Screening method and application of phaffia rhodozyma strains

Abstract

The invention discloses a screening method and application of phaffia rhodozyma strains. The phaffia rhodozyma strain HSASTA is obtained through screening evolution and has characteristic sequences of five key enzyme genes in the anabolism process of astaxanthin. When the phaffia rhodozyma is used for producing the astaxanthin, the yield can reach 8000 mg / kg or above. The strain can be applied to various industrial fields of health care products, food, food additives and the like, and has wide development and application prospects.

Description

Technical Field

[0001] The present invention relates to the fields of industrial microorganisms and the food and feed industries, and particularly relates to a screening method for a Phaffia rhodozyma strain and its application in the production of astaxanthin. Background Art

[0002] Astaxanthin, also known as astaxanthin, is a kind of carotenoid, naturally purple-red, chemically named 3,3'-dihydroxy-4,4'-diketo-β,β'-carotene, and its molecular formula is (C 40 H 52 O4). Astaxanthin has extremely strong antioxidant effects. Its molecular structure contains conjugated double bonds and a β-ionone ring structure. The unique hydroxy-ketone group makes its electronic effect active, and it can provide more electrons, thereby playing a role in scavenging free radicals. The antioxidant ability of astaxanthin is 10 times that of β-carotene, 200 times that of lutein, and 550 times that of vitamin E. Astaxanthin also has various biological activities, such as anti-inflammatory, anti-tumor, anti-diabetic, preventing cardiovascular diseases, protecting nerves, etc., and has become an effective ingredient in medicine and health foods. Astaxanthin is also a good coloring agent. In the aquaculture industry, after adding astaxanthin to feed, organisms such as salmon, rainbow trout, and salmon can improve the color of their meat by eating astaxanthin. In the livestock breeding industry, it can improve the immunity and survival rate of animals, and increase the nutritional value and color of animal products. Research shows that after feeding laying hens with feed containing astaxanthin, the egg yolk color of poultry can be effectively increased, and the nutritional value is also improved. Therefore, astaxanthin has great application prospects and development value in the health food, feed breeding, pharmaceutical, and cosmetics industries.

[0003] At present, the sources of astaxanthin are mainly divided into three types. First, chemically synthesized astaxanthin has poor safety and applicability, and is prone to chemical pollution. Second, natural astaxanthin is synthesized in microalgae and yeast, and then accumulates in plankton or fish after being eaten, so it can be extracted from aquatic organisms (such as krill, shrimp, and crabs), but the extraction is limited by limited raw materials, low yield, and high cost. Third, the microbial fermentation method directly uses strains such as the microalgae Rhodococcus rhodochrous and Phaffia rhodozyma for fermentation production of natural astaxanthin, which has good safety, stability, and practicability. Due to the improvement of people's health awareness, the demand for natural astaxanthin is also increasing.

[0004] Xanthophyllomyces dendrorhous (also known as Phaffia rhodozyma), a basidiomycetous yeast, can produce astaxanthin as its main carotenoid. Due to its advantages such as fast growth rate, short fermentation cycle, easy cultivation, ability to utilize various carbon sources, and high-density cultivation in fermenters, it is considered a microorganism with great commercial development value. Since the yield of astaxanthin in wild-type Xanthophyllomyces dendrorhous is very low and cannot compete with chemical synthesis, improving the strain to increase the astaxanthin yield has become the top priority. Currently, methods for obtaining high-yield strains include traditional methods such as physical and chemical mutagenesis and protoplast fusion, as well as many transgenic technologies. For example, CN113789322B provides a high-astaxanthin-producing Xanthophyllomyces dendrorhous strain and its breeding method, which is obtained by ARTP (atmospheric and room temperature plasma) mutagenesis and β-ionone screening using Xanthophyllomyces dendrorhous as the starting production strain. The strain is cultured in a YPD medium in a shake flask for 5 days, and the highest astaxanthin yield and content reach 35.37 mg / L and 5248 mg / kg (dry cell weight), respectively. CN114561304B effectively promoted the accumulation of astaxanthin in the cells by adding appropriate precursors during the fermentation cycle of the cells and then matching the optimized fermentation process, and the yield reached 5.75 mg / g astaxanthin. CN106318878B obtained the Xanthophyllomyces dendrorhous engineering strain SXD by transforming the host with a gene element expression cassette that improves the synthesis of intracellular acetyl-CoA in Xanthophyllomyces dendrorhous, reduces the synthesis of sterol substances, and increases the metabolic flux of the astaxanthin synthesis pathway. The highest astaxanthin content of the obtained Xanthophyllomyces dendrorhous engineering strain SXD is 4.4 mg / g dry cell weight, and the astaxanthin content after optimized cultivation is 7.1 mg / g. CN106676019B used the already constructed model organism Yarrowia lipolytica capable of biosynthesizing astaxanthin, and after optimized cultivation, an astaxanthin yield of 450 mg / L was obtained in a fermenter. CN106701880A is a method for improving the high-astaxanthin-producing ability of Xanthophyllomyces dendrorhous strains. While stimulating the high-yield production of astaxanthin by the strains, it also promotes the synthesis of astaxanthin during the fermentation process, and the astaxanthin production rate is 81.56 mg / L. CN117701407A provides a novel Xanthophyllomyces dendrorhous strain, which contains rich astaxanthin and β-glucan. When this strain is applied to aquaculture, the astaxanthin yield of this strain can reach more than 700 mg / L. CN117363502B provides a high-astaxanthin-producing Rhodotorula rubra strain and a method for preparing astaxanthin from it. The natural astaxanthin yield of the high-astaxanthin-producing Rhodotorula rubra strain (OKS) after cultivation is 152 mg / L.

