Chovulochloris zoffithii mutant strain for producing astaxanthin and canthaxanthin and application thereof

Abstract

The invention relates to the field of microbial engineering and genetic breeding, and discloses a Chovulochroma zoffii mutant strain capable of producing astaxanthin and canthaxanthin and an application of the Chovulochroma zoffii mutant strain, including a Chovulochroma zoffii mutant strain named as Cz-R1 and an application of the Chovulochroma zoffii mutant strain in production of astaxanthin and canthaxanthin, and particularly relates to a Chovulochroma zoffii mutant strain named as Cz-R1 and an application of the Chovulochroma zoffii mutant strain in production of astaxanthin and canthaxanthin. The mutant strain of the Zhovuchroma viridis retains the growth attribute of a wild strain of the Zhovuchroma viridis under a glucose heterotrophic condition, has the capability of high-density fermentation, and can improve the yield and accumulation amount of astaxanthin and canthaxanthin.

Description

Technical Field

[0001] This invention relates to the fields of microbial engineering and genetic breeding, and in particular to a mutant strain of Zoff's green algae that produces astaxanthin and canthaxanthin and its applications. Background Technology

[0002] Astaxanthin and canthaxanthin are both secondary carotenoids, with canthaxanthin being a precursor to astaxanthin. Both possess strong antioxidant capabilities and are widely used in food, medicine, cosmetics, health products, and aquaculture. Astaxanthin and canthaxanthin can be obtained artificially or from natural organisms. Artificially synthesized astaxanthin and canthaxanthin contain impurities, and their quality and antioxidant capacity are far inferior to those of naturally obtained astaxanthin and canthaxanthin. Due to the high market value and continuously growing demand in the nutritional supplement market, natural astaxanthin and natural canthaxanthin are destined to become the main players in the high-end pigment market.

[0003] Haematococcus pluvialis is a major source of natural astaxanthin. The primary cultivation mode for Haematococcus pluvialis is photoautotrophy, which results in slow growth and requires independent high-light induction for astaxanthin accumulation, leading to a production cycle of several weeks. Furthermore, the open cultivation environment makes it susceptible to biological contamination by rotifers, chytrids, and other algae, causing significant yield reductions. The inherent biological characteristics of Haematococcus pluvialis severely hinder innovation in its cultivation technology and industrial upgrading, keeping the production cost of natural astaxanthin and the prices of related end products high.

[0004] Canthaxanthin, an intermediate in the synthesis of astaxanthin, is generally present in low amounts in organisms in nature, making the industrial-scale production of natural canthaxanthin through biological systems still challenging.

[0005] Reference document 1: Chinese patent application number 202410574992.2 discloses a method for producing natural astaxanthin by fermenting Zoff's green algae. The method involves inoculating Zoff's green algae into the basic culture medium of a fermenter for fermentation culture. The fermentation culture process is divided into two stages: cell growth stage and induction of astaxanthin accumulation stage. Finally, astaxanthin is synthesized and accumulated.

[0006] Reference document 2: Chinese patent application number 202211427752.7 discloses a process and optimization method for extracting canthaxanthin from Zov's green algae. First, the natural eutectic solvent with the highest extraction efficiency for canthaxanthin from Zov's green algae is screened. Based on partial factorial design experiments, the main effect factors affecting the extraction efficiency are identified and applied to central composite design experiments to obtain the extraction efficiency of canthaxanthin under different parameter conditions. Finally, the extraction process parameters are optimized by combining response surface model and experimental verification is carried out.

[0007] Based on comparative documents 1 and 2, it is demonstrated that *Zovella zovis* possesses an innate ability to synthesize astaxanthin and canthaxanthin, making it a strong competitor to *Haematococcus pluvialis* in natural astaxanthin production. It also holds the potential for large-scale canthaxanthin production, potentially enabling the green bio-manufacturing of natural astaxanthin and canthaxanthin through fermentation. However, the astaxanthin and canthaxanthin content produced by *Zovella zovis* is relatively low, making it difficult to meet the demands of large-scale production. Summary of the Invention

[0008] The first objective of this invention is to address the shortcomings of the low astaxanthin and canthaxanthin content produced by Zoff's green algae in the prior art, and to provide a Zoff's green algae mutant strain that can increase the production of astaxanthin and canthaxanthin.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A mutant strain of Chromochloris zofingiensis producing astaxanthin and canthaxanthin, named Chromochloris zofingiensis Cz-R1, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46621 on October 16, 2025.

