Use of desmodesmus armatus and its actin in removing cadmium from the environment

Abstract

The present application relates to the application of Paffia dura and its actin in removing cadmium in the environment; and a method for removing cadmium in the environment, comprising the following steps: exposing Paffia dura or microorganisms overexpressing actin from Paffia dura to an environment containing cadmium for incubation; removing Paffia dura from the environment. The present application finds that Paffia dura has high tolerance to cadmium stress and excellent cadmium enrichment capacity, and further finds that the main substance in Paffia dura cells combined with cadmium is actin. Therefore, Paffia dura and microorganisms overexpressing the actin can be used to treat cadmium-polluted environments to remove cadmium pollution. In addition, we also unexpectedly find that cadmium stress can up-regulate the expression of actin in Paffia dura, which is a new way to up-regulate the expression of housekeeping genes.

Description

Technical Field

[0001] This invention relates to the field of microalgae and environmental protection, and more particularly to the application of *Durantalum bafuriae* and its actin in the removal of cadmium from the environment. Background Technology

[0002] Cadmium pollution, due to its persistence, bioaccumulation, and migration, poses a significant risk to humans and other organisms, becoming a global problem. Cadmium migrates more quickly in soil, water, and air than other heavy metals and is easily absorbed by plants and microorganisms. It can inhibit enzymes that repair DNA double-strand breaks, leading to diseases such as emphysema, osteoporosis, and cancer. Furthermore, cadmium has a significant harmful effect on water bodies and soil; high concentrations can harm soil organisms, accumulate in the soil, enter the food chain, and ultimately enter the human body. Therefore, the remediation of cadmium pollution is a crucial issue in agriculture and the environment.

[0003] Microbial enrichment is an important remediation strategy for toxic metal pollution in the environment, and it is widely used due to its effectiveness, low cost, and environmental friendliness. Therefore, microbial enrichment can be considered for treating cadmium pollution. However, the low survival rate and low bioremediation efficiency of a large number of microorganisms under cadmium stress limit the widespread adoption of this method. Summary of the Invention

[0004] During our research, we discovered that *Dunaliella pafus* is the fastest-growing member of the Dunaliaceae family, exhibits high tolerance to cadmium, and can effectively accumulate cadmium within its cells. Further investigation revealed that actin from *Dunaliella pafus* can form a complex with cadmium, thereby reducing its toxicity to algal cells.

[0005] Based on the above findings, this invention provides the application of Dunaliella parva in the removal of cadmium from the environment.

[0006] The present invention also provides the application of actin derived from *Duranta erecta* in the removal of cadmium from the environment.

[0007] The present invention also provides a method for removing cadmium from the environment, comprising the following steps:

[0008] S1: Incubate *Pavlova bafdurella* or microorganisms overexpressing actin from *Pavlova bafdurella* in an environment containing cadmium.

[0009] S2: Remove the *Pavlova* from the environment.

[0010] In one specific embodiment, in S1, the *Pavlova* algae or microorganisms overexpressing actin derived from the *Pavlova* algae are incubated in the cadmium-containing environment for 1-14 days, or longer.

[0011] In one specific implementation, the cadmium content in the environment does not exceed 50 mg / L.

[0012] In one specific embodiment, the amino acid sequence of the actin derived from *D. pavlova* is shown in SEQ ID NO: 1, or the nucleic acid coding sequence is shown in SEQ ID NO: 2.

[0013] In one specific implementation, the microorganism is Escherichia coli.

[0014] The present invention also provides a method for upregulating the expression of actin in microorganisms, comprising subjecting the microorganisms to cadmium stress.

[0015] In one specific implementation, the microorganism is *Pavlova pufferfish*.

[0016] In one specific implementation, the cadmium stress concentration is 1-50 mg / L.

[0017] This invention reveals the high tolerance of *Pavlova bafdurella* to cadmium stress and its excellent cadmium accumulation capacity. It also identifies actin as the main substance binding cadmium within *Pavlova bafdurella* cells. Therefore, *Pavlova bafdurella* and microorganisms overexpressing this actin can be used to remediate cadmium-contaminated environments to remove cadmium pollution. Furthermore, we unexpectedly discovered that cadmium stress can upregulate actin expression in *Pavlova bafdurella*, representing a novel way to upregulate housekeeping genes. Attached Figure Description

[0018] Figure 1 The growth curves of *D. pavlova* under different concentrations of cadmium stress are shown.

[0019] Figure 2 This is a statistical chart showing the content of chlorophyll a, chlorophyll b, and carotenoids in algal cells after being cultured for a certain period of time under cadmium exposure of 1, 5, and 25 mg / L.

