Schizochytrium sp. mutant strains by complex mutagenesis of ntg-artp, mutagenesis method and application, and method for producing eicosapentaenoic acid

Abstract

The present application relates to the field of microbial breeding and biofermentation engineering, and discloses a NTG-ARTP compound mutagenesis Aureobasidium pullulans mutant strain, a mutagenesis method and application thereof, and a method for producing eicosapentaenoic acid. The preservation number of the mutant strain is CCTCC NO: M 20253077. The NTG-ARTP compound mutagenesis Aureobasidium pullulans mutant strain provided by the present application with the preservation number of CCTCC NO: M 20253077 can realize the synergistic high production of EPA and DHA, and has excellent genetic stability. The mutant strain is suitable for a conventional industrial fermentation system, and can further improve the production capacity of EPA and DHA by combining a time sequence plant hormone induction strategy, thereby providing a high-quality strain support for large-scale production of high-purity EPA.

Description

Technical Field

[0001] This invention relates to the field of microbial breeding and bio-fermentation engineering, specifically to an NTG-ARTP composite mutagenized Schizochytrium mutant strain, its mutagenization method and application, and a method for producing eicosapentaenoic acid. Background Technology

[0002] Eicosapentaenoic acid (EPA), with the molecular formula C 20 H 30 O2, with a relative molecular weight of 302.45, is a polyunsaturated fatty acid with 20 carbon atoms and 5 non-conjugated double bonds. Because the first double bond in its molecule is located on the third carbon atom at the methyl end, it also belongs to the ω-3 series of unsaturated fatty acids. EPA is a pale yellow oily liquid at room temperature and is readily soluble in nonpolar solvents such as n-hexane and diethyl ether. EPA has many important physiological functions, including lowering cholesterol, triglycerides, and low-density lipoprotein levels in the blood, reducing blood viscosity, improving blood circulation, and preventing cardiovascular and cerebrovascular diseases such as hypertension, cerebral thrombosis, and arteriosclerosis. It is known as a "vascular cleanser."

[0003] Traditionally, EPA is extracted from fish oil. Currently, industrial EPA production relies on fish oil. The main process for separating EPA from fish oil involves removing non-essential components such as pigments, cholesterol, and saturated fatty acids, thereby separating PUFAs (polyunsaturated fatty acids) like DHA and EPA. Fish oil and its products rich in EPA on the market are mainly divided into two categories, differing in the EPA to DHA ratio and oil type: one is the triglyceride form (DHA + EPA content 20%-40%), and the other is the ethyl ester form (DHA + EPA content 50%-70%). Over the past decade, global fish oil supply has stabilized at around 1 million metric tons per year, and it is projected to continue growing in the coming years.

[0004] In recent years, microbial fermentation has become one of the sources for the industrial production of EPA. Various microorganisms, including microalgae, marine bacteria, and fungi, can produce EPA. Microalgae are photosynthetic organisms that grow in lakes, rivers, and oceans, and can be cultivated through photoautotrophic, heterotrophic, or mixed culture modes. Marine microalgae have advantages such as strong environmental tolerance, high lipid and EPA content, and not competing with humans for fertile land or freshwater, making them suitable producers of EPA.

[0005] However, with increasing living standards and enhanced health awareness, the market demand for EPA fish oil has surged, leading to a supply shortage. Overfishing has resulted in a scarcity of fish oil resources, and coupled with marine pollution and seasonal supply fluctuations, the sustainability and availability of EPA production from fish oil sources are limited. Furthermore, due to the slow growth rate of most algae, their total biomass and oil content are relatively low, making the commercial production of EPA from algae costly, and large-scale production of EPA from algae is considered challenging. In addition, fish oil suffers from drawbacks such as complex purification processes, easy accumulation of heavy metals, and unsustainable production. Existing Schizochytrium strains have significant technical limitations: on the one hand, most strains have low natural EPA yields, and DHA content is difficult to guarantee, making it difficult to achieve synergistic high yields of EPA and DHA. Some strains require complex post-processing to increase EPA content, significantly increasing industrialization costs. On the other hand, strains obtained through conventional breeding methods have poor genetic stability, and their product synthesis ability is prone to degradation after subculturing. The yields of EPA and DHA fluctuate greatly in batch production, failing to meet the needs of large-scale, standardized industrial production. Therefore, cultivating Schizochytrium strains that possess both high EPA and DHA production characteristics and excellent genetic stability is of great significance for promoting the development of related industries. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of scarce traditional EPA sources (fish oil) and high pollution risk, as well as low yield and high cost of EPA fermentation by microorganisms (algae). This invention provides an NTG-ARTP composite mutagenized Schizochytrium mutant strain, its mutagenization method and application, and a method for producing eicosapentaenoic acid. This mutant strain has the advantages of high yield of both EPA and DHA, excellent genetic stability, and strong industrial adaptability.

