Genetically modified algae that secretes astaxanthin and related methods of isolating and purifying astaxanthin

Abstract

The disclosure related to water-miscible forms of astaxanthin produced by transgenic red algae strains transformed to express β-carotene ketolase and hydroxylase genes from a green algae. These water-miscible forms of astaxanthin are aggregations of astaxanthin secreted by the transformed red algae. The particles accumulate in the extracellular space and are enriched in stationary phase of the algae culture, which enables simple and efficient isolation and purification of astaxanthin from algae. Thus, also disclosed herein are methods of isolating and / or purifying astaxanthin from transgenic algae comprising separating algae cells from its culture solution to collect the supernatant and then centrifuging the supernatant to pellet astaxanthin for collection and optionally further purification.

Description

Agent Reference: 11157-202WO-PCTGENETICALLY MODIFIED ALGAE THAT SECRETES ASTAXANTHINAND RELATED METHODS OF ISOLATING AND PURIFYINGASTAXANTHINRELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application 63 / 735,781 filed December 18, 2024, titled “Genetically Modified Algae that Secretes Astaxanthin and Related Methods of Isolating and Purifying Astaxanthin”, the entirety of the disclosure of which is hereby incorporated by this reference.TECHNICAL FIELD

[0002] This document relates to a genetically modified algae that secretes astaxanthin.BACKGROUND

[0003] Carotenoid compounds are long chain, lipophilic hydrocarbons produced from isoprene monomers in plants, algae and other microorganisms. They have strong antioxidant properties responsible for a wide variety of beneficial uses and growing demand in a variety of markets. The lipophilic nature of carotenoids complicates their isolation from cell lysates and formulation of the purified compounds into fish and animal feeds, cosmetics and nutraceuticals.

[0004] Astaxanthin is a keto-carotenoid and is a metabolite of zeaxanthin and canthaxanthin. Astaxanthin is a hydrophobic pigment with high antioxidant activity provides a red color. This pigment is naturally present in salmon, lobsters, and the flowers and leaves of a variety of plants. Accordingly, astaxanthin is used in the production of farmed salmon, shrimp, eggs and other foodstuffs. It is also heavily utilized in cosmetics as an antioxidant and in food supplements and nutraceuticals. Thus, astaxanthin is a pigment of high commercial value and needed.

[0005] Despite its commercial importance, large-scale production of astaxanthin remains problematic. The richest sources of astaxanthin are algal strains like Hematococcus pluvialis, but such algae can only be grown seasonally at commercial scale. Accordingly, the seasonality of algae maturation further complicates astaxanthin isolation at a commercial scale. Furthermore, the extraction of the astaxanthin creates challenges to the commercialization of astaxanthin from H. pluvialis. The cyst stage for these microalgae has a rigid cell wall that requires significant disruption for efficient extraction of the astaxanthin.Agent Reference: 11157-202WO-PCTSUMMARY

[0006] The methods of isolating and / or purifying astaxanthin from algae is based on the discovery that suspensions of astaxanthin aggregates are produced and secreted by red algae transformed to express P-carotene ketolase (BKT) and hydroxylase (CHYB) genes from green algae. In some aspects, the red algae transformed is Cyanidioschyzon merolae, and the BKT and CHYB genes are from Chlamydomonas reinhardtii . The secreted astaxanthin form aggregates in the extracellular space when the transgenic algae culture reaches the stationary phase (preferably the late stationary phase).

[0007] In some embodiments, the method of isolating astaxanthin from algae comprises providing an algae culture system comprising red algae transformed to express BKT and CHYB genes from a green algae and a culture solution; culturing the transgenic algae to stationary phase of growth; separating the transgenic algae from the culture solution during the stationary phase to collect resulting supernantent; and collecting astaxanthin from the supernantent thereby isolating astaxanthin from the transgenic algae. In other embodiments, the method of isolating astaxanthin from algae comprises providing a continuous, high-density culture system comprising red algae transformed to express BKT and CHYB genes from a green algae and a culture solution; harvesting a portion of the transgenic algae when dilution rate of the continuous, high-density culture system equals growth rate of the transgenic algae collected in outflow of the continuous, high-density culture system; separating the transgenic algae from the culture solution to collect resulting supernantent; and collecting astaxanthin from the supernantent thereby isolating astaxanthin from the transgenic algae. For these methods, the culture solution may comprise wastewater.

