High-yield astaxanthin induced mutation of Haematococcus pluvialis and mutation cultivation method of Haematococcus pluvialis

Abstract

The invention relates to a high-yield astaxanthin-mutated Haematococcus pluvialis and a mutagenesis cultivation method of Haematococcus pluvialis, belonging to the technical field of Haematococcus pluvialis cultivation. The high-yield astaxanthin-mutated Haematococcus pluvialis of the invention is preserved in China Center for Type Culture Collection, with a preservation number of CCTCC No: M20232109 and a preservation date of November 1, 2023. The algae strain has stable performance, fast expansion rate, stable astaxanthin yield and high density of pipeline proliferation cells. The mutagenesis cultivation method of Haematococcus pluvialis of the invention obtains an excellent algae strain with high astaxanthin yield by jointly mutagenizing Haematococcus pluvialis and selecting the mutated Haematococcus pluvialis. The obtained algae strain has good genetic stability and stable performance, and the astaxanthin yield is significantly improved compared with the original algae strain.

Description

Technical Field

[0001] The invention belongs to the technical field of Haematococcus pluvialis cultivation, and specifically relates to a high-yield astaxanthin-induced Haematococcus pluvialis and a mutation cultivation method of Haematococcus pluvialis. Background Art

[0002] Haematococcus pluvialis is the main source of astaxanthin. In order to increase the yield of astaxanthin, technical workers have been committed to the cultivation of Haematococcus pluvialis strains to obtain Haematococcus pluvialis with higher astaxanthin production.

[0003] By screening and cultivating excellent microalgae germplasm resources, the large-scale high-density cultivation of microalgae can be improved, and the diversity of commercial varieties can be increased. The screening and cultivation of algae species are necessary means for the development of excellent germplasm, and are also one of the foundations for the sustainable and stable development of microalgae industrialization. At present, the means of algae selection and breeding include natural selection, mutation breeding, cell fusion, genetic engineering, etc. Single natural selection and mutation breeding have disadvantages such as large randomness and unstable genetic traits; cell fusion technology is difficult to fuse or restore cell activity after fusion due to the unique life history of Haematococcus pluvialis; genetic engineering has not been able to obtain perfect technology due to the bottleneck of Haematococcus pluvialis genome sequencing; the original algae strains used in basic research are experimental algae strains rather than production application algae strains, and their own traits are not outstanding.

[0004] At present, there have been many achievements in the basic and applied research on the breeding of excellent germplasm of Haematococcus pluvialis. Many research teams at home and abroad have obtained many new algae strains with specific excellent traits through natural selection, mutation breeding, cell fusion and other means. However, most of the excellent algae strains obtained by applied research institutes are completed and verified under laboratory or indoor conditions, lacking step-by-step screening, evaluation and optimization in the entire process of large-scale breeding, which makes it often appear that the advantageous traits are not prominent or even cannot be cultivated in outdoor large-scale breeding.

[0005] At present, the astaxanthin content of Haematococcus pluvialis that can be cultivated on a large scale is generally 2-6%. Improving the astaxanthin content and yield of Haematococcus pluvialis has always been the research direction of Haematococcus pluvialis cultivation technology. Summary of the invention

[0006] In order to overcome the problems existing in the background technology, the first purpose of the present invention is to provide a high-yield astaxanthin induced Haematococcus pluvialis strain, the algae strain has stable performance, fast expansion rate, stable astaxanthin yield, high density of pipeline proliferation cells, astaxanthin content of more than 5.16%, up to 7.21%, astaxanthin yield of more than 2.97 mg / L / d. The second purpose of the present invention is to provide a method for induced cultivation of Haematococcus pluvialis, the Haematococcus pluvialis obtained by the cultivation method has stable performance, can still maintain performance advantages in outdoor large-scale cultivation, and the astaxanthin content and yield are significantly higher than the original strain.

[0007] The high-yield astaxanthin-induced Haematococcus pluvialis strain is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No: M20232109 and a deposit date of November 1, 2023.

[0008] Furthermore, the mutated Haematococcus pluvialis strain is obtained by mutating and cultivating the Haematococcus pluvialis strain S / HP-3.

