Method for increasing content of triglyceride-type eicosapentaenoic acid in marine microspheroidic cells

Abstract

The invention relates to the technical field of genetic engineering algae, in particular to a method for increasing the content of triglyceride-type eicosapentaenoic acid in marine globoid cells. The method comprises the following steps: constructing an expression vector comprising a delta 5 desaturase gene, an omega 3 desaturase gene and a delta 6 elongase gene; the expression vector adopts a Ribi bidirectional promoter, one end of the Ribi bidirectional promoter is connected with a delta 6 elongase gene, a delta 5 desaturase gene and an omega 3 desaturase gene are connected through a 2A peptide sequence, a polycistron is formed in a single open reading frame, and the polycistron is connected to the other end of the Ribi bidirectional promoter; the base sequence of the delta 6 elongase gene is as shown in SEQ ID NO. 1; the base sequence of the delta 5 desaturase gene is as shown in SEQ ID NO. 2; and the omega3 desaturase gene is as shown in SEQ ID NO. 3. The expression vector is transferred into marine nannochloropsis oculata chassis cells, so that the content of triglyceride eicosapentaenoic acid in the marine nannochloropsis oculata cells is increased.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering algae, in particular to a method for increasing the content of triglyceride-type eicosapentaenoic acid in marine microsphere cells. Background Art

[0002] Eicosapentaenoic acid (EPA) is a well-known omega-3 polyunsaturated fatty acid (PUFA) that has attracted much attention due to its significant health benefits. Traditionally, EPA is mainly derived from marine fish oil, but in recent years, the production of EPA through biotechnological methods using marine microalgae has gradually become a more sustainable and scalable alternative. Among them, Nannochloropsis oceanica is considered to be an ideal candidate microalgae for industrial production of EPA due to its high lipid content, rapid growth, and strong EPA synthesis ability. Studies have shown that different forms of EPA have their own unique physiological functions. Phospholipid-type EPA is closely related to improved metabolism, enhanced cognitive function, and neural repair; glycolipid-type EPA exhibits anti-inflammatory properties and plays a role in lipid regulation; while triglyceride-type EPA has attracted attention for its functions in regulating blood lipids, enhancing intestinal absorption, anti-inflammatory and anti-tumor activities. However, in marine Nannochloropsis cells, under nitrogen deficiency conditions, membrane lipids such as monogalactosyldiacylglycerol (MGDG) and phosphatidylglycerol (PG) are degraded, and the released fatty acids are redirected for the synthesis of triacylglycerol (TAG). This lipid remodeling process reduces the availability of EPA precursors, resulting in a significant decrease in EPA levels.

[0003] The biosynthesis pathway of EPA in microalgae includes complex reactions mediated by Δ12 desaturase (Δ12D), Δ6 desaturase (Δ6D), Δ6 elongase (Δ6E), Δ5 desaturase (Δ5D) and ω3 desaturase (ω3D). Among them, key enzymes such as Δ6 desaturase (Δ6D), Δ5 desaturase (Δ5D) and Δ6 elongase (Δ6E) play a central role in converting precursor fatty acids to EPA. In marine Nannochloropsis fulva, EPA is believed to be generated by desaturation of arachidonic acid (ARA) via the omega-6 pathway. Traditional genetic engineering strategies usually focus on single enzymes and fail to fully explore the combinatorial potential of multiple biosynthetic elements. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for increasing the content of triglyceride-type eicosapentaenoic acid in marine microspheroid cells, mainly by overexpressing the cDNA sequences of Δ5 desaturase (Δ5D) and ω3 desaturase (ω3D) in a 1:1 ratio in marine microspheroid cells, and co-expressing Δ6 elongase (Δ6E), thereby increasing the content and yield of triglyceride-type EPA in marine microspheroid cells under nitrogen-deficient conditions, which solves the problem that the EPA level in marine microspheroid algae decreases significantly under nitrogen-deficient conditions.

[0006] (II) Technical solution

[0007] The present invention provides an expression vector for increasing the content of triglyceride-type eicosapentaenoic acid in marine microspheroid cells, wherein the expression vector comprises a Δ5 desaturase gene, an ω3 desaturase gene and a Δ6 elongase gene;

[0008] The expression vector adopts a Ribi bidirectional promoter, one end of the Ribi bidirectional promoter is connected to a Δ6 elongase gene; the Δ5 desaturase gene and the ω3 desaturase gene are connected through a 2A peptide sequence to form a polycistron in a single open reading frame, and the polycistron is connected to the other end of the Ribi bidirectional promoter; the base sequence of the Δ6 elongase gene is shown in SEQ ID NO.1; the base sequence of the Δ5 desaturase gene is shown in SEQ ID NO.2; and the ω3 desaturase gene is shown in SEQ ID NO.3.