[0020] Figure 3 A statistical graph showing the adsorbed and accumulated cadmium content in *Durantalum bafica* cells cultured under cadmium exposure.

[0021] Figure 4 Subcellular localization of cadmium accumulated within *D. pavlova* cells. A represents the distribution of cadmium in different subcellular structures; B represents the quantity of cadmium in different forms; and C represents the molecular weight distribution of cadmium and its complexes within the algal cells.

[0022] Figure 5This image shows the separation of cadmium-binding proteins. A represents the peak diagrams of cadmium ions and cadmium-binding proteins obtained by GE-I CP-MS; B represents the electrophoresis diagrams of fractions at different time points: lanes 1-3 represent the 60-70 min fraction, lanes 4-6 represent the 75-90 min fraction, and lanes 7-9 represent the 91-105 min fraction; C represents the electrophoresis diagrams of cadmium-binding proteins obtained by elution with cadmium ion-chelating magnetic beads: lanes 1-4 represent samples eluted with 100 mM imidazole from magnetic beads that did not chelate cadmium ions, lanes 5-7 represent samples eluted with 300 mM imidazole from magnetic beads that did not chelate cadmium ions, lanes 8-10 represent samples eluted with 500 mM imidazole from magnetic beads that did not chelate cadmium ions, lanes 11-13 represent samples eluted with 300 mM imidazole from magnetic beads that chelate cadmium ions, and lane 14 represents a sample eluted with 100 mM imidazole from magnetic beads that chelate cadmium ions.

[0023] Figure 6 This experiment was conducted to detect the relative expression level of actin under cadmium exposure. In Figure A, electrophoresis images of all samples after concentration adjustments show that the total protein content is the same; Figure B is an immunoblot image targeting actin.

[0024] Figure 7 This study investigated the construction of *E. coli* strains overexpressing *D. pavlova* actin and their cadmium uptake. A shows the total protein electrophoresis image after IPTG induction; lane 1 is the control strain (BL21 transformed with pET-28a empty vector), and lanes 2-5 are the four overexpressing strain clones. B shows the immunoblotting detection of actin in the overexpressing strains; lane 1 is the total protein sample, lane 2 is the soluble protein sample, and lane 3 is the inclusion body sample. C is a statistical graph showing the amount of cadmium accumulated in the overexpressing strains after exposure to different concentrations of cadmium. D is a statistical graph showing the amount of cadmium accumulated in the overexpressing strains after cadmium exposure for different durations. Detailed Implementation

[0025] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0026] 1. High tolerance of Duprise pavlova (Dp) to cadmium

[0027] Dp were inoculated into a culture medium supplemented with 0-100 mg / L cadmium ions and cultured for 16 days. OD was measured daily. 680 Plot the growth curve. The results are as follows: Figure 1 As shown, cadmium exposure at concentrations of 1–25 mg / L did not affect algal cell growth. However, when cadmium concentrations exceeded 37.5 mg / L, algal cell growth was significantly lower than the control group, and the higher the cadmium concentration, the greater the impact on algal cell growth. Therefore, we consider the maximum tolerable concentration (MTC) for Dp to be 25 mg / L.

[0028] During the cultivation process, samples were taken and tested for chlorophyll-like substances (Chl a and b) and caroloids. The results are as follows: Figure 2 As shown, exposure to cadmium at concentrations of 1-25 mg / L had no significant effect on the chlorophyll and carotenoid content of Dp, further indicating that Cd ≤ 25 mg / L is insufficient to impair pigment synthesis and photosynthesis, and therefore has no effect on nutrient accumulation.

[0029] After 96 hours of incubation, its EC50 value was 128.5 mg / L Cd. 2+ It was much higher than that of the reported cadmium-tolerant Dunaliella salina (48.9 mg / L) and reinhardtii Chlamydomonas (12.48 ± 1.30 mg / L).

[0030] These results confirm that Dp has a high tolerance to Cd.

[0031] 2. Accumulation and localization of cadmium in cells

[0032] After exposure, algal cells were digested and Cd adsorption on the cell surface and accumulation in the cell were analyzed by ICP-MS.

[0033] like Figure 3 As shown, the adsorption rate of Cd on the Dp surface is very fast. The adsorption dose is 3.47±0.23 fg / cell within 1 hour, reaches a peak at 24 hours (21±3.39 fg / cell), decreases slightly at 96 hours (14.5±1.48 fg / cell), and remains relatively stable on day 7 (17.62±2.22 fg / cell) and day 14 (18.82±1.54 fg / cell).

