A method for constructing acid-tolerant microalgae based on ferredoxin

Abstract

The application discloses a kind of acid-tolerant microalgae construction methods based on ferredoxin, belong to biology and molecular biology field.The specific method is: the sequence structure as shown in SEQ ID No.1 Phatr3_J33543, sequence structure as shown in SEQ ID No.2 Phatr3_J50516 or sequence structure as shown in SEQ ID No.3 Phatr3_Jdraft1806 is used as target gene, the stop codon of target gene is removed, and the starting codon of fluorescent protein gene is connected by the DNA sequence of glycine coding, to obtain recombination gene;Recombination gene is inserted into the multiple cloning site of pPhaNR plasmid, to obtain vector plasmid;Vector plasmid is introduced into diatom cell by electroporation and then inoculated on ESW solid selection medium containing bleomycin, and brown algae appear during growth;Algae with fluorescent protein signal in algae are selected, which is acid-tolerant engineering algae.Compared with wild algae, the acid-tolerance ability of the obtained engineering algae is significantly improved.The application has important reference significance for acid-tolerant diatom cultivation.

Description

[0001] This application is a divisional application of the invention patent with application number 202311052310.3, application date August 21, 2023, and invention title "A method for constructing acid-resistant engineered algae". Technical Field

[0002] This invention belongs to the fields of biology and molecular biology, and specifically relates to a method for constructing acid-resistant microalgae based on ferricyanides. Background Technology

[0003] Microalgae, as single-celled microorganisms capable of photosynthesis, possess biomass production and carbon fixation efficiencies far exceeding those of terrestrial plants. Microalgae production is considered a highly efficient way to reduce carbon dioxide emissions while simultaneously converting them into high-value-added products; therefore, methods for producing microalgae have attracted widespread attention. Current research focuses on combining microalgae production with waste gas and wastewater treatment, thereby generating economic benefits while reducing carbon dioxide emissions and purifying wastewater. However, a technological bottleneck in this production strategy lies in the fact that the acidic environment caused by greenhouse gases hinders microalgae growth, leading to low yields. Therefore, it is necessary to improve algal strains to increase microalgae yields in acidic environments.

[0004] Major methods for microbial breeding and improvement include random mutagenesis, adaptive laboratory evolution (ALE), and genetic engineering. ALE utilizes artificially controlled stress environments to screen for beneficial spontaneous mutations, thereby accumulating variants in the population that are better able to withstand stress. This method is simple and effective, and can systematically optimize the fitness of microorganisms under stress, thus increasing biomass yield under adverse conditions. Based on the principle of screening for spontaneous beneficial random mutations in the population, ALE can cultivate algal strains with strong stress resistance. However, different populations may theoretically have different evolutionary pathways. If a single, constant selection pressure leads to the selection of variants with similar stress-resistant phenotypes in different populations, then the same gene mutations and differential regulation appearing in different populations are more likely to affect their similar phenotypes. We use transcriptomics to reveal the same gene regulation in different populations to guide genetic engineering.

[0005] *Phaeodactylum tricornutum* is a marine diatom. It grows rapidly and is rich in high-value bioactive components, including unsaturated fatty acids (such as EPA) and carotenoids (such as fucoxanthin), showing potential applications. However, marine algae thrive best in slightly alkaline conditions (generally, seawater has a pH of around 8.0), while acidic conditions inhibit diatom growth, limiting its application in directly utilizing industrial waste carbon dioxide to produce high-value-added algal biomass. Therefore, the microalgae field urgently needs to develop methods to obtain acid-resistant engineered algae. Summary of the Invention

[0006] The purpose of this invention is to solve the problem of diatom growth being inhibited by acidic environments in industrial production, and to provide a method for constructing acid-resistant microalgae based on ferruginin. According to this method, acid-resistant domesticated species can be obtained through adaptive evolution, and acid-resistant engineered algal species can be constructed through genetic engineering.