[0010] Using the above method, the purchased wild-type Chromochloris strain was mutated to form a mutant strain of Chromochloris that can produce high levels of astaxanthin and canthaxanthin, named Chromochloris zofingiensis Cz-R1.

[0011] The second objective of this invention is the application of the aforementioned Zoff's green algae mutant strain in the production of astaxanthin and canthaxanthin.

[0012] As a preferred method, a method for producing astaxanthin and canthaxanthin from the Zoffingiensis mutant strain is provided: take the activated cultured Zoffingiensis (Chromochloris zofingiensis) Cz-R1 seed culture at 3000×g, centrifuge for 2 min, remove the supernatant, resuspend the precipitate in BG11 + 20 g / L glucose medium, and culture continuously in the dark for 15 days at 25 ℃ and 150 rpm in a shaker. The synthesis of astaxanthin and canthaxanthin is induced by the nutrient stress formed after the depletion of carbon and nitrogen sources.

[0013] Using the above scheme, the Zoff-colored green algae mutant strain was cultured to the plateau phase under heterotrophic culture conditions of BG11 + 20 g / L glucose medium, and then cultured for another 7 days, i.e., continuously cultured for 15 days. The nutritional stress caused by the depletion of carbon and nitrogen sources induced the synthesis of large amounts of astaxanthin and canthaxanthin.

[0014] As preferred, the astaxanthin accumulation was 2.6308 mg / g; the canthaxanthin accumulation was 13.718 mg / g.

[0015] Using the above scheme, after the carbon and nitrogen sources were depleted, the Zov's green algae mutant strain could be induced to accumulate astaxanthin up to 2.6308 mg / g and canthaxanthin up to 13.718 mg / g. The astaxanthin content of the mutant strain Cz-R1 was 421% higher than that of the wild strain Cz-WT (0.50476 mg / g), and the canthaxanthin content of the mutant strain Cz-R1 was 728% higher than that of the wild strain Cz-WT (1.6565 mg / g).

[0016] The present invention has significant technical effects due to the adoption of the above technical solutions: the Zov-colored green algae mutant strain, which is obtained by mutagenesis of Zov-colored green algae Chromochloris zofingiensis UTEX32, retains the growth attributes of the wild strain Cz-WT under glucose heterotrophic conditions, has the ability to ferment at high density, and can obtain high contents of astaxanthin and canthaxanthin, which is conducive to realizing the green biomanufacturing of astaxanthin and canthaxanthin by fermentation. Attached Figure Description

[0017] Figure 1 This is a diagram showing the growth status of wild-type Cz-WT and mutant Cz-R1 in BG11 + 20 g / L glucose medium. Figure 2 The growth curves of wild-type Cz-WT and mutant Cz-R1 in BG11+20 g / L glucose medium are shown. Figure 3 The values ​​represent the astaxanthin and canthaxanthin content of wild-type Cz-WT and mutant Cz-R1 after culturing in BG11 + 20 g / L glucose medium for 15 days. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0019] Example Obtaining the Zoff-colored green algae mutant Using *Chromochloris zofingiensis* UTEX32 as the starting strain, in this application, wild-type *Chromochloris zofingiensis* or wild-type strain Cz-WT or Cz-WT are used to refer to *Chromochloris zofingiensis* UTEX32. *Chromochloris zofingiensis* UTEX32 was purchased from the UTEXCulture Collection of Algae.

[0020] S1: Cz-WT was inoculated into 20 mL of BG11 medium and incubated at 25 ℃ under a light intensity of 40 μmol / (m²). 2 The algae were activated and cultured in a light-controlled shaker at 150 rpm until the algal cell concentration reached the OD value. 680 =1.0. OD of algal cell concentration 680 The method of obtaining the value is based on existing technology and will not be elaborated here.

[0021] The preparation of BG11 medium involved adding 1.5 g NaNO3, 0.04 g K2HPO4, 0.075 g MgSO4·7H2O, 0.036 g CaCl2·2H2O, 0.02 g Na2CO3, 6.0 mg C6H8O7·H2O (citric acid), 6.0 mg ferric ammonium citrate, 1.0 mg Na2EDTA·2H2O, 0.02 g Na2CO3, 2.86 mg H3BO3, 1.81 mg MnCl2·4H2O, 0.39 mg Na2MoO4·2H2O, 0.22 mg ZnSO4·7H2O, 0.08 mg CuSO4·5H2O, and 0.049 mg Co(NO3)2·6H2O sequentially to 1 L of distilled water. After stirring and dissolving, the pH was adjusted to 7.1-7.4 using NaOH or HCl.

