Method for improving yield of astaxanthin in phaffia rhodozyma by using iron reductase gene
By overexpressing the iron reductase gene in Red Fav yeast, an efficient iron reductase gene overexpression vector was constructed, and the problem of low astaxanthin yield in Red Fav yeast was solved, achieving a significant improvement in astaxanthin yield and a balance of cell growth.
Patent Information
- Application Number
- CN202510595980.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-09
- Publication Date
- 2025-08-15
AI Technical Summary
In the prior art, red FAF yeast has problems in the astaxanthin synthesis process, which is low astaxanthin yield and is difficult to balance yeast cell growth and synthesis.
By overexpressing the iron reductase gene in red yeast cells, using overlap extension PCR or chemical synthesis to construct the iron reductase gene overexpression vector, and the genome was assisted by the CRISPR-Cas9 system to screen out positive transformants to obtain the high-yield astaxanthin-yielding Red yeast mutant.
The astaxanthin yield and cellular biomass of Red Favre yeast were significantly improved, and the astaxanthin yield increased by 96.2% to 147%, and a high-yield astaxanthin strain with stable hereditary traits were obtained.
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Figure CN120485322A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for increasing the astaxanthin production of Phaffia rhodozyma by overexpressing an iron reductase gene. Background Art
[0002] Astaxanthin is a carotenoid with a keto structure and a molecular formula of C 20 H 52 O4. Astaxanthin's antioxidant capacity is 550 times that of vitamin E and 10 times that of beta-carotene. It can cross cell membranes and the blood-brain barrier, effectively scavenging oxidative free radicals and thus protecting the brain. Astaxanthin can treat macular degeneration, with superior efficacy compared to lutein. It can also eliminate coagulation disorders caused by antiplatelet drugs. In the food industry, astaxanthin has immune-enhancing and anti-aging effects, making it a functional ingredient in health supplements. Due to its strong antioxidant properties, astaxanthin protects food nutrients from degradation and can be used as a food preservative, flavor enhancer, and quality enhancer. Furthermore, astaxanthin is a high-quality pigment. In aquaculture, astaxanthin, as a hormone, can promote fertilization of fish eggs, accelerate growth, increase the maturation rate of fish, and aid in the coloration of crustaceans.
[0003] Phaffia rhodozyma is a cold-resistant basidiomycete yeast. Astaxanthin is its primary carotenoid. Studies have found that fermentation of astaxanthin by P. rhodozyma offers advantages such as rapid growth, short fermentation cycles, and easy extraction. However, stress conditions that favor astaxanthin synthesis often affect yeast cell growth, resulting in low production of the intracellular product, astaxanthin. Therefore, balancing yeast cell growth and astaxanthin synthesis is crucial for increasing astaxanthin production in P. rhodozyma. Summary of the Invention
[0004] To solve the above technical problems, the present invention provides a method for increasing the astaxanthin production of Phaffia rhodozyma by utilizing an iron reductase gene, which can increase the astaxanthin production of Phaffia rhodozyma and balance yeast cell growth and astaxanthin synthesis to increase the astaxanthin production of Phaffia rhodozyma.
[0005] The solution adopted by the present invention to solve the above technical problems is:
[0006] 1. Application of an iron reductase gene in increasing the astaxanthin production of Phaffia rhodozyma.
[0007] 2. A method for increasing astaxanthin production in Phaffia rhodozyma using an iron reductase gene:
[0008] The method introduces a ferric reductase (FR) gene into Phaffia rhodozyma cells to express the ferric reductase gene alone, and then uses Phaffia rhodozyma to ferment and produce astaxanthin, thereby increasing yeast cell biomass and astaxanthin production, thereby increasing the astaxanthin yield, which is the astaxanthin content per liter of fermentation broth.
[0009] The method specifically comprises the steps of constructing an iron reductase gene overexpression vector by using overlap extension PCR (overlap-PCR) or gene synthesis or chemical synthesis, and introducing the vector into rhodozyma cells, screening positive transformants, i.e., rhodozyma cells into which the positive introduction is successful, and obtaining a rhodozyma iron reductase gene overexpressing strain; and then performing conventional fermentation treatment using the rhodozyma iron reductase gene overexpressing strain to produce astaxanthin.
[0010] The iron reductase gene overexpression vector is the iron reductase gene overexpression plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown or Pgdp-G418-Tgdp-Padh4-FR-Tact;
[0011] The 18sup, Pgdp, Tgdp, Padh4, Tact and 18sdown genes are SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 respectively; the G418 gene is SEQ ID No.7; and the iron reductase FR gene is SEQ ID No.9.
[0012] The iron reductase gene overexpression vector is prepared in the following manner:
[0013] S1. First, the 18sup, Pgdp, Tgdp, Padh4, ferroreductase FR, Tact, and 18sdown gene fragments were amplified by the first round of PCR using the rhodozyma genome as a template;
[0014] S2, then using plasmid pPIC9K as a template, a second round of PCR was performed to amplify the G418 resistance gene fragment;
[0015] S3, then using the gene fragments of 18sup, Pgdp, G418, Tgdp, Padh4, FR, Tact and 18sdown as templates, the gene fragments of the templates were connected in sequence by the third round of PCR to obtain the iron reductase gene overexpression plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown or Pgdp-G418-Tgdp-Padh4-FR-Tact as the iron reductase gene overexpression vector; the plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown is composed of 18sup, Pgdp, G418, Tgdp, Padh4, FR, Tact and 18sdown. h4, iron reductase FR, Tact and 18sdown gene fragments are sequentially connected in order in step S3; the plasmid Pgdp-G418-Tgdp-Padh4-FR-Tact is formed by sequentially connecting Pgdp, G418, Tgdp, Padh4, iron reductase FR, and Tact gene fragments in step S3.
