Method for producing astaxanthin with high yield by haematococcus pluvialis strain

Abstract

The invention discloses a method for producing astaxanthin with high yield by haematococcus pluvialis strains, and belongs to the technical field of bioengineering. According to the method, firstly, salt adaptive culture is performed to enhance the stress resistance of algae cells, then low-salt pre-stress culture is performed, and then high-salt is applied and is coupled with TOR accelerant MHY1485 culture to induce haematococcus pluvialis to improve the adaptability so as to promote efficient accumulation of astaxanthin. According to the method, the adaptability, the astaxanthin content, the yield and the stress resistance of the haematococcus pluvialis can be remarkably improved, the method is simple in process, easy to operate and low in dependence degree on the environment, the ecological environment cannot be harmed in the culture process, and the method has high application potential in the field of large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, and particularly relates to a method for high-yield astaxanthin production of Haematococcus pluvialis strain. BACKGROUND

[0002] As a kind of carotenoids, astaxanthin has super strong antioxidant property due to its special molecular structure, and relevant researches have confirmed that its antioxidant activity is 30-40 times of that of beta-carotene and 6000 times of that of vitamin C, so it is called super antioxidant. It has been widely used in many fields. Studies have shown that many algae have the ability to synthesize astaxanthin, among which Haematococcus pluvialis has the highest astaxanthin yield. Haematococcus pluvialis can synthesize and accumulate trans-astaxanthin under stress conditions, and the accumulation amount can reach 8% of the dry cell weight. However, there is a key problem in the process of industrialized cultivation of Haematococcus pluvialis. The stress resistance of algal cells is relatively low during the vegetative growth stage, and under the stress environment of high salinity and strong light required during astaxanthin synthesis, the algal cells are easily damaged, metabolic disorder occurs, and even death occurs, resulting in low synthesis efficiency and final yield of astaxanthin.

[0003] Therefore, developing a culture method that can both enhance the stress resistance of Haematococcus pluvialis cells and achieve high-efficiency accumulation of astaxanthin has become the key to breaking through the bottleneck of industrial development of Haematococcus pluvialis, and has important value for promoting the sustainable development of astaxanthin industry. SUMMARY

[0004] In order to solve the problems of low stress resistance of Haematococcus pluvialis cells and low synthesis efficiency of astaxanthin, the present application provides a method for high-yield astaxanthin production of high-salt adaptive evolution Haematococcus pluvialis strain. The method can regulate the metabolic pathways of substances in Haematococcus pluvialis cells, enhance their adaptability, better cope with environmental factors such as nitrogen source change, light intensity and abiotic stress, and thus promote the synthesis and accumulation of astaxanthin by adaptive evolution of Haematococcus pluvialis and coupling of TOR promoter.

[0005] The method for high-yield astaxanthin production of Haematococcus pluvialis strain according to the present application is as follows: (1) Adaptive culture: salt adaptive culture of Haematococcus pluvialis is carried out to obtain an algal strain subjected to adaptive culture.

[0006] (2) First stage culture: the algal strain subjected to adaptive culture is inoculated into a culture medium to culture to logarithmic growth phase, and then the algal strain in logarithmic growth phase is inoculated into a culture medium containing low concentration of NaCl for low-salt pre-stress culture.

[0007] (3) the second stage culture: the Haematococcus pluvialis treated by low salt pre-stress in step (2) is inoculated into the culture medium containing high concentration of NaCl, and is cultured by coupling TOR promoter to obtain the Haematococcus pluvialis strain with high astaxanthin yield. Preferably, the culture medium for culturing Haematococcus pluvialis in step (1), step (2) and step (3) of the present application is BG-11 culture medium.

[0008] Preferably, the salt adaptation culture in step (1) of the present application is specifically as follows: the Haematococcus pluvialis is acclimated and cultured for 180 days by 1 g / L NaCl, wherein the single algae is re-picked and plated every 15 days, the environmental temperature is 24-26℃, and the light intensity is 2900-3100 lux.

[0009] Preferably, the inoculation amount of Haematococcus pluvialis cells in step (2) of the present application is 1.6×10 5 cells / mL.

[0010] Preferably, the concentration of NaCl in the low concentration NaCl culture medium in step (2) of the present application is 50 mg / L.

