A Schizochytrium strain producing astaxanthin and DHA and its application

Abstract

The present invention discloses a Schizochytrium strain for producing astaxanthin and DHA and its application. The Schizochytrium strain is Schizochytrium AST32-IEIZW-12, which is classified and named as: Schizochytrium Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. AST32‑IEIZW‑12 is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee with the deposit number: CGMCC No.41544, the deposit date: October 11, 2024, and the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The present invention realizes the combined production of astaxanthin and DHA in the genus Thraustocystis, and the DHA yield can reach 2.8 g / L, and the astaxanthin yield can reach 19.58 mg / L (2.04 mg / g).

Description

Technical Field

[0001] The invention belongs to the technical field of schizochytrium genetic engineering and relates to a schizochytrium strain for producing astaxanthin and DHA and application thereof. Background Art

[0002] Astaxanthin is a natural carotenoid widely found in nature and the marine environment. It is also a high-value carotenoid derivative pigment with a deep red color. Astaxanthin has a high antioxidant property and can effectively inhibit lipid peroxidation caused by free radicals. At the same time, it has many physiological effects such as inhibiting tumor occurrence, enhancing immunity, and removing free radicals in the body. It has a good therapeutic effect on skin cancer and also has a preventive and therapeutic effect on eye diseases caused by diabetes. It has broad application prospects in health products, medicine, cosmetics, food additives, and aquaculture.

[0003] The sources of astaxanthin are mainly chemical synthesis, waste from shrimps and crabs and their processed waste, algae and red Phaffia yeast. Chemically synthesized astaxanthin has a different configuration from natural astaxanthin, and its antioxidant capacity is only 1 / 3 of that of natural astaxanthin. The astaxanthin content of shrimps and crabs and their processed waste is low, and the waste itself is easy to corrupt and is not conducive to transportation and processing. Haematococcus pluvialis and red Phaffia yeast are currently important strains for the industrial production of astaxanthin. Haematococcus pluvialis is a commonly used astaxanthin production strain in the market. Its production of astaxanthin is affected by many environmental factors, such as light intensity, temperature, pH, salt concentration, nutrients and various plant hormones and their derivatives. Although red Phaffia yeast can be produced at high density in a fermentation tank, its astaxanthin antioxidant activity is poor and is usually used as a pigment.

[0004] Schizochytrium sp. is a marine eukaryotic microalgae rich in fatty acids. Its oil content can reach 50% to 60% of the dry weight of the fungus. Docosahexaenoic acid (DHA) accounts for 20% to 40% of the total oil content, and there are small amounts of α-linolenic acid and docosapentaenoic acid (EPA). Schizochytrium sp. also has a natural astaxanthin synthesis pathway, which is relatively long and requires the enhancement of a series of genes. In addition, there are few gene editing tools in Schizochytrium sp., the transformation time is long, and there is less resistance. Summary of the invention

[0005] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide a Schizochytrium strain that produces astaxanthin and DHA.

[0007] Another object of the present invention is to provide the use of the Schizochytrium strain in producing astaxanthin and / or DHA.

[0008] To this end, the technical solution provided by the present invention is:

[0009] A Schizochytrium strain producing astaxanthin and DHA, wherein the Schizochytrium strain is Schizochytrium AST32-IEIZW-12, and the classification name of the Schizochytrium AST32-IEIZW-12 strain is: Schizochytrium Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. AST32-IEIZW-12 is deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short), with the deposit number: CGMCC No.41544, the deposit date: October 11, 2024, and the address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The method for producing astaxanthin and DHA comprises the following steps:

[0011] The Schizochytrium AST32-IEIZW-12 strain is cultured in a liquid culture medium, fermented cells are obtained after the culture, and astaxanthin is obtained from the fermented cells.

[0012] Preferably, in the method for high production of astaxanthin and DHA, the culture temperature is 26-30°C.

[0013] Preferably, in the method for high production of astaxanthin and DHA, the culture temperature is 28°C.

[0014] Preferably, in the method for high production of astaxanthin and DHA, the culture time is 5 to 9 days.

[0015] Preferably, in the method for high production of astaxanthin and DHA, the culture time is 7 days.

