Method for selecting algae having strong light resistance

Abstract

[Problem] To provide a method for selecting algae having strong light resistance. [Solution] This method for selecting algae having strong light resistance comprises determining the fluorescent intensities of multiple kinds of algae and separating a portion of the multiple kinds of algae on the basis of the fluorescent intensities, such that the separated algae and / or the progeny thereof include algae having strong light resistance.

Description

Method for selecting high light-tolerant algae

[0001] The present disclosure relates to a method for selecting high-light-tolerant algae.

[0002] Algae are widely used in various industrial fields, including as aquaculture feed, energy sources, and dietary supplements, and are an important industrial resource. Because algae grow by photosynthesis, a supply of light energy is essential for their cultivation. However, excessively strong light energy (strong light) can damage photosynthesis-related proteins and stunt growth (Non-Patent Document 1). Therefore, techniques for breeding or selecting algae that are tolerant to strong light are needed.

[0003] Many conventional techniques use genetic modification techniques to confer high-light tolerance by improving the ability to repair damaged proteins (Non-Patent Document 1). Genetic modification requires the identification and modification of the target gene, which requires a great deal of time and effort to obtain algae with the desired traits. Therefore, there is a need for a method to obtain high-light tolerance algae with less effort and operating time.

[0004] Success in "strengthening" photosynthesis: Development of a method to enhance photosynthesis's tolerance to strong light, Saitama University website, https: / / www.saitama-u.ac.jp / topics_archives / 2023 / 2023-1121-1352-16.html

[0005] The present disclosure has been made to solve the above-mentioned problems, and aims to provide a method for selecting high-light-tolerant algae.

[0006] One aspect of the present disclosure is a method for selecting high-light-resistant algae, comprising measuring the fluorescence intensity of multiple types of algae and separating a portion of the multiple types of algae based on the fluorescence intensity, wherein the separated algae and / or their progeny include high-light-resistant algae.

[0007] According to the present disclosure, a method for selecting high-light-tolerant algae can be provided.

[0008] Figure 1 shows the change in cell density over time when cultured under strong light for the highly autofluorescent sample and the control sample (1). Figure 2 shows the change in cell density over time when cultured under strong light for the highly autofluorescent sample and the control sample (2).

[0009] Non-limiting embodiments of the present disclosure will be described below. The present disclosure is not limited to the examples in the following embodiments.

[0010] In one embodiment, a method for selecting high-light-resistant algae is provided, which includes measuring the fluorescence intensity of multiple types of algae and separating a portion of the multiple types of algae based on the fluorescence intensity, wherein the separated algae and / or their progeny include high-light-resistant algae.

[0011] The mechanism by which high-light-tolerant algae are selected by the method of the embodiment is not limited. However, photosynthetic organisms have the ability to dissipate excess energy generated by strong light into pathways other than photosynthesis, including fluorescence. Because the dissipation of fluorescent energy can be measured by the intensity of autofluorescence, it is believed that algae resistant to high light can be obtained by selectively separating cells with high autofluorescence intensity. This method, which can quantify autofluorescence using commercially available equipment, does not require the identification and modification of target genes, thereby reducing the effort and time required to obtain high-light-tolerant algae.

[0012] In the present disclosure, "strong light" refers to a light intensity at which light saturation occurs, and may refer to conditions including a light intensity at which photosynthesis does not increase even if the light is made stronger than this intensity. In the embodiment, strong light is 250 μmol photons / m 2 / s or more, 500 μmol photons / m 2 / s or more, or 1000 μmol photons / m 2 / s or more, and 3000 μmol photons / m 2 / s or less, 2500μmol photon / m 2 / s or less, or 2000 μmol photons / m 2 In an embodiment, the intense light may be 250 to 3000 μmol photons / m ２ / s, and may include a light amount of 500 to 2500 μmol photons / m ２ / s, or 1000 to 2000 μmol photons / m ２ The light intensity may include a light intensity of 1 / s. These light intensities may be the maximum light intensity or the average light intensity during the culture step. Furthermore, these light intensities may be the light intensity temporarily irradiated during the culture step or the light intensity constantly irradiated.