[0011] Preferably, the culture conditions of logarithmic growth phase and low salt pre-stress in step (2) of the present application are as follows: the environmental temperature is 24-26℃, the sterile air ventilation amount is 1 vvm, and the light intensity is 2900-3100 lux.

[0012] Preferably, the low salt pre-stress culture time in step (2) of the present application is 4 days.

[0013] Preferably, the concentration of NaCl in the high concentration NaCl culture medium in step (3) of the present application is 1 g / L.

[0014] Preferably, the culture conditions in step (3) of the present application are as follows: the environmental temperature is 27-29℃, the sterile air ventilation amount is 1 vvm, and the light intensity is 21500-22500 lux.

[0015] Preferably, the TOR promoter in step (3) of the present application is MHY1485.

[0016] Preferably, the TOR promoter solution in step (3) of the present application is as follows: the TOR promoter is dissolved in dimethyl sulfoxide to prepare a TOR promoter solution with a concentration of 8 mmol / L.

[0017] Preferably, the concentration of TOR promoter in the culture medium in step (3) of the present application is 0.25-1 μmol / L.

[0018] Preferably, the culture time of Haematococcus pluvialis in step (3) of the present application is 13 days.

[0019] The TOR promoter and the TOR inhibitor used in the present application are both commercially available.

[0020] The reagents used in the present application are commercially available analytical pure unless otherwise specified.

[0021] The mechanism of the present application: The present application improves the survival rate of Haematococcus pluvialis in extreme environment and the astaxanthin synthesis capacity of Haematococcus pluvialis by adaptive evolution of Haematococcus pluvialis, forming the adaptability to the environment, thereby improving the survival ability or the synthesis efficiency of target product (such as astaxanthin). Rapamycin target protein (Target of rapamycin, TOR) regulates the growth, development and stress response mechanism of plants through the synergistic effect of nutrition and environmental factors, which embodies the core role of TOR in maintaining the stability of plant growth and development. The evolved algae strain further inhibits autophagosome-lysosome fusion under the action of TOR promoter MHY1485, reduces intracellular material degradation, preferentially directs carbon flow to the astaxanthin synthesis pathway, and enhances its adaptability. The present application significantly improves the synthesis efficiency and stability of astaxanthin by the synergistic effect of adaptive evolution of Haematococcus pluvialis and TOR promoter.

[0022] The technical scheme provided by the present application can include the following beneficial effects: (1) The results of the present application show that the salt-adapted evolution of Haematococcus pluvialis strain for high-yield astaxanthin significantly improves the stress resistance of Haematococcus pluvialis and the production potential of astaxanthin, and improves the biomass and astaxanthin accumulation of Haematococcus pluvialis. The highest biomass of Haematococcus pluvialis reaches 2.63 g / L, which is significantly improved by 23.44% compared with that without adding TOR promoter, and is significantly improved by 16.18% compared with that with adding TOR inhibitor. The highest astaxanthin content reaches 46.46 mg / g, which is significantly improved by 42.58% compared with that without adding TOR promoter, and is significantly improved by 96.57% compared with that with adding TOR inhibitor. At the same time, the astaxanthin yield reaches 9.33 mg / (L·d), which is significantly improved by 74.76% compared with that without adding TOR promoter, and is significantly improved by 128.60% compared with that with adding TOR inhibitor.

[0023] (2) The present application creates an optimal environment for the growth of Haematococcus pluvialis biomass and the accumulation of astaxanthin by regulating the culture conditions (such as temperature, light intensity, aeration amount, etc.), which helps to improve the astaxanthin output rate of algae.

[0024] (3) The application helps to promote Haematococcus pluvialis to improve astaxanthin production efficiency, and is expected to reduce dependence on natural resources and impact on the environment, thereby promoting sustainable development of the environment.

[0025] (4) The application provides an efficient culture strategy for large-scale production of astaxanthin of Haematococcus pluvialis, and has high commercial application value.

[0026] (5) The process involved in the application has the characteristics of simplicity, easy operation and high efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a flowchart of the application.

[0028] Figure 2 is a Haematococcus pluvialis biomass diagram of Examples 1-3 and Comparative Examples 1-2 of the application.

[0029] Figure 3 is a Haematococcus pluvialis astaxanthin content diagram of Examples 1-3 and Comparative Examples 1-2 of the application.

