issatchenkia orientalis chassis strains as described in our Design.

3.1 pYD1-TFD plasmid construction

The pYD1 plasmid is an expression vector designed to express, secrete and display proteins on the extracellular surface of S. cerevisiae. Gene components such as AGA2 and polyhistidine (6×His) tags have been designed on the plasmid.

Firstly, the codon-optimized TFD gene sequence of Saccharomyces cerevisiae was sent to Azenta for gene synthesis, and cloned into the position of the C terminus of the AGA2 anchor protein gene on the pYD1 plasmid vector to achieve the fusion expression of AGA2 and TFD genes (Figure 3-1 A and B). After receiving the strain of E. coli containing the pYD1-TFD plasmid, we amplified it, extracted the plasmid, and verified it by sequencing, which showed that the sequencing results were correct (Figure 3-1 C).

Figure 3-1. A. The pYD1-TFD plasmid. B. AGA2-TFD gene fusion expression on pYD1-TFD plasmid. C. The sequencing results showed successful insertion of the TFD gene into the pYD1 plasmid.

In addition, we used AlphaFold3 to predict the spatial structure of the Aga2-TFD fusion protein and found that the spatial structure of the TFD can still be well maintained (Figure 3-2).

Figure 3-2. Spatial structure of the Aga2-TFD fusion protein predicted by AlphaFold3

3.2 pYD1-TFD, pYD1 plasmids were transformed into S. cerevisiae

The constructed pYD1-TFD plasmid and the control pYD1 plasmid were respectively transformed into Saccharomyces cerevisiae EBY100 by the lithium acetate method. The transformed yeast was coated after 2 days of culture on SC-TRP agar plates (Figure 3-3).

Figure 3-3 The EBY100 (pYD1) and EBY100 (pYD1-TFD) positive monoclones obtained on the SC-TRP plates

3.3 Induce surface display protein expression

Single colonies of EBY100 (pYD1-TFD), EBY100 (pYD1), and EBY100 (as a control group) were inoculated into 5 mL of SC-TRP, SC-TRP, SC liquid medium, respectively, and cultured at 30℃ overnight. Take medium from the amplified tubes, measure absorbance at 600 nm, then inoculate to a shake flask containing 50 mL SC-TRP or SC medium, initial OD₆₀₀ = 0.1, and continue for 24–36 h. Yeast was centrifuged, washed, and inoculated into the same volume of YPG liquid medium containing 2% galactose. After 48–60 h of oscillatory incubation, the displayed protein expression was predicted to reach a maximum. Cells were centrifuged and cells were resuspended in the same volume of PBS buffer and stored for 4℃.

Figure 3-4. Inducing yeast display protein expression in S. cerevisiae

3.4 Examined the cell surface display protein, TFD

3.4.1 SDS-PAGE test for surface display

To test whether the TFD protein was successfully displayed extracellularly, we performed SDS-PAGE electrophoresis. The Aga2-TFD fusion protein expressed in the extracellular was anchored by two disulfide bonds to the cell wall surface protein Aga1, so we could test by recovering the fusion protein Aga2-TFD from the supernatant after breaking the disulfide bond.

We treated S. cerevisiae EBY100 transferred into the pYD1 plasmid and pYD1-TFD plasmid respectively with dithiothreitol (DTT) and used wild-type EBY100 as a control. After centrifugation, supernatants were removed to concentrate target proteins by ultrafiltration and verified by SDS-PAGE.

While the EBY100 strain transferred into the pYD1 plasmid retained the Aga2p-Xpress Tag-V5 tag-6 × His Tag fragment (about 18.66 kDa) in the supernatant after disulfide disconnection, the EBY100 strain transferred into the pYD1-TFD plasmid retained the Aga2p-Xpress Tag-TFD-V5 tag-6 × His Tag fragment (about 54.41 kDa), while the control EBY100 strain did not have the target fragment.

Results：

Since we did not purify the protein, the bands on the SDS-PAGE were not obvious. However, EBY100, EBY100 transferred into pYD1 plasmid and EBY100 transferred into pYD1-TFD plasmid all showed target bands (Figure 3-4), indicating that the target protein had been successfully displayed on the surface of yeast cells.