[0013] The type of algae in the present disclosure is not limited. The algae may be non-sessile algae. The algae may be, for example, unicellular algae or microalgae. Examples of algae that may be used include Aurantiochytrium, Chlamydomonas, Chlorella, Schizon algae, Spirulina, Botryococcus, Euglena, Haptophyte, Prasinophyte, Chlorophyte, Phaeophyte, Rhodophyte, Cyanobacteria, Diatoms, Xanthophyte, Chrysophyte, Dinoflagellates, and Seaweed. More specific examples of algae include algae of the genera Chaetoceros, Isochrysis, Pavlova, Pyramimonas, and Tisochrysis. Among these, algae of the Chaetoceros genus, such as Chaetoceros gracilis, are preferred. The algae in the present disclosure may be a single species of algae or a population containing multiple species of algae. For example, a population of the same species of algae with genetic variation may be used. The algae of the present disclosure can be derived from isolated algae.

[0014] The algae in the present disclosure can be cultured in closed culture facilities, including bioreactors, flasks, culture tanks, and other closed culture vessels, or the algae can be cultured in open culture facilities, including open ponds.

[0015] Depending on the properties of the algae being cultured, algae can be cultured using algae culture media known to those skilled in the art, seawater, other environmental waters, diluted seawater, artificial seawater, mixtures of these, or solutions with a partial composition similar to these. Any aqueous liquid capable of growing natural algae can be used as the algae culture medium, and an aqueous algae culture medium with a defined composition can also be used. The medium can be any medium for algae culture known to those skilled in the art, such as a medium containing nutrients, carbon sources, rare metals, etc. Specific examples of the aqueous medium include IMK medium, SWM-3 medium, modified SWM-3 (mSWM-3) medium, modified versions of these media, and mixtures of these media or modified media. IMK medium contains 200mg / L NaNO3, 1.4mg / L Na2HPO4, 5mg / L K2HPO4, 2.68mg / L NH4Cl, 5.2mg / L Fe-EDTA, 0.332mg / L Mn-EDTA, 37.2mg / L Na2-EDTA, 0.023mg / L ZnSO4・7H2O, 0.014mg / L CoSO4・7H2O, 0.0073mg / L Na2MoO4・2H2O, 0.0025mg / L CuSO4・5H2O, 0.0017mg / L H2SeO3, 0.2mg / L Thiamin-HCl, 0.0015mg / L Biotin, 0.0015mg / L Vitamin B12 0.18mg / L A medium consisting of MnCl2・4H2O and the balance seawater. In particular, when culturing diatoms, 0.2 to 1 mM Na2SiO3 may be added to the IMK medium in addition to the above components.mSWM-3 medium contained 17 mg NaNO3, 1.56 mg NaH2PO4・2H2O, 5.68 mg Na2SiO3・9H2O, 1.12 mg Na2EDTA・2H2O, 0.084 mg Fe-EDTA, 0.0346 μg Na2SeO3, 1 ml P-1 metal solution (618.3 mg H3BO4, 69.25 mg MnCl2・4H2O, 5.45 mg ZnCl2, 238 μg CoCl2・6H2O, 100 ml distilled water), 0.2 μg Vitamin B12, 1 mL Vitamin mixed solution S3 (5 mg Thiamine HCl, 1 mg Nicotinic acid, 1 mg Calcium pantothenate, 0.1 mg p-Aminobenzoic acid, 0.01 mg Biotin, 50 mg Inositol, 0.02 mg Folic acid, 30 mg Thymine, 100 mL The medium (pH 7.7-7.8) is prepared by mixing 50 mg of Tris(hydroxymethyl)aminomethane, 50 mg of distilled water, and 98 mL of seawater.

[0016] The algae of the present disclosure may be cultured under natural light and / or artificial light. For example, when culturing under natural light, light may be blocked using a light-shielding sheet. The light-blocking rate of the light-shielding sheet may be about 30%, about 40%, about 50%, about 60%, about 70%, or about 75%, or may be 30-75%, 40-65%, or 45-60%. Such light-blocking using a light-shielding sheet may be performed only at the beginning of the culture, or at other times. The algae may be cultured indoors or outdoors. The algae may be cultured in an atmosphere of 0.5% or more, 1% or more, or 2% or more CO . ２ This may involve ventilation.

[0017] In the present disclosure, "high-light-tolerant algae" generally refers to algae whose growth rate is relatively resistant to decline when placed under high-light conditions, but is not limited to this. A comparison of growth rates can be appropriately performed by those skilled in the art by culturing under the same conditions, including high-light conditions, and comparing the algae concentration after a certain period of time has elapsed since the start of culturing. In one embodiment, high-light-tolerant algae sorted based on fluorescence intensity exhibit a growth rate of 250 μmol photons / m compared to control algae that have not undergone such sorting. 2 After culturing for 2 to 5 days under constant light conditions of 1000 / s (e.g., 2 or 5 days after the start of culturing), a higher number of proliferated cells can be observed. In an embodiment, it will be understood by those skilled in the art that high-photo-tolerant algae can have better or more normal physiological function under high-photo-tolerant algae than control algae, or can be less susceptible to death under high-photo-tolerant algae. When algae become high-photo-tolerant algae, the growth of the algae can be improved under high-photo-tolerant conditions that were previously unsuitable for growth, which can lead to more efficient algae production and the production of useful substances using algae.