[0007] To achieve the above objectives, the present invention provides an NTG-ARTP combined mutagenesis strain of Schizochytrium, the preservation number of which is CCTCC NO: M 20253077.

[0008] A second aspect of this invention provides a method for inducing mutagenesis of an NTG-ARTP combined mutant strain of Schizochytrium, the method comprising the following steps: S1. The starting strain, Schizochytrium, was contacted with nitrosoguanidine to construct an NTG mutagenesis library; S2. After staining the mutant library obtained in step S1 with lipids, strains with fluorescence intensity greater than 1.5 times that of the starting strain are screened to obtain intermediate mutant strains. S3. The intermediate mutant strain obtained in step S2 was treated with ambient pressure room temperature plasma to construct an NTG-ARTP mutagenesis library.

[0009] A third aspect of the present invention provides the application of the NTG-ARTP combined mutagenized Schizochytrium mutant strain as described above and / or the NTG-ARTP combined mutagenized Schizochytrium mutant strain as described above in the production of eicosapentaenoic acid.

[0010] The fourth aspect of the present invention provides a method for producing eicosapentaenoic acid, the method comprising: fermenting and culturing the NTG-ARTP-mutated Schizochytrium mutant strain as described above and / or the NTG-ARTP-mutated Schizochytrium mutant strain mutated by the method described above.

[0011] Through the above technical solutions, the NTG-ARTP combined mutagenesis strain of Schizochytrium provided by this invention, with accession number CCTCC NO: M 20253077, can achieve synergistic high yield of EPA and DHA and has excellent genetic stability. This mutant strain is adapted to conventional industrial fermentation systems, and combined with a sequential plant hormone induction strategy, it can further improve the production capacity of EPA and DHA, providing high-quality strain support for the large-scale production of high-purity EPA and DHA.

[0012] Biological Preservation The Schizochytrium mutant strain provided by this invention is classified as Schizochytrium ( Schizochytrium sp. GL-124 is deposited at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M20253077; deposit date: December 31, 2025). Attached Figure Description

[0013] Figure 1 This is a graph showing the effect of NTG concentration and treatment time on the lethality of Schizochytrium in Example 1. Figure 2 This is a fluorescence intensity distribution diagram of different mutant strains after Nile Red staining in Example 3; Figure 3 This is a comparison of oil yield and biomass of the 7 high-fluorescence mutant strains after FACS screening in Example 3; Figure 4 This is a graph showing the effect of ARTP mutagenesis treatment time on the lethality of the Schizochytrium intermediate mutant strain NA-60 in Example 4. Figure 5 This is a graph showing the effect of different concentrations of clethodim on the lethality of Schizochytrium in Example 5. Figure 6 This is a comparison chart of biomass, lipid content, and EPA content of the starting strain, intermediate mutant strain, and the strain screened after ARTP mutagenesis in Example 6. Figure 7 This is a comparison chart of the EPA content of various strains with different hormone addition methods in Example 7. Detailed Implementation

[0014] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0015] The first aspect of this invention provides an NTG-ARTP combined mutagenesis strain of Schizochytrium, the preservation number of which is CCTCC NO: M 20253077.

[0016] The NTG-ARTP combined mutagenesis strain of Schizochytrium provided in this invention, with accession number CCTCC NO: M 20253077, can achieve synergistic high production of EPA and DHA and exhibits excellent genetic stability. This mutant strain is adapted to conventional industrial fermentation systems, and combined with a time-series plant hormone induction strategy, it can further enhance the production capacity of EPA and DHA, providing high-quality strain support for the large-scale production of high-purity EPA and DHA.

[0017] The Schizochytrium mutant strain ( Schizochytrium sp. GL-124 is deposited at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M 20253077; deposit date: December 31, 2025).

[0018] According to the present invention, preferably, the starting strain of the mutant strain is Schizochytrium (…). Schizochytrium sp. The strain is designated CCTCC NO: M 209059. The inventors discovered in their research that using *Schizochytrium* CCTCC NO: M 209059 as the starting strain allows for the utilization of its excellent industrialization characteristics—rapid growth, no need for light, tolerance to high sugar levels and strong mechanical stirring—ensuring that the mutant strain after compound mutagenesis retains its high-efficiency mass production potential while further achieving synergistic high yields of EPA and DHA.

