Phaffia rhodozyma mutant strain with high astaxanthin yield as well as screening method and application thereof

Abstract

The invention provides a phaffia rhodozyma mutant strain for high yield of astaxanthin as well as a screening method and application of the phaffia rhodozyma mutant strain, belongs to the technical field of fermentation microorganism screening, and can solve the problems of low astaxanthin yield, high cost and low growth temperature of wild type phaffia rhodozyma existing in synthesis of astaxanthin from a phaffia rhodozyma strain. The invention provides a phaffia rhodozyma mutant strain capable of producing astaxanthin at high yield, the phaffia rhodozyma mutant strain is a phaffia rhodozyma AA0529 mutant strain, the phaffia rhodozyma AA0529 mutant strain is preserved in China Center for Type Culture Collection in Wuhan, Hubei on September 7, 2023, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M20231637. According to the method, the yield of the astaxanthin can be increased, and the yield of the astaxanthin is increased to 3.3 mg / g at 26 DEG C.

Description

Technical Field

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

[0002] Astaxanthin is a natural red carotenoid widely found in marine organisms such as shrimp, crabs, and crustaceans. Astaxanthin possesses important physiological and health benefits, including antioxidant, anti-inflammatory, and immune-boosting properties, and is currently widely used in health products, food, beverages, cosmetics, and pharmaceuticals.

[0003] Red Pfaff yeast ( Xanthophyllomyces dendrohous *Pharmacospora rubrum* is the only yeast strain in nature capable of naturally synthesizing 3R,3'-dextral astaxanthin. Due to its short lifespan and rapid astaxanthin synthesis rate, *Pharmacospora rubrum* can utilize various glucose sources for rapid heterotrophic metabolism and can undergo high-density fermentation in fermenters, making it a high-quality strain for industrial astaxanthin production. Different *Pharmacospora rubrum* strains exhibit varying astaxanthin production capabilities, but due to inherent limitations, they generally suffer from low yields and high costs. Furthermore, the optimal temperature for cell growth and pigment synthesis in wild-type *Pharmacospora rubrum* is typically 17°C–21°C. Lower growth temperatures require more cooling water and energy to maintain fermentation temperatures, and large-scale production may even necessitate refrigeration equipment, significantly increasing production costs. Therefore, providing a high-yield *Pharmacospora rubrum* strain is crucial for astaxanthin production. Summary of the Invention

[0004] This invention addresses the technical problems of low astaxanthin yield, high cost, and low growth temperature of wild-type Pharfogel's yeast in the synthesis of astaxanthin by Pharfogel's yeast strains. It proposes a high-yield Pharfogel's yeast mutant strain, its screening method, and its application, which can increase astaxanthin yield to 3.3 mg / g at 26°C.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: a high-astaxanthin-producing mutant strain of *Phaeodactylum rubrum*, wherein the strain is *Phaeodactylum rubrum* (…). Phaffia rhodozyma The mutant strain AA0529 was deposited on September 7, 2023, at the China Center for Type Culture Collection in Wuhan, Hubei Province, with accession number CCTCC NO: M20231637.

[0006] In one embodiment, the CDS sequence of the crtYB gene in the high-astaxanthin-producing Pharfogel's red yeast mutant strain is shown in SEQ ID No. 1, and the CDS sequence of the crtI gene is shown in SEQ ID No. 2.

[0007] In one embodiment, the high-astaxanthin-producing Pharfogel's red yeast mutant strain was obtained by using Pharfogel's red yeast as the starting strain and combining atmospheric pressure room temperature plasma mutagenesis with a high-throughput micro-level droplet culture omics system and temperature stress screening.

