Phaffia rhodozyma strain with high astaxanthin yield and application thereof

Abstract

The invention discloses a phaffia rhodozyma strain with high astaxanthin yield and application of the phaffia rhodozyma strain. The phaffia rhodozyma strain capable of producing the astaxanthin at the high yield is named as phaffia rhodozyma CL17, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 21, 2025, and the preservation number is CGMCC No.39010. The phaffia rhodozyma strain can be used for producing the astaxanthin at the high yield. The strain obtained through screening has good temperature tolerance (15-45 DEG C) and hereditary stability, after fermentation culture, the astaxanthin content in thalli reaches 5.13 mg / g of dry cell weight, and the strain has excellent application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a high-yield astaxanthin-producing strain of Pharfogel's red yeast and its applications. Background Technology

[0002] Astaxanthin (molecular formula C) 40 H 52 O4, CAS number 472-61-7, is a ketocarotenoid with strong antioxidant activity. Its chemical name is 3,3'-dihydroxy-4,4'-diketo-β-carotene. It is a tetraterpenoid oxygen-containing derivative composed of eight isoprene units, with 11 conjugated double bonds and two ionone rings in its molecular structure. It is the terminal product in the carotenoid biosynthesis pathway. Among the more than 600 known carotenoids, astaxanthin has the most outstanding antioxidant capacity. Its free radical scavenging efficiency is 10 times that of β-carotene, zeaxanthin, and canthaxanthin, and about 100 times that of α-tocopherol (vitamin E). Therefore, it is known as "super vitamin E" and has irreplaceable application value in health products, pharmaceuticals, cosmetics, food additives, and animal husbandry. Astaxanthin has particularly significant advantages in animal husbandry. For aquatic animals such as shrimp, crab, salmon, large yellow croaker, and grouper, astaxanthin can effectively improve the color of their body surface and muscles, enhancing their appearance and market value. It can also enhance their antioxidant capacity, reduce lipid oxidation damage, and extend shelf life. Simultaneously, astaxanthin can increase the accumulation of crude protein, crude fat, and unsaturated fatty acids in aquatic animals, strengthening their stress resistance (such as low-temperature tolerance and dew tolerance) and reducing mortality during long-distance transportation. Furthermore, as a feed additive, astaxanthin can improve animal immunity and stress resistance, reduce the use of antibiotics and other drugs, and increase survival rate and feed utilization. It also has a positive effect on the development of the reproductive system of parent animals, improving egg and sperm quality, and enhancing larval survival. In poultry farming, it can improve egg yolk color and taste, further expanding its application scenarios.

[0003] Currently, the industrial production of astaxanthin mainly relies on four technical pathways: chemical synthesis, extraction from shellfish waste, cultivation of Haematococcus pluvialis, and microbial fermentation. However, these methods still have some limitations: chemical synthesis requires multiple complex reactions, and the products are mostly mixtures of cis and trans isomers, with bioactivity far lower than that of natural astaxanthin. Furthermore, there is a risk of chemical reagent residues, making it difficult to meet the stringent safety standards of the food and pharmaceutical industries. Shellfish waste extraction uses byproducts such as shrimp and crab shells as raw materials, resulting in extremely low astaxanthin content (usually less than 0.01%). The extraction process requires large amounts of organic solvents, easily causing residual pollution. Additionally, the supply of raw materials is greatly affected by fluctuations in the aquatic product processing industry, making large-scale, stable production difficult. While Haematococcus pluvialis, a classic source of natural astaxanthin, produces products with high activity, its cultivation cycle is long (requiring a green nutrient period and a red dormancy period, taking 15-20 days in total), and it is highly sensitive to light, temperature, and pH. The demanding environmental conditions and susceptibility to contamination by algae and microorganisms lead to unstable yields and high production costs (over $3,000 per kilogram of astaxanthin), limiting its large-scale application. Microbial fermentation, due to its low raw material costs, easily controllable cultivation conditions, and high product safety, has become a core direction in astaxanthin production technology research and development in recent years. Among these methods, *Phaefflera rubiginii* (red Phaefflera yeast) is a key component. Phaffia rhodozyma As one of the few yeasts capable of synthesizing free natural astaxanthin, *Phaeodactylum rubrum* is considered a highly promising astaxanthin-producing strain due to its relatively clear genetic background, light-free cultivation process, and availability of inexpensive carbon sources (such as glucose and sucrose). *Phaeodactylum rubrum* possesses its own mevalonic acid (MVA) metabolic pathway, enabling the stepwise synthesis of astaxanthin precursors via acetyl-CoA. Theoretically, this eliminates the need for complex heterologous synthetic pathways, simplifying the strain modification process. Furthermore, its intracellular metabolic environment is well-suited for storing fat-soluble astaxanthin, providing a natural advantage for product accumulation. However, wild-type... P. rhodozyma The astaxanthin yield is generally low, with the astaxanthin content in dried bacterial cells typically only 0.1–0.5 mg / g, far from meeting the needs of industrial production.