[0019] A second aspect of this invention provides a method for inducing mutagenesis of an NTG-ARTP combined mutant strain of Schizochytrium, the method comprising the following steps: S1. The starting strain, Schizochytrium, was contacted with nitrosoguanidine to construct an NTG mutagenesis library; S2. After staining the mutant library obtained in step S1 with lipids, strains with fluorescence intensity greater than 1.5 times that of the starting strain are screened to obtain intermediate mutant strains. S3. The intermediate mutant strain obtained in step S2 was treated with ambient pressure room temperature plasma to construct an NTG-ARTP mutagenesis library.

[0020] The mutagenesis method provided by this invention achieves complementary mutagenesis mechanisms by combining NTG chemical mutagenesis and ARTP plasma mutagenesis, which greatly increases the probability of beneficial mutations, and the strains obtained by the final mutagenesis can stably achieve synergistic high production of EPA and DHA.

[0021] According to the present invention, preferably, the starting strain *Schizochytridactylogyrus* is designated as CCTCC NO: M 209059. The inventors discovered in their research that selecting *Schizochytridactylogyrus* CCTCC NO: M 209059 as the starting strain allows for the utilization of its excellent industrialization characteristics—rapid growth, no need for light, tolerance to high sugar levels and strong mechanical stirring—ensuring that the mutant strain after compound mutagenesis retains its high-efficiency mass production potential while further achieving synergistic high yields of EPA and DHA.

[0022] According to the present invention, preferably, the concentration of the nitrosoguanidine is 200-400 μg / mL. The inventors have found that selecting this preferred concentration can ensure sufficient mutagenic pressure on the starting strain to induce effective gene mutations, while avoiding the problem that excessively high concentrations can lead to a strain lethality rate exceeding the suitable range and the inability of beneficial mutants to survive. This lays a reliable mutagenic foundation for subsequent screening of high-oil-yielding intermediate mutants.

[0023] According to the present invention, preferably, the contact time is 80-120 min. The inventors have found that under this preferred embodiment, the mutagen can fully induce gene mutations in the strain without causing a large number of strains to die due to prolonged action. This provides a sufficient candidate bacterial population for obtaining mutant strains with high levels of both EPA and DHA. Furthermore, the strains mutated within this time range are more likely to maintain stable traits in subsequent generations, ensuring genetic stability.

[0024] According to the present invention, preferably, the reagent used for lipid staining is Nile Red. The inventors have discovered that, under this preferred embodiment, intracellular lipids of the strain can be rapidly and accurately labeled, enabling efficient screening of intermediate strains with high lipid accumulation. This lays a solid foundation for subsequent mutagenesis to obtain strains with high EPA and DHA content. Furthermore, this staining method causes minimal damage to the strains, and the high-yield traits of the screened strains are not easily lost during subsequent passages, maintaining good genetic stability.

[0025] According to the present invention, preferably, the parameters of the ambient pressure room temperature plasma are: power supply of 100-140 W, carrier gas flow rate of 8-12 SLM, irradiation distance of 1-3 mm, and treatment time of 30-50 s. The inventors have found that, under this preferred embodiment, the mechanism can be complementary to NTG mutagenesis, significantly increasing the probability of obtaining EPA and DHA dual-high mutant strains. Furthermore, the genetic background of the strains mutated by these parameters is stable, meeting the stability requirements for large-scale production.

[0026] A third aspect of this invention provides the application of the NTG-ARTP-mutated Schizochytrium mutant strain described above and / or the NTG-ARTP-mutated Schizochytrium mutant strain induced by the method described above in the production of eicosapentaenoic acid (EPA). The inventors discovered in their research that applying this mutant strain to EPA production can achieve synergistic high yields of EPA and DHA, while the strain exhibits excellent genetic stability, ensuring the stability and continuity of mass production. Furthermore, this application, combined with a time-series plant hormone induction process, can significantly reduce production costs and meet the large-scale raw material needs of the pharmaceutical and health food industries.

[0027] A fourth aspect of this invention provides a method for producing eicosapentaenoic acid (EPA), the method comprising: fermenting and culturing an NTG-ARTP-mutated Schizochytrium mutant strain as described above and / or an NTG-ARTP-mutated Schizochytrium mutant strain induced by the method described above. The inventors have found that this method for producing EPA can achieve synergistic high yields of EPA and DHA by relying on the mutant strain, while the mutant strain exhibits excellent genetic stability, ensuring stable mass production. This method can also be combined with a fermentation optimization strategy involving the sequential addition of plant hormones to further improve the biomass of the strain and the efficiency of product synthesis, significantly reducing production costs and effectively addressing the resource and cost pain points of traditional EPA and DHA production.

[0028] According to the present invention, preferably, the fermentation culture conditions include: a temperature of 25-32℃, a time of 100-140 h, and a rotation speed of 150-200 r / min. The inventors have found that this condition range can adapt to the growth and metabolic characteristics of the mutant strain, ensuring stable proliferation of the strain to maintain a high biomass, while also providing sufficient time for lipid synthesis, achieving high accumulation of both EPA and DHA. Furthermore, the high-yield trait of the strain cultured under these conditions is not easily degraded after subculturing, maintaining good genetic stability.