[0009] Furthermore, the induced Haematococcus pluvialis strain had a dry weight increased by 27.2%, an astaxanthin content increased by 18.6%, and an astaxanthin yield increased by 50.3% compared to the original algae strain cultured during the same period.

[0010] (1) Mutagenesis plate screening

[0011] Ethyl methanesulfonate was added to the suspension of Haematococcus pluvialis and mixed and shaken for mutagenesis, and then washed and resuspended for ultraviolet mutagenesis.

[0012] (2) Orifice plate screening

[0013] Condition screening was performed using BG11 solid plates containing diphenylamine and / or carbendazim.

[0014] (3) Indoor strong light stress

[0015] The algae strain in step (2) is first proliferated, and after proliferation, red conversion medium is added and the light intensity is increased to 150-300 μmolphotons / (m2·s) for stress.

[0016] Furthermore, the strong light stress in step (3) is carried out in an indoor column reactor; the red-transforming medium added is a conventional red-transforming medium currently used in astaxanthin culture, and the light intensity is 150-300 μmol photons / (m 2 ·s).

[0017] Furthermore, the method for inducing the cultivation of the high-yield astaxanthin Haematococcus pluvialis strain continues to cultivate the following steps after strong light stress:

[0018] (4) Outdoor coercion

[0019] The algae strain obtained in step (3) is first proliferated in an outdoor columnar reactor, and after proliferation, red transformation culture medium is added and cultured under normal light stress.

[0020] (5) Single-cell algae selection and algae strain purification and domestication.

[0021] Furthermore, the algae strains with a fast growth rate in step (1) are selected for screening in step (2); and the algae strains with a fast algae colony formation rate and a large algae colony in step (2) are selected for strong light stress in step (3).

[0022] Furthermore, the UV intensity of step (1) is 120-200 μW / cm 2 The irradiation was performed for 8-12 min at a strength of 0.5-2.5% (v:v) ethyl methanesulfonate.

[0023] The induced Haematococcus pluvialis strain was bred using the above-mentioned mutagenesis breeding method.

[0024] The original algae strains are mutated Haematococcus pluvialis strains obtained by the mutagenesis breeding method of the present invention from S / HP-3, SJ-7 and SL-1.

[0025] Beneficial effects of the present invention:

[0026] The high-astaxanthin-producing induced Haematococcus pluvialis strain of the present invention has stable performance, fast culture expansion rate, stable astaxanthin yield, high density of channel proliferation cells, astaxanthin content of more than 5.16%, up to 7.21%, astaxanthin yield of more than 2.97 mg / L / d, and is the Haematococcus pluvialis strain with the highest astaxanthin content reported so far.

[0027] The mutation breeding method of the Haematococcus pluvialis strain of the present invention can breed Haematococcus pluvialis mutants with good genetic stability, stable performance, fast expansion rate, stable astaxanthin yield, and significantly improved dry weight, astaxanthin content, yield and expansion rate compared with the original strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a comparison of the differential gene expression numbers between the algae strain of Example 1 of the present invention and the original algae strain;

[0029] Figure 2 is a volcano plot of differential gene expression between the algae strain of Example 1 of the present invention and the original algae strain;

[0030] Figure 3 This is a radar chart of the structural differences between the algae strain of Example 1 of the present invention and the original algae strain;

[0031] Figure 4 This is a GO enrichment analysis diagram of down-regulated genes of the algae strain in Example 1 of the present invention and the original algae strain;

[0032] Figure 5 This is a GO enrichment analysis diagram of up-regulated genes of the algae strain in Example 1 of the present invention and the original algae strain;

[0033] Figure 6 is a GO enrichment analysis diagram of differential genes between the algae strain of Example 1 of the present invention and the original algae strain;

[0034] Figure 7 is a comparison of the initial screening results of the well plate growth of the mutant strain in Example 2 of the present invention and the corresponding wild type;

[0035] Figure 8 This is the result of culturing the mutant strain in an indoor column reactor according to Example 2 of the present invention;