[0004] In related technologies, targeting P. rhodozymaModifications often focus on overexpressing single genes or optimizing simple metabolic pathways, such as enhancing the expression of key enzymes in the MVA pathway (e.g., HMG-CoA reductase, isopentenyl pyrophosphate isomerase) or knocking out genes competing for metabolic pathways. However, these methods have failed to systematically address the imbalance in metabolic flux distribution in the astaxanthin synthesis pathway. Astaxanthin synthesis involves multiple steps, including acetyl-CoA → mevalonic acid → isopentenyl pyrophosphate → geraniol → phytopene → lycopene → β-carotene → astaxanthin, involving the synergistic effects of more than 10 enzymes. Modification of a single gene easily leads to the accumulation of intermediate products (e.g., β-carotene, lycopene), resulting in low astaxanthin conversion rates. Furthermore, P. rhodozyma The fermentation process also has some bottlenecks: on the one hand, it is sensitive to the concentration of carbon and nitrogen sources. High concentrations of glucose can easily cause osmotic pressure inhibition, resulting in slow cell growth, while low concentrations of carbon sources cannot meet the carbon flux requirements for astaxanthin synthesis; on the other hand, it is not tolerant to temperature. At higher temperatures, the strain grows slowly, affecting astaxanthin production and making it difficult to cope with the complex and variable culture conditions in industrial fermentation.

[0005] Therefore, it is necessary to screen for a variety that not only produces high levels of astaxanthin but also can withstand adverse environments such as high osmotic pressure and temperature fluctuations. P. rhodozyma The development of strains has become a key requirement for breaking through the bottleneck of existing microbial fermentation methods for astaxanthin production and achieving large-scale and stable production. Summary of the Invention

[0006] The first objective of this invention is to provide a high-yield astaxanthin-producing strain of *Phaeophyte rubrum*, named *Phaeophyte rubrum*. Phaffia rhodozyma CL17.

[0007] The second objective of this invention is to provide a method for breeding the above-mentioned high-astaxanthin-producing Pharfogel's yeast strain.

[0008] A third aspect of the present invention is to provide a microbial agent.

[0009] The fourth aspect of this invention is to provide a method for culturing a strain of Pharfogel's rubrum.

[0010] The fifth aspect of this invention aims to provide the application of the above-mentioned Pharfovia rubescens strain, inoculum, or culture method in the preparation of astaxanthin-containing products.

[0011] The sixth aspect of this invention aims to provide a method for obtaining astaxanthin.

[0012] The seventh aspect of this invention aims to provide the application of the above-mentioned Pharfovia rubescens strain and inoculum in the preparation of animal feed, food additives or health products.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a high-yield astaxanthin-producing strain of *Phaeophyte rubrum*, named *Phaeophyte rubrum*. Phaffia rhodozyma CL17 was deposited on October 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39010.

[0014] The high-astaxanthin-producing Pharfovia rubescens strain of the present invention has at least the following beneficial effects: The strains screened in this invention have good temperature tolerance (15~45℃) and genetic stability. After fermentation, the astaxanthin content in the cells reaches 5.13 mg / g dry cell weight, showing excellent application prospects.

[0015] In some embodiments of the present invention, the amino acid sequence of the lycopene cyclase-hydrolycopene synthase of the red Paffia yeast strain is shown in SEQ ID NO.2.

[0016] In some embodiments of the present invention, the amino acid sequence of the geraniol geraniol pyrophosphate synthase of the red Pharfia strain is shown in SEQ ID NO.4.

[0017] In some embodiments of the present invention, the amino acid sequence of the farnesyl pyrophosphate synthase of the red Pharfia strain is shown in SEQ ID NO. 6.

[0018] A second aspect of the present invention provides a method for breeding a high-astaxanthin-producing Pharfovia strain as described in the first aspect, comprising: The strain was obtained by sequentially passing it through nitrogen-restricted subculture, high-intensity light mutagenesis, and high-osmotic environmental stress mutagenesis.

[0019] In this invention, a mutagenic starting strain was first obtained by screening and enriching *Alnus rubrum* trees in Suqian City, Jiangsu Province. Then, the strain was passaged under high-intensity light to stimulate light stress response and the expression of astaxanthin synthesis-related genes. A nitrogen-limited culture medium was used for passage to force the cell metabolism to shift from growth and reproduction to the accumulation of secondary metabolites such as astaxanthin. The strain was then repeatedly passaged in a hyperosmolar environment containing 10–30 g / L potassium chloride solution and sorbitol to screen for robust strains that could resist osmotic stress by enhancing astaxanthin synthesis. Finally, a high-astaxanthin-producing *Phaefflera rubrum* strain was obtained. P. rhodozyma CL17.

[0020] In some embodiments of the present invention, the wild-type Pharfovia rubescens was isolated from the aquatic environment of alder forests.

[0021] In some embodiments of the present invention, the nitrogen-limited subculture includes screening using a nitrogen-limited culture medium.

[0022] In some embodiments of the present invention, the carbon-to-nitrogen ratio of the nitrogen-limiting culture medium is 30-50:1, preferably 35-45:1.

[0023] In some embodiments of the present invention, the carbon source in the nitrogen-limited culture medium includes glucose.

[0024] In some embodiments of the present invention, the nitrogen source in the nitrogen-limiting culture medium includes one or a combination of yeast extract and ammonium sulfate.

[0025] In some embodiments of the present invention, the nitrogen-limited culture medium further comprises inorganic salts. Preferably, the inorganic salts include KH₂PO₄ and MgSO₄. 7H2O, FeSO4 At least one of 7H2O.