[0008] In some embodiments, a method of isolating astaxanthin from algae from wastewater is described. In some implementations, the method comprises providing an algae culture system comprising red algae transformed to express BKT and CHYB genes from a green algae and a culture solution comprising wastewater; harvesting a portion of the transgenic algae when dilution rate of the continuous, high-density culture system equals growth rate of the transgenic algae collected in outflow of the continuous, high-density culture system; separating the transgenic algae from the culture solution to collect resulting supernantent; and collecting astaxanthin from the supernantent thereby isolating astaxanthin from the transgenic algae. In other implementations, the method comprises providing an algae culture system comprising red algae transformed to express BKT and CHYB genes from a green algae and a culture solution comprising wastewater; administering a water-immiscile and non-polar solvent to theAgent Reference: 11157-202WO-PCT continuous, high-density culture system after the transgenic algae reaches stationary phase of growth, wherein the stationary phase of growth is when dilution rate of the continuous, high- density culture system equals growth rate of the transgenic algae collected in outflow of the continuous, high-density culture system; collecting an amount of outflow from the continuous, high-density culture system after administration of the immiscile and non-polar solvent, wherein the collected outflow comprises an organic phase and a water phase; and collecting astaxanthin from the organic phase of the collected outflow thereby isolating astaxanthin from the transgenic algae in the continuous, high-density culture system.

[0009] In implementations of the method of isolating astaxanthin from algae where the culture solution comprsies waste, the wastewater is from salmon farming or shrimp farming.

[0010] In some aspects of the methods described, the green algae is C. reinhardtii. In some aspects, the red algae is C merolae, for example 10D strain or a population produced from mating C. merolae 10D with C. merolae MS 10 haploid.

[0011] For all implementations of the described methods of isolating astaxanthin from algae, the method may further comprise purifying the collected astaxanthin with ultrafiltration, an water-immiscile and non-polar solvent, a supercritical fluid, or magnetic beads densely coated with hydrophobic binding sites. The water-immiscile and non-polar solvent may be dodecane. The supercritical fluid may be supercritical CO2.

[0012] In certain implementation, the step of collecting separating the transgenic algae from the culture solution to collect the resulting supernatant for isolating astaxanthin comprises centrifugation. For example, the step comprises centrifuging the culture solution comprising the transgenic algae at 4,000 x g. In some embdoiments, the culture solution is centrifuged at 4,000 x g for 5 minutes. In some implementations, the step of collecting astaxanthin from the supernantant comprises allowing the astaxanthin in the supernatant to naturally settle or further centrifugation to pellet the astaxanthin.

[0013] The foregoing and other aspects, features, and advantages will be apparent from the DESCRIPTION and DRAWINGS, and from the CLAIMS if any are included.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0015] Implementations will hereinafter be described in conjunction with the appended and / or included DRAWINGS, where like designations denote like elements.Agent Reference: 11157-202WO-PCT

[0016] FIG. l is a representation of negative stain transmission electron microscopy image of spheroid particles of the Cyanidioschyzon merolae cell-free complexes, which contain secreted astaxanthin. These spheroid particles have a diameter of ~60 nm.

[0017] FIG. 2 is a representation photograph of three cultures of C. merolae that has been pelleted. The tube labeled 1 contains wild-type C. merolae 10D. The tube labeled 2 contains C. merolae 10D expressing Chlamydomonas reinhardtii 0-carotene ketolase (BKT), which produces canthaxanthin. The tube labeled 3 contains C. merolae 10D expressing C. reinhardtii P-carotene ketolase (BKT) and hydroxylase (CHYB), which produces astaxanthin.

[0018] FIG. 3 is a representative UV-Vis spectra from astaxanthin aggregates isolated by centrifugation and dissolved in DMSO.

[0019] FIG. 4 depicts, in accordance with certain embodiments, a media solution from a culture of the described transgenic algae culture producing water-miscible forms of astaxanthin (left) and the media solution with the astaxanthin aggregates pelleted (right).DETAILED DESCRIPTION

[0020] Detailed aspects and applications of the disclosure are described below in the following drawings and detailed description of the technology. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.

[0021] In the following description, and for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various aspects of the disclosure. It will be understood, however, by those skilled in the relevant arts, that embodiments of the technology disclosed herein may be practiced without these specific details. It should be noted that there are many different and alternative configurations, devices and technologies to which the disclosed technologies may be applied. The full scope of the technology disclosed herein is not limited to the examples that are described below.

[0022] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a step” includes reference to one or more of such steps.