[0036] Fig. 9 This is the result of evaluating the genetic stability of the mutation in Example 2 of the present invention;

[0037] Fig.10 The results of the outdoor columnar reactor growth and astaxanthin accumulation evaluation experiment of Example 2 of the present invention;

[0038] Fig.11 is the increase or decrease in growth and astaxanthin accumulation under outdoor culture conditions compared to indoor culture conditions in Example 2 of the present invention;

[0039] Fig.12 It is a comparison of the cultivation results of the outdoor columnar reactor before and after the optimization of the algae strain in Example 2 of the present invention;

[0040] Fig.13 Comparison of the results of the outdoor columnar reactor simulation pipeline culture process of the mutant strain in Example 2 of the present invention

[0041] Fig.14 The increase or decrease of dry weight and astaxanthin accumulation in the outdoor simulated pipeline of Example 2 of the present invention compared with the conventional culture conditions

[0042] Fig.15 This is the production verification and result comparison of four excellent algae strains in Example 2 of the present invention;

[0043] Remark:

[0044] Figure 1 Among them, group2 is MT1012, and group3 is the original strain;

[0045] Figure 2 In the figure, the left side of the double dashed line represents down-regulated gene expression, and the right side of the double dashed line represents up-regulated gene expression; the larger the value on the horizontal axis, the higher the differential expression fold, and the larger the value on the vertical axis, the more significant the differential expression; the points marked in the figure are the 10 genes with the largest differential expression up- and down-regulated folds;

[0046] Figure 3 In the figure, the 30 genes with the most significant differential expression between the two algae strains, the first circle indicates the log2 (FC) value of the up- and down-regulation of the genes; the second circle indicates the average expression of the genes in the experimental group and the control group; the third circle is a radar chart of the average expression of each gene in the experimental group and the control group;

[0047] Figure 4-6The enrichment of differentially expressed genes between the two algae strains in the three major items (BiologicaLprocess on the top, Cellular composition in the middle, and Molecular Function on the bottom); GO items corresponding to PopHits≥5 in the three categories were screened, and 10 items were sorted from large to small according to the -log10p-value corresponding to each item. The smaller the p-value, the longer the bar graph, and the more significant the difference; the more significant the enrichment, the more important the function corresponding to the differentially expressed genes in this item;

[0048] Figure 7 In the figure, A shows the OD yield; B shows the dry weight;

[0049] Figure 8 Figure A shows the dry weight and cell number after 10 days of proliferation; Figure B shows the dry weight-based cell number after 10 days of stress; Figure C shows the content and yield of phytochemicals after 10 days of stress;

[0050] Fig. 9 In the figure, A shows the increase or decrease fold of dry weight of mutant strains compared with wild type; B shows the increase or decrease fold of cell number of mutant strains compared with wild type;

[0051] Fig.10 Figure A shows the dry weight and cell number results after 10 days of proliferation; Figure B shows the dry weight and cell number results after 10 days of stress; Figure C shows the astaxanthin content and yield results after 10 days of stress. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0053] In order to more clearly illustrate the present invention, it is described in detail through the following examples.

[0054] Example 1

[0055] A mutagenized Haematococcus pluvialis strain (named MT1012) is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No: M20232109 and a deposit date of November 1, 2023.

[0056] The algae strain (MT1012) has a dry weight of more than 1.32 g / L, an astaxanthin content of more than 5.16%, with a maximum of 7.21%, and an astaxanthin yield of more than 2.97 mg / L / d.

[0057] The MT1012 algae strain was obtained by mutagenesis of the Haematococcus pluvialis strain S / HP-3 (derived from the production strain of Yunnan Boxin Biotechnology Co., Ltd. in Qujing City, Yunnan Province).