[0026] In some embodiments of the present invention, the pH value of the nitrogen-limiting culture medium is 5.0 to 5.5.

[0027] In some embodiments of the present invention, the nitrogen-limiting culture medium comprises 50-100 g / L glucose, 1-10 g / L yeast extract, 0.1-2 g / L ammonium sulfate, 0.5-5 g / L KH₂PO₄, and MgSO₄. 7H2O 0.1~2 g / L, FeSO4 7H2O 0.01~0.2 g / L.

[0028] In some embodiments of the present invention, the light source for the high-intensity light-induced mutation is a red-blue composite light source with an illumination intensity of 2000-3000 Lux.

[0029] In some embodiments of the present invention, the high-intensity light-induced mutation treatment time is 48-96 hours, preferably 65-80 hours.

[0030] In some embodiments of the present invention, the temperature of the high-intensity light-induced mutation treatment is 20~22°C.

[0031] In some embodiments of the present invention, the osmotic pressure of the hypertonic culture medium used for the hypertonic environmental stress mutagenesis is 800-1200 mOsm / kg.

[0032] In some embodiments of the present invention, the hypertonic culture medium contains 10-30 g / L KCl and 120-180 g / L sorbitol.

[0033] In some embodiments of the present invention, the pH value of the hypertonic culture medium is 5.0 to 5.5.

[0034] In some embodiments of the present invention, the time for the high-permeability environment stress-induced mutagenesis is 72-128 h, preferably 80-100 h.

[0035] In some embodiments of the present invention, the selection process further includes primary screening and secondary screening.

[0036] In some embodiments of the present invention, the carbon-to-nitrogen ratio of the culture medium used for the initial screening is 30-50:1, preferably 35-45:1.

[0037] In some embodiments of the present invention, the culture medium used for the initial screening comprises 10-30 g / L glucose, 0.1-2 g / L yeast extract, 10-30 g / L KCl, 80-120 g / L sorbitol, and 15-25 g / L agar.

[0038] In some embodiments of the present invention, the pH value of the culture medium used for the primary screening is 5.2 to 5.8.

[0039] In some embodiments of the present invention, the temperature of the initial screening is 20~25°C.

[0040] In some embodiments of the present invention, the light intensity of the initial screening is 1200~1800 Lux.

[0041] In some embodiments of the present invention, the secondary screening includes inoculating the secondary screened strains into a seed culture medium at 20-25°C, and then inoculating them into a fermentation culture medium at 2-15% (v / v), followed by screening after cultivation.

[0042] In some embodiments of the present invention, the seed culture medium comprises 20-50 g / L glucose, 2-8 g / L yeast extract, 0.5-2 g / L KH₂PO₄, and MgSO₄. 7H2O 0.1~2 g / L.

[0043] In some embodiments of the present invention, the fermentation medium comprises glucose, corn steep liquor, KH2PO4, MgSO4 and FeSO4.

[0044] In some embodiments of the present invention, the fermentation medium comprises 50-100 g / L glucose, 1-5 g / L corn steep liquor, 1-5 g / L KH2PO4, and MgSO4. 7H2O 0.1~2 g / L, FeSO4 7H2O 0.01~0.2 g / L.

[0045] In a third aspect, the present invention provides a microbial agent comprising the high-astaxanthin-producing Pharfovia strain described in the first aspect.

[0046] A fourth aspect of the present invention provides a method for culturing a Pharfovia strain, comprising: inoculating a high-astaxanthin-producing Pharfovia strain as described in the first aspect into a culture medium and culturing it.

[0047] In some embodiments of the present invention, the culture temperature is 15~45°C. Preferably, the culture temperature is 20~32°C.

[0048] In a fifth aspect, the present invention provides the application of the *Pharbitis purpureus* strain described in the first aspect, the inoculum agent described in the third aspect, or the culture method described in the fourth aspect in the preparation of astaxanthin-containing products.

[0049] In a sixth aspect, the present invention provides a method for obtaining astaxanthin, comprising inoculating the Pharrellis strain described in the first aspect and the bacterial agent described in the third aspect into a fermentation medium for fermentation culture, and then collecting the bacterial cells and extracting them.

[0050] In some embodiments of the present invention, the carbon-to-nitrogen ratio of the fermentation medium is 30-50:1, preferably 35-45:1.

[0051] In some embodiments of the present invention, the fermentation medium comprises glucose, corn steep liquor, KH2PO4, MgSO4 and FeSO4.

[0052] In some embodiments of the present invention, the fermentation medium comprises 50-100 g / L glucose, 1-5 g / L corn steep liquor, 1-5 g / L KH2PO4, and MgSO4. 7H2O 0.1~2 g / L, FeSO4 7H2O 0.01~0.2 g / L.

[0053] In some embodiments of the present invention, the fermentation culture temperature is 15~45°C.

[0054] In some embodiments of the present invention, the photosynthetic photon flux density of the fermentation culture is 50~300 μmol / m 2 s.

[0055] A seventh aspect of the present invention provides the use of the Pharbitis erythrosporum strain as described in the first aspect and the microbial agent as described in the third aspect in the preparation of animal feed, food additives or health products.

