CN115044474B

CN115044474B - Microalgae mutant with high tocopherol content, screening method and application thereof

Info

Publication number: CN115044474B
Application number: CN202210688627.5A
Authority: CN
Inventors: 苟元元; 杨璐; 郭建琦; 牛永洁; 孟永宏; 张佳
Original assignee: Shaanxi Healthful Biological Engineering Co ltd
Current assignee: Shaanxi Healthful Biological Engineering Co ltd
Priority date: 2022-06-17
Filing date: 2022-06-17
Publication date: 2023-05-16
Anticipated expiration: 2042-06-17
Also published as: CN115044474A

Abstract

The invention provides a chlorella aBST2-3, which is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 45167. The invention also provides an application of the chlorella aBST2-3 in the fermentation production of alpha-tocopherol. The invention carries out ARTP mutagenesis on microalgae twice, then compares the expression quantity of key genes in the synthesis process of alpha-tocopherol and determines the content of the alpha-tocopherol, initially screens mutant strains with higher alpha-tocopherol yield, screens mutant strains capable of being inherited stably, and finally screens mutant strains with high yield of alpha-tocopherol and capability of being inherited stably by comparing biomass, grease content, alpha-tocopherol content and sugar consumption conditions of the microalgae, wherein the highest yield of the mutant strains reaches 420.3 mug/gDCW, and the mutant strains are improved by 84% on the basis of original strains, thus the mutant strains are effective sources for preparing the alpha-tocopherol and have good alpha-tocopherol development prospects.

Description

Microalgae mutant with high tocopherol content, screening method and application thereof

[ field of technology ]

The invention relates to the technical field of microorganisms, in particular to a mutant strain of microalgae with high tocopherol content, application thereof and a method for screening and obtaining the mutant strain.

[ background Art ]

VE has high physiological activity, and natural VE is widely used as a nutritional supplement and an antioxidant in medicines, health products, foods, nutraceuticals and cosmetics, and is an indispensable substance in human life activities. In recent years, microalgae synthesis of VE has become a hotspot for research. VE is a generic term for tocopherols, where α -tocopherol is the most biologically active. The microalgae capable of accumulating tocopherol include spirulina, dunaliella, synechocystis, and Chlorella. The most widely studied species of algae are Euglena with tocopherol concentrations of 1.12-7.35 mg per gram of cells. The best tocopherol productivity of Euglena gracilis was found, and more than 97% of the synthesized tocopherols are alpha-type. However, the use of microalgae to synthesize VE is currently generally low, and subsequent studies have been made to increase total tocopherol content by expressing Homogentisate Phytyltransferase (HPT) and hydroxyphenylpyruvate dioxygenase (HPPD) in the synthesis precursor pathway.

The microalgae mutation breeding refers to a breeding technology for producing mutation of the genetic characters of algae by adopting various physical and chemical mutation methods, and obtaining the required excellent mutant algae strains through directional screening and cultivation. A great deal of literature reports at home and abroad that microalgae mutation breeding technology is used for successfully obtaining algae seeds with high active substance content, high oil yield, low temperature resistance, high temperature resistance and other excellent properties, and microalgae mutation breeding also becomes a main means for improving breeding efficiency and obtaining excellent microalgae germplasm. However, the research on mutagenesis and screening methods for improving the VE or alpha-tocopherol content of microalgae is less, a reference basis is lacking, and the types of microalgae which can be cultivated in a large scale are very limited, and the instability of the phenotype character can also influence the production of the microalgae, so that the process of the microalgae on VE industrialized production is accelerated by obtaining stable genetic microalgae with high tocopherol content through a mutagenesis technology and an effective screening method.

[ invention ]

The invention aims to provide a mutant strain with high alpha-tocopherol content and stable inheritance, which aims at the problems of low alpha-tocopherol content of microalgae and unstable inheritance of the mutant strain.

The invention is characterized in that ARTP mutagenesis is carried out on chlorella BST-2, mutant strains are screened through real-time fluorescent quantitative PCR, the expression quantity of 6 key genes in the synthetic pathway of tocopherol is compared, high-expression mutant strains are screened, the biomass, the grease content, the sugar consumption condition and the alpha-tocopherol content of the high-expression mutant strains in the fermentation process are compared after stability genetic analysis, and finally, a mutant strain which grows rapidly, has high grease content and high tocopherol content and can be inherited stably is screened out and used as a subsequent research.