Figure 3-5. SDS-PAGE validation of the TFD protein. The target band of EBY100 (pYD1) is 18.66 kDa, and that of EBY100 (pYD1-TFD) is 54.41 kDa.

3.4.2 Cell surface display detection by flow cell technology

Flow cytometric technology can be used to quantitatively analyze the proteins displayed on the surface of yeast cells, assessing the efficiency of protein display by combining fluorescent group-conjugated antibodies and detecting fluorescence intensity using flow cytometry.

We incubated Saccharomyces cerevisiae EBY100 with the pYD1-TFD plasmid with the his tag mouse monoclonal antibody primary antibody (against the 6 × His tag on the surface-displayed protein fragment) and the fluorescent group FITC-conjugated goat anti-mouse IgG secondary antibody to label the surface-displayed protein fragments. We subsequently analyzed the fluorescence intensity on the cell surface, thus assessing the surface display level of TFD protein. Wild-type EBY100 cells were gated to distinguish populations of expressed and unexpressed surface display proteins, and the percentage of expressed cells and mean fluorescence intensity (Mean) were calculated to assess display efficiency.

Results：

The proportion of cells in Saccharomyces cerevisiae EBY100 with pYD1-TFD plasmid was significantly higher than the control wild-type EBY100, with the maximum display efficiency (percentage of the cells displayed) reaching 9.86% (Figure 3-6 A). The average fluorescence intensity (Mean) of the cell also showed the success of the surface display strain construction.(Figure 3-6 B)

Figure 3-6 A and B. Saccharomyces cerevisiae EBY100 cells carrying the pYD1-TFD plasmid were labeled with a 6 × His (FITC) -labeled fluorescent group-conjugated antibody and analyzed by flow cytometry.

3.5 Quantitative detection of rare earth ions adsorption by surface display strains

Having confirmed that the lanthanide-adsorbed protein TFD had been successfully demonstrated on the cell surface, we need to further test the ability of the engineered yeast to adsorb rare earth ions. Add 100 μM of TbCl₃ to the EBY100 (pYD1-TFD) cell culture obtained from section 3.3. After shaking incubation for one day, centrifuge to precipitate the cells and measure the concentration of free Tb(III) in the supernatant. Cell culture medium without TbCl₃ was used as a blank control. Three parallel experiments were done for each data point.

Due to the high price of ICP-MS or ICP-OES, here we adopted the Arsenazo III dye-based assay method, which can be used to roughly determine the rare earth ion content in the solution, see our Protocols page for more details. The absorbance values at 650 nm can reflect the rare earth ion content within a certain range.

Results：

After the adsorption process, the TbCl₃ content in the solution decreased significantly, which was manifested in the obvious color depth difference of the Arsenazo III – REE complex formed before and after adsorption (Figure 3-7 A), which was also reflected by the value of A₆₅₀.

However, due to the limitation of the Arsenazo III assay and the extremely low concentration of Tb(Ⅲ) ions in the solution after adsorption, we could not quantitatively detect the exact concentration of rare earth ions by drawing a standard curve. However, we could roughly estimate it by the "two-point interval method" according to the value of A₆₅₀: For the solution after adsorption, three parallel data points were measured in a microplate reader, and the A₆₅₀ obtained was between that of 2 μM and 10 μM TbCl₃ standard solution (Figure 3-7 B), so it could be preliminarily judged that c(Tb³⁺) in the solution after the adsorption was also in this range. Since we initially added c(Tb³⁺) = 100 μM (before adsorption), it could be considered that our surface display strain had a strong adsorption capacity for rare earth ions.

Figure 3-7 A. The absorbance at 650 nm of the solution before and after biosorption was determined by Arsenazo III assay, and obvious color depth difference was observed. B. A₆₅₀ value of standard solution of TbCl₃ from 0 to 150 μM.