[0056] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0057] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 For the present invention P. rhodozyma HPLC analysis of intracellular astaxanthin synthesis by CL17 strain; Figure 2 This invention is wild-type P. rhodozyma and induced P. rhodozyma Results of astaxanthin content detection in strain CL17; Figure 3 For the temperature of the present invention P. rhodozyma Results of the detection of the effect on the growth of CL17 strain.

[0058] Figure 4 For the present invention P. rhodozyma Results of astaxanthin content detection for strain CL17 at high temperature. Detailed Implementation

[0059] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0060] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0061] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0062] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0063] In the description of this invention, the term "amino acid" refers to the basic unit that constitutes a protein, giving the protein a specific molecular structure and enabling its biochemical activity. For example, the "amino acid" used in this invention includes the following 20 natural amino acids (L-configuration amino acids): alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y).

[0064] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0065] Example 1: Starting strain Pharfia redis P. rhodozyma Screening and activation 1. Screening of starting strains: This invention isolates and purifies wild-type Pharfogel's yeast from a natural low-temperature aquatic environment in a red poplar forest in Suqian City, Jiangsu Province. Phaffia rhodozyma The strains were then isolated by streak plating, examined for morphological characteristics under a microscope, and screened for astaxanthin content (using high performance liquid chromatography to determine intracellular astaxanthin content) to obtain single colonies with stable growth status.

[0066] Microscopic examination revealed that the strain was oval or elliptical in shape, measuring 3–5 μm × 5–8 μm, and its basic astaxanthin yield was 0.1–0.4 mg / g dry cell weight (DCW).

[0067] The wild-type strain was subsequently used as the starting strain for mutagenesis.

[0068] 2. Strain activation: Use a sterile pipette to draw 0.3–0.5 mL of sterile liquid culture medium (20 g / L glucose, 5 g / L yeast extract, 1 g / L KH₂PO₄, MgSO₄). Resuspend the wild-type cells obtained from the above screening in 7H2O 0.5 g / L, pH 5.5. P. rhodozymaThe strain was then inoculated onto an activated slant culture medium (the above liquid culture medium with 20 g / L agar added), and incubated statically in a constant temperature incubator at 22°C for 48–72 h. After the slant was covered with orange-red bacterial growth, a single colony was picked and transferred to a fresh slant. This process was repeated 3–10 times to ensure the stability of the strain's activity, and then stored for later use.

[0069] Example 2: Composite Mutagenesis Treatment This invention employs a three-factor composite mutagenesis system of "nitrogen-limited subculture - high-intensity light - KCl + sorbitol hyperosmotic environment" for screening, with the following phased treatments: 1. Nitrogen source restriction pretreatment for subculture: The activated wild-type strain was inoculated into a nitrogen-limiting medium at a rate of 5% (v / v) and cultured at 22°C and 180 rpm for 48 hours to complete the first generation culture. Then, the first generation culture was transferred to fresh nitrogen-limiting medium at the same inoculation rate, and the culture was repeated 4–6 times. The bacterial concentration (OD) was measured after each subculture. 600 The amount of intracellular carotenoid precursors and other microbial accumulations should be monitored to ensure the strain adapts to low nitrogen stress and develops a metabolic orientation. Contamination by other microorganisms must be avoided during subculturing, and streak plating should be performed after each generation for purification.

[0070] The nitrogen-limited culture medium includes a carbon source (80 g / L glucose), a nitrogen source (a mixture of 2.8 g / L yeast extract and 0.7 g / L ammonium sulfate, with a precise C:N ratio of 40:1), and inorganic salts (2 g / L KH₂PO₄ and MgSO₄). 7H2O 0.8g / L, FeSO4 7H2O (0.05 g / L), adjust pH to 5.0–5.5 with 1 mol / L HCl or NaOH, and autoclave at 121°C for 20 min for later use.

[0071] 2. High-intensity light-induced mutation treatment: Take the strains obtained after subculturing with the above nitrogen source restriction, and first adjust the cell concentration to 1×10⁻⁶. 7 ~5×10 7 The concentration of CFU / mL was dispensed into sterile transparent Erlenmeyer flasks (filling the flasks with liquid to 1 / 4 to 1 / 3 of their volume to ensure sufficient contact with light), and then placed in a light incubator for mutagenesis treatment for 72 hours.

[0072] The illumination system uses a red-blue composite light source (red light RGB:255,0,0 and blue light RGB:0,0,255 mixed in a 1:2 ratio), and the light intensity is controlled at 2000-3000 Lux (corresponding to a photosynthetic photon flux density of 100-150 μmol / m²). (s) A continuous light mode (without dark cycle) is achieved through a programmable light incubator, while the temperature inside the incubator is controlled to be stable at 20-22℃ to avoid temperature fluctuations caused by heat generated by light.

[0073] During the treatment process, the Erlenmeyer flask was shaken once every 12 hours to ensure that the bacteria were exposed to light evenly. At the same time, samples were taken to detect the bacterial survival rate (plate counting method) and the survival rate was maintained within the effective mutagenesis range of 30% to 50%.

[0074] 3. KCl-sorbitol hyperosmolar environment stress-induced mutagenesis: The bacterial culture treated with the above high-intensity light was transferred to hypertonic medium with different KCl concentrations at an inoculation rate of 5% (v / v) and cultured under stress at 22℃ and 160r / min for 96h.