Based on the above, the invention provides a chlorella aBST2-3, wherein the chlorella aBST2-3 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 5 months and 23 days in 2022, the preservation address is China national academy of sciences of China, having a preservation number of CGMCC No.45167.

The invention also provides application of the chlorella aBST2-3 in the fermentation production of alpha-tocopherol.

The invention also provides a chlorella culture medium, which comprises the following components:

the water was made up to 1000mL.

Further, the invention also provides a method for selecting microalgae with high tocopherol content by mutagenesis, which comprises the following steps:

(1) Taking microalgae, separating and purifying, and measuring the content of tocopherols in the microalgae strain and the expression quantity of key genes in the tocopherol synthesis pathway, wherein the microalgae strain with high expression quantity is taken as a starting microalgae strain;

(2) Carrying out ARTP mutagenesis on the algae strain obtained in the step (1), and picking up the living algae strain after mutagenesis;

(3) Determining the tocopherol content of the algae strain in the step (2) and the expression quantity of key genes in the tocopherol synthesis path, and selecting the algae strain with high tocopherol content and high expression quantity;

(4) Carrying out genetic stability analysis on the algae strain in the step (3), and selecting the genetically stable algae strain;

(5) Carrying out ARTP mutagenesis on the algae strain obtained in the step (4), and picking up the living algae strain after mutagenesis;

(6) Determining the tocopherol content of the algae strain in the step (5) and the expression level of key genes in the tocopherol synthesis path, and selecting the algae strain with high tocopherol content.

In the present invention, the microalgae is chlorella.

According to a preferred embodiment, step (1) comprises:

s1, collecting water samples in different water areas, and then separating and purifying algae strains;

s2, detecting the expression level of key genes in the tocopherol synthesis pathway in the algae strain, and performing primary screening of the algae strain with high tocopherol content;

s3, measuring biomass and grease content of the algae strains after primary screening, then measuring tocopherol content of microalgae with high grease content, and carrying out secondary screening of the algae strains with high tocopherol content.

According to a preferred embodiment, in step (1), the collected microalgae are cultivated in the chlorella medium at a temperature of 27℃and a pressure of 200r/min.

The ARTP mutagenesis treatment conditions in the steps (2) and (5) are that the output power of a power supply is 100W, the irradiation distance is 2mm, the temperature of plasma is less than 35 ℃, the air flow is 10L/min, and the irradiation time is 25s.

Key genes in the tocopherol synthesis pathway of steps (1), (3) and (6) include 4-hydroxyphenylpyruvate dioxygenase gene (HPPD), homogentisate Phytyltransferase (HPT), geranylgeranyl diphosphate reductase gene (GGH), 2-methyl-6-phytylbenzoquinone methyltransferase (MPBQMT), tocopherol cyclase gene (TC) and γ -tocopherol methyltransferase gene (γ -TMT).

According to a particularly preferred embodiment, the method further comprises step (7): and (3) measuring biomass, sugar consumption, oil content and alpha-tocopherol content in the fermentation process of the algae strain in the step (6), and screening mutant strains which grow rapidly, have high oil content and high alpha-tocopherol content.

The invention compares the expression quantity of key genes in the synthesis process of alpha-tocopherol by using real-time fluorescence quantitative PCR after carrying out twice ARTP mutagenesis on microalgae, simultaneously determines the content of the alpha-tocopherol, initially screens mutant strains with higher alpha-tocopherol yield, analyzes the genetic stability of the mutant strains, screens out mutant strains capable of being inherited stably, finally screens out a mutant strain with high alpha-tocopherol yield and capability of being inherited stably according to the variation of the mutant strains in the fermentation process, wherein the highest yield reaches 420.3 mug/gDCW, and the mutant strain is an effective source for preparing the alpha-tocopherol and has good alpha-tocopherol development prospect.

The present invention relates to the following microorganisms:

chlorella sp. ABST2-3, wherein the mutant strain aBST2-3 is preserved in China general microbiological culture Collection center (China center for type culture Collection) for 5 months and 23 days in 2022, and the preservation address is CGMCC No.45167, national institute of microorganisms, national academy of sciences of China, no. 3, north Chen West Lu 1, the Korean area of Beijing.

Chlorella sp BST-2 is preserved in China general microbiological culture Collection center (China center for type culture Collection) for 5 months and 23 days in 2022, and the preservation address is CGMCC No.45168, which is the national institute of microbiology, national academy of sciences No. 3, north Chen West Lu No. 1, the Korean region of Beijing.