[0016] The iron reductase gene overexpression vector can also be obtained by chemical synthesis, and the chemically synthesized FR gene overexpression plasmids 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown and Pgdp-G418-Tgdp-Padh4-FR-Tact are obtained.
[0017] In the iron reductase gene overexpression vector, the plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown is introduced and inserted into the 18SrDNA site of the rhodozyma genome, and the CRISPR-Cas9 system is used to assist in the insertion into the 18SrDNA site. The plasmid Pgdp-G418-Tgdp-Padh4-FR-Tact is introduced and randomly inserted into the rhodozyma genome.
[0018] There is no fixed site for random insertion, and the introduction method includes electroporation. In a specific implementation, the above-mentioned overexpression vector is introduced into the rhodozyma yeast cells by electroporation.
[0019] Specifically, the FR protein gene overexpression plasmids 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown and Pgdp-G418-Tgdp-Padh4-FR-Tact were inserted into the genomic 18SrDNA site and randomly inserted into the genome by electroporation into rhodozyma cells, and positive transformants were screened using G418 plates to obtain rhodozyma FR gene overexpression strains.
[0020] The G418 plate is a plate added with geneticin. The protein encoded by the G418 gene can degrade G418, so that cells can grow on G418.
[0021] The iron reductase gene overexpression plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown is inserted into the genome of the rhodozyma yeast cell using the improved Cas9-sgRNA.
[0022] According to the 18S rDNA sequence of the insertion site of the overexpression vector on the rhodozyma genome, the crRNA sequence was set, and the crRNA gene sequence was SEQ ID No. 8. In vitro sgRNA preparation and Cas9-sgRNA ribonucleoprotein (RNP) complex were used to form a gene overexpression strain, specifically:
[0023] D1, synthesize crRNA, the crRNA sequence is SEQ ID No.8, i.e. TTACCCGTTGAAA CCATGGT;
[0024] D2. Synthesize the Cas9-sgRNA ribonucleoprotein (RNP) complex; this is a guide protein in CRISPR technology that helps find the cutting site.
[0025] D3. Finally, the Cas9-sgRNA ribonucleoprotein complex and the plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown of the iron reductase gene overexpression vector were introduced into the genome of the rhodozyma cells. Positive transformants were screened using G418 plates. G418 is equivalent to an antibiotic culture medium for screening. The positive rhodozyma cells that can grow are regarded as positive transformants, thereby obtaining a rhodozyma iron reductase gene overexpression strain, that is, an iron reductase gene overexpression rhodozyma mutant.
[0026] Specifically, the present invention constructs or synthesizes a ferric reductase (FR) gene overexpression vector, transforms the gene overexpression vector into Phaffia rhodozyma cells, and screens the mutant of Phaffia rhodozyma overexpressing the ferric reductase gene using G418 plates.
[0027] The original strain of Phaffia rhodozyma and the gene overexpression mutant (gene overexpression strain into which the target plasmid has been introduced) were cultured.
[0028] More preferably, the overexpressing strain of Phaffia rhodozyma (i.e., mutant) after the gene is introduced is cultured, and the culture is divided into two stages of cultivation process, namely, seed culture and fermentation culture.
[0029] During the seed culture period, the rhodozyma overexpressing strain was first inoculated into the seed culture medium under the following culture conditions: culture temperature 22°C, shaker speed 300 rpm, and culture time 96 h;
[0030] During the fermentation culture period, Phaffia rhodozyma cells occupying 2% of the volume of the fermentation medium were extracted from the seed culture results and inoculated into the fermentation medium. The culture conditions were: culture temperature 22° C., shaker speed 300 rpm, and culture time 96 h.
[0031] More specifically, the seed culture medium composition during cultivation (g / L) is: 20g glucose, 10g yeast powder, 20g peptone, and 20g agar powder is added to the solid culture medium. The fermentation medium composition for shake flasks (g / L) is: 50g glucose, 10g maltose extract, 1g yeast powder, and 0.8g peptone. Culture medium sterilization conditions: autoclave at 115°C for 30min.
[0032] The mutant strain is cultured in the seed culture medium. At this stage, the strain is most likely to be activated and cultured to the logarithmic growth phase with the strongest growth potential.
[0033] According to an embodiment of the present invention, the method for extracting the produced astaxanthin is as follows: the fermentation broth obtained after fermentation culture is centrifuged at 8000rpm for 10min, the supernatant is removed, the bacteria are collected, and after washing twice with sterile water, the washed bacteria are placed in -80℃ and frozen for 12h, and then placed in a freeze dryer for freeze drying for 24h. The freeze-dried bacteria are added to dimethyl sulfoxide preheated to 60℃, shaken and evenly mixed, heated in a water bath at 50℃ for 5min to break the cell wall, 3ml of anhydrous ethanol is added for extraction for 20min, ultrasonicated in an ultrasonic cleaning machine for 10min, centrifuged at 8000rpm for 10min, the supernatant obtained by centrifugation is transferred to a new centrifuge tube, and centrifuged under the same conditions. The bacterial precipitate is repeatedly subjected to cell wall extraction with dimethyl sulfoxide preheated to 60℃ until the bacteria are white, and the extract is pooled to obtain astaxanthin.
[0034] The fermentation broth is a mixture of the bacterial strain and fermentation medium from the fermentation stage, which is the stage with the highest potential for astaxanthin production. The extract is an astaxanthin-containing solution extracted by cell wall breaking with dimethyl sulfoxide, excluding the bacterial cells.