[0009] According to a preferred embodiment of the present invention, the expression vector further comprises a screening gene, wherein the screening gene is connected to the end of the polycistronic composed of the Δ5 desaturase gene and the ω3 desaturase gene via a 2A linker sequence.

[0010] According to a preferred embodiment of the present invention, the 2A linker sequence is P2A, F2A or T2A.

[0011] According to a preferred embodiment of the present invention, the base sequence of F2A is shown as SEQ ID NO.4, and the base sequence of P2A is shown as SEQ ID NO.5.

[0012] According to a preferred embodiment of the present invention, the screening gene is the hygromycin resistance gene Hygr, and its base sequence is shown in SEQ ID NO.6.

[0013] According to a preferred embodiment of the present invention, the expression vector comprises a Ribi bidirectional promoter, one end of the Ribi bidirectional promoter is connected to a Δ6 elongase gene, and the end of the Δ6 elongase gene is connected to a terminator; the other end of the Ribi bidirectional promoter is sequentially connected to a Δ5 desaturase gene, a 2A linker sequence, an ω3 desaturase gene, a 2A linker sequence and a hygromycin resistance gene Hygr.

[0014] Preferably, a 6×His tag can be connected to the end of the three regulatory genes, namely the Δ5 desaturase gene, the ω3 desaturase gene and the Δ6 elongase gene, to facilitate purification, detection and protein localization.

[0015] According to a preferred embodiment of the present invention, the full-length sequence of the expression vector is shown as SEQ ID NO.7.

[0016] In a second aspect, the present invention provides a marine Nannochloropsis algae that produces high levels of triglyceride-type eicosapentaenoic acid, comprising the expression vector described in any one of the above embodiments.

[0017] In a third aspect, the present invention provides a method for increasing the content of triglyceride-type eicosapentaenoic acid in marine microspheroid cells, comprising: transferring the expression vector into marine microspheroid chassis cells to construct an overexpression strain, and culturing the overexpression strain under nitrogen-deficient conditions to thereby increase the content of triglyceride-type eicosapentaenoic acid in the marine microspheroid cells.

[0018] According to a preferred embodiment of the present invention, the marine Nannochloropsis chassis cell is marine Nannochloropsis IMET1 or other Nannochloropsis algae strains. Marine Nannochloropsis chassis cell strain IMET1 (from the Institute of Marine and Environmental Technology, University of Maryland System) can be obtained by contacting the relevant person in charge.

[0019] (III) Beneficial effects

[0020] The present invention constructs an expression vector, transfers the expression vector into the marine Nannochloropsis chassis cells, and overexpresses the cDNA sequences of Δ5 desaturase (Δ5D) and ω3 desaturase (ω3D) in the marine Nannochloropsis cells at a ratio of 1:1, and co-expresses Δ6 elongase (Δ6E), thereby increasing the content and yield of triglyceride EPA in the marine Nannochloropsis cells under nitrogen deficiency conditions, thereby solving the problem that the EPA level in marine Nannochloropsis decreases significantly under nitrogen deficiency conditions. The present invention achieves the purpose of increasing the content of triglyceride EPA in marine Nannochloropsis by combining key genes in the EPA synthesis pathway, so that marine Nannochloropsis can be better applied to the production industry of triglyceride EPA, and provides good algae species resources for regulating blood lipids, enhancing intestinal absorption, anti-inflammatory and anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a schematic diagram of the connection structure of an expression vector according to a preferred embodiment of the present invention.

[0022] Figure 2 The present invention is a screening process for transferring the expression vector of the present invention into the marine Nannochloropsis algae chassis cells.

[0023] Figure 3 The figure shows the changes in oil content when the expression vector is overexpressed in marine Nannochloropsis algae; (A) is the content of total fatty acids in dry weight; (B) is the content of triglycerides in dry weight; (C) is the proportion of EPA in triglycerides; (D) is the content of triglyceride-type EPA; EV is the control group strain, and OE is the overexpression strain.