[0023] The word “exemplary,” “example,” or various forms thereof are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” or as an “example” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Furthermore, examples are provided solely for purposes of clarityAgent Reference: 11157-202WO-PCT and understanding and are not meant to limit or restrict the disclosed subject matter or relevant portions of this disclosure in any manner. It is to be appreciated that a myriad of additional or alternate examples of varying scope could have been presented but have been omitted for purposes of brevity.

[0024] When a range of values is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0025] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to”, and are not intended to (and do not) exclude other components.

[0026] As used herein, the term “bulk solution” refers to water surrounding the algae, which contains nutrients such as provided sugars. As used herein, the term encompasses the liquid component of an algae culture, which can contain algae cells and their secreted extracellular matrix.

[0027] As used herein, the term “media solution” refers to the liquid component of an algae culture without the algae cells. Accordingly, in some aspects, the media solution is the supernatant result from separating algae from the culture solution in which the algae is grown.

[0028] As required, detailed embodiments of the present disclosure are included herein. It is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limits, but merely as a basis for teaching one skilled in the art to employ the present invention. The specific examples below will enable the disclosure to be better understood. However, they are given merely by way of guidance and do not imply any limitation.

[0029] The present disclosure may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific materials, devices, methods, applications, conditions, or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed inventions. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of theAgent Reference: 11157-202WO-PCT antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0030] Reported for the first time herein is an algae culture and a method of culturing algae that secretes astaxanthin. While traditionally hydrophobic, the disclosed transgenic algae can be cultured to produce a water-miscible form of astaxanthin. Thus, a method of inducing secretion of astaxanthin and a method of isolating astaxanthin from an algae culture are described.

[0031] Current methods of astaxanthin purification require cell lysis and subsequent separation from proteins, carbohydrates, and lipids. This requires organic solvents or supercritical fluids, followed by solvent recovery and evaporation followed by high performance liquid chromatograph (HPLC), flash chromatography or centrifugal partition chromatography. The disclosed methods of isolating and purifying astaxanthin eliminate the need for cellular disruption and purification from the other cellular components, which comprise roughly 94% of the disrupted biomass in red phase Hematococcus pluvialis biomass.

[0032] It was discovered that a transgenic algae culture produces water-miscible forms of astaxanthin, which once the culture grows to a certain stage, are enriched in the extracellular space as aggregates of about approximately 20-60 nm in diameter. These astaxanthin aggregates do not contain any proteins. The highest enrichment of astaxanthin aggregates in the extracellular space takes place when the transgenic algae culture reaches the stationary phase. In other words, when the algae culture reaches a net growth rate of zero in spite having sufficient exposure to light and CO2, the net production of astaxanthin produced by the transgenic algae is at maximum and forms aggregates in the culture solution that can be easily isolated. In a continuous, high-density culture system, the stationary phase corresponds to the dilution rate equaling the growth rate with the cells collected in the outflow. At this stage of growth in the continuous, high-density culture system, the productivity of astaxanthin by the transformed red algae is maximized. In this context, “productivity” is defined as the mass of astaxanthin per culture volume (or cell mass) per unit time. Thus, the aforementioned transgenic algae culture enables easy isolation of astaxanthin from the algae culture.

[0033] The methods of characterizing astaxanthin aggregates are described in Lu et al, (2017). Secretion of astaxanthin was observed in red algae transformed to express P-carotene ketolase (BKT) and hydroxylase (CHYB) genes from a green algae (FIGs. 1-3). Introduction of BKT and CHYB genes from green algae into red algae resulted in production of orange pigment canthaxanthin and red pigment astaxanthin by the transgenic algae (FIG. 2). Introduction of BKT alone yielded only canthaxanthin, which was retained inside cells (FIG. 2). Thus,Agent Reference: 11157-202WO-PCT disclosed herein is a method of inducing secretion of astaxanthin by algae comprising culturing red algae transformed to express both BKT and CHYB genes from a green alga, wherein the transformed red algae is cultured to the stationary phase of growth. In some aspects, the BKT and CHYB genes are from Chlamydomonas reinhardtii. In certain implementations, the BKT and CHYB genes are introduced into the red algae using homologous recombination, for example, as described in Seger et al., 2023, to produce the transformed red algae.