[0030] Figure 4 is a Haematococcus pluvialis astaxanthin yield diagram of Examples 1-3 and Comparative Examples 1-2 of the application. DETAILED DESCRIPTION

[0031] The application will be further described in detail below in combination with the drawings and specific examples, but the scope of protection of the application is not limited to the content described.

[0032] The TOR promoter used in the examples and comparative examples of the application is MHY1485, and the TOR inhibitor is AZD8055; both the TOR promoter and the TOR inhibitor are dissolved in dimethyl sulfoxide to prepare a solution with a concentration of 8 mmol / L for use.

[0033] Example 1 A method for improving astaxanthin production of salt-adapted evolution Haematococcus pluvialis strains, the specific culture method is as follows: (1) Adaptive culture: Haematococcus pluvialis is domesticated and cultured with 1 g / L NaCl for 180 days, and single algae are picked and plated every 15 days, the environmental temperature is 24-26℃, and the light intensity is 2900-3100 lux, to obtain algae strains after adaptive culture.

[0034] (2) First stage culture: 2.5 L of BG-11 medium was added to a 3 L round bottom flask, and the algae strain adapted to culture was inoculated into the medium and cultured to the logarithmic growth phase. The culture environment temperature was set to 24-26°C, the light intensity was 2900 lux, and the sterile air ventilation volume was 1 vvm. NaCl was added to 400 mL of BG-11 medium, and the concentration of NaCl in the medium was 50 mg / L. The algae strain in the logarithmic growth phase was inoculated into the medium containing NaCl for low-salt pre-stress treatment to enhance the stress resistance of the algae cells. The inoculation amount of Haematococcus pluvialis was 1.6 x 10 5 cells / mL, the culture environment temperature was maintained at 24-26°C, the light intensity was 2900-3100 lux, the sterile air ventilation volume was 1 vvm, and continuous culture was carried out for 4 days to obtain Haematococcus pluvialis subjected to low-salt pre-stress treatment.

[0035] (3) Second stage culture: Haematococcus pluvialis subjected to low-salt pre-stress treatment in step (2) was inoculated into BG-11 medium added with NaCl for stress culture. The concentration of NaCl in the medium was 1 g / L, and MHY1485 solution was added to the medium (the concentration of MHY1485 in the medium was 0.25 μmol / L). The culture environment temperature was maintained at 27-29°C, the light intensity was 21500-22500 lux, the sterile air ventilation volume was 1 vvm, and continuous culture was carried out for 13 days to obtain Haematococcus pluvialis rich in astaxanthin.

[0036] Example 2 A method for improving the high-yield astaxanthin of salt-adapted evolution Haematococcus pluvialis strain, and the specific culture method is as follows: (1) Adaptation culture: Haematococcus pluvialis was cultured for 180 days by 1 g / L NaCl acclimation, and single algae was picked and plated every 15 days. The culture environment temperature was 24-26°C, and the light intensity was 2900-3100 lux to obtain the algae strain subjected to adaptation culture.

[0037] (2) First stage culture: 2.5 L of BG-11 medium was added to a 3 L round bottom flask, and the algae strain adapted to culture was inoculated into the medium and cultured to the logarithmic growth phase. The culture environment temperature was set to 24-26°C, the light intensity was 2900 lux, and the sterile air ventilation volume was 1 vvm. NaCl was added to 400 mL of BG-11 medium, and the concentration of NaCl in the medium was 50 mg / L. The algae strain in the logarithmic growth phase was inoculated into the medium containing NaCl for low-salt pre-stress treatment to enhance the stress resistance of the algae cells. The inoculation amount of Haematococcus pluvialis was 1.6 x 10 5cells / mL, the culture environment temperature was maintained at 24-26℃, the light intensity was 2900-3100 lux, the sterile air ventilation volume was 1vvm, and the continuous culture was carried out for 4 days to obtain the Haematococcus pluvialis subjected to low-salt pre-stress treatment.

[0038] (3) Second stage culture: the Haematococcus pluvialis subjected to low-salt pre-stress treatment in step (2) was inoculated into the BG-11 culture medium added with NaCl to carry out stress culture, the concentration of NaCl in the culture medium was 1g / L, and the culture medium was added with the MHY1485 solution (the concentration of MHY1485 in the culture medium was 0.5μmol / L), the culture environment temperature was maintained at 27-29℃, the light intensity was 21500-22500 lux, the sterile air ventilation volume was 1vvm, and the continuous culture was carried out for 13 days to obtain the Haematococcus pluvialis rich in astaxanthin.