[0016] Preferably, in the method for high production of astaxanthin and DHA, the liquid culture medium comprises the following components: 80 g / L glucose, 5 g / L yeast extract, 0.3 g / L sodium chloride, 1 g / L potassium sulfate, 0.1 g / L potassium dihydrogen phosphate, 4 g / L crystalline magnesium sulfate and 0.05 g / L calcium chloride.

[0017] Preferably, in the method for high-yield astaxanthin and DHA, DHA is also harvested from the fermented bacteria.

[0018] The application of the Schizochytrium strain in producing astaxanthin and / or DHA.

[0019] The present invention has at least the following beneficial effects:

[0020] The present invention discloses a method for transforming the genus Thraustochytrium by combining rational and irrational engineering strategies in Schizochytrium. First, the precursor supply of the MVA pathway is strengthened through the irrational transformation strategy of electroshock mutagenesis, thereby achieving the purpose of activating the endogenous MVA pathway of the genus Thraustochytrium. Secondly, the effect of strengthening the endogenous MVA flux of the genus Thraustochytrium is achieved through the rational transformation method of overexpression. It also achieves the combined production of astaxanthin and DHA in the genus Thraustochytrium, wherein the DHA yield can reach 2.8 g / L, and the astaxanthin yield can reach 19.58 mg / L (2.04 mg / g).

[0021] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Schizochytrium AST32-IEIZW-12, the classification name of the Schizochytrium AST32-IEIZW-12 strain: Schizochytrium Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. AST32-IEIZW-12 is deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short), with the deposit number: CGMCC No.41544, the deposit date: October 11, 2024, and the address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] FIG. 1 is a diagram of the plasmid construction process in an embodiment of the present invention.

[0024] Figure 2 Graph showing astaxanthin production and biomass of the engineered strain HTT28 and the enhanced strain of a single gene in the astaxanthin biosynthesis pathway in the examples of the present invention.

[0025] Figure 3 This is a color diagram of the engineered strain HTT28 and the enhanced strain of a single gene in the astaxanthin synthesis pathway in the embodiment of the present invention, wherein the diagram shows: 1. mutant strain HTT28; 2. engineered strain HTT28-HMGS; 3. engineered strain HTT28-HMGR; 4. engineered strain HTT28-IDI; 5. engineered strain HTT28-CrtE; 6. engineered strain HTT28-CrtIBY; 7. engineered strain HTT28-CrtW; 8. engineered strain HTT28-CrtZ.

[0026] Figure 4 To enhance theIDI, CrtE, CrtIBY Astaxanthin production and biomass of the genetic strains, and color chart of the initial screening of the strains.

[0027] Figure 5 To enhance the IDI, CrtE, CrtIBY, CrtW, CrtZ Astaxanthin production and biomass chart, and strain screening color chart.

[0028] Figure 6 To enhance the IDI, CrtE, CrtIBY Astaxanthin production and biomass diagram, and strain screening color diagram.

[0029] Figure 7 To enhance the IDI, CrtE, CrtIBY, CrtW, CrtZ Astaxanthin production and biomass chart, and strain screening color chart.

[0030] Figure 8 In the embodiment of the present invention, in the engineering strain HTT28-IEI-3, overexpression CrtW, CrtZ, CrtW Astaxanthin production and biomass chart, and strain screening color chart.

[0031] Figure 9 This is the composition of the oil components in the engineered strain AST32-IEI-3-WZW in the embodiment of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0033] It should be understood that terms such as “having”, “including” and “comprising” used herein do not specify the existence or addition of one or more other elements or combinations thereof.

[0034] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0035] The production characteristics of strains can be improved to a certain extent by mutagenesis and screening of strains through physical or chemical means, but because it is random, it is difficult to achieve precise regulation. Metabolic engineering of strains through genetic manipulation has uncertainties in metabolic regulation. If strains with improved fermentation characteristics are first screened through mutagenesis and then exogenous genes are further introduced on this basis, the efficiency of strain transformation can be significantly improved.

[0036] The present invention uses Schizochytrium GCD2032 as a wild chassis, and uses an electric shock mutagenesis method to screen a mutant strain HTT28, whose astaxanthin content is improved; on this basis, after multiple rounds of gene conversion screening, the strain AST32-IEIZW-12 with the highest astaxanthin content is obtained, wherein during the transformation process, a comparison is made with Schizochytrium GCD2032 and HTT28, and it is found that the astaxanthin content of the chassis of Schizochytrium HTT28 is the highest, which shows that the chassis obtained after mutagenesis is very suitable for producing astaxanthin. This is the core of the present invention.