[ description of the drawings ]

FIG. 1 is a graph showing the mortality curves of Chlorella BST-2 at different treatment times of ARTP.

FIG. 2 shows the results of real-time fluorescent quantitative PCR of 6 key genes of 8 mutants and original algae strains.

FIG. 3 is a comparison of the alpha-tocopherol content of 8 mutants and original algal strains.

FIG. 4 shows the real-time fluorescent quantitative PCR results of the 6 key genes of the rescreened 10 mutants and the original algal strains.

Figure 5 is a comparison of the alpha-tocopherol content of 10 mutants and the original strain.

FIG. 6 growth differences and content changes of two mutants from the original strain.

[ detailed description ] of the invention

The present invention is described in further detail below with reference to examples.

The invention relates to the following media:

chlorella medium:

the water is filled to 1000mL, and the preparation method refers to the BG-11 culture medium, with the difference that Na in the BG-11 culture medium ₂ CO ₃ The glucose was replaced with 15g/L.

In the invention, the steps of measuring the tocopherol content of microalgae are as follows:

(1) Preparation of the test sample: 1ml of the algal strain culture solution was centrifuged at 12000rpm for 5min in a 1.5ml centrifuge tube, and the supernatant was collected in a new centrifuge tube, and 40ul of glacial acetic acid was added thereto, followed by filtration through a 0.22 μm aqueous pinhole filter head.

(2) HPLC detection conditions: mobile phase: 0.01M KH ₂ PO ₄ Solution (a) and methanol (B); proportion: 90% A/10% B; flow rate: 0.8ml/min; detection wavelength: 290nm; chromatographic column: YMC-Pack ODS-AQ (4.6X1250 mm).

(3) The standard curve preparation method is as follows:

preparation of alpha-tocopherol standard curve: 10mg of alpha-tocopherol is weighed by a ten-thousandth balance, dissolved in 10ml of pure methanol solution, the concentration of the mother solution is set to be 1g/L, the mother solution is sequentially diluted by the pure methanol solution in a gradient way, the alpha-tocopherol solutions with the concentrations of 250mg/L,100mg/L,50mg/L,20mg/L and 5mg/L are respectively obtained by dilution, two batches of alpha-tocopherol solutions with the same concentration are released again by the same method, and all samples are filtered by a 0.22 mu m organic system pinhole filter head and then subjected to HPLC analysis.

In the invention, the method for calculating the qRT-PCR relative expression quantity of 6 key genes of microalgae comprises the following steps:

extracting microalgae RNA, performing reverse transcription to obtain cDNA, and then performing reverse transcription according to

qPCR/>

Green Master Mix (Without ROX) kit instructions primers were designed using Primer 5.0 software and GAPDH was selected as the reference gene for qRT-PCR analysis (see Table 1 for sequences of the gene primers). Real-time fluorescent quantitative PCR (qRT-PCR) in LineGene 9600Plus fluorescent quantitative PCR instrument(Bo Ri, hangzhou) on a substrate, and the quantitative reverse transcription step was performed according to PrimeScriptTM RT regent kit with gDNA Eraser instructions; fluorescent quantitative PCR procedure was followed +.>

qPCR

The description of the Green Master Mix (Without ROX) kit was performed. Three biological replicates and three technical replicates were performed for each sample. By 2 ^-ΔΔCt The method of (2) calculates the relative quantitative qRT-PCR expression quantity of the genes.

TABLE 1 Source and primer information for Key genes and internal reference genes in the alpha-tocopherol Synthesis pathway

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In the invention, microalgae biomass measurement is carried out by taking samples, centrifuging to obtain algae, and freeze-drying in a freeze dryer to obtain dry algae powder, wherein the dry algae powder is called as mass, and biomass=dry algae powder mass/algae liquid volume.

And (3) measuring the content of oil: extracting oil from the dry algae powder by chloroform-methanol method to obtain microalgae oil, drying at 50deg.C to constant mass, calculating oil ratio, performing 3 parallel tests, and taking average value. The mass fraction of fat and the total fat mass concentration were calculated according to the following formula:

oil mass fraction (%) = (ml/0.1) ×100

Total lipid mass concentration (g.L) ^-1 ) =microalgae algal mass concentration×oil mass fraction

Wherein m1 represents the mass (g) of oil extracted from 0.1g of algae powder; microalgae mass concentration (g.L) ^-1 ) The amount of microalgae harvested per unit volume of culture medium.

