Screening method of Rhodosporidium toruloides space mutant strain with high carotenoid yield, strain and application of strain in carotenoid production

Abstract

The invention belongs to the technical field of biological medicines, provides an efficient screening method for a high-yield carotenoid rhodosporidium toruloides space mutant strain, and belongs to the technical field of biological medicines, aiming at the problems that existing carotenoid high-yield strain screening depends on color observation, the positive mutation rate is low, the screening workload is large and the like. According to the core scheme, a YPD solid basic culture medium and a compound growth inhibitor (containing D-glucose, D-xylose, vanillin and other components) are mixed according to a certain volume ratio to prepare a screening culture medium, and the space mutation rhodosporidium toruloides mutant strain for high-yield carotenoid is efficiently screened out. Furthermore, the screened high-yield carotenoid rhodosporidium torulosporidium space mutant strain is subjected to fed-batch fermentation to produce carotenoid, the yield can reach 48.7 mg / L, and the method has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a high-carotenoid-producing Rhodotorula buergerianum (Rhodotorula buergerianum). Rhodosporidium toruloides Screening methods for space-induced mutagenesis strains, strains, and their application in the production of carotenoids. Background Technology

[0002] Carotenoids are a class of polyene compounds that are yellow, orange-red, or red in color, with over 700 different species. They are a collective term for carotene and its oxidized derivatives, such as β-carotene, lycopene, erythromycin, strophanthidin, and astaxanthin. In recent years, with the increasingly widespread application of carotenoids in clinical medicine, clinical pharmacology, food, health products, and cosmetics, they have become important pigments with nutritional, coloring, and health-promoting functions, holding significant value in multiple industrial sectors including food, feed, cosmetics, pharmaceuticals, and chemicals. Currently, carotenoids are mainly obtained through three methods: extraction from plants, microbial fermentation, and chemical synthesis. However, microbial fermentation for carotenoid production faces challenges such as low strain yields and a large workload in screening high-yielding mutant strains. In particular, it is difficult to screen mutant strains with similar colony colors, and mutants selected based on similar colors have a low positive mutation rate.

[0003] The patent "A High-Yield Fermentation Method for a High-Yield Carotenoid-Producing Strain of *Saccharomyces cerevisiae* and Its Application" (CN111793569 A) proposes that during strain screening, the strain with the reddest color and / or the largest growth zone on the solid fermentation medium plate should be selected as the starting strain for the next UV mutagenesis. The patent "High-Yield Astaxanthin Strain of *Rhodotorula rubra*" (CN115176024 A) proposes that when selecting high-yield astaxanthin-producing strains of *Rhodotorula rubra*, the colony morphology, including color and diameter, of the strains after 6 and 10 days of cultivation on standard YM agar plates should be observed and compared. Furthermore, the patent "High-Yield Carotenoid-Producing Strains and Their Application" (CN 104073457 A) proposes that after mutagenesis, the bacterial solution should be spread on TGY solid medium, and by observing the colony morphology, strains with a darker color than the starting strain should be selected to preliminarily determine their high-yield potential for carotenoid production. The patent "A Strain Producing Unsaturated Fatty Acids and Carotenoids and Its Application" (CN 113308387 A) requires dilution to different concentrations and plating onto YPD solid culture plates during the initial screening of strains induced by ion beam mutagenesis, necessitating the plating of a large number of YPD solid plates. The patent "A Strain of Rhodotorula glutinis and Its Application in Fermentation Production of Carotenoids and Oils" (CN104130952 B) utilizes ARTP mutagenesis followed by plating and inoculation, obtaining the strain KC8-AR62 with the highest carotenoid yield after multiple rounds of screening. In summary, current methods for screening high-carotenoid-producing strains have certain shortcomings. Existing methods mainly rely on visual observation of colony color intensity (compared to the starting strain) to preliminarily determine carotenoid yield. This method selects mutant strains with low positive mutation rates and struggles to effectively distinguish mutant strains with similar colors, resulting in a large workload, low efficiency, and long time consumption in obtaining high-yielding strains.