[0037] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following embodiments are provided for illustration:

[0038] 1. Materials and Methods

[0039] 1.1 Culture medium

[0040] (1) Seed medium: glucose (30 g / L), yeast extract (8 g / L), artificial sea crystal (20 g / L).

[0041] (2) Solid culture medium (PU): glucose (30 g / L), yeast extract (8 g / L), artificial sea crystal (20 g / L), and agar powder (2 g / L).

[0042] (3) Fermentation medium (Fer): glucose (80 g / L), yeast extract (5 g / L), sodium chloride (0.3 g / L), potassium sulfate (1 g / L), potassium dihydrogen phosphate (0.1 g / L), crystalline magnesium sulfate (4 g / L), and calcium chloride (0.05 g / L).

[0043] The above culture media were sterilized by high pressure steam at 115°C for 20 minutes.

[0044] 1.2 Electroporation mutagenesis and screening

[0045] The original wild mushroom is a strain of Schizochytrium Schizochytrium sp. GCD2032 was stored in the Industrial Biosystems Engineering Laboratory of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. Preparation of competent cells: The stored bacterial solution was inoculated into Seeds medium and incubated at 28°C, 180 r / min -1 Cultivate for about 18 h; collect the culture solution from the seed shake bottle and shake at 4000 r·min -1 Centrifuge for 8 min, discard the supernatant, take the precipitate, wash the precipitate with sterile water, and then -1 Centrifuge for 8 min, discard the supernatant, wash again, wash the precipitate with 1 M sorbitol solution, and then spin at 4000 r·min-1 Centrifuge for 8 min, discard the supernatant, wash again; add 500 μL 1 M sorbitol solution to suspend the cells in the solution and place on ice for 2 min.

[0046] The prepared competent cells were transferred into the electrode instrument cup, placed in the electric shock device, and shocked with high voltage. 1 mL of incubation culture medium was added to the competent cell cup after electric shock, and incubated for 5 h. The incubated cells were spread on the screening medium and cultured at 28°C for 2 days. After mutagenesis screening, the strain was obtained. Schizochytrium sp. HTT28.

[0047] 1.3 Culture conditions

[0048] Culture conditions of Schizochytrium 200 μL of the preserved strain was taken from the -80℃ ultra-low temperature freezer on the clean bench and inoculated into a 250 mL conical flask containing 50 mL of seed culture medium. The conical flask was placed in a constant temperature shaker at 28℃, the speed was set to 180 rpm, and cultured continuously for 19-25 h until the OD600 reached nm 0.5~0.6. Schizochytrium sp. GCD2032 seed liquid was inoculated into the fermentation medium at a ratio of 8% and fermented for 7 days.

[0049] 1.4 Plasmid construction

[0050] Escherichia coli DH5α was used for cloning and plasmid propagation. Schizochytrium sp. Hydroxymethylglutaryl-CoA synthase ( HMGS )、3-hydroxy-3-methylglutaryl-CoA reductase ( HMGR )、isopentenyl diphosphate isomerase; FDPPS( IDI )、Geranylgeranyl pyrophosphate synthase;( CrtE) , β-carotene synthase ( CrtIBY ) and β-carotene hydroxylase ( CrtZ ) 6 genes were amplified from Haematococcus pluvialis to β-carotene ketolase ( CrtW )(GFH09688.1) gene. Gene primers were amplified from the strain genome by PCR amplification as shown in Tables 1 to 3.

[0051] The pGEM plasmid containing the actin promoter PE, GUS protein marker gene and G418 resistance gene constructed in this experiment was used as the initial plasmid. Using the reserved EcoRI restriction enzyme cutting site, the elongation factor promoter PA and the terminator cyc were combined using 2X MultiF Seamless Assembly Mix (ABclonal) to construct the vector pGEM-PA-(KpnI)cyc-PE-GUS-G418. We used 2A peptide to connect to construct a gene expression vector containing the above five genes (CrtIBY, CrtE, IDI, CrtW and CrtZ). First, E2A and P2A were used to connect IDI, CrtE, and CrtIBY to form pGEM-IEI, and P2A was used to connect CrtZ and CrtW to form pGEM-ZW. Primers were used to amplify pGEM-IEI and pGEM-ZW to obtain two fragments. They were connected together to obtain the expression vector pGEM-IEIZW. See the plasmid construction process. Figure 1 PCR primer synthesis and sequencing services were provided by Suzhou Genewise Biotechnology Co., Ltd. The specific primers are shown in Tables 1 to 3.