[0007] The specific technical solution adopted in this invention is as follows:

[0008] This invention provides a method for constructing acid-resistant microalgae based on ferricyanides, the specific steps of which are as follows:

[0009] S1: Genes related to annotation functions, ion channels, and pH homeostasis are selected as target genes; the stop codon of the target gene is removed, and a fluorescent protein gene with the start codon removed is ligated through a DNA sequence encoding glycine to obtain a recombinant gene; the target gene is Phatr3_J33543 with the sequence structure shown in SEQ ID No. 1;

[0010] S2: Insert the recombinant gene into the multiple cloning site of the pPhaNR plasmid containing the antibiotic resistance gene to obtain the vector plasmid;

[0011] S3: Mix the vector plasmid and salmon sperm DNA solution to obtain a mixture; after the mixture is placed on an ice bath, introduce it into diatom cells that have undergone desalination pretreatment through electroporation; then place the diatom cells in the dark for the first culture; inoculate the diatom cells after the first culture onto ESAW solid medium containing bleomycin for the second culture until algal colonies appear on the solid medium;

[0012] S4: Select algal colonies with fluorescent protein signals from the algal colonies obtained in step S3, which are acid-resistant engineered algae.

[0013] Preferably, the pPhaNR plasmid in step S2 contains the bleomycin resistance gene (ZeoR / bleoR); the inserted recombinant gene is located between the endogenous nitrate reductase promoter and the terminator, and its gene expression is regulated by the nitrate reductase promoter (pNR).

[0014] Preferably, the vector plasmid described in step S2 is amplified by introducing Escherichia coli.

[0015] Preferably, the electroporation in step S3 is performed using an electroporator with the following parameters: field strength of 0.5kV, capacitance of 25μF, and resistance of 400Ohm.

[0016] Preferably, the mass ratio of the vector plasmid to salmon sperm DNA in the mixture described in step S3 is 1:10.

[0017] Preferably, the diatom cells mentioned in step S3 are wild-type Phaeodactylum tricornutum cells.

[0018] Preferably, the desalination pretreatment step S3 is as follows: After centrifuging and collecting the cells of *Phaeodactylum tricornutum* in the logarithmic growth phase, the precipitated *Phaeodactylum tricornutum* cells are washed with sorbitol solution to remove salt and then resuspended in sorbitol solution after repeated centrifugation and resuspension.

[0019] Furthermore, the concentration of the above sorbitol solution is 375 mM.

[0020] As a preferred method, the first culture is carried out in the dark for 24 hours, and the second culture is carried out for 2 to 3 weeks.

[0021] Preferably, the concentration of bleomycin in the ESAW solid selective medium described in step S3 is 100 μg / L.

[0022] Preferably, the intensity of the fluorescent protein signal in step S4 is 6 to 10 times that of the green fluorescence in wild-type algal cells.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] This invention provides a method for constructing acid-tolerant engineered algae, which enhances the acid tolerance of marine diatoms and improves their growth in low-pH environments. The invention constructs engineered algae by inserting three target genes into wild-type *Phaeodactylum tricornutum*. Experiments show that all three groups of engineered algae exhibit significantly improved tolerance to acidic stimuli, and their maximum growth rate is 1.7 to 1.8 times that of the wild type. This method has significant implications for the cultivation of acid-tolerant diatoms. Attached Figure Description

[0025] Figure 1 This is the curve of the adaptive evolution process of wild-type *Phaeodactylum tricornutum* under low pH environmental pressure in Example 1;

[0026] Figure 2 The specific growth rates of wild-type and domesticated *Phaeodactylum tricornutum* under different acidic conditions in Example 1 are shown in Figure 1 (**, p < 0.01; *, p < 0.05).

[0027] Figure 3 The diagram shows the construction of acid-resistant engineered algae in Examples 2-4 (a) and the comparison diagram of the relative intensity of green fluorescence signals (b).

[0028] Figure 4The growth performance of the acid-resistant engineered algae and wild-type algae constructed in Examples 2-4 under pH=5.0 stimulation is shown in (a) as growth curve, (b) as fluorescence quantum yield, (c) as maximum specific growth rate under optimal conditions and low pH pressure, and (d) as the fold increase in maximum specific growth rate of the transformant relative to the wild type (compared with the control group: NS, no significant difference, ***, p<0.001; **, p<0.01). Detailed Implementation

[0029] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0030] Example 1: Breeding acid-tolerant diatoms and obtaining the target gene for acid tolerance

[0031] (1) Under artificially controlled constant environmental pressure, long-term subculture was carried out to domesticate acid-resistant algae species.