[0004] In summary, mutation breeding produces a large number of mutant strains. The key to obtaining high-yielding strains lies in rapidly and efficiently screening for positive mutants. Under specific nutrient limitations (such as nitrogen restriction), *Rhodotorula glutinis* (…) Rhodosporidium toruloidesYeasts are capable of efficiently accumulating various high-value biological products, such as carotenoids and lipids, and are considered important producers of natural metabolites. However, current methods for selecting mutant strains based on colony color are difficult to effectively distinguish between mutants with similar colors. Phenolic compounds (vanillin) mainly inhibit yeast growth by disrupting intracellular redox balance, inducing reactive oxygen species accumulation, and disrupting cell membrane transport protein activity. 5-Hydroxymethylfurfural (HMF) is a major component in furanaldehyde derivative inhibitors, and its inhibitory effect on yeast growth and metabolism is more significant. These inhibitors interfere with physiological processes such as central carbon metabolism and lipid metabolism by inhibiting the activity of key enzymes in the glycolysis pathway. When *Rhodotorula buergerianum* is subjected to stress from vanillin, HMF, and levulinic acid, the yeast's own protective mechanisms are triggered. When cells are subjected to stress from these components, mitochondrial oxidoreductase activity is inhibited. To maintain normal cell function, cells produce large amounts of ATP-metabolizing inhibitors, leading to a significant increase in acetyl-CoA participation in metabolism. In the carotenoid metabolic pathway, acetyl-CoA is an important precursor. At the same time, carotenoids have antioxidant activity that can accelerate the removal of ROS or inhibit their generation, protect the structure of the endoplasmic reticulum, promote the removal of reactive oxygen species, and protect against oxidative stress damage. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a compound growth inhibitor and its application in high-throughput screening of carotenoid-producing *Rhodotorula glutinis* mutant strains induced by space mutagenesis, thereby efficiently obtaining high-carotenoid-producing mutant strains. The original starting strain used is *Rhodotorula glutinis* (… Rhodosporidium toruloides The *Rhodotorula rubrum* Y strain was purchased from the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC 2.1389. Its freeze-dried bacterial powder was subjected to space-induced mutagenesis aboard the Shijian-19 recoverable experimental satellite. To address the challenge of screening large quantities of mutant strains, YPD solid basal medium was mixed with a compound growth inhibitor at a specific ratio (e.g., 6:1, adjustable based on the yield of the starting strain) to prepare screening plates. On these plates, mutant strains with similar colony colors but low carotenoid yields failed to grow, while high-carotenoid-producing mutant strains did. This method is simple to operate, easily distinguishable colonies, and significantly reduces workload. After initial screening, the mutant strains were re-screened in shake flasks using fermentation medium to obtain high-carotenoid-producing mutants. These were then fed-batch fermented in a 5 L fermenter to produce carotenoids.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0007] The preparation method of the compound growth inhibitor and culture medium specifically includes the following steps:

[0008] Compound growth inhibitor and its preparation: Weigh 1-10 g / L glucose, 70-100 g / L D-xylose, 0.5-3 g / L vanillin, 1-4 g / L 5-hydroxymethylfurfural (HMF), and 6-10 g / L levulinic acid, dissolve them in distilled water, adjust the pH to 6.0, stir thoroughly to dissolve, and then filter the solution through a 0.22 μm aqueous microporous membrane to obtain a sterile compound growth inhibitor, which is stored at 4℃ in the dark for later use.

[0009] The preparation method of the YPD liquid / solid culture medium specifically includes the following steps:

[0010] YPD liquid / solid culture medium preparation: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, and solid culture medium also need to add agar 20 g / L. Dissolve in an appropriate amount of distilled water, stir thoroughly to dissolve, and autoclave at 115 ℃ for 30 min. After sterilization, cool to 50~55 ℃ for use.

[0011] The preparation method of the solid screening culture medium specifically includes the following steps:

[0012] Preparation of solid screening medium: Under aseptic conditions, YPD solid medium cooled to 50~55 ℃ is mixed with sterile compound growth inhibitor at a certain volume ratio, and immediately poured into sterile petri dishes. After solidification, the medium is obtained. The preferred volume ratio of YPD solid medium to sterile compound growth inhibitor is 6:1.

[0013] The fermentation medium was prepared as follows: 50 g / L glucose, 0.75 g / L yeast extract, 0.1 g / L ammonium sulfate, 0.4 g / L potassium dihydrogen phosphate, and 1.5 g / L magnesium sulfate heptahydrate were dissolved in an appropriate amount of distilled water, the pH was adjusted to 6.0, and the medium was autoclaved at 115 °C for 30 min. Glucose was sterilized separately and mixed before use.

[0014] The screening method for high-carotenoid mutant strains includes the following steps:

[0015] (1) Activation of space-mutated freeze-dried bacterial powder: Take the space-mutated Rhodotorula rubra spores Y freeze-dried bacterial powder into a centrifuge tube for later use. Use a sterile pipette to add YPD liquid culture medium into the centrifuge tube, gently shake until the freeze-dried bacterial powder is completely dissolved, and let it stand at room temperature for 10 min to activate and obtain a bacterial suspension for later use.