[0052] E2A amino acid sequence (SEQ ID NO: 1) GSGQTCNYALLKLAGDVESNPGP

[0053] P2A amino acid sequence (SEQ ID NO: 2) GSGATNFSLLKQAGDVEENPGP

[0054] 1.5 Biolistic transformation of Schizochytrium

[0055] The constructed vector was transformed into Schizochytrium using particle bombardment. First, 30 ml of Schizochytrium Schizochytrium sp.The culture was centrifuged at 7000 r / min for 5 min, washed twice with 20 ml of sterile water, and centrifuged at 7000 r / min for 5 min. The bacteria were resuspended in 1 ml of sterile water, and 200 μL of the bacterial solution was spread on a PU plate and air-dried to fix. Microparticle preparation: 5 mg 0.6 µm Gold Microcarriers (Bio-Rad, USA), 4~6 μg plasmid (c.1~2μg / μl), 20μL 0.1 M spermidine and 50 μL 2.5 M CaCl2. The mixture was then washed twice with 200 μl of anhydrous ethanol and centrifuged at 6000 g to obtain DNA-coated gold powder microparticle carriers. Next, the gold powder microparticle carriers were resuspended in 70 μL of ethanol as bullets for particle bombardment. About 7 μL of the solution containing DNA-coated gold microcarriers was spread on the microparticle carrier disk and introduced into the cells using the Biolistic PDS-1000 / He system (Bio-Rad, USA) with parameters set to 1100 psi pressure and 6 cm target distance. After bombardment, the plate was incubated at 28 °C for 16-24 hours. Then, the cells were spread on PU plates containing 150 mg / L G418 and incubated at 28 °C for 5-7 days. Transformants were initially screened by staining using a GUS staining kit (Coolaber, SL7160). To further confirm the transformation, the color change of the bacteria was observed by using an optical microscope.

[0056] The screened colonies were cultured on Seeds plates supplemented with G418 for at least three generations and verified using a GUS staining kit and PCR amplification of marker genes.

[0057] 1.6 Determination of biomass and lipid content

[0058] The biomass was determined by dry cell weight (DCW). First, 30 mL of fermentation broth was added to a 50 mL centrifuge tube and centrifuged at 6000 rpm and 4°C for 10 minutes, and the supernatant was discarded; then the cells were washed twice with distilled water and collected by centrifugation. The centrifuged sample was placed in a -80°C ultra-low temperature refrigerator for overnight freezing, and then dried in a freeze dryer until the cell weight was constant; after freeze drying, the cell weight was weighed to calculate the cell dry weight.

[0059] The extraction of oil is firstly carried out by acid-heat breaking, and then by n-hexane extraction. The specific method is as follows: first, weigh 0.1-0.2 g of freeze-dried bacterial powder and add it to an anaerobic bottle, add 5 ml of distilled water and 5 ml of concentrated hydrochloric acid to the anaerobic bottle and mix well, then place it in a 75℃ constant temperature water bath and heat it for 2 hours. During this period, shake and mix it every once in a while to make the cell wall breaking effect sufficient; cool it to room temperature, add 10 ml of analytical pure n-hexane to the anaerobic bottle, shake and mix it, and then let it stand for 1 hour. Use a separatory funnel to collect the upper organic phase in a test tube, and continue to extract the lower liquid. Repeat the operation 3 times until the upper organic phase is colorless. Collect all the upper organic phases into a test tube, use a nitrogen blower to heat at 65℃ and use nitrogen protection to volatilize the n-hexane. When the test tube is at a constant weight, calculate the percentage of bacterial oil content.