[0032] The pH of the artificial seawater culture medium was maintained at a stable slightly acidic level of 6.0 using 40 mM MES buffer. Wild-type *Phaeodactylum tricornutum* cells were subcultured in this medium under continuous low pH pressure conditions: light intensity 100 μmol / m². 2 The culture was carried out at a temperature of 22±1℃, with a subculture cycle of 5 days. At the end of each cycle, appropriate cells were collected by centrifugation and resuspended in the same volume (75 mL) of fresh culture medium to maintain a consistent initial cell concentration for each cycle. After 16 subcultures, the average growth rate of the three algal solutions significantly increased and stabilized at five days. Figure 1 As shown, the three *Phaeodactylum tricornutum* populations ALE1, ALE2, and ALE3, which exhibited higher growth rates under acidic conditions, were obtained.

[0033] To test the stability of three domesticated *Phaeodactylum tricornutum* groups (ALE1, ALE2, and ALE3), all three groups were restored to their optimal growth conditions and cultured for two weeks. The culture conditions were: light intensity 100 μmol / m². 2At a temperature of 22±1℃, 40mM Tris-HCl buffer was added to maintain the pH of the artificial seawater culture medium at 8.0 during the culture process. Then, appropriate amounts of domesticated *Phaeodactylum tricornutum* cells were collected by centrifugation. Each group of domesticated *Phaeodactylum tricornutum* cells was divided into three aliquots, washed once with acidic culture media at pH 6.5, pH 6.0, and pH 5.5 respectively, and then collected by centrifugation. The aliquots were then inoculated into culture media at the corresponding pH values ​​for stability testing. After 5 days of culture, the growth performance of domesticated and wild-type *Phaeodactylum tricornutum* under acidic stimulation was compared. The results are as follows: Figure 2 As shown, the growth rate of the domesticated *Phaeodactylum tricornutum* was higher than that of the wild type under all test conditions, with an increase of 27.5% to 110.4%.

[0034] (2) The acid-tolerant target gene common to the above-mentioned domesticated brown finger algae was identified by transcriptome sequencing.

[0035] Both domesticated and wild-type algal strains were transferred from their optimal growth conditions of pH 8.0 to a low-pH stress environment of pH 6.0. The culture conditions were: light intensity 100 μmol / m². 2 The culture was carried out at a temperature of 22±1℃, with 40mM MES buffer added to maintain the pH at 6.0 during the culture process. Cells were collected on day 5, and total RNA was extracted for next-generation transcriptome sequencing. The transcriptome of wild-type *Phaeodactylum tricornutum* under acidic stimulation was used as a control group, and differential expression analysis was performed on the transcriptomes of the three groups of domesticated *Phaeodactylum tricornutum* under the same conditions.

[0036] Among genes showing significant differential expression (fold change > 1) in two or more domesticated *Phaeodactylum tricornutum* species, several genes related to annotation functions and ion channels and pH homeostasis were selected as target genes. The selected target genes were Phatr3_J33543 (SEQ ID No. 1), Phatr3_J50516 (SEQ ID No. 2), and Phatr3_Jdraft1806 (SEQ ID No. 3).

[0037] Example 2: Construction of engineered algae HI3310

[0038] This embodiment provides a method for constructing acid-resistant engineered algae using the sequence structure Phatr3_J33543 as shown in SEQ ID No. 1. The specific steps are as follows:

[0039] (1) The stop codon at the 3' end of the Phatr3_J33543 gene, whose sequence structure is shown in SEQ ID No. 1, was removed, and the fluorescent protein (GFP) gene, which encodes a DNA sequence of 5 glycine residues, was removed by ligation to obtain the recombinant gene. Since the recombinant protein encoded by this recombinant gene contains a fluorescent protein sequence, it is convenient to screen it using a fluorescence microscope.

[0040] The recombinant gene plasmid was inserted into the multiple cloning site of the pPhaNR plasmid (NCBI ID: JN180663.1) to obtain the vector plasmid. The pPhaNR plasmid is an amplification-capable plasmid containing a gene that resists bleomycin. Bleomycin is an antibiotic that can kill wild-type *Phaeodactylum tricornutum* cells.

[0041] The constructed vector plasmid was introduced into *E. coli*, allowing it to amplify within the bacteria. The amplified vector plasmid was then purified for later use.