[0029] According to the present invention, preferably, the fermentation culture method further includes: adding a first plant hormone at a concentration of 5-15 mg / L after 8-16 h of fermentation; and adding a second plant hormone at a concentration of 1-3 mg / L after 32-40 h of fermentation. The inventors have found that this sequential addition of plant hormones can synergistically regulate the metabolic pathways of the strain, avoiding the growth inhibition problem associated with single hormone addition, and directionally enhancing the synthesis of unsaturated fatty acids, thus promoting high yields of both EPA and DHA. Simultaneously, this culture mode can stabilize the metabolic characteristics of the strain and ensure its genetic stability.

[0030] According to the present invention, preferably, the first plant hormone is methyl jasmonate. The inventors have found that, under these preferred conditions, the addition of methyl jasmonate can activate the strain's stress response system in advance, preparing it metabolically for subsequent oil and unsaturated fatty acid synthesis, effectively enhancing the synthesis basis of EPA and DHA, and the induction of this hormone does not affect the strain's genetic characteristics, ensuring the stable inheritance of high-yield traits.

[0031] According to the present invention, preferably, the second plant hormone is salicylic acid. The inventors have discovered that, under these preferred conditions, the addition of salicylic acid can form a synergistic effect with methyl jasmonic acid in the early stage, strongly guiding the metabolic flow to the lipid synthesis pathway, achieving high production of both EPA and DHA. At the same time, the hormone has a mild effect and will not cause instability at the gene level of the strain, thus maintaining the genetic stability of the strain.

[0032] The present invention will be described in detail below through examples. In the following examples, *Schizochytrium* was obtained by our laboratory from isolation and screening in coastal areas and is now deposited at the China Center for Type Culture Collection (CCTCC) with the number CCTCC NO: M 209059, which can be found in patent CN 114958932A; nitrosoguanidine was purchased from Shanghai Maclean Biotechnology Co., Ltd.; Nile red was purchased from Shanghai Sangon Biotech Co., Ltd.; methyl jasmonate was purchased from Shanghai Sangon Biotech Co., Ltd.; salicylic acid was purchased from Shanghai Sangon Biotech Co., Ltd.; other raw materials and reagents were all commercially available products.

[0033] Example 1: Nitroguanidine mutagenesis method for Schizochytrium (1) Activation of strains: The strains stored at -80℃ will be activated. Schizochytrium sp CCTCC NO: M 209059 Glycerol tubes were streaked onto solid medium and incubated at 28°C to activate the strain. Once colonies grew, single colonies were picked and inoculated into seed medium and incubated at 28°C and 170 rpm for 48 h to reach the logarithmic phase.

[0034] (2) Activation and seed culture medium (g / L): Contains 50 g glucose, 15 g yeast extract, 10 g monosodium glutamate, 0.6 g malic acid, 1 g potassium dihydrogen phosphate, 12 g sodium sulfate, 0.6 g potassium sulfate, 2 g magnesium sulfate, 2 g ammonium sulfate, 0.5 g potassium chloride, and 0.2 g calcium chloride. The solution is brought to a final volume of 1 L with deionized water. The solution is autoclaved at 121 °C for 30 min. The vitamin solution is filtered through a 0.22 μm pore size filter membrane. Under aseptic conditions, vitamin B1, vitamin B6, and vitamin B12 are added to the culture medium at a ratio of 0.1% (v / v).

[0035] (3) Preparation of bacterial suspension: Take 5 mL of bacterial suspension cultured to the logarithmic growth phase, centrifuge at 3000 rpm for 5 min to collect cells, and use 0.2 mol·L⁻¹ solution. -1 Wash three times with PBS buffer and dilute to OD200. 540 Approximately 0.6-0.7 (PBS as reference).