[0060] 1.7 Fatty acid derivatization

[0061] The fatty acids in lipids were converted into fatty acid methyl esters by methyl esterification. The specific method is as follows: First, according to the weight of oil in the test tube blown to constant weight by nitrogen in the previous step, 0.5M KOH / methanol solution (0.05 g oil: 1 ml KOH / methanol solution) was added in proportion, mixed thoroughly, and placed in a constant temperature water bath at 60°C for saponification for 15 minutes, shaking the test tube several times during the period. After the reaction, the test tube was cooled to room temperature, 2 times the volume of 14% boron trifluoride / methanol solution was added to the test tube, mixed evenly, and placed in a constant temperature water bath at 60°C for esterification for 2 minutes. After cooling to room temperature, 2 ml of chromatographically pure alcohol was added, mixed thoroughly, and then 2 ml of saturated sodium chloride solution was added, allowed to stand for 2 minutes, and a small amount of anhydrous sodium sulfate was added to remove moisture. Take the supernatant, filter impurities with an organic filter membrane, and put it into a gas phase vial for the next analysis.

[0062] 1.8 Analysis of fatty acid composition and content

[0063] The qualitative analysis of fatty acid composition was performed by gas chromatography-mass spectrometry (Agilent 7200Q-TOF GC / MS) to detect all fatty acid components in Schizochytrium lipids. In subsequent experiments, gas chromatography was used for quantitative analysis, and the fatty acid composition and content of Schizochytrium were identified and calculated by referring to the peak time of Supelco® 37 fatty acid methyl ester component mixed standard or fatty acid methyl ester standards such as DHA methyl ester.

[0064] Fatty acid composition and content analysis was performed by gas chromatography. A gas chromatograph equipped with an sp-2560 column (100mx0.25mmx0.20μm) and a flame ionization detector was used for analysis by comparing the confidentiality time of the standard. The instrument setting parameters were as follows: the temperatures of the injector port and the flame ionization port were set to 250℃ and 260℃, respectively. The oven of the gas chromatograph was initially set at 150℃, maintained for 5 min, then increased to 180℃ at a rate of 3℃ / min, maintained for 5 min, and then gradually increased to 240℃ at a rate of 8℃ / min and maintained for 16 min. High-purity nitrogen was used as the carrier gas, the split ratio was 30:1, and the injection volume was 1 µl.

[0065] 1.9 Astaxanthin extraction and content detection

[0066] Weigh 50 mg to 100 mg of dried bacteria accurately, place in a dry mortar, add 2 mL of dichloromethane-methanol solution (1:3) and grind thoroughly to break the cell wall, transfer to a 50 mL centrifuge tube, wash the mortar twice with 3 mL of dichloromethane-methanol solution, and combine the extracts. Ultrasonic extraction for 10 min, centrifuge at 8500 r / min for 5 min at 4°C, transfer the supernatant to a 50 mL volumetric flask, add 5 mL of dichloromethane-methanol solution to the centrifuge tube, repeat the above steps for more than 3 times until the extracted bacterial residue is white, combine the supernatants, and dilute to 50 mL with dichloromethane-methanol solution.

[0067] Use ultraviolet spectrophotometer to detect the content of astaxanthin in Schizochytrium. For the preparation of standard solution, weigh 1 mg of all-trans astaxanthin standard, dilute to 25 ml with dichloromethane-methanol solution, and prepare 40 µg / mL stock solution. Dilute with dichloromethane-methanol solution to concentrations of 0.1 µg / mL, 0.5 µg / ml, 1.0 µg / mL, 2.0 µg / ml, 5.0 µg / ml, and 10.0 µg / ml standard working solution, measure the absorbance of the solution at a wavelength of 480 nm to draw a standard curve, and prepare it for use immediately. Accurately pipette 1 mL of sample solution into a 1 mL quartz cuvette, and measure the absorbance of the solution at a wavelength of 480 nm.