[0042] (2) The expression vector plasmid obtained in step (1) was introduced into wild-type *Phaeodactylum tricornutum* cells using an electroporator, as follows:

[0043] First, centrifuge at 1000x g for 10 minutes to collect approximately 2×10⁻⁶ g of the sample. 8 CFU-containing *Phaeodactylum tricornutum* cells in logarithmic growth phase. The precipitate after centrifugation was washed three times with 375 mM sorbitol solution, repeatedly centrifuged and resuspended for desalting pretreatment. Finally, the pretreated cells were resuspended in 100 μL of 375 mM sorbitol solution, and 4 μg of the vector plasmid obtained in step (1) and 40 μg of salmon sperm DNA were added to obtain a mixture. The mixture was incubated in an ice-water bath for 10 minutes and then transferred to a 2 mm electroporation cuvette. Salmon sperm DNA facilitates the entry of the vector plasmid into the algal cells.

[0044] The above mixture was introduced into wild-type *Phaeodactylum tricornutum* cells using a bio-Rad perforator with the following parameters: field strength 0.5 kV, capacitance 25 μF, and resistance 400 Ohm. After incubating the wild-type *Phaeodactylum tricornutum* cells with the introduced vector plasmid in the dark for 24 hours, they were inoculated into ESAW solid medium containing 100 μg / mL bleomycin. After 2–3 weeks, brown algal colonies appearing on the medium were selected and transferred to test tubes containing artificial seawater culture medium. The culture conditions were: light intensity 100 μmol / m². 2 The temperature was 22±1℃, and 40mM Tris-HCl buffer was added to maintain the pH at 8.0 during the incubation process.

[0045] ESAW solid culture medium was prepared according to the following formula: the pH was adjusted to 8.0, 1% (w / v) agar powder was added, and the medium was sterilized at 121°C for 30 minutes. The specific components of the ESAW artificial seawater culture medium are shown in Table 1.

[0046] Table 1 Composition of ESAW artificial seawater culture medium

[0047]

[0048]

[0049] To avoid false positives in the brown algal colonies selected from the bleomycin-containing ESAW solid medium, the algal cells also need to be observed using a fluorescence microscope. The excitation wavelength of the fluorescence microscope is 488 nm, and the detection wavelength is 525 nm. Algal species with fluorescent protein (GFP) signals are selected as the constructed acid-resistant engineered algae, denoted as HI3310.

[0050] Example 3: Construction of engineered algae HI5021

[0051] This embodiment provides a method for constructing acid-resistant engineered algae using the sequence structure Phatr3_J50516 as shown in SEQ ID No. 2. The specific steps are as follows:

[0052] (1) The stop codon at the 3' end of the Phatr3_J50516 gene, whose sequence structure is shown in SEQ ID No. 2, was removed, and a fluorescent protein (GFP) gene encoding a 5-glycine DNA sequence was ligated to remove the start codon, resulting in a recombinant gene. Since the recombinant protein encoded by this recombinant gene contains a fluorescent protein sequence, it is convenient for subsequent screening using a fluorescence microscope.

[0053] The recombinant gene was inserted into the multiple cloning site of the pPhaNR plasmid (NCBI ID: JN180663.1) to obtain the vector plasmid. The pPhaNR plasmid is an amplification-capable plasmid containing a gene that resists bleomycin. Bleomycin is an antibiotic that can kill wild-type *Phaemodum triangularis* cells.

[0054] The constructed vector plasmid was introduced into *E. coli*, allowing it to amplify within the bacteria. The amplified vector plasmid was then purified for later use.

[0055] (2) The expression vector plasmid obtained in step (1) was introduced into wild-type *Phaeodactylum tricornutum* cells using an electroporator, as follows:

[0056] First, centrifuge at 1000x g for 10 minutes to collect approximately 2×10⁻⁶ g of the sample. 8CFU-containing *Phaeodactylum tricornutum* cells in logarithmic growth phase. The precipitate after centrifugation was washed three times with 375 mM sorbitol solution, repeatedly centrifuged and resuspended for desalting pretreatment. Finally, the pretreated cells were resuspended in 100 μL of 375 mM sorbitol solution, and 4 μg of the vector plasmid obtained in step (1) and 40 μg of salmon sperm DNA were added to obtain a mixture. The mixture was incubated in an ice-water bath for 10 minutes and then transferred to a 2 mm electroporation cuvette. Salmon sperm DNA facilitates the entry of the vector plasmid into the algal cells.

[0057] The above mixture was introduced into wild-type *Phaeodactylum tricornutum* cells using a bio-Rad perforator with the following parameters: field strength 0.5 kV, capacitance 25 μF, and resistance 400 Ohm. After incubating the wild-type *Phaeodactylum tricornutum* cells with the introduced vector plasmid in the dark for 24 hours, they were inoculated into ESAW solid medium containing 100 μg / mL bleomycin. After 2–3 weeks, brown algal colonies appearing on the medium were selected and transferred to test tubes containing artificial seawater culture medium. The culture conditions were: light intensity 100 μmol / m². 2 The temperature was 22±1℃, and 40mM Tris-HCl buffer was added to maintain the pH at 8.0 during the incubation process.