[0022] S2: Inoculate the activated Cz-WT into 20 mL of BG11 medium to achieve an initial concentration of OD. 680 =0.1, at 25 ℃ and light intensity of 40 μmol / (m 2 The algae were cultured in a light-controlled shaker at 150 rpm until the algal cell concentration reached an OD value of 150 rpm. 680 =0.5.

[0023] S3: Take 1 mL of S2 when the algal cell concentration reaches OD 680 The algal solution with an OD value of 0.5 was centrifuged at 3000×g for 2 min, the supernatant was removed, and the precipitate was resuspended in 2 mL of BG11 medium. At this point, the concentration of the algal solution was OD0.5. 680 =0.25, then Zeocin was added to make the concentration of Zeocin in the resuspended algae solution 100 μg / mL; finally, the resuspended algae solution containing Zeocin was placed in a shaker at 25℃ and 150 rpm for 2 h in the dark. Zeocin was purchased from Thermo Fisher Scientific.

[0024] S4: Take the algal solution from S3 after Zeocin mutagenesis and dark treatment for 2 h, centrifuge at 3000×g for 2 min, remove the supernatant, wash with 1 mL of BG11 medium, centrifuge at 3000×g for 2 min, remove the supernatant, repeat the washing process once more, then add 20 mL of BG11 medium to resuspend the precipitate, so that the algal cell concentration in the algal solution is OD. 680 =0.025.

[0025] S5: Take the algal solution from S4 with an OD680 concentration of 0.025 and spread it at a rate of 200 μL / plate onto a 90 mm diameter agar plate containing BG11 + 20 g / L glucose. Incubate in the dark at 25 ℃ for 30 days. Preparation of agar plates containing BG11 + 20 g / L glucose: Add 10 g of agar and 20 g of glucose to BG11 medium, and sterilize at 115 ℃ for 30 min after preparation.

[0026] S6: Compare the color differences of algal colonies on agar plates containing BG11 + 20 g / L glucose. Visually inspect and select algal colonies that are redder than their surrounding colonies. Streak these colonies on agar plates containing BG11 + 20 g / L glucose for separation. Incubate in the dark at 25 ℃ for 30 days. Repeat step S6 for a second streak separation. Finally, select the red algal strain for preservation, named Chromochloris zofingiensis Cz-R1, or simply Cz-R1. In this protocol, it is also referred to as mutant strain Cz-R1. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46621 on October 16, 2025.

[0027] 18S rRNA gene sequence: .

[0028] Methods for inducing the synthesis of astaxanthin and canthaxanthin A1: Cz-R1 was inoculated into BG11 + 20 g / L glucose medium and activated in the dark at 25 ℃ and 150 rpm in a shaker until the algal concentration was 5 g / L, thus forming Cz-R1 seed culture.

[0029] Preparation of culture medium containing BG11 + 20 g / L glucose: Add 20 g of glucose to BG11 culture medium and sterilize at 115 °C for 30 min after preparation.

[0030] The algal solution concentration is calculated as follows: Weigh the original 0.45 μm filter membrane as m0 (mg), take the activated algal solution volume as V (mL), filter the algae onto the 0.45 μm filter membrane using a vacuum filtration device, dry it and weigh it as m1 (mg), and calculate the algal solution concentration according to the formula: C (g / L) = (m1-m0) / V.

[0031] A2: Cz-WT was inoculated into BG11 + 20 g / L glucose medium and activated in the dark at 25 ℃ and 150 rpm in a shaker until the algal concentration was 5 g / L, thus forming Cz-WT seed culture.

[0032] A3: Take 6 mL of Cz-WT and Cz-R1 seed culture (5 g / L concentration) from A1 and A2 respectively, centrifuge at 3000×g for 2 min, remove the supernatant, and resuspend in 150 mL of BG11 + 20 g / L glucose medium. Culture in the dark for 15 days at 25 ℃ and 150 rpm in a shaker. The nutrient stress caused by prolonged culture leading to carbon and nitrogen depletion induces the synthesis of astaxanthin and canthaxanthin. The growth status of wild-type Cz-WT and mutant Cz-R1 in BG11 + 20 g / L glucose medium is as follows. Figure 1 As shown.