[0016] (2) Initial screening on screening medium plates: The bacterial suspension prepared in (1) was serially diluted to obtain bacterial suspensions of different concentrations. 100 μL of bacterial suspensions of different concentrations were spread on screening medium mixed with YPD solid medium and compound growth inhibitors. The suspensions were cultured at 28 ℃ and the colony growth was observed. Mutant strains with large colony diameter and obvious red color were selected for the next step of shake flask screening. The mutant strains after initial screening were inoculated into 5 mL of YPD liquid medium and cultured on a shaker at 28 ℃ and 220 r / min for 24 h. The strains were then stored at 80 ℃ with a final concentration of 25% glycerol.

[0017] (3) Shake flask screening: The mutant strain of Rhodotorula rubra obtained from the initial screening of the screening medium plate in step (2) was inoculated into test tubes containing 5 mL of YPD liquid medium and cultured at 28 ℃ and 220 r / min for 24 h. Then, it was inoculated into shake flasks containing 50 mL of fermentation medium at an inoculation rate of 10% (v / v) and cultured at 28 ℃ and 220 r / min for 168 h. Carotenoids were extracted using the acid-heat method and the carotenoid content was determined, thereby obtaining a high-yield carotenoid space mutant strain.

[0018] This invention also provides the application of the high-yield carotenoid space mutant strain in the preparation of carotenoids. The high-yield carotenoid mutant strain obtained by shake-flask re-screening is subjected to batch-fed fermentation in a 5 L fermenter to obtain a fermentation broth, from which carotenoids are extracted.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) The inhibitors in the compound growth inhibitors (vanillin, 5-hydroxymethylfurfural, organic acids, etc.) can inhibit the growth of low carotenoid-producing strains, while high carotenoid-producing mutant strains can tolerate and survive. This overcomes the shortcomings of traditional screening, which relies solely on color difference to distinguish mutant strains with similar colors but different yields. Furthermore, the mutant strain library is large and the positive mutation rate is low, which leads to a large workload and long screening time. Using compound growth inhibitors can significantly improve the positive mutation rate of mutant strain screening, thereby reducing the workload and efficiently screening high carotenoid-producing mutant strains.

[0021] (2) On the other hand, the present invention provides a high-yield carotenoid-producing Rhodotorula buergerianum space-mutated strain for fermentation production of carotenoids, which can be applied to multiple fields such as food, medicine and daily chemical products. Attached Figure Description

[0022] Figure 1To determine the mixing ratio of YPD solid culture medium and optimal concentration of compound growth inhibitor in the embodiments of the present invention, the volume ratio of YPD solid culture medium to optimal concentration of compound growth inhibitor is 2:1, 4:1, 6:1, 8:1 and 10:1 respectively.

[0023] Figure 2 The above are comparison images of the colony growth of *Rhodotorula buergerianum* Y-freeze-dried yeast powder after space mutagenesis at different dilutions on different culture media in the embodiments of the present invention. A: YPD solid culture medium, B: low concentration of compound growth inhibitor screening medium, C: optimal concentration of compound growth inhibitor screening medium, D: high concentration of compound growth inhibitor screening medium.

[0024] Figure 3 This is a comparison chart of carotenoid yields during shake-flask fermentation and secondary screening of the starting strain Y and the carotenoid primary screening mutant strains (Y-1~Y-9) in the embodiments of the present invention.

[0025] Figure 4 This is a comparison of the colony growth of *Rhodotorula buergerianum* on YPD solid medium and screening medium with optimal concentration of compound growth inhibitors in the embodiments of the present invention.

[0026] Figure 5 The images show a comparison of fermentation in 24-well plates of mutant strains of Rhodotorula buergerianum on YPD solid medium and screening medium with optimal concentration of growth inhibitor. A: Fermentation of a single colony of mutant strain on YPD solid medium in 24-well plate. B: Fermentation of a single colony of mutant strain on screening medium with optimal concentration of growth inhibitor in 24-well plate.

[0027] Figure 6 In this embodiment of the invention, the starting strain Y and the high-carotenoid mutant strain Y-5 were subjected to fed-batch fermentation in a 5 L fermenter to measure the carotenoid yield and cell concentration (OD). 600 Comparison chart.

[0028] Figure 7 The images show a comparison of the appearance of carotenoids, β-carotene standards, and lycopene standards produced by the high-yield carotenoid mutant strain Y-5 in this invention. A: Carotenoids produced by the high-yield carotenoid mutant strain Y-5; B: β-carotene standard (HPLC 96%); C: Lycopene standard (HPLC 98%).

[0029] Figure 8The images show the color and qualitative results of the chloroform extract of the high-yield carotenoid mutant strain Y-5, the β-carotene standard, and the chloroform solution of the lycopene standard in the embodiments of the present invention. A: Color of the chloroform extract of the high-yield carotenoid mutant strain Y-5 (①) and its reaction with concentrated sulfuric acid (②) are compared. B: Color of the chloroform solution of the β-carotene standard (HPLC 96%) (①) and its reaction with concentrated sulfuric acid (②) are compared. C: Color of the chloroform solution of the lycopene standard (HPLC 98%) (①) and its reaction with concentrated sulfuric acid (②) are compared.