[0075] The basic components of the hyperosmolar stress culture medium included: glucose 30 g / L, peptone 15 g / L, yeast extract 8 g / L, glycerol 100-300 g / L (preset gradient: 100 → 150 → 200 → 250 → 300 g / L), ammonium sulfate 5.5 g / L, corn steep liquor powder 3.0 g / L, and citric acid–sodium citrate buffer 0.1 mol / L (pH 5.0), with 20 g / L agar added. The acclimatization process involved shaking-flask inoculation of the starting strain at 20°C and 180 rpm, with each generation extended to 48-72 h. The glycerol concentration was gradually increased according to the preset gradient, and this process was repeated for 10 generations.

[0076] The KCl concentration gradient used was set to 10 g / L, 20 g / L, and 30 g / L, and the sorbitol concentration was uniformly set to 150 g / L (based on the optimized value of osmotic pressure synergistic effect). The osmotic pressure of the mixed culture medium was 800–1200 mOsm / kg. After autoclaving at 121℃ for 20 min, the medium was cooled for later use.

[0077] During the culture process, the pH of the culture medium was monitored daily, and the pH was maintained at 5.0–5.5 by adjusting with sterile buffer. At the same time, changes in cell morphology (to avoid cell rupture) and stress tolerance were detected.

[0078] Example 3: Screening and Purification of High-Yielding Strains This example demonstrates further screening of strains cultured under hyperosmolar stress to obtain strains with high astaxanthin production. The specific screening process is as follows: 1. Initial screening: The bacterial culture after hyperosmolar stress was serially diluted and spread onto screening plates (containing 20 g / L glucose, 0.5 g / L yeast extract, 20 g / L KCl, 100 g / L sorbitol, and 20 g / L agar, C:N=40:1, pH 5.5) and incubated at 22℃ under 1500 Lux blue light for 72 h. Target colonies were screened based on colony color intensity (orange-red intensity) and size, selecting 30-50 single colonies with the darkest color and a diameter greater than 1 mm, which were then transferred to slant agar for storage.

[0079] 2. Secondary screening The candidate strains obtained from the initial screening were inoculated into seed culture media (30 g / L glucose, 5 g / L yeast extract, 1 g / L KH2PO4, MgSO4). After culturing at 22℃ and 180 rpm for 24 h with 0.5 g / L 7H2O (pH 5.5), the culture was transferred to fermentation medium (60 g / L glucose, 3 g / L corn steep liquor, 2 g / L KH2PO4, MgSO4) at a 10% (v / v) inoculation rate. 7H2O 0.8g / L, FeSO4 7H2O 0.05g / L, C:N=40:1, pH 5.0), 22℃, 180r / min, 100μmol / m 2 Fermentation was carried out under light conditions for 96 h. After fermentation, the cells were collected, freeze-dried, and astaxanthin was extracted using acetone ultrasonic extraction. The content was determined by high performance liquid chromatography (HPLC).

[0080] The parameters for HPLC detection of astaxanthin were set as follows: Wavelength range (λ): 210–520 nm. Mobile phase: 1% phosphoric acid solution, pure water, pure methyl tert-butyl ether, pure methanol. Pure water was used only as a washing mobile phase solution. Column: SHIMSEN VD C30 column, Dim: 4.6*250 mm, 3 μm, P / N: 380-01000-01, column temperature: 25℃. Chromatographic conditions: SHIMSEN VD C30 column (4.6*250 mm, 3 μm), flow rate: 0.5 mL / min; mobile phase A: 1% phosphoric acid solution, mobile phase B: pure water, mobile phase C: 100% pure methyl tert-butyl ether, mobile phase D: 100% methanol. Detection wavelength: 450 nm, injection volume: 10 μL.

[0081] Gradient elution conditions: 1-15 min: Phase A 4%, Phase C 30%, Phase D 65%; 16-27 min: Phase A 4%, Phase C 80%, Phase D 16%; 28-35 min: Phase A 4%, Phase C 15%, Phase D 81%.

[0082] The candidate strain with the highest astaxanthin yield was selected and passaged 10 times consecutively. Fermentation verification was performed after each passage to detect fluctuations in astaxanthin yield. A genetically stable mutant strain with high astaxanthin yield was obtained. The HPLC results of its intracellular astaxanthin synthesis are shown below. Figure 1 As shown, the structural formula of astaxanthin is as follows: .

[0083] The mutant strain was named *Phaeophyte*. P. rhodozyma CL17 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39010, on October 21, 2025.

[0084] Example 4 P. rhodozyma Astaxanthin content detection in CL17 This example refers to the aforementioned wild type. P. rhodozyma (i.e., the starting strain for mutagenesis) and the strains obtained through screening P. rhodozyma The astaxanthin production of strain CL17 was detected using the following method: Equal amounts of wild-type strains and P. rhodozyma CL17 strain was inoculated into seed culture medium (30 g / L glucose, 5 g / L yeast extract, 1 g / L KH2PO4, MgSO4) and other media. After culturing at 22℃ and 180 r / min for 24 h with 0.5 g / L 7H2O and pH 5.5, the culture was transferred to fermentation medium (60 g / L glucose, 3 g / L corn steep liquor, 2 g / L KH2PO4, and MgSO4) at a 10% (v / v) inoculation rate. 7H2O 0.8g / L, FeSO4 7H2O 0.05g / L, C:N=40:1, pH 5.0), 22℃, 180r / min, 100μmol / m² Fermentation was carried out under light conditions for 96 h. After fermentation, the cells were collected, freeze-dried, and astaxanthin was extracted using acetone ultrasonic extraction. The content was determined by high performance liquid chromatography (HPLC), with 5 replicates per group.

