Mutagenized chlorella with high yield of phytoene and its application

Abstract

The application provides a high-yield phytoene mutagenic chlorella and an application thereof, and belongs to the technical field of chlorella. Auxenochlorella pyrenoidosa The mutagenic chlorella (Chlorella vulgaris) of the application is a mutagenic algae strain SZU-C49 of a mutagenic chlorella (Chlorella vulgaris) wild-type common chlorella starting strain, which is preserved in the Guangdong Microbial Culture Collection Center, has a preservation number of GDMCC No:66780, a preservation time of July 30, 2025, and a preservation address of No. 59 Building, 5th Floor, 100, Martyr's Road, Guangzhou. The algae strain is obtained by chemical mutagenesis and screening on the mutagenic chlorella wild-type common chlorella starting strain. The mutagenic chlorella of the application is cultured for 72 h under optimized culture conditions, the content of phytoene in the cells of the strain reaches 25799 mg / g (dry weight), which is more than 100 times that of the original starting strain, and can be widely applied to the fields of functional food, health products, cosmetics and medicines and the like.

Description

Technical Field

[0001] This invention relates to the field of Chlorella technology, and more particularly to a mutagenic Chlorella strain that produces high levels of phytoene and its applications. Background Technology

[0002] Phytoene is a colorless carotenoid precursor composed of four isoprene units (molecular formula C). 40 H 64 This compound is synthesized in vivo via the mevalonic acid pathway. It exhibits a characteristic absorption peak in the UVB band (approximately 285 nm), endowing it with natural UV filtering capabilities. Clinical studies have confirmed that oral administration for eight weeks can reduce skin erythema area by 35% and inhibit UV-induced collagenase MMP-1, significantly improving photoaging. Its potent antioxidant capacity can quench singlet oxygen (more than twice the potency of β-carotene). Simultaneously, it intervenes in cardiovascular disease risk by reducing inflammatory factors such as TNF-α and IL-6. Animal models also show that it can cross the blood-brain barrier to slow β-amyloid protein deposition, demonstrating neuroprotective potential. Natural sources are mainly found in fruits and vegetables such as tomatoes (0.5-3 mg / 100g fresh weight) and guavas. While genetically modified microbial fermentation (such as genetically engineered yeast) can theoretically achieve efficient synthesis, actual production faces a dual bottleneck: plant extraction requires processing tons of material to obtain kilogram-level products due to extremely low raw material content (<0.01% dry weight); microbial synthesis is limited by enzyme activity inhibition and intracellular feedback inhibition, while also involving the poor stability of engineered bacteria genes. Furthermore, cell wall disruption extraction further increases costs. With the continued rise in global market demand for carotenoids, how to increase the cumulative yield of phytopenic oleoresin and its derivatives has become a key issue for industrialization.

[0003] Traditional methods for producing carotenoids and their precursors primarily rely on chemical synthesis and plant extraction. These methods suffer from high production costs, significant resource consumption, and severe environmental pollution. In particular, chemical synthesis can generate isomer mixtures, leading to lower bioactivity and potential safety risks. Therefore, utilizing microalgae as "cell factories" to biosynthesize carotenoids and their precursors is gradually becoming a more sustainable alternative. Microalgae are diverse, possess high photosynthetic efficiency, short growth cycles, and the ability to accumulate large quantities of unique carotenoids and their precursors, making them the preferred source for biosynthesis.

[0004] Currently, the production of phytoene from microalgae still faces bottlenecks such as limited yield and high production costs. Wild-type microalgae strains typically accumulate low levels of phytoene, making it difficult to meet the demands of industrial production. Therefore, providing a high-yield phytoene microalgae strain and optimizing its culture conditions has become a key focus and challenge in this field. Summary of the Invention

[0005] In view of this, the present invention provides a mutagenic Chlorella strain that produces high levels of phytoene and its applications to solve the above problems.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a high-yield phytoene-producing mutagenic Chlorella strain, taxonomically named *Auxenochlorella pyrenoidosa*, deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 66780), with a deposit date of July 30, 2025, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The present invention also provides the application of the mutagenic microspheres in the preparation of phytoene.

[0009] The present invention also provides a method for preparing phytoene, wherein the mutagenic Chlorella is inoculated into a culture medium for fermentation culture.

[0010] Preferably, the fermentation culture temperature is 26~30℃, the rotation speed is 180~220rpm, the aeration rate is 0.4~0.6vvm, and the fermentation culture time is 72~96 h.