[0024] Figure 4 The figures show the changes in total fatty acid production (A), triglyceride production (B), and triglyceride-type EPA production (C) when the expression vector is overexpressed in marine Nannochloropsis algae; EV is the control group strain, and OE is the overexpression strain. DETAILED DESCRIPTION

[0025] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0026] The present invention first provides an expression vector for increasing the content of triglyceride-type eicosapentaenoic acid in marine microspheroid cells, wherein the expression vector comprises a Δ5 desaturase gene, an ω3 desaturase gene and a Δ6 elongase gene; the expression vector adopts a Ribi bidirectional promoter, one end of the Ribi bidirectional promoter is connected to the Δ6 elongase gene, the Δ5 desaturase gene and the ω3 desaturase gene are connected through a 2A peptide sequence, and a polycistron is formed in a single open reading frame, and the polycistron is connected to the other end of the Ribi bidirectional promoter; the base sequence of the Δ6 elongase gene is shown in SEQ ID NO.1; the base sequence of the Δ5 desaturase gene is shown in SEQ ID NO.2; and the ω3 desaturase gene is shown in SEQ ID NO.3.

[0027] Polycistronic proteins can be translated from one mRNA molecule into multiple independent proteins. They can break the newly formed polypeptide chain at the junction sequence through a specific mechanism during the translation process, thereby releasing independent functional proteins.

[0028] The 2A linker sequence is P2A, F2A or T2A. The base sequence of F2A is shown in SEQ ID NO.4, and the base sequence of P2A is shown in SEQ ID NO.5. The 2A linker sequence is a short protein coding sequence that automatically "cuts" itself during translation when it connects two protein coding regions, releasing two mature proteins. This mechanism does not cause any additional burden on the host cell because it occurs during the translation stage rather than relying on later protease treatment.

[0029] Preferably, the expression vector further comprises a selection gene, which is connected to the end of the genome composed of the Δ5 desaturase gene or the ω3 desaturase gene with a 2A connection sequence. This method ensures that in the algae strains screened on the resistance plate, other genes located in the same polycistronic as the resistance gene can also be effectively expressed.

[0030] Preferably, the screening gene is the hygromycin resistance gene Hygr, and its base sequence is shown in SEQ ID NO.6.

[0031] Preferably, the expression vector is constructed using a pRibi vector.

[0032] The above gene names, sequence numbers and base sequences are shown in Table 1.

[0033] Table 1:

[0034]

[0035]

[0036]

[0037]

[0038] Example 1

[0039] like Figure 1As shown, it is a schematic diagram of the connection structure of an expression vector of a preferred embodiment of the present invention. The expression vector includes a Ribi bidirectional promoter, one end of the Ribi bidirectional promoter is connected to the Δ6 elongase gene, and the end of the Δ6 elongase gene is connected to a 6×His tag and a terminator; the other end of the Ribi bidirectional promoter is sequentially connected to the Δ5 desaturase gene and the 6×His tag, the F2A connection sequence, the ω3 desaturase gene and the 6×His tag, the P2A connection sequence and the hygromycin resistance gene Hygr, and the 3' untranslated region. Among them, the Δ5 desaturase gene and the ω3 desaturase gene can be connected at interchangeable positions, but they must be connected with a 2A connection sequence, and the molar ratio of the Δ5 desaturase gene to the ω3 desaturase gene is 1:1. The Δ5 desaturase gene and the ω3 desaturase gene are connected through the 2A connection sequence to form a polycistronic in a single open reading frame. Preferably, the hygromycin resistance gene Hygr and two genes including the Δ5 desaturase gene and the ω3 desaturase gene are in the same polycistronic, and are also connected using the 2A linker sequence, so as to ensure that in the algae strains screened on the resistance plate, other genes located in the same polycistronic as the resistance gene can also be effectively expressed.

[0040] In the following embodiments, Figure 1 The expression vector is constructed by using the connection structure shown in the figure, and the expression vector is transferred into the marine Nannochloropsis algae chassis cell IMET1 by electroporation. After screening, positive clones, i.e., overexpression strains, are obtained; the overexpression strains are cultured under nitrogen deficiency conditions, and the lipids in the overexpression strains, including the content and yield changes of total fatty acids, triglycerides, and triglyceride-type EPA, are studied and analyzed. The expression vector is constructed with the pYES2 / CT vector to obtain a vector with a full-length sequence as shown in SEQ ID NO.7, which is represented as pRIBI-HyG-Δ5D-ω3D-Δ6E. Then, the expression vector shown in SEQ ID NO.7 was transformed into the marine Nannochloropsis algae chassis cell IMET1 by electrotransformation. The specific steps are as follows: 50 mL of marine Nannochloropsis algae cells in the logarithmic phase were collected by centrifugation at 3000×g for 10 min, the algae cells were resuspended with 1.5 mL of pre-cooled 7% sorbitol solution and transferred to a 2 mL centrifuge tube, the supernatant was discarded after centrifugation at 3000×g for 2 min, and then washed three times with 1.5 mL of pre-cooled 7% sorbitol solution, and finally resuspended in 300 μL of sorbitol solution; 3 μg of plasmid and 5 μL of denatured salmon sperm DNA were added thereto, and the cells were electroporated after ice bathing for 30 min. The parameters were set as follows: 50 μFD, 600Ω, 2400V; ice bathing for 5 min after electroporation, and then incubated with 10 mL of f / 2 medium for 24 h and then coated on a hygromycin-resistant f / 2 plate. A single clone can grow in about 3-4 weeks.