[0058] ESAW solid culture medium was prepared according to the following formula: the pH was adjusted to 8.0, 1% (w / v) agar powder was added, and the medium was sterilized at 121°C for 30 minutes. The specific components of the ESAW artificial seawater culture medium are shown in Table 1.

[0059] To avoid false positives in the brown algal colonies selected from the bleomycin-containing ESAW solid medium, the algal cells also need to be observed using a fluorescence microscope. The excitation wavelength of the fluorescence microscope is 488 nm, and the detection wavelength is 525 nm. The algal species with fluorescent protein (GFP) signals are selected as the constructed acid-resistant engineered algae, denoted as HI5021.

[0060] Example 4: Construction of engineered algae HI1831

[0061] This embodiment provides a method for constructing acid-resistant engineered algae using the sequence structure Phatr3_Jdraft1806 as shown in SEQ ID No. 3. The specific steps are as follows:

[0062] (1) The stop codon at the 3' end of the Phatr3_Jdraft1806 gene, whose sequence structure is shown in SEQ ID No. 3, was removed, and the fluorescent protein (GFP) gene, which had its start codon removed, was ligated through a DNA sequence encoding 5 glycine residues to obtain a recombinant plasmid. Since the recombinant protein encoded by this recombinant gene contains a fluorescent protein sequence, it is convenient to screen it using a fluorescence microscope.

[0063] The recombinant gene was inserted into the multiple cloning site of the pPhaNR plasmid (NCBI ID: JN180663.1) to obtain the vector plasmid. The pPhaNR plasmid is an amplification-capable plasmid containing a gene that resists bleomycin. Bleomycin is an antibiotic that can kill wild-type *Phaemodum triangularis* cells.

[0064] The constructed vector plasmid was introduced into *E. coli*, allowing it to amplify within the bacteria. The amplified vector plasmid was then purified for later use.

[0065] (2) The expression vector plasmid obtained in step (1) was introduced into wild-type *Phaeodactylum tricornutum* cells using an electroporator, as follows:

[0066] First, centrifuge at 1000x g for 10 minutes to collect approximately 2×10⁻⁶ g of the sample. 8 CFU-containing *Phaeodactylum tricornutum* cells in logarithmic growth phase. The precipitate after centrifugation was washed three times with 375 mM sorbitol solution, repeatedly centrifuged and resuspended for desalting pretreatment. Finally, the pretreated cells were resuspended in 100 μL of 375 mM sorbitol solution, and 4 μg of the vector plasmid obtained in step (1) and 40 μg of salmon sperm DNA were added to obtain a mixture. The mixture was incubated in an ice-water bath for 10 minutes and then transferred to a 2 mm electroporation cuvette. Salmon sperm DNA facilitates the entry of the vector plasmid into the algal cells.

[0067] The above mixture was introduced into wild-type *Phaeodactylum tricornutum* cells using a bio-Rad perforator with the following parameters: field strength 0.5 kV, capacitance 25 μF, and resistance 400 Ohm. After incubating the wild-type *Phaeodactylum tricornutum* cells with the introduced vector plasmid in the dark for 24 hours, they were inoculated into ESAW solid medium containing 100 μg / mL bleomycin. After 2–3 weeks, brown algal colonies appearing on the medium were selected and transferred to test tubes containing artificial seawater culture medium. The culture conditions were: light intensity 100 μmol / m². 2 The temperature was 22±1℃, and 40mM Tris-HCl buffer was added to maintain the pH at 8.0 during the incubation process.

[0068] ESAW solid culture medium was prepared according to the following formula: the pH was adjusted to 8.0, 1% (w / v) agar powder was added, and the medium was sterilized at 121°C for 30 minutes. The specific components of the ESAW artificial seawater culture medium are shown in Table 1.

[0069] To avoid false positives in the brown algal colonies selected from the bleomycin-containing ESAW solid medium, the algal cells also need to be observed using a fluorescence microscope. The excitation wavelength of the fluorescence microscope is 488 nm, and the detection wavelength is 525 nm. Algal species with fluorescent protein (GFP) signals are selected as the constructed acid-resistant engineered algae, denoted as HI1831.