[0011] Preferably, the fermentation culture medium is: 3 g / L KNO3, 0.62 g / L NaH2PO4·H2O, 0.089 g / L Na2HPO4·2H2O, 0.247 g / L MgSO4·7H2O, 14.7 mg / L CaCl2·2H2O, 6.95 mg / L FeSO4·7H2O, 0.061 mg / L H3BO3, 0.169 mg / L MnSO4·H2O, 0.287 mg / L ZnSO4·7H2O, 0.0025 mg / L CuSO4·5H2O, 0.01235 mg / L (NH4)6Mo7O 24 • 4H2O and 10 g / L glucose, pH=6.5.

[0012] Preferably, the fermentation culture medium is composed of: 10 g / L KNO3, 1.25 g / L KH2PO4, 1 g / L MgSO4·7H2O, 0.5 g / L EDTA-2Na, 0.1142 g / L H3BO3, 0.111 g / L CaCl2·2H2O, 0.0498 g / L FeSO4·7H2O, 0.0882 g / L ZnSO4·7H2O, 0.0142 g / L MnCl2·4H2O, 0.0157 g / L CuSO4·5H2O, 0.0049 g / L Co(NO3)2·6H2O, 0.0071 g / L MoO3, and 30 g / L glucose, with a pH of 6.1.

[0013] By adopting the above technical solution, the present invention has the following beneficial effects: The mutant strain SZU-C49 of *Auxenochlorella pyrenoidosa* of the present invention is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66780, deposited on July 30, 2025, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. This strain was obtained by chemical mutagenesis and screening of the wild-type common *Auxenochlorella* strain. After culturing under optimized conditions for 72 hours, the mutant strain of the present invention achieved an intracellular phytoene content of 2.5799 mg / g (dry weight), which is more than 100 times that of the original strain. The phytoene produced by the mutant strain of the present invention can be widely used in functional foods, health products, cosmetics, and pharmaceuticals. Attached Figure Description

[0014] Figure 1 The bar chart shows the content of phytoene in the Chlorella SZU-C49 mutant strain and wild Chlorella after 72, 84, and 96 hours of culture.

[0015] Figure 2 The bar chart shows the yield of phytoene from the SZU-C49 mutant strain of Chlorella and wild Chlorella after 72, 84, and 96 hours of culture.

[0016] Figure 3 Line graphs showing the content of phytoene in Chlorella SZU-C49 mutant strain of the present invention after fermentation in a changed culture medium for 12, 24, 36, 48, 60, and 72 hours.

[0017] Figure 4 Line graphs showing the yield of phytoene from the Chlorella SZU-C49 mutant strain of the present invention after fermentation in a different culture medium for 12, 24, 36, 48, 60, and 72 hours.

[0018] Biological Preservation Instructions

[0019] The taxonomic name of the mutagenic Chlorella SZU-C49 that produces high levels of phytoene in this invention is Auxenochlorella pyrenoidosa. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo: 66780, deposited on July 30, 2025, at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1. Preparation method of mutagenic Chlorella with high phytoene production

[0022] The preparation method of mutagenic Chlorella that produces high levels of phytoene includes the following steps:

[0023] Wild-type Chlorella pyrenoidosa was cultured to a concentration of 1×10⁻⁶. 7 For the logarithmic growth phase, take 1 mL of algal culture, centrifuge at 5000 rpm for 5 min, discard the supernatant, and add Kuhl's medium containing different concentrations of ethyl methane sulfonate (EMS) (EMS concentration gradient: 0, 0.1, 0.2, 0.3, 0.4 M). Resuspend and incubate in the dark for 60 min. Wash the treated *Auxenochlorella pyrenoidosa* three times with fresh Kuhl's medium to remove residual EMS. Finally, add 500 μL of Kuhl's medium and shake overnight in the dark (150 rpm, approximately 16 h). After treatment, spread the algal culture on solid Kuhl's medium plates and incubate at 25°C in the dark for 7 days. Select single algal colonies with significant phenotypic differences (wild *Chlorella* is green, this mutant is white) for purification, subculture, and screening to obtain a high-yielding phytoene-producing mutagenized *Chlorella*, named *Chlorella SZU-C49*.

[0024] The obtained mutant Chlorella SZU-C49 was deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66780.