[0030] Figure 9 The images shown are full-wavelength scans of the acetone extract, Torularhodin, and Torulene from the high-carotenoid mutant strain Y-5 in this invention embodiment. A: Full-wavelength scan of the acetone extract of the high-carotenoid mutant strain Y-5; B: Full-wavelength scan of Torularhodin; C: Full-wavelength scan of Torulene. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0032] The first part of this invention is to prepare screening culture media with different concentrations of compound growth inhibitors; the second part is to establish the optimal screening effect of the compound growth inhibitor culture media by performing primary and secondary screening on the space-mutated Rhodotorula circophylla Y lyophilized yeast powder to obtain a high-carotenoid-producing Rhodotorula circophylla mutant strain; the third part is to use the high-carotenoid-producing space mutant strain Rhodotorula circophylla Y-5 to produce carotenoids through batch feeding fermentation in a 5 L fermenter.

[0033] Experimental equipment: conical flask, disposable petri dishes, pipette, constant temperature shaker, constant temperature incubator, 5 L fermenter, ultraviolet spectrophotometer.

[0034] Example 1: Preparation of compound growth inhibitors and screening culture medium

[0035] (1) Preparation of the optimal compound growth inhibitor: Weigh 6 g / L glucose, 90 g / L D-xylose, 1.8 g / L vanillin, 3 g / L 5-hydroxymethylfurfural (HMF) and 9 g / L levulinic acid, dissolve them in an appropriate amount of distilled water, adjust the pH to 6.0, stir thoroughly to dissolve, and then filter the solution through a water-based microporous membrane with a pore size of 0.22 μm to obtain a sterile compound growth inhibitor. Store it at 4 ℃ in the dark for later use, and place it at room temperature for 30 min before use.

[0036] (2) Preparation of high-concentration compound growth inhibitor: Weigh 10 g / L glucose, 100 g / L D-xylose, 3 g / L vanillin, 4 g / L 5-hydroxymethylfurfural (HMF) and 10 g / L levulinic acid and dissolve them in an appropriate amount of distilled water. Adjust the pH to 6.0 and stir thoroughly to dissolve. Then filter the solution through a water-based microporous membrane with a pore size of 0.22 μm to obtain sterile high-concentration compound growth inhibitor. Store it at 4 ℃ in the dark for later use. Let it stand at room temperature for 30 min before use.

[0037] (3) Preparation of low-concentration compound growth inhibitor: Weigh 1 g / L glucose, 70 g / L D-xylose, 0.5 g / L vanillin, 1 g / L 5-hydroxymethylfurfural (HMF), and 6 g / L levulinic acid. Dissolve in an appropriate amount of distilled water, adjust the pH to 6.0, stir thoroughly to dissolve, and then filter sterilize using a 0.22 μm aqueous microporous membrane to obtain a sterile low-concentration compound growth inhibitor. Store at 4 ℃ in the dark for later use, and let stand at room temperature for 30 min before use.

[0038] (4) YPD medium preparation: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose. For solid medium, 20 g / L agar should also be added. After dissolving in distilled water, autoclave at 115 °C for 30 min and cool to 50-55 °C for later use.

[0039] (5) Preparation of screening medium for compound growth inhibitors: Under aseptic conditions, YPD solid basal medium cooled to 50~55 ℃ is mixed with the optimal concentration of compound growth inhibitors at a certain volume ratio (2:1~10:1), and immediately poured into a sterile petri dish. After solidification, the medium is obtained.

[0040] Example 2: Establishment of a Highly Efficient Screening Method for Compound Growth Inhibitors

[0041] (1) Determination of the proportion of the screening medium for compound growth inhibitors

[0042] One indicator of the screening method described in this invention is that the starting strain *Rhodotorula glutinis* Y cannot grow on a plate after mixing YPD solid medium with the optimal concentration of the compound growth inhibitor in a certain ratio, thus achieving a highly efficient screening effect. To explore the optimal ratio, different screening plates were prepared by mixing YPD solid medium with the optimal concentration of the compound growth inhibitor at volume ratios of 2:1, 4:1, 6:1, 8:1, and 10:1. A single colony was picked from a YPD solid medium plate containing the cultured starting strain Y and resuspended in 1 mL of sterile water. 100 μL of the bacterial suspension was then spread onto screening media with different ratios of compound growth inhibitors. The plates were incubated at 28 ℃ for 72 h, and colony growth was observed periodically. Figure 1 It can be seen that when YPD solid culture medium is mixed with the optimal concentration of growth inhibitor at a volume ratio of 6:1, the starting strain *Rhodotorula buergerianum* Y does not grow at all, thus achieving the effect of inhibiting the growth of the starting strain.