[0008] In another aspect, this invention provides a method for screening high-astaxanthin-producing Pharfovia rubescens mutant strains, comprising the following steps: S1. After inoculating the freeze-dried Pharbitis rubescens powder dissolved in sterile water into a shake flask for culture, it was diluted and spread on a slide for ARTP mutagenesis. S2. After collecting the mutagenized bacterial culture into the fermentation medium, droplets were generated using a high-throughput micro-level droplet culture omics system for offline culture. The droplets were then detected, and droplets with a high single-packet ratio were selected as the initial screening strains. S3. Spread the initially screened strains onto the substrate containing β-lactone. After ionone was cultured in YM solid medium, it was transferred to deep well plates for further culture. Biomass and astaxanthin content were measured, and secondary screening strains were obtained. S4. The rescreened strain was continuously passaged in shake flasks for five generations, and the biomass and astaxanthin content were repeatedly measured. The strain with the highest astaxanthin yield per unit cell was selected as the high-astaxanthin Pharf yeast mutant strain.

[0009] In this invention, a MISS cell device is used for miniaturized high-throughput single-cell culture, and simultaneously coupled with an OD detection device for droplet sorting, to achieve the isolation and culture of Pharfogel's erythrophyll at the single-cell level. The target droplets are sorted and stored in multi-well plates, which greatly reduces the workload of manual bacterial picking. Through shake-flask subculturing, the biomass and astaxanthin content are repeatedly measured to ensure the stability of the high-astaxanthin-producing Pharfogel's erythrophyll strain.

[0010] In one embodiment, in steps S1 and S4, the culture medium for shake flask culture is 5.0 g / L peptone, 3.0 g / L malt extract, 3.0 g / L yeast extract, and 10.0 g / L glucose, with the initial pH adjusted to 6.2 ± 0.2; the shake flask culture temperature is 22°C, the rotation speed is 180 rpm, and the culture time is 48 h.

[0011] In one embodiment, the ARTP mutagenesis step in step S1 includes: diluting the bacterial solution using a volume ratio of bacterial solution:sterile water = 1:5, and adjusting the bacterial solution concentration to 10. 6 ~10 8 CFU / mL; take the diluted bacterial solution, spread it evenly on the surface of the slide, and perform ARTP mutagenesis for 40~100s.

[0012] In one embodiment, in step S2, the fermentation temperature is 26°C, the rotation speed is 180 rpm, and the culture time is 48 h; the offline culture temperature is 26°C, and the culture time is 48 h.

[0013] In one embodiment, in step S3, the solid culture medium is cultured at a temperature of 26°C for 48 hours; the deep well plate is cultured at a temperature of 26°C, a rotation speed of 180 rpm, and a culture time of 48 hours.

[0014] In another aspect, the present invention provides the application of the high-astaxanthin-producing Pharfogel's mutant strain of red Pharfogel's yeast in increasing astaxanthin production.

[0015] In one embodiment, the fermentation temperature was 26°C, and the astaxanthin yield was 3.3 mg / g.

[0016] Compared with existing technologies, the advantages and positive effects of this invention are as follows: the TPM (Transcrips Per Million Reads) of the crtYB and crtI genes in the mutant strain of this invention are significantly higher than those of the original strain, indicating that the *Phaefflera rubrum* mutant strain obtained by the screening method of this invention can improve the expression of related enzymes, and the expression is positively correlated with astaxanthin synthesis, thereby improving the conversion rate of astaxanthin and achieving high astaxanthin production. In this invention, the initial strain is first subjected to room temperature plasma mutagenesis, and then high-throughput droplet screening of high-yielding mutant strains is performed using MISS cell technology combined with temperature stress. Subsequently, the screened droplets are subjected to primary plate screening and secondary screening using deep-well plates. The ability of each cell to produce astaxanthin is used to determine the optimal high-yielding mutant strain. Fermentation is carried out at 26℃, and the astaxanthin yield in the fermentation broth is 3.3 mg / g, which greatly improves the astaxanthin yield. Attached Figure Description