[0068] result

[0069] 2.1 Mutation screening and evaluation of bacterial strains

[0070] Preparation of competent cells: The preserved bacterial liquid was inoculated into the seed culture medium and cultured to OD600nm (0.5~0.6), the bacteria were collected by centrifugation, the precipitate was washed with 20 mL 1 M sorbitol solution, the bacteria were collected by centrifugation, and the cells were suspended in the solution using 500 μL 1 M sorbitol solution, and placed on ice for 2 min. The competent cells were placed in an electroporation cup, and the electric shock was performed three times with a voltage of 2000 V and 3 ms. The bacteria were first spread on a 60 μm / L bleomycin PU plate for initial screening, and then subcultured on the bleomycin PU plate for 5 times. The single bacteria that can continue to grow were transferred to a solid culture medium (PU) plate containing 340 μm / L iodoacetic acid and subcultured for 5 times. The last surviving strain was evaluated by shake flask fermentation to detect the astaxanthin content.

[0071] Finally, the high-yield astaxanthin strain was obtained and the original strain Schizochytrium sp Compared with GCD2032, its astaxanthin content increased by 4 times, from 7.5 μg / L to 30 μg / L. The strain was named Schizochytrium Schizochytrium sp . HTT28.

[0072] The present invention obtains mutants by electroporation mutation method Schizochytrium sp. The astaxanthin production of HTT28 reached 1.2 mg / L, and the DHA production was 3.46 g / L.

[0073] 2.2 Single gene screening of astaxanthin pathway

[0074] Transformants obtained by gene gun transformation have different intracellular astaxanthin content due to the influence of the insertion site. It is necessary to isolate high-yield strains from a large number of microalgae, spread the screened microalgae cells on solid Seeds plates to form colonies, and then pick out orange single colonies from the solid plates. In a 24-well plate filled with seed culture medium, by comparing the ratio of ODnm480 to ODnm600, select strains with higher ratios and transfer them to Seeds shake flask culture medium to culture until OD600 is between 0.5 and 0.6 to maintain bacteria.

[0075] from Schizochytrium The genomes of the genus were predicted by comparing them with the annotated genes of related strains. hmg-s , hmgr , idi , crtE , crtIBY and crtZ and CrtW from Haematococcus pluvialis were introduced into Schizochytrium sp . In HTT28. In HTT28-CrtIBY-2, 3.47 mg / L (390 μg / g) of astaxanthin was obtained. By comparing the effects of enhancing the key enzymes of the mevalonate pathway and carotenoids on theSchizochytrium sp The effects of astaxanthin in HTT28 were screened out and ranked as follows: CrtIBY>CrtE>IDI>CrtW>CrtZ>HMGR>HMGS ( Figure 2 ). Figure 3 Indicates the color of the engineered bacteria after expression.

[0076] 2.3 In Schizochytrium sp. Enhanced carotenoid pathway in GCD2032

[0077] The plasmid containing IDI, CrtE, and CrtIBY was transferred into Schizochytrium sp. The carotenoid metabolic pathway in GCD2032 is enhanced to β-carotene. The accumulation of β-carotene affects the endogenous β-carotene carboxylase and ketolase, converting β-carotene into astaxanthin. Finally, GCD2032-EI-6 was screened and the highest astaxanthin yield of 8.4 mg / L (326.87 μg / g) was obtained. Figure 4 ).

[0078] 2.4 Schizochytrium sp. Enhanced astaxanthin pathway in GCD2032

[0079] The plasmid containing IDI, CrtE, CrtIBY, CrtW, and CrtZ genes was transferred into Schizochytrium Schizochytrium sp. The carotenoid metabolic pathway in GCD2032 was enhanced to astaxanthin. The highest astaxanthin yield of 8.6 mg / L (346.4 μg / g) was obtained in GCD2032-IEIZW-12 ( Figure 5 ).

[0080] 2.5 Enhancement of the carotenoid pathway in the mutant strain HTT28

[0081] The plasmid containing IDI, CrtE, and CrtIBY genes was transferred into Schizochytrium sp. HTT28, AST32-IEI-3 was screened to obtain astaxanthin up to 15.2 mg / L (1555.2 μg / g) Figure 6 ).

[0082] 2.6 Enhancement of astaxanthin pathway in mutant strains.

[0083] The plasmid containing IDI, CrtE, CrtIBY, CrtW, and CrtZ genes was transferred into Schizochytrium Schizochytrium sp. HTT28, AST32-IEIZW-12 was obtained through screening, and the astaxanthin production was 13.2 mg / L (1701.3 μg / g) ( Figure 7). After 5 rounds of fermentation stability evaluation, the astaxanthin production of the strain was 19.58 mg / L (2040 μg / g), of which the DHA production was 2.8 g / L.