[0043] (2) High-efficiency primary screening of Rhodotorula circophylla Y space-induced mutant strain library using compound growth inhibitor screening medium.

[0044] Weigh 1.3 mg of *Rhodotorula glutinis* Y-type lyophilized bacterial powder, which was induced by spaceborne mutagenesis aboard the Shijian-19 satellite, and place it in a 1.5 mL centrifuge tube. Add 1 mL of sterile YPD liquid culture medium to dissolve the powder, and let it stand at room temperature for 10 min. Then, perform serial dilutions of the bacterial suspension. -1 10 -2 10 -3 Take 100 μL of bacterial suspension from each centrifuge tube at diluted concentration and spread it onto YPD solid medium ( Figure 2 A) Low-concentration compound growth inhibitor screening medium ( Figure 2 B) Optimal concentration of growth inhibitor screening medium ( Figure 2 C) and high-concentration compound growth inhibitor screening medium ( Figure 2 In step D), each dilution concentration was performed in triplicate, and the plates were then incubated in a 28 °C incubator for 72 h.

[0045] By comparing the four types of solid culture plates (Table 1), the number of colonies on the optimal concentration of compound growth inhibitor screening medium was significantly lower than that on YPD solid culture medium. The high concentration of compound growth inhibitor screening medium completely inhibited the growth of colonies, while the number of colonies on the low concentration of compound growth inhibitor screening medium was not significantly reduced compared with YPD solid culture medium, indicating poor screening effect.

[0046] To achieve good bacterial screening results, this invention selects... Figure 2The optimal concentration of the growth inhibitor screening medium shown in Figure C was used for the initial screening of *Rhodotorula turcica* Y strain induced by space travel. The initial screening procedure was as follows: 100 μL of the *Rhodotorula turcica* Y suspension after space travel mutation was added dropwise to a plate containing the optimal concentration of the growth inhibitor screening medium and spread evenly. The plate was then incubated at 28 ℃ for 72 h. Single colonies of the *Rhodotorula turcica* mutant strain after space travel mutation were compared with colonies of the original strain Y, and mutant strains with larger diameters and darker colors than the original strain Y were selected.

[0047] Table 1. Colony count of Rhodotorula glutinis Y space-mutated freeze-dried bacterial powder on different individual culture medium plates.

[0048]

[0049] Example 3: Shake-flask fermentation and secondary screening of high-yield carotenoid mutant strains

[0050] The *Rhodotorula glutinis* mutant strains obtained from the initial screening using the compound growth inhibitor medium in Example 2 were subjected to shake-flask secondary screening: 5 mL of YPD liquid medium was added to a test tube, and the initially screened mutant strains were inoculated and cultured at 28 °C and 220 r / min for 24 h. Then, at a 10% (v / v) inoculation rate, each mutant strain was inoculated into a 250 mL shake flask containing 50 mL of fermentation medium and fermented at 28 °C and 220 r / min for 168 h. After fermentation, the carotenoid content of each mutant strain was measured, and high-carotenoid-producing *Rhodotorula glutinis* mutant strains were screened.

[0051] Figure 3 The shake flask carotenoid production of 10 tested strains (including the original strain Y and mutant strains Y1 to Y9 that are darker or similar in color to the original strain Y) was compared. Among them, the mutant strain Y-5 had the highest carotenoid production of 10.78 mg / L, which was 57% higher than that of the original strain Y under the same conditions.

[0052] The components of YPD liquid culture medium and shake flask fermentation medium are as follows:

[0053] (1) YPD liquid culture medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L.

[0054] (2) Shake flask fermentation medium: glucose 50 g / L, yeast extract 0.75 g / L, ammonium sulfate 0.1 g / L, potassium dihydrogen phosphate 0.4 g / L, magnesium sulfate heptahydrate 1.5 g / L, adjust pH to 6.0.

[0055] Example 4: Validation of the screening effect of compound growth inhibitor culture medium

[0056] (1) Plate screening and shake-flask fermentation yield verification of compound growth inhibitors

[0057] Strains Y and mutants Y1-Y9 from Example 3 were inoculated into YPD solid medium ( Figure 4 A) and the optimal concentration of growth inhibitor screening medium ( Figure 4 B) Observe the growth of each strain. It can be seen that in the selection medium with the optimal concentration of the compound growth inhibitor, the starting strain Y, and the mutant strains Y-1, Y-2, Y-7 and Y-8 do not grow, while the mutant strain Y-5 has the best growth.