[0018] In the method for selecting high-light-tolerant algae of the present embodiment, the multiple types of algae may be multiple types of algae with different genotypes. In this sense, the "multiple types" of algae may be, for example, multiple algae individuals that are taxonomically the same species but contain diverse genotypes. The origin of the algae with different genotypes is not limited. The algae with different genotypes may be a population of multiple types of algae or their progeny contained in a sample collected from a field, or a population of algae containing algae treated with a mutagen and / or their progeny. In the present embodiment, the mutagen may include one or more selected from radiation, ultraviolet light, and a chemical mutagen. In the present embodiment, the mutagen may be neutron radiation. In the present embodiment, the radiation may be neutron radiation, alpha radiation, or gamma radiation. Examples of chemical mutagens in the present embodiment include ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), and N-methyl-N-nitrosourea (MNU).

[0019] The type of neutron beam used in the embodiment is not limited, but preferably, a high-energy neutron beam can be used. As will be understood by those skilled in the art, a high-energy neutron beam is also called a fast neutron beam, and is typically a neutron beam containing a large number of neutrons with energies of 0.1 megaelectron volts or more.

[0020] The method for generating neutrons in the embodiments is not limited, but as known to those skilled in the art, neutrons can be generated by, for example, bombarding charged particles accelerated by a particle accelerator with a neutron generation target, such as beryllium, tungsten, lithium, lead, or mercury.

[0021] In an embodiment, the neutron irradiation conditions can be adjusted in different parts of the irradiation device. For example, the neutron beam intensity can be changed by changing the current value of the accelerator. For example, the neutron beam intensity can be adjusted by setting the current value to 10 μA, 65 μA, 100 μA, or 120 μA.

[0022] Alternatively, the energy of the neutron beam can be adjusted by using a moderator between the neutron generation target and the algae culture solution to be irradiated. For example, by changing the type and thickness of the moderator, it is possible to generate a neutron beam that is mainly composed of high-energy neutrons or thermal neutrons. Examples of moderators that can be used include water, heavy water, polyethylene, and methane.

[0023] Alternatively, the absorbed dose can be adjusted by varying the distance between the neutron generating target and the algal culture to be irradiated.

[0024] It will be understood by those skilled in the art that by changing or combining the various irradiation conditions described above, it is possible to generate neutron beam species with appropriate characteristic values ​​such as energy and intensity.

[0025] In an embodiment, neutron irradiation of algae can be carried out after transferring algae cultured to the logarithmic growth phase into a container such as a centrifuge tube, etc. The material of the container such as a centrifuge tube can be, for example, glass, polypropylene, polycarbonate, polyethylene, polystyrene, polyethylene terephthalate, etc., or a combination thereof, but is not limited to these.

[0026] The absorbed dose of the irradiated neutron beam may be, for example, in the range of 0.1 Gy to 100 Gy (0.1 Gy or more and 100 Gy or less), and preferably in the range of 0.2 Gy to 50 Gy (0.2 Gy or more and 50 Gy or less). It will be understood by those skilled in the art that the absorbed dose of the irradiated neutron beam can be adjusted to a desired value for both thermal neutron beams and high-energy neutron beams by adjusting one or more of the irradiation time, the distance between the neutron generation target and the algae culture solution, and the beam current value of the accelerator.

[0027] The method for selecting high-light-tolerant algae disclosed herein includes measuring the fluorescence intensity of multiple types of algae and separating a portion of the multiple types of algae based on the fluorescence intensity. The measured fluorescence of the algae may be correlated (usually positively correlated) with its ability to dissipate high-light energy. In embodiments, the fluorescence may include autofluorescence of the algae. In embodiments, the fluorescence may include chlorophyll fluorescence, which may be chlorophyll a, chlorophyll b, or chlorophyll c. These types of fluorescence may be positively correlated with the algae's ability to dissipate excess energy generated by high light into pathways other than photosynthesis. In embodiments, the fluorescence may be measured using, for example, an excitation wavelength of 488 nm and a 695 / 50 nm bandpass filter for detection. A flow cytometer or a cell sorter integrated therewith may be suitably used to measure the fluorescence intensity. The algae and / or their progeny separated by this method may include high-light-tolerant algae.