[0035] According to an embodiment of the present invention, the extract was used to determine the astaxanthin content by high performance liquid chromatography, and the chromatographic elution conditions were as follows: chromatographic column: C 18 Chromatographic column; column temperature: 30℃; UV detection wavelength: 478nm; mobile phase: methanol:acetonitrile = 9:1 (V / V); injection volume: 10μL; flow rate: 1mL / min.
[0036] The present invention constructs a single-gene overexpression vector for ferroreductase (FR) based on genomic 18SrDNA site insertion, genomic random insertion, and CRISPR-Cas9-assisted genomic 18SrDNA site insertion. Through electroporation and G418 plate screening, single-gene overexpression mutants such as Phaffia rhodozyma FR (M4, M5, and M6) are obtained.
[0037] The beneficial effects of the present invention are:
[0038] The present invention increases the astaxanthin yield in Phaffia rhodozyma by overexpressing the iron reductase (FR) gene alone in the yeast. Mutants M4 to M6 of Phaffia rhodozyma that overexpress the FR gene achieve a biomass of 6.4 to 6.8 g / L, a 23.1% increase over the original strain, and astaxanthin production reaches 3.1 to 3.9 mg / L, a 96.2% to 147% increase over the original strain.
[0039] The present invention constructs a high-astaxanthin-producing Phaffia rhodozyma strain through gene overexpression technology, which has the characteristics of stable genetic traits and astaxanthin fermentation yield and is highly feasible. BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Figure 1 Comparison of the biomass, astaxanthin content, and yield of Phaffia rhodozyma after 96 h of culture for the iron reductase gene-overexpressing strain constructed in the present invention, i.e., a comparison of the biomass, astaxanthin content, and astaxanthin yield of mutant strains M4, M5, and M6 and the original strain (WT). DETAILED DESCRIPTION
[0041] The present invention will be further described below with reference to the accompanying drawings and specific implementations.
[0042] The embodiments of the present invention are as follows:
[0043] Example 1:
[0044] 1) Construction of Ferric Reductase (FR) Gene Overexpression Vector: Using the Phaffia rhodotorula genome as a template, PCR was used to amplify the 18sup, Pgdp, Tgdp, Padh4, ferric reductase (FR), Tact, and 18sdown gene fragments. The G418 gene fragment was amplified using the plasmid pPIC9K as a template. Using these fragments as templates, a second round of PCR was performed to ligate the fragments in a fixed sequence, generating the FR protein gene overexpression plasmids 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown. PCR conditions: 98°C for 3 minutes, 30 cycles of (98°C for 10 seconds, 55°C for 1 minute, and 72°C for 3 minutes), and 72°C for 10 minutes.
[0045] 2) Gene transformation and positive transformant screening: The plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown was transformed into Phaffia rhodozyma cells by electroporation, and positive transformants M4 were obtained by screening on G418 plates.
[0046] 3) Activation Culture: Remove positive transformant M4 strains from a glycerol stock in a -80°C freezer and aseptically inoculate onto fresh solid culture medium. Incubate at room temperature for 96 hours. Aseptically select well-growing colonies from the plate using an inoculating loop and inoculate into 50 mL of liquid culture medium. Incubate at 22°C, 300 rpm, and shake for 96 hours. Extract from the seed culture and inoculate 0.2% of the volume into 50 mL of fermentation medium. Incubate at 22°C, 300 rpm, and shake for 96 hours.
[0047] 4) Culture medium composition (g / L): 20g glucose, 10g yeast extract, 20g peptone, plus 20g agar powder for solid culture. Shake flask fermentation medium composition (g / L): 50g glucose, 10g maltose extract, 1g yeast extract, 0.8g peptone. Culture medium sterilization conditions: Autoclave at 115°C for 30min.
[0048] 5) Biomass Determination: The fermentation broth was centrifuged at 8000 rpm for 10 minutes. The supernatant was removed and the cells were collected. After washing twice with sterile water, the washed cells were frozen at -80°C for 12 hours and then freeze-dried in a freeze dryer for 24 hours. The total weight of the cell pellet and the centrifuge tube was weighed and subtracted from the previous centrifuge tube weight to obtain the cell weight and biomass.
[0049] 6) Extraction of astaxanthin: The freeze-dried bacteria were added to dimethyl sulfoxide preheated to 60°C, shaken and heated in a water bath at 50°C for 5 minutes to break the bacterial cell wall. 3 ml of anhydrous ethanol was added for extraction for 20 minutes. After ultrasonic cleaning for 10 minutes, the cells were centrifuged at 8000 rpm for 10 minutes. The supernatant obtained by centrifugation was transferred to a new centrifuge tube and centrifuged under the same conditions. The bacterial cell pellet was repeatedly subjected to cell wall breaking and extraction using dimethyl sulfoxide preheated to 60°C until the cells turned white. The extracts were then pooled.
[0050] 7) HPLC elution method is as follows: Chromatographic column: C 18 Chromatographic column; column temperature: 30℃; UV detection wavelength: 478nm; mobile phase: methanol:acetonitrile = 9:1 (V / V); injection volume: 10μL; flow rate: 1mL / min.
[0051] 8) Specific operation of HPLC determination: Take a sample, filter it through a 0.22μm organic filter membrane, and prepare for determination. Start the HPLC instrument and establish a new elution method according to the above elution conditions. Use pure water to wash the system for 30 minutes at a flow rate of 1mL / min. Use mobile phase to wash the system for 30 minutes at a flow rate of 1mL / min. Install C 18 Run the column and run the baseline. Inject the sample. Wash the system with pure methanol for 30 minutes at a flow rate of 1 mL / min to protect the column. Wash the system with pure water for 30 minutes at a flow rate of 1 mL / min.