3.6 Measure the effect of rare earth ions on yeast growth

Finally, given that high concentrations of rare earth ions may be detrimental to the growth of the engineered yeast itself in actual industrial production, we measured the effect of Tb(III) ions in the culture medium on the growth of surface display strains. We cultured EBY100 and EBY100 (pYD1-TFD) in 50 mL SC and SC-TRP liquid medium for 24 h, and then replaced them with YPG liquid medium containing galactose for induction of TFD protein expression. Each group controlled the same initial OD₆₀₀ = 2 ~ 3. For induced expression for 18 h, TbCl₃ was added to a concentration of 100 μM, and the culture was continued until 84 h. OD₆₀₀ was measured every 12 h to obtain the growth curves of both strains in the medium containing Tb(III) and without Tb(III).

Results：

The obvious difference in the growth curves between EBY100 and EBY100 (pYD1-TFD) strains may be due to the different growth status of the two strains themselves, thus lacking comparative significance. However, after adding a higher concentration of Tb(III) (100 μM) at 12 h, there was no obvious effect on the growth of both strains regardless of the addition of Tb(III) (The shapes of the two growth curves were similar).

Therefore, we believe that the high concentration of rare earth ions will not have a significant effect on the growth status of yeast if put into the actual biological mining when used. Of course, the actual working environment may also involve harsh conditions such as lower pH, as our final design goal is to build within the Issatchenkia orientalis chassis strain, which can be overcome by the excellent acid tolerance properties of Issatchenkia orientalis.

Figure 3-8. Effects of the addition of 100 μM Tb(III) ions on the growth of surface display strains

Outlook

Finally, due to time constraints, we only used S. cerevisiae strain EBY100 for proof of concept in the experiments of this module, with preliminary demonstration that TFD protein display on the yeast cell surface can construct a whole-cell bio-adsorbent with strong biosorption capacity for rare earth ions. However, in future industrial applications, we expect to integrate AGA2-linker-TFD gene expression cassette into the Issatchenkia orientalis genome to achieve constitutive expression to achieve more stable and continuous expression of lanthanide binding protein TFD, and thus better application to bio-adsorption in acid rare earth mine wastewater.

Module 4: Biofilm Module

Overview

Here, we used the constitutive strong promoter P_TEF1 to overexpress the Saccharomyces cerevisiae surface adhesion gene FLO11 (BBa_K5261015, BBa_K5261017) to construct yeast biofilms in a hydrophobic carrier filler of MBBR-MABR membrane bioreactor. We used the CRISPR-Cas9 system to insert the P_TEF1 promoter upstream of the coding region of the endogenous gene FLO11 in Saccharomyces cerevisiae BY4741. Subsequently, we performed the surface adhesion ability characterization of the Saccharomyces cerevisiae BY4741 FLO11+ strain.

4.1 The P_TEF1 promoter was integrated by using the CRISPR-Cas9 system

4.1.1 Construction of the CRISPR-Cas9 system’s integration tool

Using the CRISPR-Cas9 system requires separate construction of plasmids containing gRNA, Cas9, and donor fragments for gene integration according to the principle of homologous recombination.

We obtained the gRNA expression plasmid p426-SpSgH-URA3 and Cas9 expression plasmid p416-Cas9-G418 from the research group of Associate Professor Lidan Ye, Zhejiang University. First, the appropriate gRNA sequence was designed according to the gene sequence of the recombination site within -200 bp upstream of the coding region of the FLO11 gene through the CRISPR assisted-design website (https://benchling.com). The p426-SpSgH-URA3 plasmid was inserted with the BsaI enzyme to generate separate expression plasmids for gRNA1-4. Then, according to the principle of homologous recombination, homologous primers with 40 bp homologous arms were designed. The gene integration fragment pTEF1 donor was obtained by PCR from pUMRI-HO-pTEF1 plasmid, and the PCR product was concentrated by ethanol precipitation to obtain a high concentration integrated fragments (Figure 4-1).

Figure 4-1. The CRISPR-Cas9 integration tool. A. The p426-SpSgH-URA3 plasmid. B. The p416-Cas9-G418 plasmid. C. gRNA1-4 is designed for gene integration just upstream of the FLO11 gene. D. Integration of the pTEF1 promoter upstream of the FLO11 gene.

Results：Electrophoresis result conveyed that the pTEF1 donor fragment (theoretical band 493 bp) was successfully obtained, and the result of sequencing showed that the gRNA expression plasmid p426-SpSgH-URA3-gRNA was successfully conducted (Figure 4-2).