[0058] RNA was extracted from MT1012 and S / HP-3 strains and transcriptome sequencing was performed. The differential gene expression numbers were compared as shown in the attached figure. Figure 1 ; Volcano plot of differential gene expression (attached Figure 2 ); Radar chart of differentially expressed genes (attached Figure 3 );GO enrichment analysis of down-regulated genes (attached Figure 4 );GO enrichment analysis of up-regulated genes (attached Figure 5 );GO enrichment analysis of differentially expressed genes (attached Figure 6 ). Figure 1-6 It can be seen that the mutant strain MT1012 has a large difference in gene expression compared with the original strain S / HP-3, among which the number of up-regulated genes is 710 and the number of down-regulated genes is 377. In the astaxanthin synthesis pathway, the genes involved in the process from β-carotene synthesis to astaxanthin in MT1012 are significantly up-regulated, followed by some genes in the carbon metabolism and terpenoid skeleton synthesis pathways.

[0059] Example 2

[0060] The method for inducing mutation and cultivating a high-yield astaxanthin Haematococcus pluvialis strain comprises the following steps:

[0061] (1) Take the original strain of Haematococcus pluvialis,

[0062] The algae strains S / HP-3 (derived from the production algae strain of Yunnan Boxin Biotechnology Co., Ltd. in Qujing City, Yunnan Province) and CJ-7 (wild algae strains collected and isolated from the water of Fuxian Lake in Chengjiang City, Yunnan Province), and SL-1 (wild algae strains collected and isolated from the natural water in Shilin Scenic Area, Shilin County, Yunnan Province) were selected.

[0063] In the present embodiment, the culture conditions involved are mainly divided into indoor culture and outdoor culture.Indoor culture is completed in clean area (sterile environment), and indoor culture is mainly divided into static culture and ventilation culture.Static culture includes from solid plate to 1L triangular flask, all using improved BG11 culture medium (Table 1).Aeration culture mainly includes 100mL colorimetric tube, 1L column reactor, 1L triangular flask and 5L round-bottom flask, all using BG11 culture medium (Table 1).Outdoor expansion culture mainly includes 1L column reactor, 150L vertical plastic plate (Vertical plastic plate), 3000L pilot pipeline reactor (Tubularphotobioreactor), 7000L production pipeline reactor.Outdoor culture is all ventilation culture, and each stage expansion culture uses BG11 culture medium (Table 1).

[0064] Table 1. Nutrient elements and concentrations used in BG11 and modified BG11 culture medium

[0065]

[0066]

[0067] The above algae strains were purified by antibiotic BG11 plate: different concentrations of ampicillin (10-200 μg / mL) were selected according to the degree of contamination to prepare purification solid plates, and algae colonies were selected and inspected after they grew out, and pure algae were obtained after multiple selections. Subsequently, algae strains with faster growth rates were selected after rejuvenation.

[0068] (2) Set up a UV mutagenesis area and a mutagenesis plate screening area in the clean area, and use UV light and ethyl methanesulfonate to carry out combined mutagenesis: 1. Prepare 0.06 mol / L phosphate buffer and prepare EMS mixed solutions of different concentrations (v:v) according to the required mutagenesis dose. Take the algae solution in the logarithmic growth phase (cell density 10 5 cells / mL) in a centrifuge tube, centrifuge at 25°C, 5000rpm for 5 minutes, discard the supernatant, add 2mL of EMS mixed buffer containing the required mutagenic dose, shake well and place in a dark place for mutagenesis for 2 hours. After the mutagenesis, centrifuge at 5000rpm to remove the supernatant and wash twice with phosphate buffer; 2. Place the algae solution washed in step 1 in a sterilized 30mm quartz covered culture dish and place under a UV lamp (200-275nm, 15W, irradiation intensity 120-200μW / cm 2 ) for different time periods. After irradiation, the culture dish was placed in a dark place for 12 hours and then transferred to a sterilized 2 mL centrifuge tube. The algae solution in the centrifuge tube was diluted to 10 3 After measuring the concentration of cells / mL, 100 μL was taken and spread on different solid plates using the spreading plate method to obtain the mortality rates of algae strains under different mutagenesis conditions as shown in the table.