[0052] Example 2:
[0053] 1) Construction of a Ferroreductase (FR) Gene Overexpression Vector: Using the Paffia rhodozyma genome as a template, PCR was used to amplify the Pgdp, Tgdp, Padh4, ferroreductase (FR), and Tact gene fragments. The G418 gene fragment was amplified using the plasmid pPIC9K as a template. Using these fragments as templates, a second round of PCR was performed to ligate the fragments in a fixed sequence to generate the FR protein gene overexpression plasmids Pgdp-G418-Tgdp-Padh4-FR-Tact. PCR conditions: 98°C for 3 minutes, 30 cycles of (98°C for 10 seconds, 55°C for 1 minute, and 72°C for 3 minutes), and 72°C for 10 minutes.
[0054] 2) Gene transformation and positive transformant screening: The plasmid Pgdp-G418-Tgdp-Padh4-FR-Tact was transformed into Phaffia rhodozyma cells by electroporation, and the positive transformant M5 was obtained by screening on G418 plates.
[0055] 3) Activation Culture: Remove positive transformant M5 strains from a glycerol stock in a -80°C freezer and aseptically inoculate onto fresh solid culture medium. Incubate at room temperature for 96 hours. Aseptically select well-growing colonies from the plate using an inoculating loop and inoculate into 50 mL of liquid culture medium. Incubate at 22°C, 300 rpm, and shake for 96 hours. Extract from the seed culture and inoculate 0.2% of the volume into 50 mL of fermentation medium. Incubate at 22°C, 300 rpm, and shake for 96 hours.
[0056] 4) Culture medium composition (g / L): 20g glucose, 10g yeast extract, 20g peptone, plus 20g agar powder for solid culture. Shake flask fermentation medium composition (g / L): 50g glucose, 10g maltose extract, 1g yeast extract, 0.8g peptone. Culture medium sterilization conditions: Autoclave at 115°C for 30min.
[0057] 5) Biomass Determination: The fermentation broth was centrifuged at 8000 rpm for 10 minutes. The supernatant was removed and the cells were collected. After washing twice with sterile water, the washed cells were frozen at -80°C for 12 hours and then freeze-dried in a freeze dryer for 24 hours. The total weight of the cell pellet and the centrifuge tube was weighed and subtracted from the previous centrifuge tube weight to obtain the cell weight and biomass.
[0058] 6) Extraction of astaxanthin: The freeze-dried bacteria were added to dimethyl sulfoxide preheated to 60°C, shaken and heated in a water bath at 50°C for 5 minutes to break the bacterial cell wall. 3 ml of anhydrous ethanol was added for extraction for 20 minutes. After ultrasonic cleaning for 10 minutes, the cells were centrifuged at 8000 rpm for 10 minutes. The supernatant obtained by centrifugation was transferred to a new centrifuge tube and centrifuged under the same conditions. The bacterial cell pellet was repeatedly subjected to cell wall breaking and extraction using dimethyl sulfoxide preheated to 60°C until the cells turned white. The extracts were then pooled.
[0059] 7) HPLC elution method is as follows: Chromatographic column: C 18 Chromatographic column; column temperature: 30℃; UV detection wavelength: 478nm; mobile phase: methanol:acetonitrile = 9:1 (V / V); injection volume: 10μL; flow rate: 1mL / min.
[0060] 8) HPLC analysis: Collect samples, filter through a 0.22 μm organic filter, and prepare for analysis. Start the HPLC instrument and establish a new elution method based on the above elution conditions. Wash the system with pure water for 30 minutes at a flow rate of 1 mL / min. Wash the system with mobile phase for 30 minutes at a flow rate of 1 mL / min. Install the C18 column and run a baseline. Inject the sample. Wash the system with pure methanol for 30 minutes at a flow rate of 1 mL / min to protect the column. Wash the system with pure water for 30 minutes at a flow rate of 1 mL / min.
[0061] Example 3:
[0062] 1) Construction of Ferroreductase (FR) Gene Overexpression Vectors: PCR amplified the 18sup, Pgdp, Tgdp, Padh4, Ferroreductase (FR), Tact, and 18sdown gene fragments using the Phaffia rhodozyma genome as a template. The G418 gene fragment was amplified using the plasmid pPIC9K as a template. Using these fragments as templates, a second round of PCR ligated these fragments in a fixed sequence to generate the FR protein gene overexpression plasmids 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown and Pgdp-G418-Tgdp-Padh4-FR-Tact, respectively. PCR conditions: 98°C for 3 minutes, 30 cycles of (98°C for 10 seconds, 55°C for 1 minute, and 72°C for 3 minutes), and 72°C for 10 minutes.
[0063] 2) sgRNA design and Cas9-sgRNA ribonucleoprotein (RNP) complex assembly: Based on the 18S rDNA sequence of the insertion site of the overexpression vector on the rhodozyma genome, the crRNA sequence TT ACCCGTTGAAACCATGGT was designed, and sgRNA was prepared in vitro and the Cas9-sgRNA ribonucleoprotein (RNP) complex was formed.
[0064] 3) Gene transformation and positive transformant screening: The plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown and the Cas9-sgRNA ribonucleoprotein (RNP) complex were transformed into Phaffia rhodozyma cells by electroporation, and positive transformant M6 was obtained by screening on G418 plates.
[0065] 4) Activation Culture: Remove positive transformant M6 strains from a glycerol stock in a -80°C freezer and aseptically inoculate onto fresh solid culture medium. Incubate at room temperature for 96 hours. Aseptically select well-growing colonies from the plates using an inoculating loop and inoculate into 50 mL of liquid culture medium. Incubate at 22°C, 300 rpm, and shake for 96 hours. Extract from the seed culture and inoculate 0.2% of the volume into 50 mL of fermentation medium. Incubate at 22°C, 300 rpm, and shake for 96 hours.