[0085] Test results as follows Figure 2 As shown, this illustrates wild-type cells after activation and fermentation culture. P. rhodozymaand induced P. rhodozyma CL17 strain of astaxanthin can synthesize and accumulate astaxanthin intracellularly, among which the wild type P. rhodozyma The strain produced 0.15 mg / g DCW of astaxanthin. [[ID=三十一]]P. rhodozyma The astaxanthin content of strain CL17 was 5.13 mg / g DCW, obtained through mutagenesis. P. rhodozyma The astaxanthin content of strain CL17 was 8.7 times that of the wild type.

[0086] Example 5 P. rhodozyma CL17 genome sequencing analysis This example focuses on the mutant strains screened above. P. rhodozyma The CL17 genome sequencing analysis was performed using the following procedure.

[0087] 1. Strain activation and culture: The above-filtered P. rhodozyma Activation of strain CL17 was performed. Specifically, 100 µL of the strain preservation solution was evenly spread on a solid culture medium (containing 2 g / L yeast extract, 3 g / L malt extract, 5 g / L peptone and 10 g / L glucose, 2% agar) and incubated in a biochemical incubator at 19–25°C for 6–9 days.

[0088] Subsequently, larger and more distinctly colored colonies were selected from individual colonies and inoculated into liquid seed culture medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, pH 6.4) and cultured at 20°C and 180 rpm for 72 hours. The bacterial culture was then inoculated into 100 mL Erlenmeyer flasks containing 30 mL of culture medium, at an inoculation concentration of 2%. After inoculation, the cultures were further cultured at 20°C and 180 rpm in fermentation medium (2.08 g / L yeast extract, 6.25 g / L peptone, 25 g / L glucose, with 5 mmol / L glutamate added) for 72 hours. Samples were then collected, and biomass and pigment yield were measured.

[0089] 2. Whole-genome sequencing and comparative analysis: High-astaxanthin-producing strains were sequenced using the Illumina NovaSeq 6000 platform. P. rhodozyma Whole-genome sequencing was performed on CL17, and a 350bp insert library was constructed. Raw sequencing data was obtained through paired-end sequencing. After quality control (filtering bases below Q30 and removing adapter sequences), approximately 500Mb of effective sequencing data was obtained, with a sequencing depth of 100× and a genome assembly integrity (BUSCO assessment) of ≥98%, ensuring the accuracy and coverage of the genome sequence.

[0090] After assembly P. rhodozyma CL17 strain genome sequence compared with wild type P. rhodozyma Alignment analysis was performed using a reference genome (e.g., NCBI accession number GCA_000149615.1).

[0091] The comparison results show P. rhodozyma Mutated genes account for 25% of the genome of strain CL17. The mutation types include single nucleotide polymorphisms (SNPs) and small fragment insertions and deletions (Indels), with SNPs accounting for over 80% and mainly distributed in metabolism-related gene regions.

[0092] 3. Analysis of key mutated genes: Based on the sequencing results, further targeted analysis was conducted on key genes in the astaxanthin synthesis pathway (starting from central metabolism, via the mevalonate pathway and carotenoid synthesis pathway to astaxanthin production). Three core genes were found to have point mutations. These three genes are the lycopenecyclase-phytoene synthase gene. crt YB), geranylgeranylpyrophosphate synthase gene, crt E) and farnesyl pyrophosphate synthase (ERG20) gene.

[0093] in, crt YB encodes a bifunctional enzyme that participates in both the formation of phytopene (the initiation step of carotenoid synthesis) and the cyclization of lycopene to β-carotene. Comparative results show that... crt The nucleotide sequence of the YB gene shows a mutation at position 381 (C to A), position 973 (G to C), and position 1267 (G to A); crt In the amino acid sequence of YB, amino acid residue I at position 127 is mutated to I (synonymous mutation), amino acid residue E at position 325 is mutated to Q, and amino acid residue A at position 425 is mutated to T.

[0094] After mutation crt The YB gene sequence information is as follows:

[0095] After mutation crt The amino acid sequence of YB is as follows: * (SEQ ID NO.2).

[0096] crt E, a key rate-limiting enzyme gene in the carotenoid synthesis pathway, encodes a product that catalyzes the polymerization of isopentenyl pyrophosphate to geranylgeranyl pyrophosphate, providing a direct precursor for astaxanthin synthesis; comparative results show... crt In the E gene, the 51st base G is mutated to A, the 245th base C is mutated to T, the 302nd base C is mutated to G, and the 563rd base T is mutated to A. crtIn the amino acid sequence of E, amino acid residue Q at position 17 is mutated to Q (synonymous mutation), amino acid residue P at position 82 is mutated to L, amino acid residue T at position 101 is mutated to S, and amino acid residue F at position 188 is mutated to Y.