[0069] Table 2. Combined mutagenic lethal dose exploration (S / HP-3 strain)

[0070]

[0071] (3) The algae strains after combined mutagenesis were spread on BG11 solid plates for growth and condition screening: 1. Preparation of diphenylamine screening plates: BG11 agar medium was sterilized and cooled to below 50°C, diphenylamine was added to the medium to make its concentration reach 12 mg / L, and the agar medium was poured into a plastic culture dish to make a diphenylamine screening plate; 2. Preparation of carbendazim screening plates: BG11 agar medium was sterilized and cooled to below 50°C, carbendazim was added to the medium to make its concentration reach 5 mg / L, and the agar medium was poured into a plastic culture dish to make a carbendazim screening plate; 3. Combined screening plates were prepared by adding diphenylamine and carbendazim at the same time according to the above steps. Finally, the better mutants under each treatment were obtained through plate algal colony formation and size and purification plate rescreening (plate purification in step (1)) (Table 3), and the mutants were selected and recorded on the BG11 plate and screened at 5 μmol photons / (m 2 ·s) Solid plate seed preservation was carried out under weak light. After algal colonies were formed, algal colonies were selected from the solid plate and resuspended in well plates. After recovery in 96-well plates and 24-well plates, they were placed in 50 mL triangular flasks for static culture and subculture storage. The initial cell concentration was 5×10 4 cells / mL, shake the bottle 4 times a day, and adjust the light intensity to 10-60μmol photons / (m 2 The culture temperature in the clean area is maintained at 22±2℃, and the clean area is kept under continuous light for 24 hours.

[0072] Table 3. Summary of the better mutants after mutagenesis and initial plate screening

[0073]

[0074] (4) Select superior mutant strains for 24-well plate growth screening and evaluation experiments, and the experimental period is 10 days ( Figure 7 ). Then the cells were expanded in 250 mL Erlenmeyer flasks: the initial cell concentration was 5×10 4 cells / mL, shake the flask 4 times a day, and adjust the light intensity to 10–60 μmol photons / (m 2 ·s). After culturing for an average of 15 days, the number of cells reached 25×10 4 cells / mL, you can proceed to the next level of screening and evaluation experiments.

[0075] (5) The cultured algae were cultured for 10 days and then 2 g / L NaCl and 0.45 g / L NaHCO were added. 3 The final concentration of red medium was used for red transformation medium stress, and the light intensity was increased to 200 μmol photons / (m 2 ·s) for 10 days, and finally harvested for testing (test results see Figure 8 ). At the end of the proliferation period, the dry weight of MT1012 also reached 1.02g / L, and the increase in dry weight after stress was the largest among all algae strains. Its dry weight of 1.463g / L was also the highest among all algae strains, followed by MT663 and SL-1, which were 1.42 and 1.412g / L respectively. These were also the three algae strains whose dry weight exceeded 1.4g / L in the column reactor experiment. In terms of astaxanthin content and yield, MT1012 had the highest values ​​of both indicators among all algae strains, reaching 3.62% and 2.649mg / (L·d) respectively, followed by SL-1, MT663, S / HP-3, MT1005, and MT1006, with astaxanthin contents of 3.4-3.5% and astaxanthin yields of 2.1-2.4mg / (L·d).

[0076] The superior mutants were divided into two categories: MT712 and MT734, which had high motility and small cells; and MT663, MT1005, MT1006, and MT1012, which had poor motility, large cells, and were easy to transform into spore cells. These superior mutants were further cultured indoors step by step for subsequent outdoor screening and evaluation.

[0077] (6) The superior mutant strains were evaluated for genetic stability: they were continuously subcultured in 50 mL Erlenmeyer flasks, and three parallel inoculations were added at the 1st, 3rd, 5th, 10th, and 15th generations, with the initial cell density of 5 × 10 4 cells / mL, and harvested after 15 days of culture for cell counting and dry weight determination. The wild type was cultured at the same time as the control, and the increase or decrease in dry weight and cell number compared with the wild type was used as the evaluation index of mutation stability ( Fig. 9 ). Comprehensive evaluation showed that the genetic stability of mutant strains was as follows: MT1012>MT712>MT1005≈MT1006≈MT663>MT734. MT734 was discarded, and the remaining excellent mutant strains entered the outdoor screening and evaluation experiment. At the same time, the wild type SL-1 also showed higher dry weight and astaxanthin content in the evaluation of the well plate and column reactor, and SL-1 was also selected as a relatively excellent algae strain for outdoor screening and evaluation experiments.