[0005] Although based on previous studies, the astaxanthin content of Xanthophyllomyces dendrorhous has been improved, to meet the requirements of industrial production, it is still necessary to further increase the biomass and astaxanthin content of Xanthophyllomyces dendrorhous. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a screening method for Phaffia rhodozyma strains and their application in astaxanthin production to meet the requirements of industrial production. Through screening, the present invention has obtained a Phaffia rhodozyma strain with significantly increased astaxanthin content, biomass, etc. This strain has the characteristic sequences of five key enzyme genes in the astaxanthin synthesis and metabolism process, which can significantly increase the astaxanthin production yield. It is expected to greatly reduce the cost of astaxanthin production by microbial fermentation and improve its application value in related fields.

[0007] On the one hand, the present invention provides a Phaffia rhodozyma strain, which is Phaffia rhodozyma strain HSASTA. It is preserved in the General Microbiology Center of the China Microbial Culture Collection Center, with the preservation address being No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is: CGMCC No. 28781, and the preservation date is October 26, 2023. The taxonomic name is Phaffia rhodozyma.

[0008] The Phaffia rhodozyma strain provided by the present invention was obtained by screening from the microbial laboratory of Xiamen Huisheng Biotech Co., Ltd., China. The strain was identified, and the identification result was Phaffia rhodozyma.

[0009] The Phaffia rhodozyma strain HSASTA provided by the present invention has the following microbiological characteristics: Colony morphology and microscopic characteristics: Cultured in malt extract liquid medium at 25 °C for three days, the cell size is (1.8~4)×(1.2~2.4) μm, oval, elliptical. Precipitation is formed. Cultured on malt extract agar slant at 25 °C for one month, the colony is cheese-like, orange-red, smooth and shiny on the surface, with neat edges. Cultured on corn agar Dalmau plate, no pseudohyphae are produced. The cells cultured in YM liquid medium for 3 days are as Figure 1 shown.

[0010] The following is the metabolic process of astaxanthin production by the Phaffia rhodozyma strain HSASTA through the mevalonate pathway. From the metabolism of glucose to generate precursor substances, to the generation of IPP and DMAPP through the MVA pathway, and then to the final synthesis of astaxanthin, the whole process involves multiple key enzymes and metabolic steps. Specifically as follows: Carotenoids in yeast are derived from the mevalonate pathway. Starting from glucose, pyruvate is generated through the Embden-Meyerhof pathway (EMP), and then oxidized and decarboxylated to obtain acetyl-CoA. Three molecules of acetyl-CoA condense to form MVA, and MVA is further phosphorylated and decarboxylated to convert into isopentenyl pyrophosphate (IPP). IPP is the synthetic precursor of all isoprenoid compounds such as astaxanthin, carotenoids, and ergosterol. Yeast synthesizes substances such as glycerol-3-phosphate (G3P), pyruvate, and acyl-CoA through the glycolytic pathway using carbon sources such as glucose and fructose. Glycerol-3-phosphate, pyruvate, and acyl-CoA flow into the next stage as precursor substances of IPP and DAMPP. At the same time, a part of acetyl-CoA enters the tricarboxylic acid cycle (TCA). In the mevalonate pathway (MVA) for the synthesis of IPP and DAMPP, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) forms mevalonate under the action of HMG-CoA reductase, and mevalonate synthesizes IPP through a series of phosphorylation reactions.