[0017] Figure 1 This illustrates the effect of different ARTP mutagenesis times on the lethality of Pharfovia rubescens in Example 1 of this invention. Figure 2 This is the detection result of the initial screening encapsulation rate of sample MISS cell in Example 1 of the present invention; Figure 3 This is the detection result of the initial screening encapsulation rate of sample two MISS cells in Example 1 of the present invention; Figure 4 This is the detection result of the initial screening encapsulation rate of sample three MISS cells in Example 1 of the present invention; Figure 5 This is the detection result of the initial screening encapsulation rate of sample four MISS cells in Example 1 of the present invention; Figure 6 This is a characterization of the astaxanthin production level of deep-well cultured Rhodophora oryzae in Example 1 of the present invention; Figure 7 This is a characterization of the mass level of Rhodopseudomonas erythrosporum cultured in deep wells according to Example 1 of the present invention; Figure 8 This is a characterization of astaxanthin production per unit cell of *Phaeodactylum rubrum* cultured in deep wells according to Example 1 of the present invention. Figure 9 The image shows a color comparison of the red phaf yeast fermentation broth obtained in Example 1 and Comparative Example 1 of the present invention. The left image is Example 1, and the right image is Comparative Example 1. Figure 10 The image shows a color comparison of the red phaf yeast fermentation broth obtained in Example 1 and Comparative Example 2 of the present invention. The left image is Example 1, and the right image is Comparative Example 2. Figure 11 The TPM values ​​of the crtI gene of the mutant strain in Example 1 and the original strain in Comparative Example 1 are shown. Figure 12 The TPM values ​​of the crtYBI gene of the mutant strain in Example 1 and the original strain in Comparative Example 1 are shown. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] This invention provides a high-astaxanthin-producing Pharrellis erythrophyll mutant strain, its screening method, and its application. The TPM (Transcrips Per Million Reads) of the crtYB and crtI genes in the mutant strain are significantly higher than those in the original strain, indicating that the Pharrellis erythrophyll mutant strain obtained through the screening method of this invention can improve the expression of related enzymes. This expression is positively correlated with astaxanthin synthesis, thereby increasing the astaxanthin conversion rate and achieving high astaxanthin production. Applying the high-astaxanthin-producing Pharrellis erythrophyll mutant strain to astaxanthin production can increase both fermentation temperature and astaxanthin yield. At a fermentation temperature of 26℃, the astaxanthin yield is 3.3 mg / g.

[0020] This invention utilizes ambient pressure room temperature plasma (ARTP) combined with a high-throughput micro-level droplet culture omics system (MISScell ​​culture omics) to achieve the isolation and culture of bacterial communities at the cellular level. A high-astaxanthin-producing *Phaefflera rubra* strain was obtained through ARTP mutagenesis screening, and a heat-resistant, high-yielding strain was obtained through MISScell ​​temperature stress screening. Finally, a *Phaefflera rubra* mutant strain with high astaxanthin yield was screened based on biomass and astaxanthin production. Continuous passage was used to ensure the stability of the mutant and improve screening efficiency. The obtained high-astaxanthin-producing *Phaefflera rubra* mutant strain achieved an astaxanthin yield of 3.3 mg / g at 26°C, making it suitable for industrial production. The obtained astaxanthin can be used as an additive in aquaculture feed to improve the body color of salmon, trout, ornamental fish, and crustaceans (such as shrimp and crab), enhance their commercial value, strengthen their antioxidant capacity and immunity, and improve their survival rate and growth performance. It can also be used in health products as a dietary supplement for anti-aging and immune enhancement. Furthermore, it can be used in the food industry as a safe coloring agent in the processing of candies, beverages, and other foods to replace synthetic pigments, or combined with eggs, poultry, etc., to produce nutritionally fortified foods rich in astaxanthin.