[0058] Combination Figure 3 and Figure 4 The results show that the screening of mutant strains with good growth using the optimal concentration of compound growth inhibitors is highly consistent with the carotenoid yield measurement results. This screening medium can inhibit the growth of mutant strains with similar carotenoid yield to the starting strain and can be used for efficient screening of high-yield carotenoid mutant strain libraries.

[0059] (2) Screening of compound growth inhibitors on plates and verification of fermentation yield in 24-well plates

[0060] Single colonies of *Rhodotorula buergerianum* mutant strains that showed good growth and dark color on YPD solid medium and a selection medium containing a compound growth inhibitor were inoculated into 24-well plates containing 3 mL of fermentation medium and cultured at 28 ℃ and 220 r / min for 168 h. The results after culture were as follows: Figure 5 As shown, A①②③④ represent single colonies of different mutant strains with significant color differences selected from YPD solid medium, while B①②③④ represent single colonies of different mutant strains with significant color differences selected from screening medium containing the optimal concentration of growth inhibitor. After fermentation, the carotenoid yield of each mutant strain in a 24-well plate was measured. The starting strain Y produced 2.5 mg / L of carotenoids during fermentation in a 24-well plate. Tables 2 and 3 show the carotenoid yield, positive mutation rate, average carotenoid yield, and highest carotenoid yield of single colonies of mutant strains screened on different solid media after fermentation in a 24-well plate, respectively. As shown in Table 3, the positive mutation rate of the mutant strains obtained by initial screening using the compound growth inhibitor screening medium was 49.1%, which was much higher than the positive mutation rate of the mutant strains obtained by screening by colony color on YPD solid medium alone (19.8%). The average and highest carotenoid yields of the mutant strains initially screened using the compound growth inhibitor screening medium were 3.82 mg / L and 8.63 mg / L, respectively, which were also higher than the average (1.96 mg / L) and highest (7.18 mg / L) carotenoid yields of the mutant strains initially screened using YPD solid medium.

[0061] Table 2. Carotenoid yield (mg / L) of mutant strains screened on different culture media using 24-well plate fermentation.

[0062]

[0063]

[0064] Table 3. Screening effect of different culture media on space-mutated Rhodotorula buergeriana mutant strains

[0065]

[0066] Example 5: Production of carotenoids from high-yield carotenoid mutant strain Y-5 via fed-batch fermentation in a fermenter.

[0067] (1) Seed culture preparation: single colonies of the well-cultured starting strain Rhodotorula buergerianum Y and the high-carotenoid mutant strain Y-5 were picked with a sterile inoculation loop and inoculated into test tubes containing 5 mL of YPD liquid medium. The culture was carried out at 28 ℃ and 220 r / min for 24 h to obtain the primary seed culture. The primary seed culture was inoculated into YPD liquid medium at an inoculation rate of 10% (v / v) and cultured at 28 ℃ and 220 r / min for 24 h to obtain the secondary seed culture.

[0068] (2) Production of carotenoids by batch feeding fermentation in a 5 L fermenter

[0069] 1.6 L of fermentation medium was added to a 5 L fermenter and sterilized at 115 °C for 30 min. Glucose from the fermentation medium was sterilized separately at 115 °C for 30 min before being added to the fermenter. The cultured secondary seed culture was inoculated into the 5 L fermenter at a rate of 10% (v / v), and the initial pH was adjusted to 6.0. During fermentation, 24% ammonia solution was automatically added to maintain the pH at around 6.0 to promote cell growth. Simultaneously, glucose (800 g / L) mother liquor was added to maintain the glucose concentration at 10-20 g / L to promote product formation. During fermentation, the temperature was maintained at 28 °C, the aeration rate was 4 L / min, and the dissolved oxygen was maintained at 30% by a coupled rotation speed. Figure 6 The figure shows the carotenoid yield and cell concentration (OD) of the starting strain Y and the high-carotenoid mutant strain Y-5 in a 5 L feed-feed fermenter. 600Process curves. The cell concentration of the high-carotenoid-producing mutant strain Y-5 in the fermenter was consistently slightly higher than that of the original strain Y. The carotenoid yield of the original strain Y was 18.6 mg / L after 168 h of fermentation and 19.2 mg / L after 192 h; the carotenoid yield of the high-carotenoid-producing mutant strain Y-5 was 46.9 mg / L after 168 h and 48.7 mg / L after 192 h, both more than 2.5 times that of the original strain Y, significantly increasing carotenoid yield and reducing production costs.

[0070] Example 6: Method for Extraction and Determination of Carotenoids

[0071] 1. Sample pretreatment:

[0072] Take 3 mL of culture medium into a 5 mL centrifuge tube, centrifuge at 8000 r / min for 10 min to collect the bacterial cells, and wash the bacterial cells three times with distilled water.