[0011] The Phaffia rhodozyma strain HSASTA provided by the present invention was found to have 5 specific gene sequence fragments of the Phaffia rhodozyma strain HSASTA through genome sequencing, all of which are closely related to the synthesis of precursor substances for astaxanthin synthesis in glycolysis and the tricarboxylic acid cycle, especially the synthesis of acetyl-CoA.

[0012] Furthermore, the Phaffia rhodozyma strain HSASTA provided by the present invention contains at least one gene related to the synthesis of precursor substances for astaxanthin synthesis in glycolysis and the tricarboxylic acid cycle. Further, the precursor substance is acetyl-CoA.

[0013] Furthermore, the gene is selected from at least one of an alcohol dehydrogenase gene, a pyruvate carboxylase gene, a lactate dehydrogenase gene, a ubiquinone monooxygenase gene, and a cytochrome c oxidase gene.

[0014] Furthermore, the Phaffia rhodozyma strain HSASTA provided by the present invention contains a special alcohol dehydrogenase gene, and its sequence is shown as SEQ ID NO:1: Furthermore, the Rhodotorula rubra yeast strain HSASTA provided by the present invention contains a special pyruvate carboxylase gene, and its sequence is as shown in SEQ ID NO:2: Furthermore, the Rhodotorula rubra yeast strain HSASTA provided by the present invention contains a special lactate dehydrogenase gene, and its sequence is as shown in SEQ ID NO:3: Furthermore, the Rhodotorula rubra yeast strain HSASTA provided by the present invention contains a special ubiquinone monooxygenase gene, and its sequence is as shown in SEQ ID NO:4: Furthermore, the Rhodotorula rubra yeast strain HSASTA provided by the present invention contains a special cytochrome c oxidase gene, and its sequence is as shown in SEQ ID NO:5: On the other hand, the present invention provides a screening and evolution method for the Phaffia rhodozyma strain HSASTA, which is obtained by screening and culturing with light radiation and monoclonal fermentation using Phaffia rhodozyma as the starting production bacterium.

[0015] Furthermore, the specific steps of the screening and evolution method are as follows: (1) Strain activation: Transfer Phaffia rhodozyma to a liquid screening medium for culture to obtain an activated strain. (2) Screening culture: Coat the activated strain on a solid screening medium, culture it under light radiation, detect the dry weight and astaxanthin content, and select strains with higher yields for monoclonal isolation. (3) Monoclonal fermentation culture: Transfer the strain to a fermentation medium, culture it under light radiation, centrifuge the cells, detect the dry weight and astaxanthin content, and select strains with higher yields. (4) Repeat steps (2) to (3) for continuous screening and evolution to obtain a strain with good growth state and high astaxanthin yield, named HSASTA.

[0016] Furthermore, the conditions for strain activation culture are a temperature of 22°C and a culture at 180 r / min for 3 days. Further, the liquid screening medium is glucose 10 - 40 g / L; yeast extract powder: 5 - 10 g / L, dissolved and prepared using 50% natural seawater, with a pH of 5.5 - 6.0.

[0017] Furthermore, the conditions for screening culture are a temperature of 22°C, light of 200 - 500 Lux for 3 days, preferably 200 Lux. Further, the solid screening medium is glucose 10 - 40 g / L; yeast extract powder: 5 - 10 g / L, dissolved and prepared using 50% natural seawater, with a pH of 5.5 - 6.0, and 20 g / L of agar powder is added. Through screening culture, it is beneficial to have a greater mutation rate under light mutagenesis conditions, which is conducive to increasing the probability of obtaining the target mutant strain in subsequent fermentation culture.