[0021] To more clearly and in detail introduce the high-astaxanthin-producing Pharfia redis mutant strain, its screening method, and its application provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0022] Example 1 S1. The screening method for high-astaxanthin-producing Pharfovia mutant strains in this embodiment includes the following steps: S1.1 Preparation of bacterial culture Dissolve 0.2g of lyophilized Pharrellis redis in 0.5mL of sterile water to obtain the starting strain. Inoculate 0.2mL of the strain into a shake flask and incubate at 22℃ and 180rpm for 48h. The fermentation medium in the shake flask consists of 5.0g / L peptone, 3.0g / L malt extract, 3.0g / L yeast extract, and 10.0g / L glucose, with the initial pH adjusted to 6.2. S1.2, ARTP mutagenesis The bacterial suspension was diluted with sterile water at a ratio of 1:5 (v / v), and its optical density at 475 nm was adjusted to 1.2, thereby controlling the corresponding bacterial suspension concentration at 10. 6 CFU / mL; 10 μL of diluted bacterial suspension was evenly spread on the surface of a slide for ARTP mutagenesis. The effect of different ARTP mutagenesis times on the lethality of *Phaefflera rubescens* was investigated. Figure 1 As shown, by Figure 1It can be seen that after ARTP irradiation for 40s, 80s, 100s, 120s, 140s, 160s, 180s, and 220s, the lethality rates were 43.44%, 89.34%, 92.62%, 96.72%, 98.36%, 100%, 100%, and 100%, respectively. The mutagenic effect was best when the lethality rate was 70%~80%. Therefore, the irradiation time of 80s and 100s, which were closest to this range of lethality rate, was selected. That is, the mutagenicity time was set at 80s and 100s, and five parallel experiments were conducted at each time point, for a total of 100µL of bacterial suspension. S1.3, Droplet primary screening The mutagenized bacterial culture was collected into 3 mL of fermentation medium and cultured at 26 °C and 180 rpm for 1 h. The sample concentration was then reduced to 10 by adding more medium. 1 10 2 10 3 10 4 The samples were recorded as Sample 1, Sample 2, Sample 3, and Sample 4, respectively. Droplets were generated using a MISS cell and incubated offline at 26℃ for 48 hours. The incubated droplets were then detected using a MISS cell at an OD of 475 nm. The detection results are as follows: Figures 2-5 As shown; according to Figures 2-5 The MISS cell detection results show that the droplet encapsulation rate of sample one is too high to be sorted. The droplet encapsulation rates of samples two, three and four are 20%, 15.55% and 1.07% respectively. The proportion of single-cell droplets in the sorted droplets is high, which is in line with the experimental expectations. The proportion of single-cell droplets refers to the proportion of single-cell droplets in the total droplets. Droplets with a high percentage of single-packet samples were retained in 96-well plates. Based on droplet OD detection data, the sorting lower limit was set at 4.50, and droplets with OD values ​​higher than 4.90 were collected. The top 50 droplets with the highest OD values ​​were used as the initial screening strains. The initially screened droplets were then spread onto plates containing β-lactone. Ionone was cultured on YM solid medium at 26°C for 48 h; S1.4, Deep-hole plate double screen The initially screened strains were transferred to 24-well plates and cultured at 26℃ and 180 rpm for 48 h before secondary screening and verification. Their astaxanthin content (OD475 value representing the astaxanthin production level of the bacteria) and biomass (OD600 value representing the bacterial cell mass) were measured. The results are as follows: Figure 6 and Figure 7 As shown, according to Figure 8 The mutant strains with higher yields (OD600 / OD475 values ​​represent astaxanthin yield per unit cell) were selected, and 12 mutant strains were obtained through screening. S1.5, Shaking Bottle Transplantation The 12 mutant strains obtained by screening were continuously passaged for five generations, and their biomass and astaxanthin content were repeatedly measured to screen out the strain with the highest OD475 / OD600 value. To more accurately characterize the astaxanthin production capacity of the mutant strains, the OD475 / OD600 values ​​of the strains were compared and analyzed (OD475 / OD600 values ​​represent the astaxanthin production per unit strain). S1.6 Stability Test The high-yield astaxanthin strain obtained from the shake-flask subculturing in step S1.5 was subjected to stability tests. The strain was fermented three times consecutively in Erlenmeyer flasks containing fermentation medium. Astaxanthin was extracted from the fermentation broth after each fermentation, and its yield was measured. The fermentation conditions were: 26℃, 180 rpm, and cultured for 48 h. The results of the three measurements showed that the astaxanthin yield was relatively stable. The high-yield astaxanthin strain obtained through mutation was labeled AA0529, and its astaxanthin yield increased to 3.3 mg / g. This strain is the selected high-yield astaxanthin Pharfogel's mutant strain. S1.6.1 Extraction and Determination Methods of Astaxanthin Centrifuge 5 mL of fermentation broth at 5000 rpm for 10 min, wash twice with deionized water, and collect the bacterial cells. Add 1 mL of dimethyl sulfoxide (DMSO) preheated at 55 °C, and vortex for 10 min. Add 4 mL of acetone, vortex at 4 °C for 20 s, then centrifuge at 5000 rpm for 10 min. After separating the supernatant, repeat the above operation with dimethyl sulfoxide and acetone on the precipitate until the bacterial cells turn white. Combine the supernatants, filter through a 0.22 μm organic filter membrane, and analyze by ultra-high performance liquid chromatography. The astaxanthin yield was 3.3 mg / g.