[0034] Compared to adsorption, cadmium accumulates more slowly within cells. Each cell contains 0.63 ± 1.54 fg Cd at 1 hour, only about 18% of the adsorbed amount. After 96 hours, the intracellular accumulation reaches 21.93 ± 0.74 fg / cell, exceeding the adsorbed amount. The maximum adsorbed amount on day 14 is 64.62 ± 1.54 fg / cell, approximately 3 * 10-1 8 The Cd / cell ratio is approximately 3.43 times that of the surface adsorption capacity.

[0035] Compared with other reported microalgae, Dp has a higher Cd accumulation capacity: E. gracilisa (12.2 fg / cell at 96 h) and Chlamydomonas sp. (3.4 fg / cell at 72 h).

[0036] Converted to dry weight, as shown in Table 1, the cumulative Cd content on day 7 and day 14 was 738±19 and 1499±65 mg / kg dry weight, respectively, which is much higher than that of microalgae in existing technologies. For example, in Mahshid's study, the cumulative dry weights of C. reinhardti and CPCC 121 at 600 mM Cd were 2.76±0.22 and 2.08±0.36 mg / kg, respectively.

[0037] Dp cells can tolerate high concentrations of Cd, indicating they must employ specific strategies to mitigate Cd toxicity. To determine whether Cd is stored in a specific space, we analyzed the subcellular locations of Cd.

[0038] Cd adsorbed on the surface was eluted with 10 mM EDTA. Then, the partitions were analyzed using sonication and differential centrifugation. The results are as follows: Figure 4 As shown in Figure A, cadmium was most abundant in heat-stable proteins (HSPs) (73.47 ± 1.78%), followed by cell debris (18.42 ± 0.91%), organelles (18.42 ± 0.91%), particles (0.31 ± 0.14%), and heat-denatured proteins (0.21 ± 0.10%). These results indicate that most Cd is captured by HSPs to mitigate its toxicity.

[0039] To further determine the chemical species of Cd, we extracted Cd in different chemical forms. The results are as follows: Figure 4 As shown in Figure B, 80% ethanol was used to extract water-soluble inorganic cadmium; deionized water was used to extract water-soluble cadmium-organic acid complexes and Cd(H₂PO₄)₂; 1M NaCl was used to extract cadmium integrated into pectin and proteins; 2% acetic acid (HAC) was used to extract insoluble cadmium phosphates, including cadmium phosphate and cadmium hydrogen phosphate; 0.6M HCl was used to extract cadmium oxalate; the remainder was present in the algal residue. It can be seen that cadmium mainly exists in the form integrated into pectin and proteins (69.6±1.9%), followed by water-soluble cadmium-organic acid complexes and Cd(H₂PO₄)₂ (22.2±1.8%). Small amounts of cadmium phosphate and cadmium hydrogen phosphate (3.95±0.3%) and water-soluble inorganic cadmium (3.9±0.3%) were present. Cadmium was not detected in the algal residue. Cadmium is stored in pectin and proteins, which are less toxic than its water-soluble form, which may explain why Dp can tolerate high concentrations of cadmium.

[0040] Ultrafiltration analysis results are as follows Figure 4 As shown in C, 53.5 ± 11.3% of cadmium was trapped by Millipore's 100 kDa cellulose acetate membrane with a pore size of approximately 10 nm. This indicates that most Cd exists in cells in a form larger than 10 nm, suggesting that the HSPs that cadmium may bind to have a relatively high molecular weight.

[0041] 3. Cadmium-binding proteins were separated and sequenced using GE-ICP-MS and cadmium chelating magnetic beads.

[0042] Cadmium-binding proteins were isolated from total Dp protein using GE-ICP-MS. Total Dp protein was extracted using an algal protein extraction kit (BestBio, China), while Cd levels were monitored. 2+ I - and Hg 2+ The cadmium ion peak appeared at 35 min, and the cadmium binding peak appeared at 75 min, which is almost identical to that of I2-labeled OVA protein (74 min). Figure 5 A). This indicates that the molecular weight of cadmium-binding protein is comparable to that of OVA.

[0043] Furthermore, the peak area of ​​the cadmium binding peak was significantly larger than that of the cadmium ion peak, further confirming that cadmium mainly exists in the form of protein binding. The fraction collected from 60 to 105 min was used for SDS-PAGE analysis. The results are as follows... Figure 5 As shown in Figure B, three protein bands were observed at 75-90 min and 91-105 min, with a molecular weight of approximately 40-45 kDa. These three bands were cut out for mass spectrometry analysis.

[0044] Simultaneously, cadmium-binding proteins were isolated by incubation with cadmium-chelating magnetic beads and total Dp protein, with empty magnetic beads serving as a control. SDS-PAGE results are shown below. Figure 5 As shown in Figure C, lane 14 contains a specific band with a molecular weight of approximately 41 kDa. No protein was found in the control group, confirming the specificity of the cadmium-binding protein. Furthermore, this band originated from a sample eluted with 100 mM imidazole, indicating that the interaction between cadmium and the protein is relatively easily disrupted.