Figure 4-2. A. Electrophoresis result showing that the pTEF1 donor was successfully obtained. B. Sequencing result showing a successful construction of p426-SpSgH-URA3-gRNA plasmid.

4.1.2 Transforming Saccharomyces cerevisiae by CRISPR-Cas9 system integration tool

The two plasmids mentioned above and the donor were introduced into Saccharomyces cerevisiae BY4741 using lithium acetate transformation to achieve gene integration. Single clones were obtained after 3 days of culture on SC-URA+G418 agar plates. Sixteen single yeast colonies were selected as templates for colony PCR and verified by agarose gel electrophoresis.

Results: Electrophoresis result showed two positive clones (The region flanking the recombination site upstream of the FLO11 coding region. A successfully integrated band theoretically has 729 bp, or 523 bp if weren’t integrated.)

Figure 4-3. Colony PCR verified the electrophoresis results of CRISPR-Cas9 gene integration. Positive results for numbers 2 and 13 (target band 729 bp)

4.1.3 The removal of the screening markers from CRISPR/Cas9 System

After verifying the correctly integrated genotype in Saccharomyces cerevisiae, removal of the transferred p426-SpSgH-URA3-gRNA plasmid and p416-Cas9-G418 plasmid were required based on the principle that the plasmids would be naturally lost during screen-free pressure, as well as on the principle of the URA3 and 5-FOA negative screens.

Results: After sequencing validation, the stably inherited Saccharomyces cerevisiae BY4741 FLO11 + strain (Figure 4-4) was obtained.

Figure 4-4. Sequencing showed that the pTEF1 promoter was successfully integrated upstream of the endogenous FLO11 gene in Saccharomyces cerevisiae BY4741.

4.2 Characterization of the surface adhesion phenotype of the BY4741 FLO11 + strain

4.2.1 Enhanced cell-cell adhesion

After growing Saccharomyces cerevisiae BY4741 and BY4741 FLO11+ strains in YPD liquid medium overnight, photographs were taken and natural sedimentation of the two strains was recorded every 30 min by simultaneously standing without shaking.

Results: Within 60 minutes, the natural sedimentation rate of Saccharomyces cerevisiae BY4741 FLO11 + strain was significantly faster than that of BY4741 (Figure 4-5. A).

Although FLO11p is thought to be associated with cell-surface adhesion, we propose that overexpression of the FLO11 gene also conferred a mild cell-cell adhesion phenotype of Saccharomyces cerevisiae BY4741 FLO11+. Therefore, we also simultaneously observed the two strains under a light microscope.

Results: At 40× magnification， more cell-cell adhesion of BY4741 FLO11+ strains in the field of view, while wild-type BY4741 cells were dispersed; At 100× magnification, BY4741 FLO11 + cells formed large clumps of 5-30 cells (Figure 4-5. B).

Figure 4-5. A. BY4741 FLO11+ strains naturally settle faster than wild-type BY4741. B. The cell-cell adhesion phenomenon was observed in the BY4741 FLO11 + strain.

4.2.2 Adherence to agar and plastic plates

We subsequently characterized the cell-surface adhesion properties, which included adhesion to plastic and agar.

(1) Characterization of plastic surface adhesion: The cultures of the two strains were overlaid on plastic dishes made of polystyrene. After one day, pour the surface culture medium and rinse with water. The BY4741 FLO11+ strain was seen to form a radiopaque biofilm at the bottom of the plastic plate (Figure 4-6. A). The biofilm was stained with the ammonium oxalate-crystal violet dye solution and was observed with a light microscope (Figure 4-6. B).

Results: At 40× magnification, the BY4741 FLO11+ strain in the visual field formed dense biofilm, while the wild-type BY4741 cells showed a loose dispersed distribution, which suggested the ability of strain BY4741 FLO11+ to adhere to the plastic surface and form biofilm.

Figure 4-6. A. BY4741 FLO11 + strain formed a biofilm at the bottom of the polystyrene plate. B. Biofilm morphology under the microscope.