[0073] 2. Carotenoid extraction:

[0074] Add 3 mL of 3 mol / L hydrochloric acid to the washed bacterial cells, vortex to mix, let stand at room temperature for 50 min, boil in a water bath for 5 min, then cool rapidly on ice, centrifuge at 8000 r / min for 10 min and discard the supernatant. Add 4 mL of acetone to the bacterial precipitate, vortex to mix, and fully dissolve the carotenoids to obtain the acetone extract (this process should be carried out in the dark).

[0075] 3. Determination of carotenoid yield (ultraviolet spectrophotometry): After appropriately diluting the acetone extract with acetone, the absorbance value was measured at a wavelength of 475 nm using a spectrophotometer. The yield was calculated using the following formula:

[0076] Carotenoid production (mg / L) = Aλmax × D × V / 0.16 × V0

[0077] Parameter in the formula: A λmax : Absorbance value of the extracted carotenoid at the maximum absorption wavelength (475 nm); V: Amount of acetone used to extract carotenoid (L); D: Dilution factor when measuring the absorbance of the extract; V0: Total volume of fermentation broth used to extract carotenoid (L); 0.16: Molecular extinction coefficient of carotenoid.

[0078] Example 7: Drying method for carotenoids

[0079] Carotenoid extraction was performed on the high-yield carotenoid mutant strain Y-5 using the fermentation broth according to the carotenoid extraction method described in Example 6 (the extraction must be performed in the dark). The acetone extract was transferred to a glass petri dish and allowed to evaporate naturally in a fume hood. After the acetone had completely evaporated, a dark red, viscous carotenoid concentrate remained in the petri dish. This concentrate was then left to stand in a fume hood for ≥12 h, followed by drying in a 30 ℃ vacuum drying oven for 48 h. The dried concentrate solid was collected. The solid was thoroughly ground in a mortar and pestle and then sieved through a 120-mesh sieve to obtain the carotenoid solid powder from the high-yield carotenoid mutant strain Y-5. Figure 7 For high-yield carotenoid mutant strain Y-5 carotenoid ( Figure 7 A) β-carotene standard (HPLC 96%) Figure 7 B) and lycopene standard (HPLC 98%) Figure 7 C) Appearance comparison diagram. The results show that the carotenoid solid powder of the high-yield carotenoid mutant strain Y-5 is red, which is consistent with the color of the carotenoid extract, but differs from the color of the β-carotene and lycopene standards.

[0080] Example 8: Qualitative analysis of fermentation products from high-yield carotenoid mutant strain Y-5

[0081] The high-yield carotenoid mutant strain Y-5 was extracted with chloroform and then qualitatively analyzed using a colorimetric method (Wang Jie, Journal of Xihua University (Natural Science Edition), 2022, 41(04):82-89+97) and a full-wavelength scanning method using ultraviolet spectrophotometer (Aburai, Journal of Biotechnology, 2018, Vol.268:6-11). Based on the structural characteristics of the conjugated polyene chain of carotenoids, their reaction with concentrated sulfuric acid is mainly protonation, accompanied by secondary reactions such as isomerization and degradation, and the intermediate products will show characteristic color changes. After dissolving β-carotene and lycopene standards in chloroform, 200 μL of concentrated sulfuric acid was added dropwise to the chloroform extract of the high-yield carotenoid mutant strain Y-5, the chloroform solution of β-carotene standard, and the chloroform solution of lycopene standard, respectively, and the color changes were observed. Figure 8 These are the chloroform extracts of the high-carotenoid mutant strain Y-5 ( Figure 8 A) β-carotene standard chloroform solution ( Figure 8 B) and lycopene standard chloroform solution ( Figure 8 The color (①) of C) and its qualitative test results (②). The chloroform extract of the high-yield carotenoid mutant strain Y-5 ( Figure 8 A①) reacts with concentrated sulfuric acid ( Figure 8 The color change of A②) and the reaction of β-carotene standard with concentrated sulfuric acid ( Figure 8 B②) and the reaction of lycopene standard with concentrated sulfuric acid ( Figure 8 The color change characteristics of C②) are basically consistent, indicating that it belongs to carotenoids. Based on the characteristic absorption peak characteristics of carotenoids in the 400-600 nm range of acetone, a full-wavelength scan analysis was performed on the target sample, and the results are as follows: Figure 9 As shown: Acetone extract of high-carotenoid-producing mutant strain Y-5 ( Figure 9 A) It exhibits the maximum absorption wavelength at 493 nm; Figure 9 The maximum absorption wavelength of Torularhodin is 500 nm (Mussagy CU, ACS Sustainable Chemistry & Engineering, 2019, 7(19):16765-16776). Figure 9 The maximum absorption wavelength of torulene is 480 nm (Mussagy CU, ACSSustainable Chemistry & Engineering, 2019, 7(19): 16765-16776). By comparing the absorption spectral characteristics, the absorption peak of the acetone extract of the high-carotenoid-producing mutant strain Y-5 simultaneously covers the absorption peak ranges of both torulene and erythromycin, and is closer to the absorption peak characteristics of erythromycin, suggesting that it contains a mixture of both. Figure 8 The characteristic colorimetric reaction results in, and Figure 9 The spectral curves of the extract of the high-yield carotenoid mutant strain Y-5 are highly consistent with those of the red yeast erythrin standard, suggesting that the pigment produced by the spaceborne Rhodotorula buergerianum mutant strain Y-5 belongs to the carotenoid class of substances, and that the red yeast erythrin content is relatively high.