[0023] Comparative Example 1 This comparative example uses the original strain to prepare astaxanthin through fermentation, specifically including the following steps: Dissolve 0.2g of lyophilized Pharrellis redis in 0.5mL of sterile water, then inoculate 0.2mL of the inoculum into an Erlenmeyer flask containing fermentation medium. Fermentation is carried out by shaking the flask three times to obtain the fermentation broth. The fermentation medium consists of 5.0g / L peptone, 3.0g / L malt extract, 3.0g / L yeast extract, and 10.0g / L glucose, with the initial pH adjusted to 6.2. The fermentation conditions are: 22℃, 180r / m, and culture for 48h. Centrifuge 5 mL of fermentation broth at 5000 rpm for 10 min, wash twice with deionized water, and collect the cells. Add 1 mL of dimethyl sulfoxide preheated at 55 °C and vortex for 10 min. Add 4 mL of acetone, vortex at 4 °C for 20 s, then centrifuge at 5000 rpm for 10 min. After separating the supernatant, add the fermentation broth to the precipitate and repeat the above operation until the cells turn white. Combine the supernatants, filter through a 0.22 μm organic filter membrane, and analyze by ultra-high performance liquid chromatography. The astaxanthin yield was determined to be 0.15 mg / g.

[0024] Comparative Example 2 This comparative example uses strains that have only undergone mutagenesis but not high-temperature stress screening to prepare astaxanthin through fermentation. The specific steps include: S1. Preparation of bacterial culture Dissolve 0.2g of lyophilized Pharrellis redis in 0.5mL of sterile water, then inoculate 0.2mL of the culture into a shake flask and incubate at 22℃ and 180rpm for 48h. The fermentation medium in the shake flask consists of 5.0g / L peptone, 3.0g / L malt extract, 3.0g / L yeast extract, and 10.0g / L glucose, with the initial pH adjusted to 6.2. S2,ARTP mutagenesis The bacterial suspension was diluted with sterile water at a ratio of 1:5 (v / v), and its optical density at 475 nm was adjusted to 1.2, thereby controlling the corresponding bacterial suspension concentration at 10. 6 CFU / mL; Take 10 μL of diluted bacterial solution, spread it evenly on the surface of the slide, and perform ARTP mutagenesis. Set the mutagenesis time to 80s and 100s, and perform five parallel experiments at each time point, for a total of 100µL of bacterial solution to be mutagenized. S3, shake-flask fermentation The mutagenized bacterial culture was coated onto a substrate containing β-lactone. Ionone was cultured on YM solid medium at 22℃ for 48 h. The dominant strains on the plates were then transferred to Erlenmeyer flasks containing fermentation medium and fermented three times using a shake-flask method to obtain the fermentation broth. The fermentation medium in the Erlenmeyer flasks consisted of 5.0 g / L peptone, 3.0 g / L malt extract, 3.0 g / L yeast extract, and 10.0 g / L glucose, with the initial pH adjusted to 6.2 ± 0.2. The fermentation conditions were 26℃, 180 rpm, and cultured for 48 h. S4. Centrifuge 5 mL of fermentation broth at 5000 rpm for 10 min, wash twice with deionized water, and collect the bacterial cells. Add 1 mL of dimethyl sulfoxide preheated at 55 °C and suspend by shaking for 10 min. Add 4 mL of acetone, vortex at 4 °C for 20 s, then centrifuge at 5000 rpm for 10 min. After separating the supernatant, add dimethyl sulfoxide and acetone to the precipitate and repeat the above operation until the bacterial cells turn white. Combine the supernatants, filter through a 0.22 μm organic filter membrane, and analyze by ultra-high performance liquid chromatography. The astaxanthin yield was determined to be 0.02 mg / g.