[0097] After mutation crt The E gene sequence information is as follows:

[0098] The amino acid sequence information of crtE after mutation is as follows: MDYANILTAIPLEFTPQDDIVLLEPYHYLGKNPGKEIRSQLIEAFNYWLDVKKEDLEVIQNVVGMLHTASLMDDVEDSSVLRCGSPVAHLIYGIPQTINSANYVYFLAYQEIFKLRPTPIPMPVIPPSSASLQSSVSSASSSSSASSENGGTSTPNSQIPFSKDTYLDKVITDEMLSLHRGQGLELYWR DSLTCPSEEEYVKMVLGKTGGLFRIAVRLMMAKSECDIDFVQLVNLISIYFQIRDDYMNLQSSEYAHNKNFAEDLTEGKFSFPTIHSIHANPSSRLVINTLQKKSTSPEILHHCVNYMRTETHSFEYTQEVLNTLSGALERELGRLQGEFAEANSRMDLGDVDSEGRTGKNVKLEAILKKLADIPL* (SEQ ID NO.4).

[0099] ERG20 is located downstream of the mevalonate pathway, encoding a product that catalyzes the formation of farnesyl pyrophosphate, a key intermediate in sterol synthesis and an important branch node in the pathway leading to carotenoid synthesis. Comparative results showed that the nucleotide sequence of the ERG20 gene had a mutation: base C at position 132 was mutated to G; base G at position 869 was mutated to A; amino acid residue N at position 44 was mutated to K; and amino acid G at position 290 was mutated to D.

[0100] The mutated ERG20 gene sequence information is as follows:

[0101] The amino acid sequence information of the mutated ERG20 is as follows: MSTTPEEKKAARAKFEAVFPVIADEILDYMKGEGMPAEALEWMKKNLYYNTPGGKLNRGLSVVDTYILLSPSGKDISEEEYLKAAILGWCIELLQAYFLVADDMMDASITRRGQPCWYKVEGVSNIAINDAFMLEGAIYFLLKKHFRKESYYVDLLELFHDVTFQTELGQLIDLLTAPED HVDLDKFSLNKHHLIVVYKTAFYSFYLPVALAMRMVGVTDDEAYKLALSILIPMGEYFQVQDDVLDAFAPPEILGKIGTDILDNKCSWPINLALSLASPAQREILDTSYGQKNSEAEARVKALYAELDIQGKFNAYEQQSYESLNKLIDSIDEEKSGLKKEVFHSFLGKVYKRSK* (SEQ ID NO.6).

[0102] Example 6 P. rhodozyma CL17 High Temperature Resistance Test This example refers to the aforementioned wild type. P. rhodozyma (i.e., the starting strain for mutagenesis) and the strains obtained through screening P. rhodozyma The high-temperature tolerance of strain CL17 was tested using the following methods: Equal amounts of wild-type strains and P. rhodozyma CL17 strain was inoculated into seed culture medium (30 g / L glucose, 5 g / L yeast extract, 1 g / L KH2PO4, MgSO4) and other media. Seed culture was obtained by culturing the seed culture at 22℃ and 180r / min for 24 hours with 0.5g / L 7H2O (pH 5.5).

[0103] Then, the seed culture was transferred to fermentation medium (60 g / L glucose, 3 g / L corn steep liquor, 2 g / L KH2PO4, MgSO4) at an inoculation rate of 10% (v / v). 7H2O 0.8g / L, FeSO4 7H2O 0.05g / L, C:N=40:1, pH 5.0), under different temperature conditions, at 180r / min, 100μmol / m² Fermentation was carried out under light conditions for 96 h. The temperature gradient was set at 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, and 45℃. The cell concentration (OD) was measured after fermentation. 600 ), with 5 repetitions per group.

[0104] Test results as follows Figure 3 As shown, compared to the wild-type P. rhodozyma strain, P. rhodozyma The CL17 strain exhibits relatively better adaptability at different culture temperatures, with the wild type showing the best performance. P. rhodozyma The strain grew normally at 30℃, but its OD600 began to decrease at 35℃, approaching 2. P. rhodozyma The OD600 of strain CL17 was significantly higher than that of the wild-type strain at temperatures ranging from 15 to 45°C. P. rhodozyma The biomass is high, reaching a maximum of 3.9 under 30℃ culture conditions, indicating that it is a high-yielding astaxanthin strain. P. rhodozyma CL17 exhibits good temperature adaptability and is more tolerant than the original strain.

[0105] Example 7 Phaffia rhodozyma Astaxanthin content detection at high temperature using CL17 This embodiment tested the results obtained from the above screening. Phaffia rhodozyma The astaxanthin content of CL17 at a relatively high temperature (30℃) was determined using the following method: Equal amounts of wild-type strains and P. rhodozyma CL17 strain was inoculated into seed culture medium (30 g / L glucose, 5 g / L yeast extract, 1 g / L KH2PO4, MgSO4) and other media. After culturing at 30℃ and 180 r / min for 24 h with 0.5 g / L 7H2O (pH 5.5), the culture was transferred to fermentation medium (60 g / L glucose, 3 g / L corn steep liquor, 2 g / L KH2PO4, MgSO4) at a 10% (v / v) inoculation rate. 7H2O 0.8g / L, FeSO4 7H2O 0.05g / L, C:N=40:1, pH 5.0), 22℃, 180r / min, 100μmol / m² Fermentation was carried out under light conditions for 96 h. After fermentation, the cells were collected, freeze-dried, and astaxanthin was extracted using acetone ultrasonic extraction. The content was determined by high performance liquid chromatography (HPLC), with 5 replicates per group.