[0082] In summary, the compound growth inhibitors prepared in this invention can efficiently screen high-carotenoid-producing space-induced mutant strains. The high-carotenoid-producing *Rhodotorula glutinis* space-induced mutant strains obtained using this method can be used for fermentation production of carotenoids and have broad application prospects.

Claims

1. A method for screening space-mutated strains of *Rhodotorula glutinis* that produce high levels of carotenoids, characterized in that... Includes the following steps: (1) Prepare a compound growth inhibitor screening medium for screening mutant strains of Rhodotorula circinata after space mutagenesis; The screening medium for the compound growth inhibitor was prepared by mixing YPD solid medium and the compound growth inhibitor at a volume ratio of 6:

1. The YPD solid culture medium components and preparation include: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 20 g / L agar, sterilized at 115 ℃ for 30 min; The compound growth inhibitor components and preparation method include: glucose 1~10 g / L, D-xylose 70~100 g / L, vanillin 0.5~3 g / L, 5-hydroxymethylfurfural (HMF) 1~4 g / L, levulinic acid 6~10 g / L, dissolved in distilled water, pH adjusted to 6.0, and filtered through a 0.22μm filter membrane for sterilization; (2) Activation of strains: Take the freeze-dried powder of Rhodotorula rubra after space mutagenesis, dissolve it in sterile YPD liquid medium and let it stand at room temperature for 10 min to activate it; (3) Initial screening: The activated bacterial solution of (2) was serially diluted, and 100 μL was spread on the compound growth inhibitor screening medium and incubated at 28℃ for 72 h. Single colonies of mutant strains with darker colony color and larger colony diameter were selected from the compound growth inhibitor screening medium. (4) Shake flask fermentation and screening: The mutant strain of Rhodotorula circophylla obtained in the initial screening in (3) was inoculated into test tubes containing 5mLYPD liquid medium and cultured at 28 ℃ and 220 r / min for 24 h. Then, it was inoculated into shake flasks containing fermentation medium at an inoculation rate of 10% (v / v) and cultured at 28 ℃ and 220 r / min for 168 h. After the fermentation was completed, carotenoids were extracted and determined, and high-producing Rhodotorula circophylla mutant strains were screened to obtain the results. The fermentation medium consists of: 50 g / L glucose, 0.75 g / L yeast extract, 0.1 g / L ammonium sulfate, 0.4 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, and pH adjusted to 6.

0.

2. The method for screening high-carotenoid-producing Rhizopus cylindrica space-mutated strains according to claim 1, characterized in that, The aforementioned method for extracting carotenoids is the acid-heat method.

3. The method for screening high-carotenoid-producing Rhizopus cylindrica space-mutated strains according to claim 1, characterized in that, The method for determining carotenoids is ultraviolet spectrophotometry.

4. A space-mutated strain of *Rhodotorula glutinis* that produces high levels of carotenoids, characterized in that... Original starting strain: Rhodotorula buergerianum ( Rhodosporidium toruloides Purchased from the China General Microbiological Culture Collection Center, accession number CGMCC 2.1389.

5. A space-mutated strain of *Rhodotorula glutinis* that produces high levels of carotenoids, characterized in that... The mutation method involved space-based mutation induced by aboard the Shijian-19 recoverable technology experimental satellite.

6. A space-mutated strain of *Rhodotorula glutinis* with high carotenoid production, characterized in that, The strain was obtained by screening using the strain screening method described in claim 1.

7. The application of the high-carotenoid-producing *Rhodotorula glutinis* space-mutated strain as described in claim 6 in carotenoid fermentation production, characterized in that... The fermentation production medium is a fermentation medium. The glucose concentration is controlled at 10~20 g / L by adding glucose (800 g / L) mother liquor, the dissolved oxygen is controlled at 30%, the fermentation temperature is 28 ℃, the culture time is 168~192 h, and the pH is maintained at 6.0.

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