[0084] The strain was sent to the General Microbiological Center of China Microbiological Culture Collection Administration for preservation. After identification, the classification name of Schizochytrium AST32-IEIZW-12 strain was: Schizochytrium Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. AST32-IEIZW-12 is deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short), with the deposit number: CGMCC No.41544, the deposit date: October 11, 2024, and the address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0085] 2.7 Increase the expression of β-carotene ketoluase and carboxylase

[0086] Based on the AST32-IEI-3 strain, CrtW and CrtZ were overexpressed. The plasmid pGEM-WZW containing two CrtW and one CrtZ was transferred into the AST32-IEI-3 strain, and the astaxanthin production increased from 15.2 mg / L to 19.01 mg / L (1999 μg / g) ( Figure 8 ). Figure 9 The lipid composition of the AST32-IEI-3 strain is shown.

[0087] The present invention discloses a Schizochytrium strain with high astaxanthin production and a construction method thereof. The present invention combines rational and irrational engineering strategies to transform the Thraustochytrium genus. The study first uses an irrational transformation strategy of electroporation mutagenesis to strengthen the precursor supply of the MVA pathway, thereby achieving the purpose of activating the endogenous MVA pathway of the Thraustochytrium genus. Secondly, through a rational transformation method of overexpression, the effect of strengthening the endogenous MVA flux of the Thraustochytrium genus is achieved. First, the electroporation mutagenesis method is used to screen a Schizochytrium mutant that produces astaxanthin by itself, and the mutant is used as a chassis to load the mutant with IDI , crtE , crtIBY , crtw , crtZThe fragments of the gene are transferred into the strain by gene gun method, the strain is inoculated into the fermentation medium, the fermentation broth is harvested, and the astaxanthin in the broth is extracted. The present invention first obtains a mutant that produces astaxanthin by electric shock mutagenesis, and on this basis, the astaxanthin synthesis gene is transformed into the mutant to obtain a high-yield astaxanthin Schizochytrium. This method combines random mutation with genetic transformation, which significantly improves its astaxanthin production. At the same time, the obtained astaxanthin contains a large amount of DHA, and compared with traditional microalgae or yeast, the nutritional composition is richer. The yield of astaxanthin can reach 19.58 mg / L (2 mg / g).

[0088] Table 1 Primers for major genes

[0089]

[0090] Table 2 Primers for major genes

[0091]

[0092] Table 3 Primers for major genes

[0093]

[0094] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A Schizochytrium strain producing astaxanthin and DHA, characterized in that: The Schizochytrium strain is Schizochytrium AST32-IEIZW-12, and the classification name of the Schizochytrium AST32-IEIZW-12 strain is: Schizochytrium sp. , strain Schizochytrium Schizochytrium sp. AST32-IEIZW-12 is deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short), with the deposit number: CGMCC No.41544, the deposit date: October 11, 2024, and the address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A method for producing astaxanthin and DHA, characterized in that: The steps include: The Schizochytrium AST32-IEIZW-12 strain as claimed in claim 1 is cultured in a liquid culture medium, fermentation cells are obtained after the culture, and astaxanthin is obtained from the fermentation cells.

3. The method for producing astaxanthin and DHA according to claim 2, characterized in that The temperature during cultivation is 26~30℃.

4. The method for producing astaxanthin and DHA according to claim 3, characterized in that The temperature during cultivation was 28°C.

5. The method for producing astaxanthin and DHA according to claim 2, characterized in that The culture time is 5 to 9 days.

6. The method for producing astaxanthin and DHA according to claim 5, characterized in that The culture time is 7 days.

7. The method for producing astaxanthin and DHA according to claim 2, characterized in that The liquid culture medium comprises the following components: 80 g / L glucose, 5 g / L yeast extract, 0.3 g / L sodium chloride, 1 g / L potassium sulfate, 0.1 g / L potassium dihydrogen phosphate, 4 g / L crystalline magnesium sulfate and 0.05 g / L calcium chloride.

8. The method for producing astaxanthin and DHA according to claim 2, characterized in that DHA is also harvested from the fermentation cells.

9. Use of the Schizochytrium strain according to claim 1 in producing astaxanthin and / or DHA.

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