[0066] 5) Culture medium composition (g / L): 20g glucose, 10g yeast extract, 20g peptone, plus 20g agar powder for solid culture. Shake flask fermentation medium composition (g / L): 50g glucose, 10g maltose extract, 1g yeast extract, 0.8g peptone. Culture medium sterilization conditions: Autoclave at 115°C for 30min.
[0067] 6) Biomass Determination: The fermentation broth was centrifuged at 8000 rpm for 10 minutes. The supernatant was removed and the cells were collected. After washing twice with sterile water, the washed cells were frozen at -80°C for 12 hours and then freeze-dried in a freeze dryer for 24 hours. The total weight of the cell pellet and the centrifuge tube was weighed and subtracted from the previous centrifuge tube weight to obtain the cell weight and biomass.
[0068] 7) Extraction of astaxanthin: The freeze-dried bacteria were added to dimethyl sulfoxide preheated to 60°C, shaken and heated in a water bath at 50°C for 5 minutes to break the bacterial cell wall. 3 ml of anhydrous ethanol was added for extraction for 20 minutes. After ultrasonic cleaning for 10 minutes, the cells were centrifuged at 8000 rpm for 10 minutes. The supernatant obtained by centrifugation was transferred to a new centrifuge tube and centrifuged under the same conditions. The bacterial cell pellet was repeatedly subjected to cell wall breaking and extraction using dimethyl sulfoxide preheated to 60°C until the cells turned white. The extracts were then pooled.
[0069] 8) HPLC elution method is as follows: Chromatographic column: C 18 Chromatographic column; column temperature: 30℃; UV detection wavelength: 478nm; mobile phase: methanol:acetonitrile = 9:1 (V / V); injection volume: 10μL; flow rate: 1mL / min.
[0070] 9) Specific operation of HPLC determination: Take a sample, filter it through a 0.22μm organic filter membrane, and prepare for determination. Start the HPLC instrument and establish a new elution method according to the above elution conditions. Use pure water to wash the system for 30 minutes at a flow rate of 1mL / min. Use mobile phase to wash the system for 30 minutes at a flow rate of 1mL / min. Install C 18 Run the column and run the baseline. Inject the sample. Wash the system with pure methanol for 30 minutes at a flow rate of 1 mL / min to protect the column. Wash the system with pure water for 30 minutes at a flow rate of 1 mL / min.
[0071] Comparative Example 1:
[0072] 1) Activation Culture: Aseptically inoculate a refrigerated strain of Phaffia rhodozyma (WT) onto fresh solid culture medium and incubate at room temperature for 96 hours. Aseptically, use an inoculating loop to pick colonies from the plate and inoculate them into 50 mL of liquid culture medium. Incubate at 22°C, 300 rpm, and shake for 96 hours. Inoculate 0.2% of the inoculum into 50 mL of fermentation medium. Incubate at 22°C, 300 rpm, and shake for 96 hours.
[0073] 2) Culture medium composition (g / L): 20g glucose, 10g yeast extract, 20g peptone, plus 20g agar powder for solid culture. Shake flask fermentation medium composition (g / L): 50g glucose, 10g maltose extract, 1g yeast extract, 0.8g peptone. Culture medium sterilization conditions: Autoclave at 115°C for 30min.
[0074] 3) Biomass Determination: The fermentation broth was centrifuged at 8000 rpm for 10 minutes. The supernatant was removed and the cells were collected. After washing twice with sterile water, the washed cells were frozen at -80°C for 12 hours and then freeze-dried in a freeze dryer for 24 hours. The total weight of the cell pellet and the centrifuge tube was weighed and subtracted from the previous centrifuge tube weight to obtain the cell weight and biomass.
[0075] 4) Obtaining astaxanthin: The freeze-dried bacteria were added to dimethyl sulfoxide preheated to 60°C, shaken and heated in a water bath at 50°C for 5 minutes to break the bacterial cell wall. 3 ml of anhydrous ethanol was added for extraction for 20 minutes. After ultrasonic cleaning for 10 minutes, the supernatant was centrifuged at 8000 rpm for 10 minutes. The supernatant was transferred to a new centrifuge tube and centrifuged under the same conditions. The bacterial cell pellet was repeatedly extracted with dimethyl sulfoxide preheated to 60°C until the bacterial cells turned white, and the extracts were pooled.
[0076] 5) HPLC elution conditions are as follows: Chromatographic column: C 18 Chromatographic column; column temperature: 30℃; UV detection wavelength: 478nm; mobile phase: methanol:acetonitrile = 9:1 (V / V); injection volume: 10uL; flow rate: 1mL / min.
[0077] The extract of Comparative Example 1 was subjected to high performance liquid chromatography elution measurement, and the biomass of the original strain of Phaffia rhodozyma was obtained to be 5.2 g / L, the astaxanthin content was 0.3 mg / g DCW, and the astaxanthin yield was 1.58 mg / L.