[0034] The astaxanthin aggregates in the extracellular space can easily be separated from the bulk solution. For example, differential, low-speed centrifugation is applied to the bulk solution to separate algae cells from the media solution, and then the astaxanthin aggregates can be collected from the media solution by pelleting of the astaxanthin aggregates. Thus, the isolation of astaxanthin from the described transgenic algae culture is a two-phase process. In an exemplary implementation, the first phase is separating the algae cells from the culture solution and the second phase is isolating the astaxanthin aggregates in the cell-free supernatant in concentrated form. In some aspects, the first phase comprises centrifugating the bulk solution at 4,000 x g for 5 minutes to isolate the algae cells from the culture solution. In some aspects, the second phase isolates astaxanthin aggregates by further low-speed centrifugation, filtration, or natural settling, which can yield highly concentrated astaxanthin aggregates ready for further purification by any means desired. Exemplary methods for further purification of astaxanthin include ultrafiltration or astaxanthin purification methods known in the art using an organic solvent or a supercritical fluid. In some aspects, the organic solvent is a non-polar solvent.

[0035] In another implementation, the astaxanthin aggregates can be be purified via a continous culture / astaxanthin removal production mode. In some aspects, this mode utilizes existing solvent extraction systems using bio-compatible, water-immiscible solvents or magnetic beads densely coated with hydrophobic binding sites to capture the astaxanthin aggregates from the bulk solution. In some aspects, the bio-compatible, water-immiscible solvent is a non-polar solvent or a supercritical fluid, for example, dodecane or supercritical CO2 or other specially formulated hydrocarbon mixtures or certain ionic liquids. Because the transgenic algae releases astaxanthin from the cells during growth, astaxanthin purification could be inexpensively performed in either batch or continuous modes. The astaxanthin isolated from such an algae culture could also be easily purifed to absolute purity.

[0036] Accordingly, in one aspect, the methods described herein comprise providing an algae culture system comprising transgenic algae, wherein the transgenic algae is red algae transformed to express BKT and CHYB genes from a green algae, and a culture solution; and culturing the transgenic algae to stationary phase of growth. The method next comprisesAgent Reference: 11157-202WO-PCT separating the transgenic algae from the culture solution during the stationary phase and collecting resulting supernantent. Then method further comprises collecting astaxanthin from the supernantent thereby isolating astaxanthin from the transgenic algae. In another aspect, the methods described herein comprise providing a continuous, high-density culture system comprising transgenic algae, wherein the transgenic algae is red algae transformed to express BKT and CHYB genes from a green algae, and a culture solution; and harvesting a portion of the transgenic algae when dilution rate of the continuous, high-density culture system equals growth rate of the transgenic algae collected in outflow of the continuous, high-density culture system. The method next comprises separating the transgenic algae from the culture solution and collecting the resulting supernantent. The method further comprises collecting astaxanthin from the supernantent thereby isolating astaxanthin from the transgenic algae. Conventional methods of purifying astaxanthin isolates could be used to further purify the astanxanthin fraction collected from the supernatant. Exemplary purifiction steps may include using ultrafiltration or using organic solvents or supercritical fluids to isolate purer fractions of astaxanthin.

[0037] In certain implementations of the disclosed methods, the culture solution comprises wastewater. In particular embodiments, the culture solution comprises wastewater from salmon or shrimp farming. In other embodiments, the methods utilize wastewaters from food and beverage manufacturing or agricultural wastewaters, such as obtained from hydroponic plant production, as the culture solution.

[0038] In a particular implementation, the method of producing astaxanthin comprises providing a continuous, high-density culture system comprising transgenic algae and a culture solution comprising wastewater, wherein the transgenic algae is red algae transformed to express BKT and HYB genes from a green algae; and harvesting a portion of the transgenic algae when dilution rate of the continuous, high-density culture system equals growth rate of the transgenic algae collected in outflow of the continuous, high-density culture system. The method next comprises separating the transgenic algae from the culture solution to collect resulting supernantent; and collecting astaxanthin from the supernantent thereby isolating astaxanthin from the transgenic algae. In some aspects, the wastewater is from salmon or shrimp farming, food and beverage manufacturing, or agricultural wastewater.Transgenic red algae

[0039] In some embodiments, the red algae that is transformed to express BKT and CHYB genes from green algae is a Cyanidioschyzon species, for example, Cyanidioschyzon merolae.Agent Reference: 11157-202WO-PCTAs a thermoacidophilic organism, C. merolae has fewer competitors and pathogens than H. pluvialis and other algae currently used for astaxanthin production at neutral pH and lower temperatures. Accordingly, C. merolae is a much easier to manage cellular machinery for production of astaxanthin.