[0106] The results are as follows Figure 4 As shown, this is at a higher temperature (30°C). P. rhodozyma CL17 had an astaxanthin content of 4.65 mg / g DCW, while the control group (wild-type) had a lower content.P. rhodozyma The astaxanthin yield was only 0.09 mg / g DCW, a 40% decrease compared to the 22℃ culture condition. These results indicate... P. rhodozyma The CL17 mutant strain still exhibits good biomass accumulation and astaxanthin production at higher temperatures, i.e. P. rhodozyma The temperature stability of CL17 is significantly higher than that of the wild-type strain.

[0107] In summary, this invention provides a high-astaxanthin-producing *Phaefflera rubrum* strain and its applications. First, a mutagenic starting strain was obtained by screening and enriching *Alnus rubrum* trees in Suqian City, Jiangsu Province. Then, the strain was passaged under high-intensity light to stimulate light stress response and the expression of astaxanthin synthesis-related genes. A nitrogen-limited culture medium was used for passage, forcing the cell metabolism to shift from growth and reproduction to the accumulation of secondary metabolites such as astaxanthin. Next, the strain was repeatedly passaged in a hyperosmolar environment containing 10–30 g / L potassium chloride solution and sorbitol to screen for robust strains that could resist osmotic stress by enhancing astaxanthin synthesis. Finally, a high-astaxanthin-producing *Phaefflera rubrum* strain was obtained. P. rhodozyma CL17.

[0108] Furthermore, through genome sequencing analysis, this invention discovered that three core genes underwent point mutations, namely... crt YB, crt E and ERG20, among which crt In the amino acid sequence translated by YB, amino acid residue I at position 127 is mutated to I (synonymous mutation), amino acid residue E at position 325 is mutated to Q, and amino acid residue A at position 425 is mutated to T; crt In the amino acid sequence translated from E, amino acid residue Q at position 17 is mutated to Q (synonymous mutation), amino acid residue P at position 82 is mutated to L, amino acid residue T at position 101 is mutated to S, and amino acid residue F at position 188 is mutated to Y; in the amino acid sequence translated from ERG20, amino acid residue N at position 44 is mutated to K, and amino acid G at position 290 is mutated to D.

[0109] The strains screened by this invention have good temperature tolerance. After fermentation, the astaxanthin content in the bacterial cells reaches 5.13 mg / g dry cell weight, showing excellent application prospects.

[0110] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A red Phaffia yeast strain with high astaxanthin production, named as Phaffia rhodozyma Phaffia rhodozyma CL17, deposited with the China General Microbiological Culture Collection Center on October 21, 2025, and given the accession number CGMCC No. 39010.

2. The Phaffia yeast strain according to claim 1, characterized in that, The amino acid sequence of the lycopene cyclase-octaprenyl diphosphate synthase of the Phaffia rhodozyma strain is shown as SEQ ID NO. 2; The amino acid sequence of the geranylgeranyl diphosphate synthase of the Phaffia rhodozyma strain is shown as SEQ ID NO. 4; The amino acid sequence of the farnesyl diphosphate synthase of the Phaffia rhodozyma strain is shown as SEQ ID NO.

6.

3. A method for breeding a high astaxanthin-producing strain of Phaffia rhodozyma according to claim 1 or 2, characterized in that, Comprising: The wild-type Phaffia rhodozyma strain is isolated from the water environment of a poplar tree forest.

4. The method of breeding according to claim 3, wherein The wild-type Phaffia rhodozyma strain is isolated from the water environment of a poplar tree forest. The high-intensity light source for the high-intensity light mutagenesis is a red-blue composite light source, and the light intensity is 2000-3000 Lux. The high-osmotic medium used in the high-osmotic environment stress mutagenesis has an osmotic pressure of 800-1200 mOsm / kg. The high-yield astaxanthin Phaffia rhodozyma strain of claim 1 or 2.

5. An inoculant characterized in that, Comprising:

6. A method for culturing a strain of Phaffia rhodozyma, characterized by, The high-yield astaxanthin Phaffia rhodozyma strain of claim 1 or 2 is inoculated into a culture medium and cultured.

7. Use of the Phaffia rhodozyma strain of claim 1 or 2, the microbial agent of claim 5, or the culture method of claim 6 in the preparation of an astaxanthin-containing product. The high-yield astaxanthin Phaffia rhodozyma strain of claim 1 or 2 or the microbial agent of claim 5 is inoculated into a fermentation medium for fermentation culture, and then the bacterial cells are collected and extracted to obtain.

8. A method for obtaining astaxanthin, characterized by, The carbon-nitrogen ratio of the fermentation medium is 30-50:

1.

9. The acquisition method of claim 8, wherein, The fermentation medium comprises glucose, corn syrup, KH2PO4, MgSO4, and FeSO4. The fermentation culture is carried out at a temperature of 15-45℃.

10. Use of the Phaffia rhodozyma strain of claim 1 or 2 or the microbial agent of claim 5 in the preparation of animal feed, food additives, or health products. and / or the photosynthetic photon flux density of the fermentation culture is 50 to 300 pmol / m 2 s. ​

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