[0036] (4) Nitrosoguanidine mutagenesis: Take 10 mL of the above bacterial suspension from 24 groups into 50 mL Erlenmeyer flasks. Prepare nitrosoguanidine (NTG) stock solution using sterile PBS buffer (pH=7.2). After sterilization by filtration through a 0.22 μm microfiltration membrane, add appropriate amounts to the Erlenmeyer flasks containing bacterial suspensions to make the final NTG concentrations 100, 200, 300, and 400 μg / mL, respectively. Each group has 3 parallel samples. Mix well and incubate at 28℃ and 170 rpm for 20, 40, 60, 80, and 100 min, respectively. After the specified time, immediately add 1% sodium thiosulfate solution to each flask to terminate the reaction. Centrifuge to remove the supernatant, add 20 mL of sodium thiosulfate solution to rinse the bacterial cells, centrifuge again, discard the supernatant, and wash the precipitate twice with PBS. After washing, the bacterial cells were resuspended in 1-2 mL of PBS. 100 μL of this precipitate was spread onto a solid culture medium and incubated at 28°C until single colonies appeared. Schizochytrium without NTG stock solution served as a control. Colony counts were performed in each mutagenized group. The lethality rate (%) was calculated as (control group colony count - mutagenized group colony count) / control group colony count × 100%. Lethality curves were plotted, and the results are shown below. Figure 1 As shown, cell lethality increased with increasing NTG concentration. A lethality of 90%-95% resulted in a higher positive mutation rate in the strain. When the NTG concentration was 300 μg / mL and the treatment time was 100 min, the lethality was 90.8%, therefore this condition was selected as the optimal NTG mutagenesis dose.

[0037] First round of NTG mutagenesis: The starting strain was treated with the optimal NTG mutagenesis parameters (300 μg / mL, 100 min). Schizochytrium spCCTCC NO: M 209059. The mutagenic bacterial culture, after initial screening by FACS, was inoculated into a novel 96-well microplate and cultured at 800 rpm and 25℃ for 48 h. After Nile Red staining, the culture was analyzed using FI / OD2000. 595 To screen for high-oil-producing strains, intermediate strain NA60 was obtained.

[0038] Second round of NTG mutagenesis: Using NA60 as the starting strain, the same NTG mutagenesis parameters were applied. After initial screening by FACS, 80 μL of bacterial suspension was spread onto a plate containing 80 μg of NTG. mL -1 Solid culture medium plates for clethodim were incubated at 28°C for 2 days. Fifty larger colonies were selected for shake-flask verification. Biomass, oil content, and EPA yield were measured, and high-yielding strain NB6 was obtained through screening.

[0039] Third round of ARTP mutagenesis: Before using the ARTP mutagenesis instrument, the workbench was sterilized by irradiating with UV light for 30 minutes. Using NB6 as the starting strain, 80 μL of the mutagenized strain was spread onto a substrate containing 100 μg of... mL -1 Solid plates containing clethodim were cultured at 28°C for 2-3 days. Strains with good growth were selected for fermentation to determine EPA yield, and the final mutant strain SCNA9 was obtained through shake-flask verification.

[0040] The Schizochytrium mutant strain (SCNA9) provided by this invention is classified as Schizochytrium ( Schizochytrium sp. GL-124 is deposited at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M 20253077; deposit date: December 31, 2025).

[0041] Example 2: Fluorescence-activated cell sorting of high-oil-yielding mutant strains The mutagenic cells prepared in Example 1 were cultured at 28°C with shaking at 170 rpm for 48 h. The cells were then collected by centrifugation, washed twice with sterile PBS buffer, and the bacterial concentration was adjusted to 10. 7 -10 8The Nile Red solution was prepared by dissolving 1 mg of Nile Red in 10 mL of acetone, filtering it through an organic filter membrane into a small brown bottle, and storing it in the dark. A certain volume of bacterial culture was taken and mixed with 20% (v / v) DMSO. Nile Red staining solution was then added to bring the final Nile Red concentration to 2 μg / mL. The mixture was mixed and stained in the dark. After staining was terminated, the cells were washed with sterile PBS and resuspended. The cells were then transferred to test tubes for flow cytometry sorting. Single-cell populations were identified by forward scattering (FSC) and side scattering (SSC) (excluding interference from cell aggregates). Nile Red fluorescence intensity (FL1 channel) was used as the sorting index. The fluorescence intensity distribution of the starting strain was determined in a preliminary experiment. A screening threshold of "fluorescence intensity > 1.5 times that of the starting strain" was set. High-fluorescent single cells were sorted and directly collected into hexagonal microtiter plates with six baffles for culture (each well containing 200 μL of sterile medium to avoid secondary cloning).

[0042] Example 3: Cultivation and screening of a hexagonal microtiter plate with six baffles A CFD-RSM model (a composite modeling and optimization technique combining computational fluid dynamics and response surface methodology) was established. By combining the three-dimensional response surface model with computational fluid dynamics, the superior performance of the six-baffle hexagonal microtiter plate in oxygen transfer efficiency and mixing performance was ultimately established. The novel microplate can achieve a 0.62s... -1 Volumetric transfer efficiency and 2364 W / m 3 The high mixing level provided sufficient conditions for the subsequent cultivation and screening of DHA-producing strains. Based on computational fluid dynamics modeling, a geometric model of the novel reactor was established, and then 3D printing technology was used to fabricate microplates for the culture of highly fluorescent single cells.