[0025] Table 1 shows the fermentation temperature and astaxanthin yield of Example 1 and Comparative Examples 1-2. The color of the Rhodopseudomonas erythrorhizon fermentation broth obtained in Example 1 and Comparative Examples 1-2 is compared. Figure 9 and Figure 10 As shown.

[0026] Table 1. Fermentation temperature and astaxanthin yield of Example 1 and Comparative Examples 1-2

[0027] As shown above, Comparative Example 1 used the original Pharfogel's yeast strain to ferment and prepare astaxanthin at 22°C, and the astaxanthin yield in the fermentation broth was 0.15 mg / g. Comparative Example 2 used a strain that had only undergone mutagenesis but not temperature stress screening to ferment and prepare astaxanthin at 26°C, and the astaxanthin yield in the fermentation broth was only 0.02 mg / g. In Example 1, the initial strain was first subjected to room temperature plasma mutagenesis, and high-throughput droplet screening of high-yield mutagenesis strains was performed using MISS cell technology combined with temperature stress. Subsequently, the screened droplets were subjected to plate primary screening and deep-well plate secondary screening to determine the optimal high-yield mutagenesis strain based on the ability of each cell to produce astaxanthin. Fermentation was carried out at 26°C, and the astaxanthin yield in the fermentation broth was 3.3 mg / g, which was 22 times that of Comparative Example 1 and 165 times that of Comparative Example 2. This indicates that the Pharfogel's yeast mutant strain screened in this invention can improve the astaxanthin yield.

[0028] Transcriptome analysis of *Phaeff's red yeast*: Fermentation broths from the mutant strain of Example 1 and the original strain of Comparative Example 1 were each divided into three groups for parallel experiments. The mutant strains of Example 1 were numbered B1, B2, and B3, and the original strains of Comparative Example 1 were numbered A1, A2, and A3. All strains were centrifuged and treated with liquid nitrogen, and transcriptome analysis was performed to identify differentially expressed genes. Significant differences were found in the expression levels of 3,4-dehydrolycopene synthase (crtI) and 15-cis-phytene synthase / lycopene β-cyclase (crtYB), as shown below. Figure 11 and Figure 12 As shown. By Figure 11 and Figure 12It can be seen that the TPM (Transcrips Per Million Reads) of the crtYB and crtI genes in the mutant strain of Example 1 is significantly higher than that of the original strain. This indicates that the Pharrellis rubrum mutant strain obtained by the screening method of this invention can improve the expression of related enzymes, and the expression is positively correlated with astaxanthin synthesis, thereby improving the conversion rate of astaxanthin and achieving the effect of high astaxanthin production. The CDS sequence of the crtYB gene in the mutant strain is shown in SEQ ID No. 1, and the CDS sequence of the crtI gene is shown in SEQ ID No. 2.