[0041] After a single clone grew out, PCR was used to verify the insertion of Δ5D and ω3D at the genomic level. Chelex-100 was used to extract crude DNA, and primers were designed based on the 5' sequence of Δ5D cDNA and the 3' sequence of ω3D cDNA, respectively, to amplify and obtain positive clones with polycistronic insertion of Δ5D-F2A-ω3D.

[0042] The primers were: 5'-ACACGCAACCGATCCATGCCTCCCCAGAACGAC-3' and 5'-CTTCGACATCTCCGGCTTGTTTCAGCAGAGAGAAGTTTGTT-3'. After screening, a total of 7 positive clones were obtained (screening results are shown in Figure 2 The positive clones were stored on plates.

[0043] Example 2

[0044] Algae were picked from the seed plate and inoculated into a conical flask containing 10 mL of f / 2 liquid culture medium at 23°C, 150 rpm, and 50 μmol / m 2 / s light intensity for 4 days, expanded to a triangular flask containing 100mL f / 2 liquid culture medium, and cultured for 4 days as seed solution. The seed solution was inoculated into a triangular flask containing 200mL f / 2 nitrogen-deficient liquid culture medium at an initial concentration of OD750=1.5, and incubated at 23°C, 150rpm, 50μmol / m 2 / s light intensity for 3 days, dry weight was measured every day during the culture process, and samples were taken for lipid analysis on the 1st and 3rd days. 200μL chloroform: methanol = 2: 1 (v / v) and 300μL 5% hydrochloric acid methanol (v / v) were added to the freeze-dried algae powder and heated in a metal bath at 85℃ for 1h. After standing and cooling to room temperature, 1mL of n-hexane was added, vortexed for 30s, and then fully shaken and mixed and allowed to stand at room temperature for 2h. 200μL was transferred to a GC loading bottle and 5μL of pentadecane internal standard was added for quantification. The fatty acid composition of the overexpression strain and the control (pRibi-Hyg empty vector without key genes was transferred into the marine Nannochloropsis chassis cells) was quantitatively analyzed using an AGIENT 7890B GC gas chromatography system (flame ionization detector FID and HP-88 column (60m*0.25mm, 0.2μm). Gas chromatography conditions: carrier gas (nitrogen) flow rate 2mL / min; no split; injector and FID temperatures of 250℃ and 280℃; the column box temperature was first maintained at 50℃ for 2min, then increased by 25℃ per minute to 175℃, maintained for 5min, and continued to increase the temperature at 7℃ per minute to 210℃, maintained for 2min, and then increased to 230℃ at a rate of 2℃ per minute. Using 37 fatty acid methyl esters as standards, a standard curve was established and the content and composition of each fatty acid in the samples were quantitatively analyzed.

[0045] See also Figure 3-4 The figure shows the effect of the expression vector on the accumulation of EPA and total fatty acids in the cells of Nannochloropsis edulis. N1 represents the first day of anoxic culture, N3 represents the third day of anoxic culture, EV represents the control algae strain (transferred with the pRibi-Hyg empty vector without key genes), and OE represents the overexpression algae strain. The total fat content (% of dry weight) and triglyceride content (% of dry weight) of the overexpression algae strain did not change significantly compared with the control on the first day of nitrogen deficiency culture ( Figure 3 , A and B). On the third day of nitrogen deficiency, the total fat content of the overexpression strain increased significantly by 11%, and the triglyceride content increased significantly by 76% (the triglyceride content was significantly increased compared with the control group on the first day of nitrogen deficiency). The EPA content in triglycerides of the overexpression strain increased significantly by 18% and 40% on the first and third days of nitrogen deficiency, respectively, compared with the control group ( Figure 3 , C). The content of triglyceride EPA in the overexpression algae strain (% of dry weight) is shown in Figure 3 The D of the control group increased significantly by 94% (1.94 times) and 150% (2.5 times) on the first and third days of nitrogen deficiency, respectively. Triglyceride EPA refers to EPA in the form of triglycerides, which are ester compounds composed of one glycerol molecule and three fatty acid molecules. When the fatty acid is EPA (C20:5, n-3), that is, eicosapentaenoic acid, triglyceride EPA is formed.