[0025] Example 2

[0026] The *Chlorella* mutant strain SZU-C49 (preservation number GDMCC No: 66780) obtained in Example 1 and wild *Chlorella* were cultured in shake flasks. The fermentation medium in the fermenter contained the following components: 3 g / L KNO3, 0.62 g / L NaH2PO4·H2O, 0.089 g / L Na2HPO4·2H2O, 0.247 g / L MgSO4·7H2O, 14.7 mg / L CaCl2·2H2O, 6.95 mg / L FeSO4·7H2O, 0.061 mg / L H3BO3, 0.169 mg / L MnSO4·H2O, 0.287 mg / L ZnSO4·7H2O, 0.0025 mg / L CuSO4·5H2O, and 0.01235 mg / L (NH4)6Mo7O 24 • 4H2O and 10 g / L glucose, pH 6.5.

[0027] The fermentation conditions in the fermenter were as follows: initial inoculum size was 5%, and the culture temperature was 28℃, initial rotation speed was 200 rpm, and aeration rate was 0.5 vvm. The phytoene content and yield were measured after 72, 84, and 96 h of culture. The phytoene content and yield of mutant Chlorella SZU-C49 and wild Chlorella were as follows: Figure 1 and Figure 2 As shown. By Figure 1 and Figure 2 It can be seen that the content of phytoene in the Chlorella mutant strain SZU-C49 of the present invention is significantly higher than that in wild Chlorella.

[0028] Example 3

[0029] Unlike Example 2, the fermentation medium for the Chlorella mutant strain SZU-C49 was replaced with the following components: 10 g / L KNO3, 1.25 g / L KH2PO4, 1 g / L MgSO4·7H2O, 0.5 g / L EDTA-2Na, 0.1142 g / L H3BO3, 0.111 g / L CaCl2·2H2O, 0.0498 g / L FeSO4·7H2O, 0.0882 g / L ZnSO4·7H2O, 0.0142 g / L MnCl2·4H2O, 0.0157 g / L CuSO4·5H2O, 0.0049 g / L Co(NO3)2·6H2O, 0.0071 g / L MoO3, and 30 g / L glucose, pH [missing value]. 6.1. Other fermentation conditions were the same as in Example 2. The plants were cultured for 12, 24, 36, 48, 60, and 72 hours, and the content and yield of phytoene were measured. The results are as follows: Figure 3 and Figure 4As shown in the figure. The results showed that after changing the culture medium, the content and yield of phytoene in the Chlorella mutant strain SZU-C49 increased.

[0030] As can be seen from the above embodiments, the present invention provides a high-yield phytoene-producing mutagenized Chlorella and its application. The content and yield of phytoene-producing Chlorella mutant strain SZU-C49 of the present invention are significantly improved compared with wild Chlorella.

[0031] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutagenic Chlorella strain SZU-C49 producing phytohexopentin, characterized in that, The taxonomic name of the mutated Chlorella is... Auxenochlorella pyrenoidosa The specimen is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66780, on July 30, 2025, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of the mutagenic Chlorella SZU-C49 described in claim 1 in the preparation of phytohexene.

3. A method for preparing phytoene, characterized in that, The mutagenic Chlorella SZU-C49 of claim 1 was inoculated into a culture medium for fermentation.

4. The preparation method according to claim 3, characterized in that, The fermentation culture temperature is 26~30℃, the rotation speed is 180~220rpm, the aeration rate is 0.4~0.6 vvm, and the fermentation culture time is 72~96 h.

5. The preparation method according to claim 3, characterized in that, The fermentation culture medium consisted of: 3 g / L KNO3, 0.62 g / L NaH2PO4·H2O, 0.089 g / L Na2HPO4·2H2O, 0.247 g / L MgSO4·7H2O, 14.7 mg / L CaCl2·2H2O, 6.95 mg / L FeSO4·7H2O, 0.061 mg / L H3BO3, 0.169 mg / L MnSO4·H2O, 0.287 mg / L ZnSO4·7H2O, 0.0025 mg / L CuSO4·5H2O, and 0.01235 mg / L (NH4)6Mo7O 24 • 4H2O and 10 g / L glucose, pH=6.

5.

6. The preparation method according to claim 3, characterized in that, The fermentation culture medium consisted of: 10 g / L KNO3, 1.25 g / L KH2PO4, 1 g / L MgSO4·7H2O, 0.5 g / L EDTA-2Na, 0.1142 g / L H3BO3, 0.111 g / L CaCl2·2H2O, 0.0498 g / L FeSO4·7H2O, 0.0882 g / L ZnSO4·7H2O, 0.0142 g / L MnCl2·4H2O, 0.0157 g / L CuSO4·5H2O, 0.0049 g / L Co(NO3)2·6H2O, 0.0071 g / L MoO3, and 30 g / L glucose, with a pH of 6.1.

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