[0018] Furthermore, the conditions for fermentation culture are a temperature of 22°C, 180 r / min, light of 200 - 500 Lux for 120 days, preferably 500 Lux. Further, the fermentation medium is: glucose 30 - 60 g / L, yeast extract powder 5 - 20 g / L, KH2PO4 5 - 15 g / L, (NH4)2SO4 10 - 15 g / L, MgSO4 1.0 - 5.0 g / L, NaCl 0.1 - 0.5 g / L, CaCl2 0.1 - 1.0 g / L, KCl 0.1 - 1.0 g / L, CuSO 4▪5H2O: 0.08 - 0.15 g / L, sodium citrate: 3.0 - 5.0 g / L. After fermentation culture and centrifugation detection, mutagenic conditions can be removed, which is beneficial to selecting strains with higher yields.

[0019] Furthermore, the continuous screening and evolution are carried out for more than 15 generations, preferably 20 generations.

[0020] By repeating steps (2) - (3) for continuous screening and evolution, the mutagenic effect can be maximally improved, and strains with good growth states and high astaxanthin yields can also be obtained to the greatest extent.

[0021] On the one hand, the present invention provides an application of the above-mentioned Phaffia rhodozyma strain HSASTA in the production of astaxanthin.

[0022] On the one hand, the present invention provides a method for producing astaxanthin, and the specific steps are as follows: inoculating the Phaffia rhodozyma strain HSASTA of the present invention or the Phaffia rhodozyma strain HSASTA obtained by the screening and evolution method into a fermentation tank for culture.

[0023] Furthermore, for the method for producing astaxanthin, the fermentation culture conditions and steps are as follows: including shake flask seed culture, seed tank culture, and fermentation tank culture.

[0024] Furthermore, the conditions for shake flask seed culture: rotation speed 150 - 200 rpm, temperature 22 - 25 °C, light intensity 2000 - 5000 Lux, culture for 48 - 72 h.

[0025] Furthermore, the conditions for seed tank culture: adjusting pH to 5.5 - 6.0 with citric acid and ammonia water, dissolved oxygen: 20 - 80%, rotation speed 120 - 270 rpm, temperature 22 - 25 °C, light intensity 2000 - 5000 Lux, culture for 48 - 72 h.

[0026] Furthermore, the conditions for fermentation tank culture: adjusting pH to 5.5 - 6.0 with citric acid and ammonia water, dissolved oxygen: 20 - 80%, rotation speed 120 - 270 rpm, temperature 22 - 25 °C, culture for 96 - 168 h, dark culture before 72 h, light on after 72 h, light intensity 5000 - 10000 Lux.

[0027] More preferably, the medium in the shake flask is glucose: 10 - 40 g / L; yeast extract powder: 4 - 10 g / L; Further preferably, the culture medium in the seed tank is glucose 20-40 g / L, yeast extract powder 5-20 g / L, KH2PO4 5-15 g / L, (NH4)2SO4 10-15 g / L, MgSO4 1.0-5.0 g / L, NaCl 0.1-0.5 g / L, CaCl2 0.1-1.0 g / L, KCl 0.1-1.0 g / L, CuSO 4▪ 5H2O 0.08-0.15 g / L; Further preferably, the fermentation tank culture medium is: glucose 30-60 g / L, yeast extract powder 5-20 g / L, KH2PO4 5-15 g / L, (NH4)2SO4 10-15 g / L, MgSO4 1.0-5.0 g / L, NaCl 0.1-0.5 g / L, CaCl2 0.1-1.0 g / L, KCl 0.1-1.0 g / L, CuSO 4▪ 5H2O g / L, citrate 3.0-5.0 g / L, sodium acetate 2.5-3.5 g / L.

[0028] Further, the inoculation amount of the Phaffia rhodozyma strain HSASTA in the shake flask, seed tank and fermentation tank is 3-10%.

[0029] On the other hand, the present invention provides the application of the Phaffia rhodozyma strain HSASTA and astaxanthin produced thereby in health foods, food additives, and feeds as described above.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: In the present invention, the Phaffia rhodozyma is irradiated by light, so that the structure of its cell membrane or cell wall is changed, the permeability is changed, and its genes are lost or changed, thereby significantly changing the gene sequence and metabolism of the Phaffia rhodozyma, and finally gene mutation occurs. By continuous screening and mutation, the probability of screening the Phaffia rhodozyma strain HSASTA is finally increased.

[0031] The Phaffia rhodozyma strain HSASTA provided by the present invention has the characteristic sequences of 5 key enzyme genes in the astaxanthin synthesis and metabolism process, which is of great significance for the production of astaxanthin.