[0043] After screening and culturing using FACS (Fluorescence Activated Cell Sorting), the strains were stained with Nile Red again. 200 μL of the stained bacterial suspension was resuspended in a 96-well black ELISA plate, and the fluorescence intensity was detected using a multi-mode microplate reader. The fluorescence intensity value of the blank medium was subtracted from the Nile Red staining value. To avoid interference from differences in inoculum size or strain growth status, the FI / OD ratio was used. 595 As a screening criterion, fluorescence intensity was positively correlated with the lipid concentration inside the strain; approximately 10 strains were obtained from a single NTG mutagenesis. 5 Mutant cells were sorted using FACS. After screening, the growth, proliferation, and fluorescence intensity of each single cell were measured, resulting in the collection of 200 highly fluorescent monoclonal strains (fluorescence intensity > 1.5 times that of the starting strain). The sorting purity was > 95% (single cell rate verified by flow cytometry), which is more than 10 times the throughput of traditional microplate screening. The screening results are as follows: Figure 2 As shown. Select FI / OD. 595Seven strains with higher values ​​were cultured in shake flasks to assess oil content. Fermentation results are as follows: Figure 3 As shown, the oil yield of the mutant strains was higher than that of the original strain, and the biomass of 6 strains was increased compared with that of the original strain, among which mutant NA-60 had the highest oil yield.

[0044] Example 4: ARTP mutagenesis method for Schizochytrium (1) Activation of strain: The high oil-producing strain NA-60 selected in Example 3 was inoculated into seed culture medium and cultured at 28℃ and 170 rpm for 48 h to the logarithmic phase.

[0045] (2) Activation and seed culture medium (g / L): The method is the same as in Example 1.

[0046] (3) Preparation of bacterial suspension: Take an appropriate amount of bacterial suspension cultured to the logarithmic phase, centrifuge at 3000 rpm for 5 min to collect cells, and use 0.2 mol·L⁻¹ solution. -1 Wash 1-2 times with PBS buffer, then dilute with 5% glycerol to OD. 600 Approximately 0.6-0.8 (PBS as reference). Used for ARTP mutagenesis treatment.

[0047] (4) ARTP mutagenesis: Before using the ARTP mutagenesis instrument, turn on the UV lamp switch on the main menu of the operation interface and irradiate the inside of the workbench for 30 min to sterilize it before conducting the experiment. Take 10 μL of the above bacterial suspension and carefully spread it on a sterile iron plate. Transfer it to the groove in the operating chamber of the ARTP mutagenesis instrument near the alcohol lamp. Treat with high-purity helium plasma for 0, 10, 20, 30, 40, 50, 60, 70, and 80 s. The mutagenesis parameters are: power supply 120W, helium flow rate 10 SLM, and irradiation distance 2mm. After ARTP mutagenesis, the iron plate was transferred to an EP tube containing 1 mL of fresh culture medium. The cells were eluted by shaking to ensure complete resuspending of the mutated cells in the EP tube. 80 μL of the bacterial suspension was spread onto a clethodim solid medium plate and incubated for 2-3 days. The number of single colonies was counted, and the lethality rate at different ARTP mutagenesis times was calculated. Lethality curves were plotted. Results are as follows: Figure 4 As shown, the cell lethality increased with the extension of ARTP treatment time, reaching 100% lethality at 60 s; the cell lethality was 94.2% at 40 s. Therefore, 40 s was selected as the ARTP mutagenesis treatment time. The high-oil-producing mutant strain NA-60 screened in Example 1 was subjected to ARTP mutagenesis treatment under these optimized conditions.

[0048] Example 5 Screening of Clethodim using Plate Solid Culture Wild-type Schizochytrium fungi were cultured in liquid medium for 30 hours. A certain volume of fermentation broth was placed in a test tube and diluted 1000 times using the 10-fold dilution method. 80 μL of the bacterial solution was then evenly spread onto solid medium with clethodim concentrations of 0, 20, 40, 60, 80, and 100 μg / mL, and incubated for 3 days. The number of mutant colonies was counted, and the lethality of clethodim against Schizochytrium fungi at each concentration was calculated. The results are as follows: Figure 5 As shown in the figure, clethodim concentrations with a lethality rate between 90% and 95% were selected for subsequent screening.