[0039] Example 3 A method for improving the astaxanthin yield of the salt-adapted evolution Haematococcus pluvialis strain, and the specific culture method is as follows: (1) Adaptation culture: the Haematococcus pluvialis was domesticated and cultured with 1g / L NaCl for 180 days, and the single algae was picked and plated every 15 days, the culture environment temperature was 24-26℃, and the light intensity was 2900-3100 lux to obtain the algae strain subjected to adaptation culture.

[0040] (2) First stage culture: 2.5L of BG-11 culture medium was added into a 3L round-bottom flask, and the algae strain subjected to adaptation culture was inoculated into the culture medium to culture to the logarithmic growth phase, the culture environment temperature was set to 24-26℃, the light intensity was 2900 lux, and the sterile air ventilation volume was 1vvm; NaCl was added into 400mL of BG-11 culture medium, the concentration of NaCl in the culture medium was 50mg / L, and the obtained algae strain in the logarithmic growth phase was inoculated into the culture medium containing NaCl to carry out low-salt pre-stress treatment, aiming to enhance the stress resistance of the algae cells, and the inoculation amount of the Haematococcus pluvialis was 1.6×10 5 cells / mL, the culture environment temperature was maintained at 24-26℃, the light intensity was 2900-3100 lux, the sterile air ventilation volume was 1vvm, and the continuous culture was carried out for 4 days to obtain the Haematococcus pluvialis subjected to low-salt pre-stress treatment.

[0041] (3) Second stage culture: the Haematococcus pluvialis subjected to low-salt pre-stress treatment in step (2) is inoculated into BG-11 medium added with NaCl to carry out stress culture, the concentration of NaCl in the medium is 1 g / L, and the medium is added with MHY1485 solution (the concentration of MHY1485 in the medium is 1.0 μmol / L), the culture environment temperature is maintained at 27-29°C, the light intensity is 21500-22500 lux, the sterile air ventilation volume is 1 vvm, and after 13 days of continuous culture, the Haematococcus pluvialis rich in astaxanthin is obtained.

[0042] Comparative Example 1 The difference between the present comparative example and Example 1 lies in that no exogenous TOR promoter is applied for treatment in the second culture stage, and the other steps are the same as those in Example 1, and the specific steps are as follows: (1) Adaptive culture: the Haematococcus pluvialis is domesticated and cultured for 180 days by 1 g / L NaCl, and the single algae is picked and plated every 15 days, the culture environment temperature is 24-26°C, and the light intensity is 2900-3100 lux, so that the algae strain subjected to adaptive culture is obtained.

[0043] (2) First stage culture: 2.5 L of BG-11 medium is added into a 3 L round bottom flask, and the algae strain subjected to adaptive culture is inoculated into the medium to culture to logarithmic growth phase, the culture environment temperature is set to 24-26°C, the light intensity is 2900 lux, and the sterile air ventilation volume is 1 vvm; 400 mL of BG-11 medium is added with NaCl, the concentration of NaCl in the medium is 50 mg / L, and the algae strain in logarithmic growth phase is inoculated into the medium containing NaCl to carry out low-salt pre-stress treatment, so as to enhance the stress resistance of the algae cells, the inoculation amount of Haematococcus pluvialis is 1.6 x 10 5 cells / mL, the culture environment temperature is maintained at 24-26°C, the light intensity is 2900-3100 lux, and the sterile air ventilation volume is 1 vvm, and after 4 days of continuous culture, the Haematococcus pluvialis subjected to low-salt pre-stress treatment is obtained.

[0044] (3) Second stage culture: the Haematococcus pluvialis subjected to low-salt pre-stress treatment in step (2) is inoculated into BG-11 medium added with NaCl to carry out stress culture, the concentration of NaCl in the medium is 1 g / L, the culture environment temperature is maintained at 27-29°C, the light intensity is 21500-22500 lux, and the sterile air ventilation volume is 1 vvm, and after 13 days of continuous culture, the Haematococcus pluvialis is obtained.

[0045] Comparative Example 2 The difference between the present comparative example and example 1 is that the TOR inhibitor AZD8055 is added during the second culture stage, and the other steps are the same as those of example 1. The specific steps are as follows: (1) Adaptive culture: Haematococcus pluvialis was acclimated and cultured in 1 g / L NaCl for 180 days, and single algae were picked and plated every 15 days. The culture temperature was 24-26°C, and the light intensity was 2900-3100 lux. The algae strain obtained by adaptive culture was obtained.