Sugar consumption assay: the method is described in the literature [ Li Jiantao ] by means of DNS colorimetry, the breeding and optimization research [ D ] university of Tianjin, 2019 ] of schizochytrium DHA high-yield strain based on ARTP mutagenesis.

Example 1 acquisition of algal strain BST-2

1. Collecting water sample in water area, separating and purifying algae strain

Water samples are respectively collected from natural water pools of Tianshan pond in Xinjiang, boston lake in Xinjiang, barilikun lake in inland, tornado lake, salt-yang lake in Shaanxi, artificial lake in wetland park in Fengqing park, artificial lake in Qinling mountain in Shaanxi, 1L of water sampler is adopted for floating microalgae, the benthic microalgae are scraped from objects such as stones and branches in water body by a brush, the collected water samples are stored by a transparent plastic bottle, and then the collected water samples are brought into a laboratory for separation and purification.

The collected water sample is separated and purified by adopting a plate streaking method, and a chlorella BST-2 culture medium is adopted for culture; and (3) coating 100 mu L of water sample into a culture medium for separation, respectively culturing for 20d at the temperature of (27+/-1), then picking up single algae, streaking and separating on a solid plate until single algae cells are obtained by microscopic examination of a water immersion tablet method, transferring the purified single algae cells into 50mL of chlorella BST-2 culture medium for heterotrophic culture, inoculating the logarithmic growth phase algae liquid into the chlorella culture medium with the inoculation amount of 10%, culturing at the temperature of (27+/-1) DEG C at 120r/min, and culturing in a shaking table in a dark place.

2. Primary screening of algal strains with high tocopherol content

And carrying out real-time fluorescent quantitative PCR on the water samples collected in 8 sampling places, comparing the expression quantity of key genes in 6 tocopherol synthesis routes in the water samples in 8 water areas, and preliminarily judging whether the water areas contain algae species capable of generating tocopherol.

As a result, 6 genes in Shaanxi Qinling natural pond and Wu Lun ancient lakes are low in expression level, and 6 genes in artificial lakes of Xinjiang Tianshan Tianchi, d wetland forest parks, salty yang lakes, fengqing park artificial lakes, xinjiang Bos Teng lake and Xinjiang Barilikun lake are high in expression level, so that the algae species possibly containing more high tocopherol content in the sampling places are primarily judged.

And then carrying out real-time fluorescence quantitative PCR on 6 key genes in the tocopherol synthesis pathway in 16 algae strains separated and purified in the water area, measuring the expression quantity of the key genes, comparing the expression quantity, and primarily screening out algae strains with high expression quantity of the key genes in the tocopherol synthesis pathway to obtain 10 algae strains with high expression quantity of the 6 key genes, wherein the expression quantity of 6 genes of the algae strains BST-2, BST-3, CB-2 and CB-3 is relatively high, and further determining that more tocopherols can be produced by the four algae strains.

3. Rescreening of algal strains with high tocopherol content

The obtained microalgae are dyed by a nile red dyeing method, the fluorescence intensity of the microalgae is measured to preliminarily screen grease-producing microalgae, and the result shows that the fluorescence values of algae strains BST-2, BST-3, CB-2, CB-3 and BLK-2 are relatively high, and the fluorescence intensity of 5 microalgae reaches over 70000 a.u. and shows that the grease content of the 5 microalgae is relatively high. And simultaneously, the biomass, the fat content and the alpha-tocopherol content are measured, and the microalgae with high fat production capacity and high alpha-tocopherol content are screened from the biomass, so that subsequent experiments are carried out. The biomass measuring method is carried out by a weighing method after centrifugal harvesting algae are frozen and dried. The method for measuring the content of the grease adopts a chloroform-methanol method to extract the grease in the dry algae powder, the obtained microalgae oil is dried to constant quality at 50 ℃, and the oil rate is calculated.

The determination result shows that the biomass of the algae strain BST-2 is maximum (0.93 g/L), the fat content is higher (33.85%), the fat yield is highest (0.315 g/L), and the alpha-tocopherol content is also highest, reaching 235.5ug/g DCW. Therefore, the strain BST-2 was used as a starting strain for subsequent studies.

EXAMPLE 2 preparation of Chlorella BST-2 high-tocopherol content mutant strains

1. Preparation of Chlorella BST-2 algae suspension

Inoculating Chlorella BST-2 into triangular flask containing 50mL Chlorella culture medium, culturing at 27deg.C under 200r/min to logarithmic phase, centrifuging 1mL culture solution, and suspending algae body with physiological saline to obtain suspension OD ₆₀₀ The value is 0.6-0.7.