[0049] In this experiment, bacterial cultures collected after ARTP mutagenesis incubation were spread on a plate and incubated at 28°C for 3 days. Single colonies with rapid growth, plump surfaces, and large diameters were selected for streak plating verification. Then, single clones of the mutant strain SCNA-9 with rapid growth and large colonies were selected from the transfer plates and cultured in shake flasks. Example 6: Re-screening after shake-flask culture The mutant strains with growth advantages and high oil yield obtained in Example 5 were used to prepare seed culture according to the method described in Example 1. These seed cultures were then inoculated into 250 mL shake flasks containing 50 mL of culture medium and cultured for 120 h in a constant-temperature shaker at 28°C and 170 r / min. Biomass and eicosapentaenoic acid (EPA) content were measured, and the results are as follows: Figure 6 As shown, the mutant strain SCNA-9 had a 1.46-fold increase in biomass and a 14.8-fold increase in EPA content compared to the original strain. Example 7: Fermentation optimization culture of mutant strains Fermentation optimization experiments were conducted on the mutant strain SCNA-9 obtained in Example 5. In experimental group S1 (mutant strain SCNA-9), methyl jasmonate was added to a final concentration of 10 mg / L after 12 h of the initial fermentation stage, and salicylic acid was added to a final concentration of 2 mg / L after 36 h of fermentation. The blank control group received no inducer. The single control group F1 received methyl jasmonate to a final concentration of 10 mg / L only after 12 h of fermentation; the single control group F2 received salicylic acid to a final concentration of 2 mg / L only after 36 h of fermentation; the simultaneous addition control group F3 received both methyl jasmonate and salicylic acid at 12 h of fermentation; and the simultaneous addition control group F4 received both methyl jasmonate and salicylic acid at 36 h of fermentation. After 96 h of fermentation in each group, glucose to a final concentration of 3 g / L was added to the fermentation system, and the fermentation temperature was adjusted to 26℃. Fermentation was continued for 120 h to complete the fermentation. The fermentation results are as follows: Figure 7As shown, the hormone addition combination based on the time-series synergistic effect showed a significant increase in both oil yield and eicosapentaenoic acid (EPA) ratio compared to the control group, with the final EPA ratio of the mutant strain SCNA-9 reaching 18.373%.

[0050] Example 8 Systematic evaluation of Schizochytrium mutant strains (1) Biomass determination Place 2 mL empty centrifuge tubes in a 65℃ oven to dry to constant weight, and record the weight of the dried centrifuge tubes. Take 1 mL of fermentation broth and put it into the dried centrifuge tubes. Centrifuge (4000 rpm, 5 min), discard the supernatant, and dry the tubes in a 65℃ oven to constant weight. Calculate the biomass in the fermentation broth as shown in the following formula:

[0051] (2) Glucose concentration determination Every so often, 1 mL of fermentation broth was taken and centrifuged (4000 rpm, 5 min). The supernatant obtained was transferred to a new centrifuge tube, diluted 100 times, and then detected using an SBA-40C biosensor to record the current glucose concentration of the fermentation broth.

[0052] (3) Screening by plate solid culture Oil extraction: Take a certain volume of fermentation broth (100 mL), adjust the pH of the fermentation broth to about 11 with 2 mol / L NaOH solution, then add 0.3% (g / L) of cell-wall-breaking enzyme, mix and shake well, and then break the cell walls at 50℃ and 170 rpm on a shaker until the cells are broken. Microscopic examination shows large oil droplets and no bacterial cells, indicating complete cell wall breaking. Then, add ethanol and n-hexane (1:1:1) to the fermentation broth successively. After precipitating the protein with ethanol, add n-hexane, stir, and let stand until the layers separate. After centrifugation at 8000 rpm for 5 min, take the n-hexane phase, and then use a rotary evaporator to remove the n-hexane to obtain crude oil. Finally, aspirate the oil into centrifuge tubes and dry them in an oven at 65℃ until constant weight, then weigh them again. The oil content in the fermentation broth is shown in the following formula:

[0053] (4) Determination of fatty acid content Methyl esterification of oils and fats: Take about 0.5 mL of total oils and fats, add 3 mL of 0.5 mol / L potassium hydroxide-methanol solution, and then heat in a water bath at 65℃ for 15-20 min, and cool to room temperature; then add 2 mL of boron trifluoride diethyl ether solution, heat in a water bath at 65℃ for 5-10 min, and then cool to room temperature; then add 2 mL each of saturated sodium chloride solution and chromatographic grade n-hexane in sequence, shake, let stand, and separate into layers. Take the upper layer solution, extract it twice with n-hexane, take a certain amount of the extract and add an equal amount of methyl nonadecanoate solution and mix well for gas chromatography analysis.

[0054] Gas chromatography analysis: Column: DB-23 (60 m * 0.25 mm * 0.25 μm); Detector: FID; Carrier gas: Nitrogen; Split ratio: 30 / 1; Injector temperature: 250℃; Detector temperature: 280℃; Injection volume: 1 μL; Temperature program: Initial column temperature 100℃, increased to 196℃ at a rate of 25℃ / min, then increased to 220℃ at a rate of 2℃ / min, held for 12 min. Column flow rate: 3.0 mL / min; Make-up flow rate: 30 mL / min; Hydrogen flow rate: 40 mL / min; Air flow rate: 400 mL / min.

