Nucleotide sequence related to EPA (Eicosapentaenoic Acid) production, engineering strain and application thereof

Abstract

The invention relates to the technical field of genetic engineering and microorganisms, and particularly discloses a nucleotide sequence related to EPA (eicosapentaenoic acid) production, an engineering strain and application of the nucleotide sequence. Part of genes are terminated or deleted in advance through mutagenesis, so that the resource consumption of cells on irrelevant metabolic pathways is reduced, the metabolic burden is relieved, and the EPA production efficiency is improved. Different from a technical route for improving the yield of EPA through overexpression of desaturase or elongase, specific structural variation is introduced into a mutagenic schizochytrium limacinum genome, including that a nucleotide sequence is inserted into a target gene coding region to cause early termination of coded protein, so that the EPA yield is improved. The fatty acid synthesis pathway is effectively reconstructed from the level of global metabolic regulation, the engineering strain is not obviously degraded in the aspects of biomass, growth rate and culture adaptability compared with an original strain, and the engineering strain has the advantages that the engineering strain can be used for preparing the fatty acid synthesis pathway of the fatty acid synthesis pathway in the aspect of global metabolic regulation, and the engineering strain can be used for preparing the fatty acid synthesis pathway of the fatty acid synthesis pathway of the fatty acid synthesis pathway of the fatty acid synthesis pathway of the fatty acid synthesis pathway. Meanwhile, the yield of EPA is remarkably increased, the technical contradiction that in the prior art, the yield of EPA is often increased along with growth inhibition is overcome, the genetic stability is high, the high yield of EPA can still be kept in the multi-generation subculture process, and the method is suitable for industrial-scale continuous fermentation or batch fermentation production.

Description

Technical Field

[0001] This invention relates to the field of nucleotide technology, and specifically discloses a nucleotide sequence, engineered strain, and its application related to EPA production. Background Technology

[0002] EPA (eicosapentaenoic acid) is a long-chain omega-3 polyunsaturated fatty acid and a key precursor in the biosynthesis of arachidic acid, which is an important signaling molecule involved in inflammation. Therefore, EPA has multiple functions in regulating physiological health, such as anti-inflammation, anti-oxidation, prevention of cardiovascular disease, anti-cancer, treatment of depression, and regulation of blood pressure, blood lipids, and blood sugar. It also has some potential beneficial effects on atherosclerotic plaque factors and weight loss; and plays a positive role in preventing Alzheimer's disease and alleviating retinopathy. However, the human body cannot synthesize EPA on its own and can only obtain it from food. Deep-sea fish oil is the main source of industrially produced EPA, but the fatty acid composition of fish oil is complex, and the content of various chain lengths and saturations directly affects its efficacy. Moreover, fish oil has a strong odor and high purification costs, leading to a supply shortage. EPA in fish oil accumulates through the food chain, so algae, lower fungi, and marine bacteria are the initial sources of EPA. Currently, the commonly used DHA-producing bacteria is Schizochytrium, while the EPA-producing bacteria are Shewanella sp. and Photobacterium profundum. EPA fermentation production has not yet achieved large-scale industrial production, and the biomass of several commonly used EPA production bacteria is far lower than that of Schizochytrium.

[0003] Schizochytrium is a type of marine microalga that produces high levels of DHA, but its EPA content is relatively low. The effects of changing the proportion of unsaturated fatty acids in Schizochytrium through methods such as culture medium optimization and fermentation regulation are limited. Researchers have improved EPA-producing strains through various means such as mutagenesis breeding, laboratory adaptive evolution, and genetic engineering, thereby obtaining high-quality and efficient EPA-producing strains.

[0004] In Schizochytrium, the biosynthesis of EPA and DHA is catalyzed by a series of enzymes in related anabolic pathways. Current research results show that there are two main biosynthetic pathways for EPA and DHA in Schizochytrium: (1) Fatty Acid Synthase (FAS) pathway, which uses acetyl-CoA and malonyl-CoA as substrates and synthesizes palmitic acid (C16:0) through multiple condensation, reduction, dehydration and reduction steps. Through the catalysis of elongation enzymes and desaturases, stearic acid (SAC18:0), the final product of the FAS pathway, is formed. Then, under the action of a series of desaturases and elongation enzymes, EPA, DPA and DHA are synthesized. (2) Polyketide synthase pathway (PKS pathway), which mainly synthesizes EPA or DHA with acetyl-CoA and malonyl-CoA as precursors under the action of polyketide synthase. However, the specific biosynthetic pathway and mechanism are currently controversial. At the same time, as a complex whole, an organism has a wide range of gene regulatory networks. Mutations in any other metabolism-related genes may also indirectly affect the production of EPA and DHA. Analysis of the genomes of high-EPA-producing strains can help to identify genes related to the biosynthetic pathways of EPA and DHA, which will provide favorable conditions for further improving the EPA production of Schizochytrium. Summary of the Invention

[0005] To address the problems existing in the prior art, the first aspect of the present invention proposes a mutant nucleotide sequence in which the nucleotide sequence SEQ ID NO:2 is inserted after the 360th position relative to SEQ ID NO:1, and the insertion causes the corresponding encoded protein to terminate prematurely.