[0046] (2) First stage culture: 2.5 L of BG-11 medium was added to a 3 L round-bottom flask, and the algae strain obtained by adaptive culture was inoculated into the medium and cultured to the logarithmic growth phase. The culture temperature was set to 24-26°C, the light intensity was 2900 lux, and the sterile air ventilation rate was 1 vvm. NaCl was added to 400 mL of BG-11 medium, and the concentration of NaCl in the medium was 50 mg / L. The algae strain obtained in the logarithmic growth phase was inoculated into the medium containing NaCl for low-salt pre-stress treatment to enhance the stress resistance of the algae cells. The inoculation amount of Haematococcus pluvialis was 1.6 x 10 5 cells / mL, the culture temperature was maintained at 24-26°C, the light intensity was 2900-3100 lux, and the sterile air ventilation rate was 1 vvm. The Haematococcus pluvialis obtained by low-salt pre-stress treatment was cultured for 4 days.

[0047] (3) Second stage culture: The Haematococcus pluvialis obtained by low-salt pre-stress treatment in step (2) was inoculated into BG-11 medium containing NaCl for stress culture. The concentration of NaCl in the medium was 1 g / L, and AZD8055 solution was added to the medium (the concentration of AZD8055 in the medium was 0.25 μmol / L). The culture temperature was maintained at 27-29°C, the light intensity was 21500-22500 lux, and the sterile air ventilation rate was 1 vvm. After 13 days of continuous culture, Haematococcus pluvialis was obtained.

[0048] The astaxanthin obtained by the present application was extracted and determined, and the specific extraction and determination methods were as follows: The culture solution obtained after culturing for 13 days in the second stage was taken 5 mL into a centrifuge tube in a sterile clean bench, centrifuged at 3500 rpm for 5 minutes, and the supernatant was discarded. The algal strain was collected, 5 mL of a mixed solution of dimethyl sulfoxide and glacial acetic acid (volume ratio of dimethyl sulfoxide to glacial acetic acid was 100:1) was added, and the mixture was placed in a 65°C water bath for 15 minutes. Then the mixture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected into a new centrifuge tube. The precipitate was added with 5 mL of the mixed solution of dimethyl sulfoxide and glacial acetic acid again, and the above water bath and centrifugation operation was repeated until the algal body turned white. The supernatants were combined, and the absorbance value was measured at 530 nm using a spectrophotometer. The astaxanthin concentration of Haematococcus pluvialis was calculated by substituting the standard curve.

[0049] The biomass of Haematococcus pluvialis is shown in Table 1, and the astaxanthin content is shown in Table 2. Figure 2 As can be seen from the figure, the biomass of Haematococcus pluvialis in Example 1 was 2.27 g / L, which was increased by 6.25% compared with Comparative Example 1; the biomass of Haematococcus pluvialis in Example 2 was 2.63 g / L, which was significantly increased by 23.44% compared with Comparative Example 1 and was significantly increased by 16.18% compared with Comparative Example 2; the biomass of Haematococcus pluvialis in Example 3 was 2.37 g / L, which was significantly increased by 10.94% compared with Comparative Example 1 and was increased by 4.41% compared with Comparative Example 2.

[0050] The astaxanthin content is shown in Table 2, and the astaxanthin yield is shown in Table 3. Figure 3 As can be seen from the figure, the astaxanthin content in Example 1 was 34.45 mg / g, which was significantly increased by 5.72% compared with Comparative Example 1 and was significantly increased by 45.76% compared with Comparative Example 2; the astaxanthin content in Example 2 was 46.46 mg / g, which was significantly increased by 42.58% compared with Comparative Example 1 and was significantly increased by 96.57% compared with Comparative Example 2; the astaxanthin content in Example 3 was 34.97 mg / g, which was significantly increased by 7.32% compared with Comparative Example 1 and was significantly increased by 47.97% compared with Comparative Example 2.