(2) Characterization of agar surface adhesion: After the simultaneous culture of Saccharomyces cerevisiae BY4741 and BY4741 FLO11 + strains on YPD solid agar plates for 2 days, the two strains grew equally well on the agar plates (before washing).

Results: When the plate was gently washed with the running water, the cells of the wild-type BY4741 were almost completely washed out, and the majority of the cells overexpressing FLO11 remained firmly adhered to the agar surface (Figure 4-7), which could indicate the ability of the BY4741 FLO11 + strain to adhere firmly to the agar surface.

Figure 4-7.BY4741 FLO11+ strain exhibited a stronger adhesion ability on YPD agar plates.

4.2.3 Quantitative characterization of adhesion in polypropylene 96-well plates

The YPD cultures of the above two strains were seeded in an inoculum of 100 μL OD600 =1 in wells of polypropylene 96-well plates, incubated for 60,120,180, and 240 minutes to allow sufficient adherence, then continued biofilm staining for 30 minutes by adding 50μ L of ammonium oxalate-crystal violet dye solution. After the staining, the cells were washed four times with PBS buffer to remove the excess dye solution, and finally 200μL of absolute ethanol was added to dissolve the dye solution on the biofilm in each well. The absorbance at 590 nm for each well was measured using a microplate reader. Three parallel experiments were done for each data point.

Results: On polypropylene 96-well plates, the BY4741 FLO11+ strain tended to be significantly more adherent than the BY4741 strain

Figure 4-8. A. After the adherent biofilms in polypropylene 96-well plates were stained with ammonium oxalate-crystal violet dye solution, the absorbance at 590nm was quantified in a microplate reader. B. Biofilm membrane-forming conditions undergoing different incubation times.

4.2.4 Dry Weight change of the adherent biofilm on K3 carrier packing of MBBR

Saccharomyces cerevisiae BY4741 and BY4741 FLO11+ strains were grown in batches in a shake flask containing 50mL YPD with an initial inoculum OD600 = 0.2, and five K3 fluidized bed fills (Figure 4-9) were added for each group. After 1,2,3 and 4 days of culture in each group, the K3 packing was removed. After naturally drying, the total dry weight change was recorded and the biofilm dry weight change curve was drawn. Three parallel experiments were done for each data point.

Figure 4-9. K3 fluidized bed carrier packing adhesion experiment.

Results: After 3 days of batch culture, obvious biofilm adhesion (Figure 4-10) appeared on the K3 fluidized bed fills cultured with the BY4741 FLO11+ group strains. The biofilm dry weight change curve shows that the amount of film reached the maximum (Figure 4-11), which may be related to the growth cycle and state of the cell. So the best bioadsorption process can be preliminarily judged.

Figure 4-10. Strain overexpressing FLO11 could adhere to form significant biofilm on the K3 fluidized bed carrier packing, while no adhesion was observed in the BY4741 strain.

Figure 4-11. The K3 fluidized bed carrier filler was added at 0 h, Biofilm dry weight change curves on carrier filler after 20 h and 24,48,72 and 96 h of batch culture.

Module 5: Biosafety

Overview

Considering the biosafety, visualization, and sensitivity of the experiment, we replaced the toxin RelE and antitoxin RelB with mCherry and EGFP, respectively, in the actual experimental design to characterize the strength of the P_CTR3 and P_CYC1 promoters. Since we designed a copper ion concentration-responsive engineered yeast suicide mechanism and were limited by time, we selected only the core genetic circuit for proof of concept. Specifically, we conducted characterization experiments on the copper-repressible promoter P_CTR3 (BBa_K5261020) using only the mCherry. Ultimately, we aimed to explore the copper ion response concentration of the P_CTR3 promoter to determine the working range of this biosafety system.

1. Construction of the P_CTR3-mCherry-T_CYC1 expression plasmid

We constructed the mCherry gene expression cassette driven by the P_CTR3 promoter on a free plasmid (BBa_K5261021). We amplified the sequence of the endogenous copper-repressible promoter P_CTR3 from the genome of Saccharomyces cerevisiae CEN.PK2-1C using PCR. Next, we amplified the mCherry gene sequence through PCR. At the same time, we amplified the plasmid vector fragment from the p416-pTEF1-Cas9-tCYC1-G418 plasmid, which carried the KanMX marker (conferring resistance to the antibiotic G418 in yeast). We verified the correctness of the three gene fragments using agarose gel electrophoresis.