[0028] The fluorescence of the embodiment may include the autofluorescence of the algae and may be fluorescence that is substantially free of fluorescence due to exogenous fluorophores. Here, the exogenous fluorophores may be fluorophores contained in exogenous fluorescent proteins, fluorescent small molecule compounds, or antibodies containing these, etc., and by being substantially free of these fluorophores that may emit fluorescence unrelated to high-light tolerance, the fluorescence of the algae may be more strongly correlated with the algae's ability to dissipate high-light energy. In the present disclosure, "substantially free of fluorescence due to exogenous fluorophores" may mean that the fluorescence of exogenous fluorophores is not present or detectable in amounts that would interfere with the fluorescence-based evaluation of high-light tolerance of the algae.

[0029] In the embodiment, separating a portion of the plurality of types of algae based on fluorescence intensity may include separation using a cell sorter. As will be apparent to those skilled in the art, a cell sorter is a device that separates particles, including algae and other cells, based on optical properties such as fluorescence intensity measured by an integrated flow cytometer or the like, and the cell sorter can separate algae having a specific fluorescence intensity.

[0030] In some embodiments, separating a portion of the plurality of types of algae based on light intensity may include separating algae that exhibit a fluorescence intensity greater than a threshold value. The threshold value may be a threshold value for separating algae that exhibit a fluorescence intensity corresponding to the top 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the fluorescence intensity distribution, or any intermediate value therebetween. Those skilled in the art will appreciate that such a threshold value can be easily determined by obtaining a distribution of fluorescence intensities of algal samples in preliminary experiments. Alternatively, in some embodiments, separating a portion of the plurality of types of algae based on light intensity may include separating algae that exhibit a fluorescence intensity equal to or greater than the n% percentile (where n is a positive number equal to or greater than 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99) in the plurality of types of algae population. For a given sample, "sorting" can include sorting all algae that fall within the fluorescence intensity range described above, or sorting only a portion of the algae that fall within that range.

[0031] The following examples of the present disclosure are provided, but the present disclosure is not limited to the examples described below. In particular, similar results may be observed with algae other than the model algae used in the specific experiments described below.

[0032] The model alga, Chaetoceros gracilis, was cultured in IMK medium, and 40 mL of the culture medium in the early logarithmic growth phase was transferred to a 50 mL centrifuge tube. The tube was then irradiated with high-energy neutrons (40 Gy absorbed dose). A parallel, unirradiated centrifuge tube was prepared as a control sample (1), which was not subjected to mutagen treatment or autofluorescence selection.

[0033] The culture medium of the irradiated sample and the control sample (1) was transferred to a flask and cultured at room temperature under weak light for about three weeks with shaking at 50 rpm. After that, the culture was cultured at 25°C under light conditions until stationary phase, and then transferred to dark conditions.

[0034] 5 x 10 culture medium from irradiated algae 5 Subcultured in IMK medium at 3x10 cells / mL. 6 When the cell density reached approximately 3 x 10 cells / mL, 5,000 cells with autofluorescence intensity above the threshold were sorted using a Sony cell sorter with an excitation wavelength of 488 nm and a 695 / 50 nm bandpass filter (FL4) for detection. These 5,000 cells were a population of relatively highly autofluorescent cells, with fluorescence intensity falling within the top 1%. The sorted highly autofluorescent population was cultured at room temperature under room light. After that, the cell density was reduced to 3 x 10 cells. 6 When the number of cells / mL reached approximately 100, the same sorting and culturing process was repeated. In parallel, a culture medium was prepared without selection by autofluorescence after neutron irradiation, and this was used as control sample (2). Control sample (2) was grown at 25°C and 50 μmol photons / m 2 The light intensity was maintained at or below 1 / s thereafter.

[0035] Algal cultures that had been selected twice based on autofluorescence were cultured at 5 x 10 5 Subcultured in IMK medium at 3 x 10 cells / mL. 6When the cell concentration reached approximately 100 cells / mL, cells with FL4 fluorescence intensity at an excitation wavelength of 488 nm higher than the threshold were sorted into a 96-well plate at 20 cells / well.