[0032] Using the Phaffia rhodozyma of the present invention to produce astaxanthin, the yield can reach more than 8000 mg / kg. After continuous subculture for 12 times, the astaxanthin content is 7000-8000 mg / kg, and the stability is consistent; the astaxanthin yield, content and corresponding biomass of the strain of the present invention all meet the requirements of industrial production and have great application prospects.

[0033] Compared with other complex mutagenesis methods, the method of the present invention is simple and efficient, and the mutagenesis effect is remarkable. The screened strains have greatly improved astaxanthin production. Moreover, the strains obtained by the present invention have a large number of consecutive passages and can maintain the stability of the strains and the stability of astaxanthin production.

[0034] Aiming at the problems of low biomass and low content in the current production of astaxanthin by fermentation method, a strain of Phaffia rhodozyma with a fast growth rate and a high astaxanthin content is provided. This strain can be applied to a variety of industrial fields such as health products, foods, and food additives, and has broad development and application prospects.

[0035] The following further elaborates the technical solution of the present invention in combination with examples. These examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Colony morphological characteristics of Phaffia rhodozyma strain HSASTA DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention will be further described below in combination with specific examples. These examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0038] Example 1 Culture Medium Screening medium (liquid, solid): Glucose 10 - 40 g / L; Yeast extract powder: 5 - 10 g / L, dissolved and prepared with 50% natural seawater, pH 5.5 - 6.0. 20 g / L agar powder is added to the solid medium. The specific screening medium (liquid, solid) can be: Glucose 40 g / L; Yeast extract powder: 8 g / L, dissolved and prepared with 50% natural seawater, pH 5.5 - 6.0. 20 g / L agar powder is added to the solid medium.

[0039] Seed medium (g / L): Glucose 20 - 40, Yeast extract powder 5 - 20, KH2PO4 5 - 15, (NH4)2SO4 10 - 15, MgSO4 1.0 - 5.0, NaCl 0.1 - 0.5, CaCl2 0.1 - 1.0, KCl 0.1 - 1.0, CuSO 4▪ 5H2O 0.08 - 0.15. The specific seed medium (g / L) can be: Glucose 30, Yeast extract powder 10, KH2PO4 5, (NH4)2SO4 10, MgSO4 1.0, NaCl 0.1, CaCl2 0.1, KCl 0.1, CuSO 4▪5H2O 0.08。

[0040] Fermentation medium (g / L): Glucose 30 - 60, yeast extract powder 5 - 20, KH2PO4 5 - 15, (NH4)2SO4 10 - 15, MgSO4 1.0 - 5.0, NaCl 0.1 - 0.5, CaCl2 0.1 - 1.0, KCl 0.1 - 1.0, CuSO 4▪ 5H2O 0.08 - 0.15, sodium citrate 3.0 - 5.0. The specific fermentation medium (g / L) can be: Glucose 40, yeast extract powder 10, KH2PO4 5, (NH4)2SO4 15, MgSO4 1.0, NaCl 0.5, CaCl2 0.5, KCl 0.8, CuSO 4▪ 5H2O 0.10, sodium citrate 4.0.

[0041] Shake flask medium (g / L): Glucose 10 - 40; Yeast extract powder: 4 - 10. The specific shake flask medium (g / L) can be: Glucose 30; Yeast extract powder: 6.

[0042] Seed tank medium (g / L): Glucose 20 - 40, yeast extract powder 5 - 20, KH2PO4 5 - 15, (NH4)2SO4 10 - 15, MgSO4 1.0 - 5.0, NaCl 0.1 - 0.5, CaCl2 0.1 - 1.0, KCl 0.1 - 1.0, CuSO 4▪ 5H2O 0.08 - 0.15. The specific seed tank medium (g / L) can be: Glucose 30, yeast extract powder 10, KH2PO4 8, (NH4)2SO4 10, MgSO4 3.0, NaCl 0.2, CaCl2 0.6, KCl 0.6, CuSO 4▪ 5H2O 0.12.