[0078] like Figure 1As shown in the comparison, the extracts of each of the above examples were subjected to high performance liquid chromatography elution measurement, and the biomasses of the red Phaffia yeast mutant M4 of Example 1, the red Phaffia yeast mutant M5 of Example 2, and the red Phaffia yeast mutant M6 of Example 3 were 6.8 g / L, 6.4 g / L, and 6.6 g / L, respectively, which were 30.7%, 23%, and 26.9% higher than those of the original strains, respectively. The astaxanthin contents of M4, M5, and M6 reached 0.54 mg / g DCW, 0.58 mg / g DCW, and 0.59 mg / g DCW, increased by 80%, 93.3% and 96.7% respectively compared with the original strain; the astaxanthin production of M4, M5 and M6 reached 3.2 mg / L, 3.1 mg / L and 3.9 mg / L respectively, increased by 101%, 95% and 145% respectively compared with the original strain, indicating that FR protein can promote the growth and astaxanthin synthesis of red yeast cells under stress conditions, thereby significantly increasing the overall astaxanthin production.
[0079] The above specific embodiments are used to illustrate the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.
[0080] The above description is only a preferred embodiment of the present invention. Therefore, any equivalent changes or modifications made according to the structure, characteristics and principles described in the scope of the patent application of the present invention are included in the scope of the patent application of the present invention.
[0081] The gene sequence involved in the present invention is as follows:
[0082] SEQ ID No. 1;
[0083] Name: 18sup gene
[0084] DNA type: other DNA
[0085] Biological origin: Artificial sequence / synthetic construct
[0086] AGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTATAAACAAATTCATACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATAGTTTATTTGATGGTACCTTGCTACATGGATAACTGTGGTAATTCTAGAGCTAATACATGCCTAAAAGCCCCGACTTCTGGAAGGGGTGTATTTATTAGATAAAAAACCAACGCGTGAAAGCGCTCCCTTGGTGATTCATAATAACTTTTCGAATCGTATGGCCTTGCGCCGACGATGCTTCATTCAAATATCTGCCCTATCAACTTTCGATGGTAGGATAGAGGCCTACCATGGTTTCAACGGGTAACGGGGAATAAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATAACAATATAGGGCTCTTTGGGTCTTATAATTGGAATGAGTACAATTTAAATCCCTTAACGAGGAACCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAGTAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAACTTCAGGCCTGGTCGGCCGGTCCGCCTCACGGTGTGTACTGGTCGGCCGGGCCTTACCTCTTGGTGAGCCGTTCGGTCGTTTACGCGGCCGGGCGGGGAACCAGGATTTTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCTATGCCCGAATACATTAGCATGGAATAATAGAATAGGACGTGCGGTTCTATTTTGTTGGTTTCTAGGATCGCCGTAATGA
[0087] SEQ ID No.2;
[0088] Name: Pgdp gene
[0089] DNA type: other DNA
[0090] Biological source: Artificial Sequence / synthetic construct
[0091] CAAGTCGAGGGAACCCGAGAGAACGCGGTGGAAAAGGAGGCAAGGCACAAGTGGGCGAACCAGATCAGGCACGCCAGGGCCAGGCACGGCAAATAGAGTTTATCCAGCCCGAGAGCCCGTGGGTGGATGGGTTGTATTTGTGTGTATGTGTGTGGGTGCGTGCGTGCTAGAGAGTGTGTGTGTATACGCAGTGGGAGACAAGCAATCAAGCATGCTTACTAATCACACACACATGCAACACACACACACACACATTCTCTTCACCCTCCCACAGATGACTCGACCACCATCTGAAAGAACCAGCCAGAAGGACGGTGGTGGGTGCATGTATGTACGTGAGTGAGTGTGGGGAAGGCGAGTACGTGTGTGTACGCGCAAGGAAGAACAACGAAGCGCACGCTATGAGCAAGCACAACTGGGCACCGAACGAGAACAGTAACTGTCGGTATCTTCCCACCGACCGAGGCGTCTCCCGGCGGCAGCCGCCGGTGCCCCCCTCCGCTTACGTCAGCCACCCAGTTTTCTTCCATCTCTTTCTCTCTCCTTCCAAAAGTCTTTCAGTTTTAAACGGCCCCCAAAAAAAGAAGAGGCGACTTTTTCTTTCCTTCTCTCCCATCATCCACAAAGATCTCTCTTCTTCAACAACAACTACTACTACTACCACCACCACCACTACTTCTCTAACACTCTTACCATC
[0092] SEQ ID No.3;
[0093] Name: Tgdp gene
[0094] DNA type: other DNA
[0095] Biological source: Artificial Sequence / synthetic construct
[0096] ACGGTTCTCTCCAAACCCTCTCCCCTTTTGCCCTGCCCATTGAATTGATTCCCTAAATAGAATATCCCACCTTCTTTTATGCTCTACCTATGATCAGTTTATCTGTCTTTTTTTTCTTTTTGTGTGTGTCGGTCGTGCGACTGTACTCACCTCTCGAGGGACAAGGCAAGAAGTAAGCAAGACATGAACAAGAACAACAAAGAGAAAGAGAGAAAGAAAAAAAAAAGATAAAAAAAAAACAATCCCCCCCTCCCCACAAAAAAAAAGTCTCTATCTTAATCTGATCAAGAGATTACACCGCATCTTTAGAATTTCCCATCGCCTTCATCCTCGCCAGAGAAAAAAGTTTCAATCACATCTGTTGACCATCACCATCATCTCCGTCATCTCTCATGACTCCCTTGGCTGTGAATCGACCAGTCCATCAGCAGCCCTTCCA