[0045] High-resolution LC-MS / MS analysis showed that actin is likely the cadmium-binding protein in Dp, and its amino acid sequence is shown in SEQ ID NO:1, while the encoded nucleic acid sequence is shown in SEQ ID NO:2.

[0046] 4. Cadmium exposure upregulates actin expression.

[0047] The relative expression levels of actin in the control group and the cadmium-exposed group were detected by Western blotting. Total protein content was used as a normalization standard, and all samples were used in equal amounts for SDS-PAGE analysis. All samples showed the same protein pattern. Figure 6 A). Immunoblot analysis further showed that in the control group, actin protein remained at a constant level over 14 days, while in the cadmium exposure group, it was significantly upregulated after 14 days of exposure. Figure 6 B), at which point the algal cells accumulated the most cadmium, at 64.6 ± 2.8 fg / cell. Figure 3 ).

[0048] This result challenges our long-held beliefs. Actin has traditionally been considered a housekeeping gene, its expression unaffected by external stimuli, maintaining basic cellular functions and thus often used as a normalization standard for gene expression. Our study is the first to demonstrate that cadmium exposure can affect ACTIN protein expression, which may be a way for algal cells to cope with cadmium toxicity.

[0049] 5. Bioaccumulation of Cd in Escherichia coli overexpressing the above-mentioned actin

[0050] The multiple cloning site of the ACTIN gene fragment expression vector pET28a was placed under the control of the T7 promoter to obtain the overexpression plasmid pET28a-ACTIN. This plasmid was then used to transform *E. coli* BL21, and positive transformants were screened to obtain ACTIN-induced overexpressing strains. SDS-PAGE analysis showed that ACTIN appeared 12 hours after IPTG induction, with a molecular weight of approximately 41 kDa. Figure 7 A) indicates that ACTIN was successfully expressed.

[0051] Immunoblotting results as follows Figure 7 As shown in Figure B, using a His-tag antibody, the ACTIN protein was fused with the His-tag, and the specific reaction band was located at approximately 41 kDa. This result further confirms the correct fusion of the ACTIN protein with the His tag of the pET-28a vector. The expression level of soluble protein was lower than that of inclusion bodies, indicating that ACTIN mainly exists in the form of inclusion bodies.

[0052] Heterogeneously expressed ACTIN in E. coli was used to evaluate the role of this protein as a potential biological factor in Cd uptake. Figure 7 C). Compared with the control group, the transgenic E. coli in all groups showed significantly enhanced cadmium absorption capacity. At 1 mg / L, the absorption capacity increased from 11.4 (ng / 10) 7 ×CFU) increased to 28.9 (ng / 10) 7 ×CFU). Absorbance at 3 mg / L is 28.3 (ng / 10). 7 ×CFU) increased to 47.3 (ng / 10) 7 ×CFU). Absorbance at 5 mg / L is 48.0 (ng / 10). 7 ×CFU) increased to 80.5 (ng / 10) 7 ×CFU).

[0053] The enrichment of cadmium by the overexpressing strain increased with increasing exposure time, reaching 95.9 ng / 10⁻⁶ at 18 h. 7 ×CFU) reached its peak, compared to 35.8 (ng / 10) at 6h.7 ×CFU) is 2.7 times higher ( Figure 7 D). The results showed that Cd removal time was relatively long. Furthermore, overexpression of ACTIN had no effect on bacterial growth, making it a promising candidate for bioremediation.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Pavlovian algae ( Dunaliellaparva Application of cadmium removal in the environment.

2. Application of actin derived from Dunaliella pavgenia in the removal of cadmium from the environment.

3. A method for removing cadmium from the environment, characterized in that, Includes the following steps: S1: Incubate *Pavlova bafdurella* or microorganisms that overexpress actin from *Pavlova bafdurella* in an environment containing cadmium. S2: Remove the *Pavdulella* from the environment; In S1, the *Pavlova* algae or microorganisms overexpressing actin from the *Pavlova* algae are incubated in the cadmium-containing environment for 1-14 days, or longer. The cadmium content in the environment shall not exceed 50 mg / L; The amino acid sequence of the actin derived from *D. bafur* is shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that, The nucleic acid coding sequence of the actin derived from *D. pavlova* is shown in SEQ ID NO:

2.

5. The method according to claim 3 or 4, characterized in that, The microorganism in question is Escherichia coli.

6. A method for upregulating the expression of actin in microorganisms, characterized in that, This includes subjecting the microorganisms to cadmium stress; The microorganism in question is *D. pavlova*, and the cadmium stress concentration is 1-50 mg / L.

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