[0043] Fermentation tank medium (g / L): Glucose 30 - 60, yeast extract powder 5 - 20, KH2PO4 5 - 15, (NH4)2SO4 10 - 15, MgSO4 1.0 - 5.0, NaCl 0.1 - 0.5, CaCl2 0.1 - 1.0, KCl 0.1 - 1.0, CuSO 4▪ 5H2O 0.08 - 0.15, citrate 3.0 - 5.0, sodium acetate 2.5 - 3.5. The specific fermentation tank medium (g / L) can be: Glucose 40, yeast extract powder 10, KH2PO4 8, (NH4)2SO4 12, MgSO4 3.0, NaCl 0.3, CaCl2 0.6, KCl 0.7, CuSO 4▪5H2O 0.09, citrate 4.0, sodium acetate 3.5.

[0044] Example 2 Astaxanthin Extraction Method (1) Pretreatment of the sample: Take 5 ml of the fermentation broth, centrifuge at 3500 r / min for 5 minutes, discard the supernatant, add another 5 ml of distilled water, shake well, and then continue centrifuging and washing. Repeat 1 - 2 times, and reserve the bacterial sludge. (2) Extraction of astaxanthin: Add approximately 4.5 ml of DMSO (dimethyl sulfoxide) solution preheated to approximately 75°C to the centrifuge tube, tighten the lid, shake for 1 - 2 minutes, and then keep it at 75°C for 5 minutes; add 3 mL of mobile phase to the centrifuge tube, centrifuge at 3500 r / min for 5 minutes, and carefully transfer the centrifuged supernatant to a 50 ml volumetric flask; repeat the above process until the sludge layer is colorless or only has a very light color, and finally make up to the scale with the mobile phase to obtain the test solution.

[0045] Determination of astaxanthin content by high performance liquid chromatography: Chromatographic column: 380 - 01238 - 43 ShimNex HEC18 - aq, 5μm, 4.6×250mm, Mobile phase: methanol:acetonitrile = 9:1 (v / v), Column temperature: 30°C, Flow rate: 1 mL / min, Detection wavelength: 478nm, Injection volume: 10 uL.

[0046] Example 3 Screening and Evolution of Phaffia rhodozyma Strain HSASTA (1) Transfer the fermentation sample preserved by Xiamen Huisheng Biotech Co., Ltd. to 5 mL of liquid screening medium, culture at 22°C and 180 r / min for 3 days. Spread it on the screening medium plate, culture at 22°C and 200 Lux light for 3 days, and then select monoclonal colonies on the plate. Transfer them to the fermentation medium, culture at 22°C, 180 r / min, and 500 Lux light for 120 days, then centrifuge the bacteria, and detect the dry weight and astaxanthin content.

[0047] (2) Select monoclonal colonies with high dry weight and astaxanthin content, repeat (1), and conduct continuous 20 - generation evolution. Finally, obtain a strain with good growth state and high astaxanthin production, named HSASTA.

[0048] (3) Conduct a stability test on HSASTA. Continuously passage it 12 times in the fermentation medium, and the astaxanthin content is all between 7000 - 8000 mg / kg, with consistent stability.

[0049] Example 4 Identification of Phaffia rhodozyma Strain HSASTA The identification of the Phaffia rhodozyma strain HSASTA was entrusted to the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences. The identification result was the Phaffia rhodozyma strain. An identification report was issued, and the report number was Weijianzi No. 2024JB090. The submission date was December 2023.

[0050] The Phaffia rhodozyma strain HSASTA provided by the present invention has the following microbiological characteristics: Colony morphology and microscopic characteristics: Cultured in malt extract liquid medium at 25°C for three days, the cell size is (1.8~4)×(1.2~2.4) μm, oval and elliptical. Precipitation is formed. Cultured on a malt extract agar slant at 25°C for one month, the colony is cheese-like, orange-red, smooth and shiny on the surface, with a neat edge. No pseudohyphae are produced when cultured on a corn agar Dalmau plate. The cells cultured in YM liquid medium for 3 days are as Figure 1 shown.

[0051] The physiological and biochemical characteristics are shown in Table 1 below It can be seen from Table 1 that the Phaffia rhodozyma strain HSASTA provided by the present invention cannot ferment glucose, maltose, galactose, lactose, sucrose, and raffinose; it can strongly assimilate glucose, sucrose, maltose, cellobiose, trehalose, and melezitose as carbon sources, but has a weak assimilation ability for glycerol, D-mannitol, raffinose, D-xylose, and L-arabinose; it cannot assimilate L-rhamnose, galactose, methanol, L-sorbose, ethanol, erythritol, ribitol, galactitol, lactose, melibiose, D-sorbitol, methyl glucoside, salicin, soluble starch, lactose, succinic acid, citric acid, D-ribose, and inositol; it can strongly assimilate nitrate and lysine as nitrogen sources and cannot assimilate cadaverine hydrochloride and sarcosine.