[0097] SEQ ID No.4;
[0098] Name: Padh4 gene
[0099] DNA type: other DNA
[0100] Organism source: Artificial Sequence / synthetic construct
[0101] GGTAAAGAAAAGAAAGTCGACCGTGTAAGAACGGAGGATCCAAGAGAAGGCGACATTCTGAAGCAACCAGACGAGGAGATAGATGACTATGGATTCACAGAGTGTTAGGGTTAACGAACTCGATTAGTCAGCTTGATAACGCGCAGTAGCCTTCACGCGCAGCAGCCACGTCATTTCGGCCCGGCGCAAACGGATGCCGAGTGTTTTTTTCGGGCCGTTCGGAAGAGAGAGGAACAACGACGATGGCAGAGAAAACATCTTGGGATGGACAACTCTTGCTGTTCACGTCAGAGGCTGAGGACCAAGGGCTGGGTATTGGGGTTTGAGATTGACTATAGAGTGATAGTAATATCTGGAGATCAGAGCTGAGACTGATAAGACTGATGCTAACGAGCTGACGCTGATAGATTACCGGTGCTGCTGAAACTGCTATCCGGACTAACGAGGTGGAAGATGACGGAACTCTTTCTTCGCCGGCTGTTCCATCTTCTTCTTCGTCTTTCTCTCATTGCCTCGTTTTTCTTCCCATCGAGGGGCTCCTCAGAAACGTCCGGTTTCCGACATCTTCTGTCAATCTTTGTTCCAGTCTGTGGTAGGTGCTTGTGTGCTGATCCTTACGGCCTCTGTTCTTCCAGGGGTTTCCCGTGGGAAATCTGTCCGGGAAGGGATCCGATTGATTCCTGACAATGTCCTTTCTTTCGAGCGATAGGCAGAAGACTGGTTTATTTAATGTTTCCCTTTTCCGTCTGCTCGCTCACGGAGGATCCAACGTTCTTTCTCTTCCTCCACTTCCGTATCGTCCGGGGCAGATCGTCATCACAGAACGGAGACTGAAATGAACCATATAAAGCAGGTGGTGTCTCGATCTCGATCTCACTCTCCTTTCTTTCCCTCACCATACAAACATCCCATCCTATCAAGCCTCAGCATCACTCCTAGCTTACTCTCACAGAGATCCTATTCAACCAACCAACCAATAACACATCATCTGAACCCCACA。
[0102] SEQ ID No.5;
[0103] Name: Tact gene
[0104] DNA type: other DNA
[0105] Organism source: Artificial Sequence / synthetic construct
[0106] ATCAACAAAGTCTTTCTATCCTTTAAGGCAGGGCGGTTTCTCTTCTTACGATAGAGGCACCCTTGGCGGTCTTTCGCCAGCGGTGGTTTTCTCACTTTTTTTTCCAATATTTTCACCGACGTTTTTGGTCGCGTGTATGTTTTTCTCTACTTGATGCTATCAATATCATGGCTTTTACGTTCCGTTGTATCATCATCTTCTGCGCCTTTCTGACTGAGATATCGAAATCGTTACAAAGTAAGACGTATAAAAATGAGATTTATTCAATTCCTAAATGACGATACATCAGACGTAATAAATACATTCATAGACGGGCGCGAATGTTGATCGATCAAAGACTTTATTCGTATTTGCGACATTCTCCAAAACCCCAAGTCGAACTCTTTCCATCATCTATTTCTTCCCAAACTTCGAAACATCTACTCTCTTTCTCAAGCTCAGTGTCACCTCGAGAACGGATCTTCAATCTTTAGAGCGTGTCTTTTTCCGGTCTTAGTTTGAATTTGTTATCGTCATTCGATATAGATCTCCCTTGTTGTATTCCATCCTTCGAACGCGTTCTATACGTACCAACCCTCGACCATGCCGTCCGACCCATCCCATCGAGCCTCACGATCTGGTCCATCAGACAAGAACAGGTCGAATACAAACGTCTCATCAGCTCAGTCTTCGTCTCTGTTCTCCGGCTCAGCTTCAGCTCCGGCAGGT
[0107] SEQ ID No.6;
[0108] Name: 18sdown gene
[0109] DNA type: other DNA
[0110] Biological origin: Artificial sequence / synthetic construct
[0111] TTAATAGGGATAGTTGGGGGCATTAGTATTCAGTTGCTAGAGGTGAAATTCTTGGATTTACTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAGGTTAGGGGATCGAAAACGATCAGATACCGTTGTAGTCTTAACAGTAAACTATGCCGACTAGGGATCGGGCGATGTTCTCTTTTGACTCGCTCGGCACCTTACGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACAATATAAGGATTGACAGATTGATAGCTCTTTCTTGATTTATTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGGCCGGCTCTTGCTGGTCGCCGGCTTCTTAGAGGGACTATCAGCATCTAGCTGATGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGACAGAGCCAGCGAGTTTCTTTCCTTGGCCGAAAGTCTGGGTAATCTTGTGAAACTCTGTCGTGCTGGGGATAGAGCATTGCAATTCTTGCTCTTCAACGAGGAATTCCTAGTAAGCGCAAGTCATCAGCTTGCGTTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTTAGTGAGATCTCCGGATTGGCTTTGGGAAGCCGGCAACAGCCTATCGTCGAGAAGCTGCTCAAACTTGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTA
[0112] SEQ ID No.7;
[0113] Name: G418 gene
[0114] DNA Type: other DNA
[0115] Organism Source: Artificial Sequence / synthetic construct
[0116] ATGAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTATAA
[0117] SEQ ID No.8;
[0118] Name: crRNA sequence
[0119] RNA type: other RNA
[0120] Biological origin: Artificial sequence / synthetic construct
[0121] TTACCCGTTGAAACCATGGT
[0122] SEQ ID No.9;
[0123] Name: Ferroreductase gene FR
[0124] DNA type: other DNA
[0125] Biological origin: Artificial sequence / synthetic construct
[0126]
Claims
1. An application of an iron reductase gene, characterized in that: Application in improving astaxanthin production of Phaffia rhodozyma.