[0036] The algae collected on the plate were exposed to light at 25°C, with a photon density of 50-250 μmol / m 2 The algae were cultured under constant light conditions of 250 μmol photons / m². 24 wells with good growth were selected from the 96-well plate, and 20 μL of each well was transferred to a 24-well plate containing 2 mL of IMK medium. The algae in the 24-well plate were cultured at 25°C under a constant light condition of 250 μmol photons / m². 2 The cells were cultured under constant light conditions with relatively strong light of 50 μmol photons / m / s. One well showing good growth was visually selected, and the entire well was subcultured into a culture flask containing 38 mL of IMK medium (high autofluorescence sample). After subculture, the high autofluorescence sample and the control sample (1) were cultured at 25°C under a constant light condition of 50 μmol photons / m / s. 2 The cells were cultured on a shaker at 75 rpm under constant light conditions of 1000 / s.

[0037] The high autofluorescence sample and the control sample (1) were diluted to 1 × 10 4 cells / mL. 2 The cells were then transferred to constant light conditions of 1000 x 1000 / s and cultured on a shaker at 75 rpm. From day 4 onwards, the cell density of the highly autofluorescent sample was higher than that of the control sample (1), reaching 1.51 times that of the control sample (1) by day 12, indicating that the highly autofluorescent sample had a higher proliferation ability under strong light (Figure 1).

[0038] Next, the high autofluorescence sample and the control sample (2) were mixed at 1 × 10 4 cells / mL. 2 The cells were then transferred to constant light conditions of 1000 x 1000 / s and cultured on a shaker at 75 rpm. On day 2, the cell density of the highly autofluorescent sample was 2.72 times that of the control sample (2), and on day 6, it was 1.12 times higher. The highly autofluorescent sample had a higher proliferation ability under strong light during the early to mid-stages of proliferation (Figure 2).

[0039] The above results demonstrate that algae with high phototolerance can be selected by separating cells with high autofluorescence intensity, significantly reducing the effort and time required for selection compared to conventional techniques that involve, for example, identifying specific genes involved in phototolerance and specific genetic manipulation.

[0040] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the examples in the above embodiments. Various modifications can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0041] The present disclosure includes the following embodiments: (Item 1) A method for selecting high-light-tolerant algae, comprising measuring the fluorescence intensity of multiple types of algae, and separating a portion of the multiple types of algae based on the fluorescence intensity, wherein the separated algae and / or their progeny comprise high-light-tolerant algae. (Item 2) The method of item 1, wherein separating a portion of the multiple types of algae based on the fluorescence intensity comprises: (a) separating algae whose measured fluorescence intensity is greater than a threshold, or (b) separating algae whose fluorescence intensity is equal to or greater than the n% percentile (where n is a positive number greater than or equal to 75) in the population of the multiple types of algae. (Item 3) The method of item 1 or 2, wherein the fluorescence includes autofluorescence of the algae and is substantially free of fluorescence due to exogenous fluorophores. (Item 4) The method of any one of items 1 to 3, wherein the fluorescence includes fluorescence of chlorophyll. (Item 5) The method of any one of Items 1 to 4, wherein sorting a portion of the multiple types of algae based on the fluorescence intensity includes sorting using a cell sorter. (Item 6) The method of any one of Items 1 to 5, wherein the multiple types of algae include algae that have been treated with a mutagen and / or their progeny. (Item 7) The method of Item 6, wherein the mutagen includes one or more selected from radiation, ultraviolet light, and a chemical mutagen. (Item 8) The method of Item 6 or 7, wherein the mutagen includes neutron beams.

Claims

1. A method for selecting high-light-tolerant algae, comprising: measuring the fluorescence intensity of multiple types of algae; and separating a portion of the multiple types of algae based on the fluorescence intensity, wherein the separated algae and / or their progeny include high-light-tolerant algae.

2. The method of claim 1, wherein separating a portion of the plurality of types of algae based on the fluorescence intensity comprises: (a) separating algae whose measured fluorescence intensity is greater than a threshold value; or (b) separating algae whose fluorescence intensity is equal to or greater than the n% percentile (where n is a positive number greater than or equal to 75) in the population of the plurality of types of algae.

3. The method of claim 1 or 2, wherein the fluorescence comprises the autofluorescence of the algae and is substantially free of fluorescence due to exogenous fluorophores.

4. The method of claim 1 or 2, wherein the fluorescence comprises chlorophyll fluorescence.

5. The method according to claim 1 or 2, wherein separating a portion of the plurality of types of algae based on the fluorescence intensity includes separating the portion using a cell sorter.

6. The method of claim 1 or 2, wherein the plurality of algae species includes algae and / or their progeny that have been treated with a mutagen.

7. The method of claim 6, wherein the mutagen comprises one or more selected from radiation, ultraviolet light, or a chemical mutagen.

8. The method of claim 6, wherein the mutagen comprises neutron radiation.

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