2. ARTP treatment of Chlorella BST-2

Taking 10 mu L of algae suspension, uniformly coating on the upper surface of a metal slide, drying at room temperature for 5min, placing the metal slide carrying algae liquid into an operation bin of an ARTP mutagenesis system by using tweezers, adopting high-purity helium gas as working gas of plasma, setting the mutagenesis operation parameter to have 100W of power output, irradiating the algae slide with the irradiation distance of 2mm, and treating the algae slide with the plasma temperature of less than 35 ℃ and the air flow of 10L/min for 0 (control), 5, 10, 15, 20, 25, 30 and 40s. After the treatment, the slide glass is transferred into an EP tube containing 0.8mL of chlorella culture medium, vortex vibration elution is carried out to form new algae suspension, 200 mu L of algae liquid is respectively coated in a solid plate of the heterotrophic chlorella culture medium, and the culture is carried out at the temperature of 27 ℃. Typically, the growth of the mutagenized microalgae on the plates takes about ten days to log phase, and the mortality is calculated by the number of colonies according to the formula:

mortality (%) = (number of surviving cells of 1-treated group/number of cells of control group) ×100%

Experiments show that the mutation rate of the algae is different at different ARTP irradiation times. Different ARTP irradiation times were used and the algae lethality was analyzed. The results are shown in FIG. 1.

The results show that there is a clear dose-response relationship between ARTP irradiation time and mortality. Within 0-15s, the death rate of the cells rapidly increased with the increase of the irradiation treatment time. After 15s, the irradiation time is prolonged, and the mortality rate is slowly increased. When the irradiation treatment time was 35s, the mortality rate reached 100%. When the irradiation treatment time was 25s, the mortality was 92%. Therefore, the mutagenesis was performed with an ARTP irradiation time of 25s.

EXAMPLE 3 screening of high tocopherol content mutant strains

1. Primary screening of high-tocopherol content mutagenized algae strains

The microalgae are subjected to teratogenic mutation by irradiating with ARTP for 25 seconds, and microalgae on a flat plate with the mortality rate of more than 90% are selected to finally obtain 8 strains of microalgae, wherein the numbers of the microalgae are aBST1, aBST2, … …, aBST7 and aBST8 respectively. The 8 microalgae are amplified and cultured by conventional operation, after one period, the microalgae are transferred into a 500mL triangular flask, and real-time fluorescence quantitative PCR comparison of alpha-tocopherol synthesis key gene expression is carried out after 3 days of culture, and the alpha-tocopherol content is measured.

And respectively calculating the relative quantitative qRT-PCR expression quantity of the genes of the 8 microalgae selected by the screening method, and comparing the relative qRT-PCR expression quantity of 6 key genes of the 8 microalgae.

As a result, as shown in FIG. 2, the expression levels of 6 genes in the 8 mutants were different, and the expression levels of 6 genes in aBST5, aBST6 and aBST8 were all lower, and it was preliminarily ascertained that the tocopherol production potential was smaller in these 3 mutants. The 6 genes showed higher expression levels in each of aBST2, aBST3, aBST4 and aBST7 and, initially, it was judged that these 4 mutants probably contained higher tocopherols.

The tocopherol content of 8 mutant microalgae was measured and the results are shown in FIG. 3. Comparing the alpha-tocopherol content of 8 strain of mutagenized microalgae, it was found that the alpha-tocopherol content of strain aBST2 was highest, being 394.2 μg/g DCW, followed by strain aBST7, the alpha-tocopherol content of which was 356.8 μg/g DCW, followed by aBST3 and aBST4, respectively, and the alpha-tocopherol content of which was 345.7 and 319.6 μg/g DCW, respectively. Among them, the lowest was aBST5, which had an alpha-tocopherol content of 79.5. Mu.g/g DCW, and the alpha-tocopherol contents of 8 microalgae were compared, and it was found that the alpha-tocopherol contents of the above 4 mutants having high expression levels of the 6 key genes were also higher, so that these 4 mutants were used as a primary screening result for the subsequent study.

2. Secondary mutagenesis of high tocopherol content mutagenized algal strains

And (3) screening microalgae on a flat plate with the lethality of more than 90% by the same method after carrying out secondary mutagenesis on 4 strains of mutagenized microalgae aBST2, aBST7, aBST3 and aBST4 obtained by primary screening by an ARTP mutagenizing instrument, carrying out transfer culture for 3 days, and carrying out real-time fluorescence quantitative PCR to compare the expression of the key genes for synthesizing alpha-tocopherol and simultaneously measuring the content of the alpha-tocopherol.