[0070] The engineered algae constructed in Examples 2-4 were tested using acidic stimulation experiments. First, the growth performance of the three engineered algae groups and the wild-type *Phaeodactylum tricornutum* was compared under optimal growth conditions. The culture conditions were: light intensity 100 μmol / m². 2 The culture time was 22±1℃, and the pH of the artificial seawater containing 40mM Tris-HCl buffer was maintained at 8.0 during the culture process.

[0071] Then, a suitable amount of cells was collected by centrifugation and washed once with acidic culture medium at pH 5.0. The cells were collected again by centrifugation and inoculated into artificial seawater culture medium at pH 5. After 5 days of culture, the growth performance of the three groups of engineered algae and wild-type *Phaeodactylum tricornutum* under acidic stimulation was compared, as detailed below. Figure 4 As shown in the figure. The experimental results showed that the wild-type *Phaeodactylum tricornutum* was strongly inhibited in an environment of pH 5.0, while the tolerance of the three groups of engineered algae to acidic stimulation was significantly improved, and the maximum growth rate of the engineered algae was 1.7 to 1.8 times that of the wild type.

[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A method for constructing acid-resistant microalgae based on ferricyanides, characterized in that, The specific steps are as follows: S1: Genes related to annotation functions, ion channels, and pH homeostasis are selected as target genes; the stop codon of the target gene is removed, and a fluorescent protein gene with the start codon removed is ligated through a DNA sequence encoding glycine to obtain a recombinant gene; the target gene is Phatr3_J33543 with the sequence structure shown in SEQ ID No. 1; S2: Insert the recombinant gene into the multiple cloning site of the pPhaNR plasmid containing the antibiotic resistance gene to obtain the vector plasmid; S3: The vector plasmid and salmon sperm DNA solution are mixed to obtain a mixture; after the mixture is placed on ice, it is introduced into diatom cells that have undergone desalination pretreatment via electroporation. The diatom cells are then placed in ESAW culture medium and kept in the dark for the first culture; the diatom cells after the first culture are inoculated onto ESAW solid selective medium containing bleomycin for the second culture until algal colonies appear on the solid selective medium; the diatom cells are wild-type Phaeodactylum tricornutum cells; S4: Select algal colonies with fluorescent protein signals from the algal colonies obtained in step S3, which are acid-resistant engineered algae.

2. The method for constructing acid-resistant microalgae based on ferrugin according to claim 1, characterized in that, The pPhaNR plasmid described in step S2 contains the bleomycin resistance gene ZeoR / bleoR; the inserted recombinant gene is located between the endogenous nitrate reductase promoter and terminator, and its gene expression is regulated by the nitrate reductase promoter pNR.

3. The method for constructing acid-resistant microalgae based on ferricyanide according to claim 1, characterized in that, The electroporation process described in step S3 uses an electroporator with the following parameters: field strength of 0.5kV, capacitance of 25μF, and resistance of 400Ohm.

4. The method for constructing acid-resistant microalgae based on ferrugin according to claim 1, characterized in that, In step S3, the mass ratio of the vector plasmid to salmon sperm DNA in the mixture is 1:

10.

5. The method for constructing acid-resistant microalgae based on ferrugin according to claim 1, characterized in that, The desalination pretreatment steps described in step S3 are as follows: After centrifuging and collecting the cells of *Phaeodactylum tricornutum* in the logarithmic growth phase, the precipitated *Phaeodactylum tricornutum* cells are repeatedly centrifuged and resuspended. The precipitated cells are then washed with sorbitol solution to remove salt and resuspended in sorbitol solution.

6. The method for constructing acid-resistant microalgae based on ferrugin according to claim 5, characterized in that, The concentration of the sorbitol solution was 375 mM.

7. The method for constructing acid-resistant microalgae based on ferrugin according to claim 1, characterized in that, The first culture was carried out in the dark for 24 hours; the second culture was carried out for 2 to 3 weeks.

8. The method for constructing acid-resistant microalgae based on ferrugin according to claim 1, characterized in that, The concentration of bleomycin in the ESAW solid selective medium described in step S3 is 100 μg / L.

9. The method for constructing acid-resistant microalgae based on ferrugin according to claim 1, characterized in that, In step S4, the intensity of the fluorescent protein signal is 6 to 10 times that of the green fluorescence in wild-type algal cells.

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