[0055] Example 9: Validation of the genetic stability of the Schizochytrium mutant strain SCNA-9 Single colonies of the mutant strain SCNA-9 obtained in Example 5 were picked and inoculated into 50 mL of seed culture medium (same as in Example 1). The culture was kept at 28°C and 170 r / min for 48 h until the logarithmic growth phase, and recorded as the first generation strain. The first generation bacterial solution was transferred to fresh seed culture medium at an inoculation rate of 5% (v / v) and cultured under the same conditions to complete the subculturing. This operation was repeated to subculture up to the 10th generation, and the bacterial solution of each generation was retained for later use.

[0056] Seed cultures of the 1st, 3rd, 5th, 7th, and 10th generation SCNA-9 strains, as well as the seed culture of the starting strain, were inoculated into 250 mL shake flasks containing 50 mL of culture medium at an inoculation rate of 10% (v / v). Three parallel samples were set up and cultured at 28℃ and 170 r / min for 120 h. The fermentation conditions were the same as in Example 6.

[0057] Following the method described in Example 8, the biomass, total lipid content, EPA content, and DHA content of the fermentation broth for each generation of strains were measured. The average value and coefficient of variation of each indicator were calculated to assess the stability of the traits. The results are shown in Table 1.

[0058] Table 1

[0059] As shown in the table above, after the mutant strain SCNA-9 provided by this invention was continuously passaged to the 10th generation, its biomass, total oil content, EPA content, and DHA content did not decrease significantly, and the coefficient of variation of each index was ≤2%. Compared with the starting strain, the EPA content of each generation of mutant strains was stable at 2.5 g / L (accounting for more than 16% by weight of total oil), the DHA content was maintained at 6.3 g / L (accounting for more than 42% by weight of total oil), and the biomass was always maintained at more than 34 g / L, without any phenotypic degeneration.

[0060] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A mutant strain of *Schizochytrium* induced by NTG-ARTP complex mutagenesis, characterized in that, The mutant strain has the accession number CCTCC NO: M 20253077.

2. The mutant strain according to claim 1, characterized in that, The mutant strain originated from Schizochytrium (Schizochytrium). Schizochytrium sp. (), numbered CCTCC NO: M 209059.

3. A method for inducing mutagenesis of an NTG-ARTP combined mutant strain of Schizochytrium, characterized in that, The method includes the following steps: S1. The starting strain, Schizochytrium, was contacted with nitrosoguanidine to construct an NTG mutagenesis library; S2. After staining the mutant library obtained in step S1 with lipids, strains with fluorescence intensity greater than 1.5 times that of the starting strain are screened to obtain intermediate mutant strains. S3. The intermediate mutant strain obtained in step S2 was treated with ambient pressure room temperature plasma to construct an NTG-ARTP mutagenesis library.

4. The method according to claim 3, characterized in that, The starting strain, Schizochytrium, is numbered CCTCCNO: M 209059; And / or, the concentration of the nitrosoguanidine is 200-400 μg / mL; And / or, the contact time is 80-120 min; And / or, the reagent used for the lipid staining is Nile Red.

5. The method according to claim 4, characterized in that, The parameters of the ambient pressure room temperature plasma are: power supply of 100-140 W, carrier gas flow rate of 8-12 SLM, irradiation distance of 1-3 mm, and processing time of 30-50 s.

6. The application of the NTG-ARTP-mutated Schizochytrium mutant strain according to claim 1 or 2 and / or the NTG-ARTP-mutated Schizochytrium mutant strain mutated by the method described in any one of claims 3-5 in the production of eicosapentaenoic acid.

7. A method for producing eicosapentaenoic acid, characterized in that, The method includes: fermenting and culturing the NTG-ARTP-mutated Schizochytrium mutant strain according to claim 1 or 2 and / or the NTG-ARTP-mutated Schizochytrium mutant strain mutated by the method described in any one of claims 3-5.

8. The method according to claim 7, characterized in that, The fermentation conditions include: a temperature of 25-32℃, a time of 100-140 h, and a rotation speed of 150-200 r / min.

9. The method according to claim 8, characterized in that, The fermentation culture method further includes: adding a first plant hormone at a concentration of 5-15 mg / L after fermentation for 8-16 h; and adding a second plant hormone at a concentration of 1-3 mg / L after fermentation for 32-40 h.

10. The method according to claim 9, characterized in that, The first plant hormone is methyl jasmonate; And / or, the second plant hormone is salicylic acid.

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