The mutant 10 strains are obtained by re-screening, and the real-time fluorescence quantitative PCR result is shown in figure 4, wherein the expression level of 6 genes in aBST2-3 and aBST7-5 is the highest.

On the other hand, the results of the alpha-tocopherol content measurement are shown in FIG. 5, the alpha-tocopherol content of the algae strain aBST2-3 and the algae strain aBST7-5 are highest, namely 417.8 mug/g DCW and 411.6 mug/g DCW, respectively, which are improved by 81% and 78% on the basis of the original strain BST-2, and the alpha-tocopherol content of the algae strain aBST2-1 and the algae strain aBST2-6 reach 398.5 mug/g DCW and 386.5 mug/g DCW, which are respectively improved by 73% and 67% on the basis of the original strain, and the alpha-tocopherol content of other mutant strains is improved by 35% -61% compared with the original strain. Based on the above results, the algal strains aBST2-3 and aBST7-5 were used as mutants to be screened by the secondary mutagenesis for subsequent studies.

3. Analysis of genetic stability

For further analysis of the potential of the mutant strains aBST2-3 and aBST7-5 obtained by mutagenesis screening for producing alpha-tocopherol, genetic stability analysis was performed on the 10 high-yield mutant strains by using a shake flask subculture method, and the alpha-tocopherol production within 1-6 generations was determined, respectively. As shown in Table 2, the alpha-tocopherol production of each strain did not change significantly during passage, suggesting that the obtained mutants aBST2-3 and aBST7-5 had genetic stability and were available for subsequent study.

TABLE 2 analysis of genetic stability of mutant aBST2-3 and aBST7-5

Example 4 comparison of biomass, sugar consumption, fat content and alpha-tocopherol content during fermentation of mutants

The selected mutants aBST2-3 and aBST7-5 were cultured with chlorella culture medium at 27℃for 4 days, and samples were taken every 12 hours to determine the biomass, fat content, sugar consumption and alpha-tocopherol content.

The results of the measurement of the difference in growth and the change in content of the mutant strains aBST2-3 and aBST7-5 from the original strain were shown in FIG. 6 by measuring the biomass, the content of alpha-tocopherol in the oil and fat, and the consumption of sugar during fermentation.

The results show that the glucose consumption rate of the mutant strain and the original strain gradually increases and the biomass accumulation gradually increases at 0-24 h; when the fermentation time is 24-60 hours, the glucose utilization rate of the three strains is the fastest, and biomass is rapidly accumulated; as can be seen from fig. 6B and C, the lipid content and alpha-tocopherol content of both mutants rapidly accumulated over a period of 0h to 60h, and did not increase after 72 h; the original algae strain grows gradually from 0h to 24h, and grows rapidly from 24h to 60h, and also does not grow after 72 h; the mutant strain is improved in the production rate and accumulation amount of grease and alpha-tocopherol based on the original strain, so that the mutant strain starts to accumulate grease and alpha-tocopherol in the early growth stage, and the alpha-tocopherol content of the mutant strains aBST2-3 and aBST7-5 is highest in 72h of fermentation.

In addition, comparing the differences between mutants aBST2-3 and aBST7-5, it was found that aBST2-3 consumed sugar faster than aBST7-5, which was also a part of the reason that aBST2-3 had higher biomass, lipid content and alpha-tocopherol content than aBST 7-5. Based on the above results, mutant aBST2-3 was selected as the best mutant according to the difference in growth and the difference in content.

According to the invention, through carrying out ARTP mutagenesis on microalgae twice, a mutant strain capable of being inherited stably is obtained, a mutant strain with high yield of alpha-tocopherol and capable of being inherited stably is finally screened out, the highest yield of the mutant strain reaches 420.3 mug/gDCW, the highest yield of the mutant strain is improved by 84% compared with that of the original strain, and the mutant strain is an effective source for preparing the alpha-tocopherol and has good alpha-tocopherol development prospect.

Claims

1. Chlorella strainChlorella sp. ) The aBST2-3 is characterized in that the chlorella aBST2-3 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 45167 in 2022, 5 and 23 days.

2. Use of chlorella aBST2-3 as defined in claim 1 for the fermentative production of alpha-tocopherol.