[0051] The astaxanthin yield is shown in Table 3, and the astaxanthin yield is shown in Table 3. Figure 4 As can be seen from the figure, the astaxanthin yield in Example 1 was 5.95 mg / (L·d), which was significantly increased by 11.34% compared with Comparative Example 1 and was significantly increased by 45.64% compared with Comparative Example 2; the astaxanthin yield in Example 2 was 9.33 mg / (L·d), which was significantly increased by 74.76% compared with Comparative Example 1 and was significantly increased by 128.60% compared with Comparative Example 2; the astaxanthin yield in Example 3 was 6.35 mg / (L·d), which was significantly increased by 18.91% compared with Comparative Example 1 and was significantly increased by 55.55% compared with Comparative Example 2.

[0052] In summary, the present application significantly improves the stress resistance and astaxanthin productivity of Haematococcus pluvialis by salt adaptive evolution and adding TOR promoter, so that the biomass reaches 2.63 g / L (increasing by 23.44% and 16.18%), the astaxanthin content reaches 46.46 mg / g (increasing by 42.58% and 96.57%), and the yield reaches 9.33 mg / (L·d) (increasing by 74.76% and 128.60%), the method has environmental friendliness and commercial value, and provides an innovative solution for the astaxanthin industry.

[0053] The above has described various embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles, practical applications, or improvements to the technology in the market of the embodiments, or to enable other ordinary skilled persons in the art to understand the embodiments disclosed herein.

Claims

1. A method for high-yield astaxanthin production from Haematococcus pluvialis strain, characterized in that, The specific method is as follows: (1) Adaptation culture: Haematococcus pluvialis was subjected to salt adaptation culture to obtain algal strains that had undergone adaptation culture; (2) First stage of culture: The algal strains that have undergone adaptive culture are inoculated into the culture medium and cultured to the logarithmic growth phase. Then, the algal strains in the logarithmic growth phase are inoculated into the culture medium containing low concentration of NaCl for low salt pre-stress culture. (3) Second stage of cultivation: Haematococcus pluvialis treated with low salt pre-stress in step (2) was inoculated into a culture medium containing high concentration of NaCl and coupled with TOR promoter solution for cultivation to obtain Haematococcus pluvialis strain with high astaxanthin production.

2. The method for high astaxanthin production by Haematococcus pluvialis strain according to claim 1, characterized in that, The culture medium used to culture Haematococcus pluvialis in steps (1), (2) and (3) is BG-11 medium.

3. The method for high astaxanthin production by Haematococcus pluvialis strain according to claim 1, characterized in that, The salt adaptation culture in step (1) is as follows: Haematococcus pluvialis is acclimatized and cultured with 1g / L NaCl for 180 days. During this period, a single algal colony is picked and plated every 15 days. The culture environment temperature is 24~26℃ and the light intensity is 2900~3100 lux.

4. The method for high astaxanthin production by Haematococcus pluvialis strain according to claim 1, characterized in that, In step (2), the inoculum size of Haematococcus pluvialis cells is 1.6 × 10⁻⁶. 5 cells / mL; the NaCl concentration in the low-concentration NaCl culture medium is 50 mg / L.

5. The method for high astaxanthin production by Haematococcus pluvialis strain according to claim 1, characterized in that, The culture conditions for the logarithmic growth phase and low salt pre-stress in step (2) are: ambient temperature of 24~26℃, sterile air ventilation of 1vvm, and light intensity of 2900~3100 lux.

6. The method for high astaxanthin production by Haematococcus pluvialis strain according to claim 1, characterized in that, In step (2), the low-salt pre-stress culture period is 4 days.

7. The method for high astaxanthin production by Haematococcus pluvialis strain according to claim 1, characterized in that, The concentration of NaCl in the high-concentration NaCl culture medium mentioned in step (3) is 1 g / L.

8. The method for high astaxanthin production by Haematococcus pluvialis strain according to claim 1, characterized in that, The culture conditions in step (3) are: ambient temperature of 27~29℃, sterile air ventilation of 1vvm, and light intensity of 21500~22500 lux.

9. The method for high astaxanthin production by Haematococcus pluvialis strain according to claim 1, characterized in that, In step (3), the TOR promoter is MHY1485; the TOR promoter solution is prepared by dissolving the TOR promoter in dimethyl sulfoxide to a concentration of 8 mmol / L; the concentration of the TOR promoter in the culture medium is 0.25~1 μmol / L.

10. The method for high astaxanthin production by Haematococcus pluvialis strain according to claim 1, characterized in that, In step (3), the Haematococcus pluvialis culture time is 13 days.

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