Results: The electrophoresis results showed bands corresponding to the p416 Vector (5000+ bp), CTR3 (700+ bp), and mCherry (700+ bp), which matched the theoretical lengths of 5733 bp, 777 bp, and 751 bp, respectively, indicating that we had successfully obtained the three fragments through PCR.

Figure 5-1. PCR results of p416 Vector, pCTR3, andmCherry gene fragments

The P_CTR3 gene sequence, mCherry gene sequence, and p416 Vector were assembled by Gibson Assembly to construct the recombinant plasmid p416-pCTR3-mCherry-tCYC1-G418 according to the following diagram.

Figure 5-2. Plasmid map of p416-pCTR3-mCherry-tCYC1-G418

The constructed recombinant plasmid was introduced into E. coli DH5α for amplification, and E. coli monoclonal colonies were obtained through transformation. Four single colonies were selected as templates for colony PCR, and verification was performed using agarose gel electrophoresis.

Figure 5-3. Electrophoresis results of colony PCR

Take the above E. coli monoclonal amplification, extract the plasmid, and sequence it.

Results: The sequencing results of plasmids 1, 3, and 4 were correct, but plasmid 2 was wrong, and discarded.

Figure 5-4. Sequencing results of p416-pCTR3-mCherry-tCYC1 plasmids 1 - 4

2. Transformation of P_CTR3-mCherry-T_CYC1 expression plasmid into Saccharomyces cerevisiae

The successfully constructed p416-pCTR3-mCherry-tCYC1-G418 plasmid was transformed into Saccharomyces cerevisiae BY4741 using the lithium acetate transformation method. After cultivating on YPD-G418 agar plates for 2-3 days, positive clone strains were obtained.

3. Characterization of the copper ion repression concentration of the P_CTR3 promoter

To characterize the copper ion repression concentration of the P_CTR3 promoter, we batch-cultivated Saccharomyces cerevisiae BY4741 strains transformed with the p416-pCTR3-mCherry-tCYC1-G418 plasmid in 50 mL YPD-G418 shake flasks, with an initial inoculation of OD₆₀₀ = 0.1. At 0 hours, we added CuCl_₂ to final concentrations of 0, 0.2, 0.5, 1, 2, 10, 20, 30, 40, 50, and 60 μM, and used Saccharomyces cerevisiae BY4741 strains as the wild-type control group.

Fluorescence measurements were performed approximately every 12 hours. We took a portion of the culture, washed it twice with ddH_₂O, and then diluted it to OD₆₀₀ = 1 with PBS. The fluorescence of mCherry was measured using a microplate reader with an excitation wavelength of 585 nm and an emission wavelength of 620 nm. We assessed the repression of the copper ion-repressible promoter P_CTR3 based on the fluorescence intensity of each group. Measurements were conducted continuously for 72 hours.

Figure 5-5. Copper ion inhibition concentration characterization of the pCTR3 promoter

Results: After adding copper ions at 0 hours, the fluorescence intensity of each group rapidly decreased to varying extents. Significant fluorescence repression was observed at a Cu(Ⅱ) concentration of 1 μM. After 72 hours of batch cultivation, groups with Cu(Ⅱ) concentrations greater than 30 μM showed more than 10-fold repression compared to the group without adding Cu(Ⅱ). These observations indicated that the P_CTR3 promoter experienced effective repression.

However, we also observed that there was still some level of leakage expression of the P_CTR3 promoter even when Cu(Ⅱ) was added, which aligned with our previous speculation. In our design, we used the constitutive weak promoter P_CYC1 to express the antitoxin gene RelB to counteract the leakage expression of RelE. Therefore, we could conclude that the design of our biosafety module was fundamentally reasonable.

Figure 5-6. Characterization of the copper ion repression concentration of the P_CTR3 promoter. A. mCherry fluorescence values detected over 0-72 hours. B. mCherry fluorescence intensity for each group at 70 hours.

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