2. A method for increasing astaxanthin production in Phaffia rhodozyma using an iron reductase gene, characterized in that: The method introduces an iron reductase gene into a Phaffia rhodozyma cell to express the iron reductase gene alone, and then uses the Phaffia rhodozyma to perform fermentation treatment to produce astaxanthin, thereby increasing yeast cell biomass and astaxanthin yield.
3. The method for increasing astaxanthin production in Phaffia rhodozyma using an iron reductase gene according to claim 2, wherein: The method specifically comprises the following steps: constructing an iron reductase gene overexpression vector by using overlap extension PCR or gene synthesis or chemical synthesis, introducing the vector into rhodozyma cells, screening positive transformants, and obtaining a rhodozyma iron reductase gene overexpression strain; and then fermenting the rhodozyma iron reductase gene overexpression strain to produce astaxanthin.
4. The method for increasing astaxanthin production in Phaffia rhodozyma using an iron reductase gene according to claim 3, wherein: The iron reductase gene overexpression vector is the iron reductase gene overexpression plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown or Pgdp-G418-Tgdp-Padh4-FR-Tact; the 18sup, Pgdp, Tgdp, Padh4, Tact and 18sdown genes are SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 respectively, the G418 gene is SEQ ID No.7; and the iron reductase FR gene is SEQ ID No.
9.
5. The method for increasing astaxanthin production in Phaffia rhodozyma using an iron reductase gene according to claim 3 or 4, wherein: The iron reductase gene overexpression vector is prepared in the following manner: S1. First, the 18sup, Pgdp, Tgdp, Padh4, ferroreductase FR, Tact, and 18sdown gene fragments were amplified by the first round of PCR using the rhodozyma genome as a template; S2, then using plasmid pPIC9K as a template, a second round of PCR was performed to amplify the G418 resistance gene fragment; S3. Then, using the above gene fragments as templates, the gene fragments of the templates were connected in sequence by a third round of PCR to obtain the iron reductase gene overexpression plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown or Pgdp-G418-Tgdp-Padh4-FR-Tact as the iron reductase gene overexpression vector; The plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown is formed by sequentially connecting 18sup, Pgdp, G418, Tgdp, Padh4, ferroreductase FR, Tact and 18sdown gene fragments in step S3; the plasmid Pgdp-G418-Tgdp-Padh4-FR-Tact is formed by sequentially connecting Pgdp, G418, Tgdp, Padh4, ferroreductase FR and Tact gene fragments in step S3.
6. The method for increasing astaxanthin production in Phaffia rhodozyma using an iron reductase gene according to claim 4 or 5, wherein: The plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown is inserted into the 18SrDNA site of the rhodozyma genome, and the CRISPR-Cas9 system is used to assist in the insertion into the 18SrDNA site. The plasmid Pgdp-G418-Tgdp-Padh4-FR-Tact is randomly inserted into the rhodozyma genome.
7. A method for increasing astaxanthin production of Phaffia rhodozyma using an iron reductase gene according to any one of claims 2 to 4, characterized in that: The iron reductase gene overexpression plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown is inserted into the genome of the rhodozyma yeast cell using Cas9-sgRNA.
8. The method for increasing astaxanthin production in Phaffia rhodozyma using an iron reductase gene according to claim 7, wherein: According to the 18S rDNA sequence of the insertion site of the overexpression vector on the rhodozyma genome, the crRNA sequence was set, and the gene overexpression strain was formed by in vitro sgRNA preparation and Cas9-sgRNA ribonucleoprotein complex, specifically: D1, synthesize crRNA, the crRNA sequence is SEQ ID No.8; D2, synthesis of Cas9-sgRNA ribonucleoprotein complex; D3. Finally, the Cas9-sgRNA ribonucleoprotein complex and the plasmid 18sup-Pgdp-G418-Tgdp-Padh4-FR-Tact-18sdown of the iron reductase gene overexpression vector were co-introduced into the genome of the rhodozyma cells. Positive transformants were screened using G418 plates to obtain the rhodozyma iron reductase gene overexpression strain.
9. The method for increasing astaxanthin production in Phaffia rhodozyma using an iron reductase gene according to claim 7, wherein: The over-expressed Phaffia rhodozyma strain after the gene introduction is cultured. The cultivation process is divided into two stages: seed culture and fermentation culture. During the seed culture period, the rhodozyma overexpressing strain was first inoculated into the seed culture medium under the following culture conditions: culture temperature 22°C, shaker speed 300 rpm, and culture time 96 h; During the fermentation culture period, 2% of the volume of the fermentation medium was inoculated with Phaffia rhodozyma cells extracted from the seed culture results. The culture conditions were as follows: culture temperature 22° C., shaker speed 300 rpm, and culture time 96 h. The fermentation broth obtained after fermentation culture was centrifuged at 8000rpm for 10 minutes, the supernatant was removed, and the cells were collected. After washing twice with sterile water, the washed cells were placed in a -80℃ freezer for 12 hours and then freeze-dried in a freeze dryer for 24 hours. The freeze-dried cells were added to dimethyl sulfoxide preheated to 60℃, shaken and evenly mixed, and heated in a 50℃ water bath for 5 minutes to break the cell wall. 3ml of anhydrous ethanol was added for extraction for 20 minutes. After ultrasonic cleaning for 10 minutes, the cells were centrifuged at 8000rpm for 10 minutes. The supernatant obtained by centrifugation was transferred to a new centrifuge tube and centrifuged under the same conditions. The cell pellet was repeatedly extracted with dimethyl sulfoxide preheated to 60℃ until the cells turned white and the extracts were pooled.