[0078] (7) Growth and astaxanthin accumulation screening and evaluation experiments were carried out in an outdoor 1L column reactor. After 10 days of proliferation, samples were taken to evaluate their growth. Then, red-transforming medium was added for stress and placed under normal light stress for 10 days. Finally, the samples were harvested for relevant index testing. The results were compared with those of the production algae strain S / HP-3 in the experiment ( Fig.10 ), and also compared with the results of indoor column reactor culture ( Fig.11 ).

[0079] After comprehensive evaluation, MT1012 and MT663 were superior to the production strains in dry weight and astaxanthin content, and entered the algae strain optimization as better mutants. At the same time, MT1006 and SL-1 were also selected as better strains for algae species optimization because their indoor and outdoor screening results were comparable to those of the production strains. In addition, although MT712 was inferior to the production strains in dry weight and astaxanthin content, it had a significant improvement compared to the indoor evaluation, and its cell number growth during the growth stage was the fastest among all algae strains, so it was also listed as a better mutant strain for algae species optimization.

[0080] (8) The selected and evaluated algae strains and the S / HP-3 algae strain as a control were subjected to natural culture stress in an outdoor column reactor, and then single-cell algae were selected, purified and domesticated. After indoor expansion, they were grown and stressed again in an outdoor column reactor, and the culture results were compared with those in step (7). Fig.12 ).

[0081] (9) The better algae strains after screening and optimization were screened and evaluated with the control group production algae strains in a 1L column reactor simulating the production pipeline reactor culture process, and the harvest parameters (dry weight, astaxanthin content and astaxanthin yield) of the better algae strains were compared with the production algae strain (S / HP-3) ( Fig.13 ). In terms of dry weight, MT712>MT1012>MT663>MT1006≈S / HP-3>SL-1; in terms of astaxanthin content, MT1012>MT663>SL-1>MT1006≈S / HP-3>MT663>MT712; and in terms of comprehensive evaluation of astaxanthin yield, MT1012>MT663>MT712>SL-1≈MT1006≈S / HP-3.

[0082] At the same time, the parameters of each algae strain are compared with the parameters in step (8) Fig.14 ). It was found that except for MT712, the dry weight of the other algae strains decreased, among which MT663 had the largest decrease of 18.03%. The astaxanthin content increased significantly, with MT663 showing the largest increase of 18.81%, but due to its large decrease in dry weight, the astaxanthin yield decreased.

[0083] (10) After the optimization of the above screening and evaluation, the algae strains were gradually expanded indoors and then screened and evaluated in outdoor pilot pipeline reactors. Three pilot screening and evaluations were completed (Table 4).

[0084] Table 4. Evaluation of three pilot screenings of superior algae strains and production algae strains

[0085]

[0086] (11) Production scale verification and evaluation

[0087] The four excellent mutant strains MT663, MT712, MT1006, and MT1012 obtained by screening and evaluation in step (10) were purified and revived on plates and then gradually expanded according to the existing S / HP-3 production and breeding process from the inside out. Finally, verification and exploration were carried out under the production scale of 7000L pipeline reactor, and the S / HP-3 algae production and breeding that was relatively close to the previous and next were selected as the same period comparison. At the same time, the S / HP-3 batch production data of the same breeding area in the same month of each experiment were collected and sorted, and the monthly average, monthly best, and monthly worst batches were selected for comparison ( Fig.15 ).

[0088] Finally, it should be noted that the above preferred 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 through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A mutant Haematococcus pluvialis strain producing astaxanthin, characterized in that: The mutated Haematococcus pluvialis strain is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No: M20232109 and a deposit date of November 1, 2023.

2. A method for producing astaxanthin, characterized in that: Firstly, the induced Haematococcus pluvialis strain described in claim 1 is revived and domesticated using a purification plate, and then the culture is expanded step by step according to the production and breeding process from indoor to outdoor.

3. Use of the mutated Haematococcus pluvialis strain as claimed in claim 1 in producing astaxanthin.

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