[0040] In some embodiments, C. merolae 10D strain (wildtype counterpart being the NIES- 3377 strain) is transformed to express BKT and CHYB genes from green algae. In certain embodiments, the red algae for genetic modification is produced from the C. merolae 10D strain and the C. merolae MSI strain. The 10D strain is haploid without a cell wall, while the MSI strain is diploid, has a cell wall, and forms haploids at pH 0.5. Thus in some aspects, the red algae that is transformed to express BKT and CHYB genes from green algae is the diploid, cell wall-containing algae population produced from mating an MSI haploid with the BKT and CHYB transformed 10D strain. Because diploid cells with cell walls are more resistant to shearing stress produced in some types of bioreactors, this diploid, cell wall-containing algae population produced from mating an MSI haploid with the BKT and CHYB transformed 10D strain is suited for culture conditions where the algal cells experience mechanical force from impellers, aeration, or fluid flow, for examples in bioreactors with stirred tanks and perfusion systems.Examples

[0041] The present disclosure is further illustrated by the following examples that should not be construed as limiting.Example 1: An exemplary method of purifying astaxanthin

[0042] Astaxanthin aggregates produced in transgenic C. merolae 10D strain (see Seger et al., Metabolic Engineering Communications, 2023, 17: e00226) are suspended in the extracellular growth medium as approximately -20-60 nm diameter spheroids (FIG. 1). The particles accumulate in the extracellular space and are enriched in stationary phase cultures. The following steps describe an exemplary method of isolating astaxanthin.

[0043] Step 1 : Growth of C. merolae 10D strain cells transformed to express P-carotene ketolase (BKT) and hydroxylase (CHYB) genes from a green algae to late stationary phase. Alternatively, one could use a continuous, high-density culture system in which the dilution rate equals the growth rate with the cells collected in the outflow passed to the next step in the protocol.Agent Reference: 11157-202WO-PCT

[0044] Step 2: Separation of cells from the growth medium via (1) centrifugation at 4,000 x g, (2) filtration through appropriate media that retain cells and allow astaxanthin aggregates to flow through, or (3) simple settling.

[0045] Step 3: Centrifugation of astaxanthin in the spent, cell-free growth medium at 12,000 x g yields a red pellet of the astaxanthin aggregates.

[0046] Step 4: Resuspension of astaxanthin aggregates followed by ultrafiltration using a 200 kDa molecular weight cut off filter to concentrate the complex and remove small molecules present in the pellet from the 12,000 x g pellet. Alternatively, the astaxanthin can be dissolved in a suitable organic solvent or supercritical fluid for additional purification.Example 2: An exemplary method of purifying astaxanthin from a continuous culture system

[0047] The following steps describe an exemplary method of isolating astaxanthin from a continuous, high-density culture system using a continous culture / astaxanthin removal production mode.

[0048] Step 1 : Growth of C. merolae 10D strain cells transformed to express BKT and hydroxylase CHYB genes from a green algae in a continuous, high-density culture system until the dilution rate equals the growth rate with the cells collected in the outflow.

[0049] Step 2: Introduction of a compatible but immiscible and non-polar solvent (for example, dodecane) into the culture system upon which the astaxanthin aggregates will dissolve generating an organic phase and a water phase.

[0050] Step 3 : Separation of the organic and water phases from the outflow of the continuous reactor and collect the organic phase.

[0051] Step 4: Performance of additional purification steps with the organic phase, as needed, to purify astaxanthin, for example, with centrifugations and / or ultrafiltration.REFERENCES CITED AND INCORPORATED BY REFERENCE o Seger et al., Metabolic Engineering Communications, 2023, 17: e00226. o Fassett and Coombes, Mar Drugs., 2011, 9(3): 447-465. o Yamashita, Foods., 2018, 7(2):50. o Ohgami et al., Invest Ophthalmol 'is Sci., 2003, 44(6): 2694-2701. o Djordjevic et al., J Sports Med Phys Fitness., 2012, 52(4):382-392. o Iwamoto et al., J Atheroscler Thromb., 2000, 7(4):216-222. o Grimmig et al., J Nutr Biochem., 2017, 52:81-86. o Chew et al., Anticancer Res., 1999, 19(6B):5223-5227.Agent Reference: 11157-202WO-PCT o Yang et al., J Diabetes Res., 2013, 2013:696158. o Lu, et al., Spectrochimica Acta Part A, 2017, 185:85-92.