[0033] (3) Detection of astaxanthin and canthaxanthin content B1: Take the Cz-WT and Cz-R1 algal solutions after continuous culture of A3 for 15 days, centrifuge at 3000×g for 2 min and remove the supernatant, wash with sterile distilled water and centrifuge at 3000×g for 2 min and remove the supernatant. Repeat the above washing process twice.

[0034] B2: The Cz-WT and Cz-R1 algal precipitates obtained in B1 were placed in a freeze dryer to evaporate the moisture. The dried Cz-WT and Cz-R1 algal powders were then thoroughly ground and the cells were broken using a ball mill.

[0035] B3: Take 50 mg each of Cz-WT and Cz-R1 cell lysates from B2 and extract them separately with 0.5 mL of a hexane / acetone / ethanol mixture (1:1:1, v / v / v) containing 0.01% BHT (g / mL). Vortex at room temperature for 20 min, then centrifuge at 12000 r / min for 5 min at 4 ℃. Collect the supernatant and repeat the above steps. Combine the supernatants obtained from the two centrifugations to form the extract. Concentrate the extract under nitrogen blowing, redissolve it with 150 μL of dichloromethane, filter it through a 0.22 μm filter membrane, and store it in a brown sample vial for LC-MS / MS analysis. BHT is 2,6-di-tert-butyl-p-cresol.

[0036] The astaxanthin and canthaxanthin contents in Cz-WT and Cz-R1 after carbon and nitrogen source depletion induction were calculated by LC-MS / MS analysis. The calculated astaxanthin content in Cz-R1 was as high as 2.6308 mg / g, an increase of 421% compared to 0.50476 mg / g in Cz-WT; the canthaxanthin content in Cz-R1 was 13.718 mg / g, an increase of 728% compared to 1.6565 mg / g in Cz-WT. For details, please refer to [reference needed]. Figure 3 .

[0037] (4) Determination of growth of Zoff's green algae mutant and wild-type Zoff's green algae H1: Take 6 mL of Cz-WT and Cz-R1 seed culture with an algal concentration of 5 g / L from A1 and A2 respectively, centrifuge at 3000×g for 2 min, remove the supernatant, and resuspend the precipitate in 150 mL of BG11 + 20 g / L glucose medium to make the initial algal concentration 0.2 g / L. Incubate continuously in the dark at 25 ℃ and 150 rpm in a shaker.

[0038] H2: Every 24 h, take 5 mL of algal solution from Cz-WT and Cz-R1 cultured in H1, filter the algae onto a 0.45 μm filter membrane using a vacuum filtration device, dry and weigh, and calculate the algal solution concentration.

[0039] Measurements showed that the growth of Cz-R1 was not significantly different from that of Cz-WT, proving that Cz-R1 retains the growth properties of Cz-WT under glucose heterotrophic conditions and can undergo high-density fermentation culture. (See attached reference for details.) Figure 2 .

[0040] The Zov-colored green algae mutant strain Cz-R1 was obtained by mutagenesis of Zov-colored green algae Chromochloris zofingiensis UTEX32. This mutant strain retains the growth properties of the wild strain Cz-WT under glucose heterotrophic conditions, has the ability to ferment at high density, and can produce high contents of astaxanthin and canthaxanthin, which is conducive to realizing the green bio-manufacturing of astaxanthin and canthaxanthin by fermentation.

[0041] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A mutant strain of Zoff's green algae producing astaxanthin and canthaxanthin, characterized in that: The alginate, named Chromochloris zofingiensis Cz-R1, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46621 on October 16, 2025.

2. The application of the Zoff's green algae mutant strain according to claim 1 in the production of astaxanthin and canthaxanthin.

3. The application according to claim 2, characterized in that: A method for producing astaxanthin and canthaxanthin using a mutant strain of Chromochloris zofingiensis is provided: After centrifuging activated Chromochloris zofingiensis Cz-R1 seed culture at 3000×g for 2 min, the supernatant is removed, and the precipitate is resuspended in BG11 + 20 g / L glucose medium. The mixture is then cultured in the dark for 15 days at 25 ℃ and 150 rpm in a shaker. The synthesis of astaxanthin and canthaxanthin is induced by nutrient stress caused by the depletion of carbon and nitrogen sources.

4. The application according to claim 3, characterized in that: The accumulation of astaxanthin was 2.6308 mg / g; the accumulation of canthaxanthin was 13.718 mg / g.

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