[0029] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, evolutions, or improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A high-astaxanthin-producing Pharfovia erythropoietin mutant strain, characterized in that, The strain is *Phaeodactylum rubrum* (…). Phaffia rhodozyma The mutant strain AA0529 was deposited on September 7, 2023, at the China Center for Type Culture Collection in Wuhan, Hubei Province, with accession number CCTCC NO: M20231637.

2. The high-astaxanthin-producing Pharfovia erythropoietin mutant strain according to claim 1, characterized in that, In the high-astaxanthin-producing Pharfia redis mutant strain, the CDS sequence of the crtYB gene is shown in SEQ ID No. 1, and the CDS sequence of the crtI gene is shown in SEQ ID No.

2.

3. The high-astaxanthin-producing Pharfovia rubescens mutant strain according to claim 1, characterized in that, The high-astaxanthin-producing Pharrell's red yeast mutant strain was obtained by using Pharrell's red yeast as the starting strain and combining atmospheric pressure room temperature plasma mutagenesis with a high-throughput micro-level droplet culture omics system and temperature stress screening.

4. The method for screening high-astaxanthin-producing Pharfovia rubescens mutant strains according to claim 1, characterized in that, Includes the following steps: S1. After inoculating the freeze-dried Pharbitis rubescens powder dissolved in sterile water into a shake flask for culture, it was diluted and spread on a slide for ARTP mutagenesis. S2. After collecting the mutagenized bacterial culture into the fermentation medium, droplets were generated using a high-throughput micro-level droplet culture omics system for offline culture. The droplets were then detected, and droplets with a high single-packet ratio were selected as the initial screening strains. S3. Spread the initially screened strains onto the substrate containing β-lactone. After ionone was cultured in YM solid medium, it was transferred to deep well plates for further culture. Biomass and astaxanthin content were measured, and secondary screening strains were obtained. S4. The rescreened strain was continuously passaged in shake flasks for five generations, and the biomass and astaxanthin content were repeatedly measured. The strain with the highest astaxanthin yield per unit cell was selected as the high-astaxanthin Pharf yeast mutant strain.

5. The method for screening high-astaxanthin-producing Pharfovia rubescens mutant strains according to claim 4, characterized in that, In steps S1 and S4, the culture medium for shake flask culture consisted of 5.0 g / L peptone, 3.0 g / L malt extract, 3.0 g / L yeast extract, and 10.0 g / L glucose, with the initial pH adjusted to 6.2 ± 0.

2. The shake flask culture temperature was 22℃, the rotation speed was 180 rpm, and the culture time was 48 h.

6. The method for screening high-astaxanthin-producing Pharfovia rubescens mutant strains according to claim 4, characterized in that, The ARTP mutagenesis step in step S1 includes: diluting the bacterial culture using a volume ratio of bacterial culture to sterile water of 1:5, and adjusting the bacterial culture concentration to 10. 6 ~10 8 CFU / mL; take the diluted bacterial solution, spread it evenly on the surface of the slide, and perform ARTP mutagenesis for 40~100s.

7. The method for screening high-astaxanthin-producing Pharfovia rubescens mutant strains according to claim 4, characterized in that, In step S2, the fermentation temperature was 26℃, the rotation speed was 180 rpm, and the culture time was 48 h; the offline culture temperature was 26℃, and the culture time was 48 h.

8. The method for screening high-astaxanthin-producing Pharfovia rubescens mutant strains according to claim 4, characterized in that, In step S3, the solid culture medium is cultured at 26℃ for 48 hours; the deep well plate culture temperature is 26℃, the rotation speed is 180 rpm, and the culture time is 48 hours.

9. The application of the high-astaxanthin-producing Pharfovia mutant strain according to any one of claims 1-3 in improving astaxanthin production.

10. In the application according to claim 9, the fermentation temperature is 26°C, and the astaxanthin yield is 3.3 mg / g.

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