[0052] Example 5 Fermentation culture of the Phaffia rhodozyma strain HSASTA 1. Flask seed culture 1.1 Flask medium (g / L): Glucose 20; Yeast extract powder: 6.

[0053] 1.2 Flask seed culture conditions: Rotation speed 170 rpm, temperature 22°C, light intensity 2000 Lux, culture for 72 h; inoculate into the seed tank at an inoculation amount of 5%.

[0054] 2. Fermentation tank seed culture 2.1 Seed tank culture medium (g / L): Glucose 30, yeast extract powder 10, KH2PO4 8, (NH4)2SO4 10, MgSO4 3.0, NaCl 0.2, CaCl2 0.6, KCl 0.6, CuSO 4▪ 5H2O 0.12.

[0055] 2.2 Seed tank culture conditions: Adjust the pH to 5.7 with citric acid and ammonia water, dissolved oxygen: 60%, rotation speed 170 rpm, temperature 22 °C, light intensity 3000 Lux, culture for 72 h, and inoculate into the fermenter at an inoculation amount of 10%.

[0056] 3. Fermenter culture 3.1 Fermenter culture medium (g / L): Glucose 40, yeast extract powder 10, KH2PO4 8, (NH4)2SO4 12, MgSO4 3.0, NaCl 0.3, CaCl2 0.6, KCl 0.7, CuSO 4▪ 5H2O 0.09, citrate 4.0, sodium acetate 3.5.

[0057] 3.2 Fermenter culture conditions: Adjust the pH to 5.7 with citric acid and ammonia water, dissolved oxygen: 80%, rotation speed 200 rpm, temperature 22 °C, culture for 168 h, culture in the dark before 72 h, turn on the light after 72 h, and the light intensity is 10000 Lux.

[0058] The biomass, carotenoid and astaxanthin contents of Phaffia rhodozyma HSASTA were analyzed by the detection method in Example 2, and the results are shown in the following table (Table 2).

[0059] Table 2. Analysis of detection indexes of the fermented cells of strain HSASTA It can be illustrated by Example 5 that the Phaffia rhodozyma strain obtained by the breeding method of the present invention still has the characteristics of stable and high-yield astaxanthin after being cultivated for 144 h. When continuously passaged to the 12th generation, the astaxanthin content in the dry cell weight can still reach 13672.05 mg / kg, indicating that the strain provided by the present invention can maintain the stability of the strain and also maintain stability in terms of astaxanthin production, meet the requirements of industrial production, and has great application prospects.

[0060] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A strain of Phaffia rhodozyma, characterized in that, It contains at least one gene related to the synthesis of precursors for astaxanthin synthesis in glycolysis and the tricarboxylic acid cycle; further preferably, the precursor is acetyl coenzyme A.

2. The Rhodotorula rubra yeast strain according to claim 1, wherein The gene is selected from at least one of alcohol dehydrogenase gene, pyruvate carboxylase gene, lactate dehydrogenase gene, ubiquinone monooxygenase gene, and cytochrome c oxidase gene.

3. The Rhodotorula rubra yeast strain according to claim 1, wherein, The alcohol dehydrogenase gene has a sequence as shown in SEQ ID NO:1; The pyruvate carboxylase gene has a sequence as shown in SEQ ID NO:2; The lactate dehydrogenase gene has a sequence as shown in SEQ ID NO:3; The ubiquinone monooxygenase gene has a sequence as shown in SEQ ID NO:4; The cytochrome c oxidase gene has a sequence as shown in SEQ ID NO:

5.

4. A screening and evolution method for the Rhodotorula rubra yeast strain HSASTA according to any one of claims 1 to 3, characterized in that, It is obtained by screening and culturing with light radiation and monoclonal fermentation screening using Phaffia rhodozyma as the starting production strain.