[0046] See also Figure 4 As shown, on the third day of nitrogen deficiency, the total fatty acid production of the overexpressing algae strain reached 197.2 mg / L, an increase of 17% over the control ( Figure 4 , A); triglycerides increased by 87% compared with the control, reaching 139.8 mg / L ( Figure 4 , B). The production of triglyceride EPA in the overexpressing algae strain reached 2.4 mg / L on the third day of nitrogen deficiency, an increase of 163% compared with the control ( Figure 4 , C).

[0047] In summary, the present invention constructs a polycistronic construct of Δ5D, ω3D and antibiotic resistance genes with 2A sequence, which can not only ensure the overexpression of Δ5D and ω3D through resistance screening, but also make Δ5D and ω3D overexpressed in a ratio of 1:1, and at the same time use the bidirectional promoter Ribi to coordinately express the upstream gene Δ6E of the EPA synthesis pathway, which can promote the co-localization of Δ6E with Δ5D and ω3D at the subcellular level, and help the influx of precursor fatty acids such as C18:3 into EPA. And from Figure 3-4According to the experimental results, the content and yield of triglycerides and triglyceride-type EPA in the transformant overexpressing the vector constructed by the present invention are significantly improved. Compared with the prior art, for example, the regulation results of a single ω3D gene recorded in the article "Modulation of an omega-3 fatty acid desaturase for eicosapentaenoic acid biosynthesis in the alga Nannochloropsis oceanica" published by Jie Zheng, Ying Shi et al., the single ω3D gene cannot effectively increase the content of triglyceride-type EPA in the overexpression strain, while the construction method of the expression vector constructed by the present invention significantly improves the content and yield of triglycerides and triglyceride-type EPA in the marine Nannochloropsis transformant, achieving an unexpected technical effect.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. These modifications or replacements, or the technical features in the above embodiments may be combined in the manner described in the embodiments if they do not conflict with each other, and these modifications, replacements or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An expression vector for increasing the content of triglyceride-type eicosapentaenoic acid in marine microsphere cells, characterized in that: The expression vector comprises a Δ5 desaturase gene, an ω3 desaturase gene and a Δ6 elongase gene; The expression vector adopts a Ribi bidirectional promoter, one end of the Ribi bidirectional promoter is connected to a Δ6 elongase gene; the Δ5 desaturase gene and the ω3 desaturase gene are connected through a 2A peptide sequence to form a polycistron in a single open reading frame, and the polycistron is connected to the other end of the Ribi bidirectional promoter; the base sequence of the Δ6 elongase gene is shown in SEQ ID NO.1; the base sequence of the Δ5 desaturase gene is shown in SEQ ID NO.2; and the ω3 desaturase gene is shown in SEQ ID NO.

3.

2. The expression vector according to claim 1, characterized in that The expression vector further comprises a selection gene, which is connected to the end of a polycistronic composed of a Δ5 desaturase gene and an ω3 desaturase gene via a 2A linker sequence.

3. The expression vector according to claim 1, characterized in that The 2A linker sequence is P2A, F2A or T2A.

4. The expression vector according to claim 3, characterized in that The base sequence of the F2A is shown in SEQ ID NO.4, and the base sequence of the P2A is shown in SEQ ID NO.

5.

5. The expression vector according to claim 1, characterized in that The screening gene is the hygromycin resistance gene Hygr, and its base sequence is shown in SEQ ID NO.

6.

6. The expression vector according to claim 1, characterized in that The expression vector comprises a Ribi bidirectional promoter, one end of which is connected to a Δ6 elongase gene, and the end of which is connected to a terminator; the other end of the Ribi bidirectional promoter is sequentially connected to a Δ5 desaturase gene, a 2A linker sequence, an ω3 desaturase gene, a 2A linker sequence and a hygromycin resistance gene Hygr.

7. The expression vector according to claim 1, characterized in that The full-length sequence of the expression vector is shown in SEQ ID NO.

7.

8. A marine Nannochloropsis algae with high production of triglyceride-type eicosapentaenoic acid, characterized in that: It comprises the expression vector according to any one of claims 1 to 7.

9. A method for increasing the content of triglyceride-type eicosapentaenoic acid in marine microsphere cells, characterized in that: include: The expression vector according to any one of claims 1 to 7 is transferred into the marine Nannochloropsis chassis cells to construct an overexpression strain, and the overexpression strain is cultured under nitrogen-deficient conditions to increase the content of triglyceride-type eicosapentaenoic acid in the marine Nannochloropsis cells.

10. The method according to claim 9, characterized in that The marine Nannochloropsis algae chassis cell is marine Nannochloropsis algae IMET1.

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