Claims

Agent Reference: 11157-202WO-PCTCLAIMSI / We Claim:

1. A method of isolating astaxanthin from algae comprising: providing an algae culture system comprising: transgenic algae, wherein the transgenic algae is red algae transformed to express P-carotene ketolase (BKT) and hydroxylase (CHYB) genes from a green algae; and a culture solution; culturing the transgenic algae to stationary phase of growth; separating the transgenic algae from the culture solution during the stationary phase to collect resulting supernantent; and collecting astaxanthin from the supernantent thereby isolating astaxanthin from the transgenic algae.

2. The method of claim 1, wherein the green algae is Chlamydomonas reinhardtii.

3. A method of isolating astaxanthin from algae comprising: providing a continuous, high-density culture system comprising: transgenic algae, wherein the transgenic algae is red algae transformed to express P-carotene ketolase (BKT) and hydroxylase (CHYB) genes from a green algae; and a culture solution; harvesting a portion of the transgenic algae when dilution rate of the continuous, high- density culture system equals growth rate of the transgenic algae collected in outflow of the continuous, high-density culture system; separating the transgenic algae from the culture solution to collect resulting supernantent; and collecting astaxanthin from the supernantent thereby isolating astaxanthin from the transgenic algae.

4. The method of claim 2, wherein the green algae is Chlamydomonas reinhardtii.

5. A method of producing astaxanthin using wastewater comprising: providing a continuous, high-density culture system comprising: transgenic algae, wherein the transgenic algae is red algae transformed to express P-carotene ketolase (BKT) and hydroxylase (CHYB) genes from a green algae; and a culture solution comprising wastewater; harvesting a portion of the transgenic algae when dilution rate of the continuous, high- density culture system equals growth rate of the transgenic algae collected in outflow of the continuous, high-density culture system;Agent Reference: 11157-202WO-PCT separating the transgenic algae from the culture solution to collect resulting supernantent; and collecting astaxanthin from the supernantent thereby isolating astaxanthin from the transgenic algae.

6. The method of claim 5, wherein the green algae is Chlamydomonas reinhardtii.

7. A method of producing astaxanthin using wastewater comprising: providing a continuous, high-density culture system comprising: transgenic algae, wherein the transgenic algae is red algae transformed to express P-carotene ketolase (BKT) and hydroxylase (CHYB) genes from a green algae; and a culture solution comprising wastewater; administering a water-immiscile and non-polar solvent to the continuous, high-density culture system after the transgenic algae reaches stationary phase of growth, wherein the stationary phase of growth is when dilution rate of the continuous, high-density culture system equals growth rate of the transgenic algae collected in outflow of the continuous, high-density culture system; collecting an amount of outflow from the continuous, high-density culture system after administration of the immiscile and non-polar solvent, wherein the collected outflow comprises an organic phase and a water phase; and collecting astaxanthin from the organic phase of the collected outflow thereby isolating astaxanthin from the transgenic algae in the continuous, high-density culture system.

8. The method of claim 7, wherein the green algae is Chlamydomonas reinhardtii.

9. The method of any one of claims 1-8, wherein the red algae is Cyanidioschyzon merolae.

10. The method of claim 9, wherein the red algae is C. merolae 10D.

11. The method of claim 9, wherein the red algae is produced from mating C. merolae 10D with C. merolae MS 10 haploid.

12. The method of any one of claims 1-4, wherein the culture solution comprises wastewater.

13. The method of claim 12, wherein the wastewater is from salmon farming or shrimp farming.

14. The method of any one of claim 5-8, wherein the wastewater is from salmon farming or shrimp farming.

15. The method of any one of claims 1-8, further comprising purifying the collected astaxanthin with ultrafiltration.Agent Reference: 11157-202WO-PCT16. The method of any one of claims 1-8, further comprising purifying the collected astaxanthin with an water-immiscile and non-polar solvent, a supercritical fluid, or magnetic beads densely coated with hydrophobic binding sites.

17. The method of any one of claims 1-6, wherein the step of separating the transgenic algae from the culture solution to collect resulting supernantent comprises centrifuging the culture solution comprising the transgenic algae at 4,000 x g.

18. The method of claim 17, wherein the culture solution comprising the transgenic algae is centrifugated for 5 minutes.

19. The method of any one of claims 1-6, wherein the step of collecting astaxanthin from the supemantant comprises allowing the astaxanthin in the supernatant to naturally settle.

20. The method of any one of claims 1-6, wherein the step of collecting astaxanthin from the supemantant comprises centrifuging the supemantant to pellet the astaxanthin.

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