5. The screening and evolution method according to claim 4, wherein, The specific steps are as follows: (1) Strain activation: Transfer Phaffia rhodozyma to a liquid screening medium for culture to obtain an activated strain; (2) Screening culture: Spread the activated strain on a solid screening medium, culture with light radiation, detect the dry weight and astaxanthin content, and select strains with higher yields for monoclonal; (3) Monoclonal fermentation culture: Transfer the strain to a fermentation medium, culture with light radiation, centrifuge the cells, detect the dry weight and astaxanthin content, and select strains with higher yields; (4) Repeat steps (2) to (3), continuously screen and evolve to obtain a strain with good growth state and high astaxanthin yield, named HSASTA.

6. The screening and evolution method according to claim 5, wherein, The conditions for strain activation culture are temperature 22°C, 180 r / min for 3 d; the conditions for fermentation culture are temperature 22°C, 180 r / min, light 200 - 500 Lux for 120 d; the conditions for screening culture are temperature 22°C, light 200 - 500 Lux for 3 d.

7. The screening and evolution method according to claim 5, characterized in that, The liquid screening medium is glucose 10 - 40 g / L; yeast extract powder: 5 - 10 g / L, dissolved and prepared with 50% natural seawater, pH 5.5 - 6.0; The solid screening medium is glucose 10 - 40 g / L; yeast extract powder: 5 - 10 g / L, dissolved and prepared with 50% natural seawater, pH 5.5 - 6.0, adding 20 g / L agar powder; The fermentation medium is as follows: glucose 30 - 60 g / L, yeast extract powder 5 - 20 g / L, KH2PO4 5 - 15 g / L, (NH4)2SO4 10 - 15 g / L, MgSO4 1.0 - 5.0 g / L, NaCl 0.1 - 0.5 g / L, CaCl2 0.1 - 1.0 g / L, KCl 0.1 - 1.0 g / L, CuSO 4▪ 5H2O 0.08 - 0.15 g / L, sodium citrate 3.0 - 5.0 g / L.

8. A method for producing astaxanthin, characterized in that, The Phaffia rhodozyma strain HSASTA described in any one of claims 1 - 3 or the Phaffia rhodozyma strain HSASTA obtained by the screening and evolution method described in any one of claims 4 - 7 is inoculated into a fermenter for culture to obtain.

9. The method for producing astaxanthin according to claim 8, characterized in that, The fermentation culture conditions and steps are as follows: Inoculation amount: The inoculation amount at each level is 3 - 10%; Shake flask seed culture conditions: Rotation speed 150 - 200 rpm, temperature 22 - 25°C, light 2000 - 5000 Lux, culture for 48 - 72 h; Seed tank culture conditions: Adjust the pH to 5.5 - 6.0 with citric acid and ammonia water, dissolved oxygen: 20 - 80%, rotation speed 120 - 270 rpm, temperature 22 - 25°C, light 2000 - 5000 Lux, culture for 48 - 72 h; Fermentation tank culture conditions: Adjust the pH to 5.5 - 6.0 with citric acid and ammonia water, dissolved oxygen: 20 - 80%, rotation speed 120 - 270 rpm, temperature 22 - 25 °C, culture for 96 - 168 h, culture in the dark before 72 h, turn on the light for culture after 72 h, light intensity 5000 - 10000 Lux; Further preferably, the medium in the shake flask is glucose 10 - 40 g / L; yeast extract powder: 4 - 10 g / L; Further preferably, the culture medium in the seed tank is 20 - 40 g / L of glucose, 5 - 20 g / L of yeast extract powder, 5 - 15 g / L of KH2PO4, 10 - 15 g / L of (NH4)2SO4, 1.0 - 5.0 g / L of MgSO4, 0.1 - 0.5 g / L of NaCl, 0.1 - 1.0 g / L of CaCl2, 0.1 - 1.0 g / L of KCl, 0.08 - 0.15 g / L of CuSO 4▪ 5H2O; Further preferably, the fermentation tank culture medium is: glucose 30-60 g / L, yeast extract powder 5-20 g / L, KH2PO4 5-15 g / L, (NH4)2SO4 10-15 g / L, MgSO4 1.0-5.0 g / L, NaCl 0.1-0.5 g / L, CaCl2 0.1-1.0 g / L, KCl 0.1-1.0 g / L, CuSO 4▪ 5H2O g / L, citrate 3.0-5.0 g / L, sodium acetate 2.5-3.5 g / L.

10. Use of the Phaffia rhodozyma strain HSASTA according to any one of claims 1 - 3 in the production of astaxanthin.

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