[0006] In some specific embodiments of the mutant nucleotide sequence described in the first aspect, the nucleotide sequence is an insertion sequence after position 360 relative to SEQ ID NO:1, and the length of the insertion sequence is 2295 bp.

[0007] In some specific embodiments of the mutant nucleotide sequence described in the first aspect, the mutant nucleotide sequence includes SEQ ID NO:3.

[0008] A second aspect of the present invention provides a nucleotide sequence having at least 95%, 96%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO:3.

[0009] The third aspect of this invention proposes a schistocytic strain for high expression of EPA production, obtained by mutagenesis of the original strain HS01, and which, compared to the original strain, has at least two structural variations in gene coding regions in its genome. The first structural variation, SV1, is a nucleotide sequence insertion that results in a frameshift mutation in the coding region associated with EPA synthesis. The second structural variant, SV2, is a deletion of a nucleotide sequence, which results in the deletion of at least three gene sequences.

[0010] In some specific embodiments of the fissuri strains described in the third aspect, the nucleotide sequence encoded by the SV1 position-related gene is shown in SEQ ID NO:1, followed by a nucleotide sequence of 2295 bp in length.

[0011] In some specific embodiments of the fissuri strains described in the third aspect, the SV1 causes partial or complete loss of function of the corresponding protein.

[0012] In some specific embodiments of the fissuri strains described in the third aspect, the SV2 mutation results in a 9322bp deletion and the deletion of three gene sequences: SV2-1, SV2-2, and SV2-3, the nucleotide sequences of which are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0013] In some specific embodiments of the fissuri strains described in the third aspect, the structural variation is an insertion variation, a deletion variation, or a combination thereof.

[0014] A fourth aspect of this invention provides a method for preparing the fissile chytrid strain described in any of the third aspects, comprising the following steps: The original strain HS01 was subjected to mutagenesis. The mutagenized strains were screened to obtain candidate strains with increased EPA production; Genome analysis of the candidate strains confirmed the presence of at least the structural variants SV1 and SV2 in their genomes. This resulted in obtaining a schistocytic strain that highly expresses EPA production.

[0015] In some specific embodiments of the preparation method described in the fourth aspect, the mutagenesis process includes first chemical mutagenesis and plasma mutagenesis.

[0016] In some specific embodiments of the preparation method described in the fourth aspect, the first chemical mutagenesis includes the original strain HS01 being mutagenized with sodium azide to obtain a first mutagenic bacterial group, the first mutagenic bacterial group being subjected to plasma mutagenesis to obtain a second mutagenic bacterial group, and the second mutagenic bacterial group being subjected to adaptive mutagenesis and screening under selective culture conditions containing 2,2′-bipyridine to obtain strain HS09.

[0017] The fifth aspect of this invention provides for the application of any of the fissuri strains described in the third aspect in the production of EPA.

[0018] The sixth aspect of the present invention provides a method for producing EPA, comprising: fermenting the strain of schistocytic bryophyte according to any one of the third aspects to obtain a fermentation broth, and separating and purifying the fermentation broth to obtain EPA.

[0019] Advantages of this invention: This invention provides an engineered strain obtained through mutagenesis. Compared with the starting strain, this engineered strain does not show significant deterioration in biomass, growth rate, and culture adaptability. At the same time, it significantly increases EPA production, overcoming the technical contradiction in existing technologies where increased EPA production is often accompanied by growth inhibition. The mining of genes regulating EPA and DHA metabolism shows that, in addition to known fatty acid synthesis-related enzymes, structural variations in other genes in the genome can also significantly affect the metabolic allocation of EPA and DHA, providing new targets for regulating polyunsaturated fatty acid synthesis. The introduced mutations are stable genomic structural variations, unlike transcriptional regulation or exogenous plasmid expression. The mutations do not depend on resistance selection or exogenous gene maintenance, exhibiting high genetic stability. They can maintain high EPA production even during multiple generations of subculture, making them suitable for industrial-scale continuous fermentation or batch fermentation production.

[0020] This strain includes a mutant gene sequence. Unlike existing techniques that increase EPA production by overexpressing desaturases or elongating enzymes, avoiding direct overexpression of key enzymes in EPA biosynthesis to reduce metabolic burden, the mutant strain exhibits premature termination or deletion of some genes. This reduces cellular resource consumption on irrelevant metabolic pathways, alleviates metabolic burden, and facilitates the targeted allocation of carbon sources to the EPA synthesis pathway, thereby increasing the EPA accumulation level per unit biomass. This strategy is not simply about regulating a single rate-limiting enzyme. Specific structural variations are introduced into the Schizochytrium genome, including the insertion of a nucleotide sequence into the coding region of a target gene leading to premature termination of the encoded protein, and another structural variation causing the deletion of a genomic fragment and inactivation of multiple genes. This effectively reconstructs the fatty acid synthesis pathway at the global metabolic regulation level, thereby significantly increasing the EPA synthesis flux. Attached Figure Description

[0021] Figure 1 This is a colony diagram of strain HS09; Figure 2 Microscopic images of the HS09 strain; Figure 3 Comparison of sequencing contig similarity between HS01 and HS09; Figure 4 Phylogenetic tree of HS09 and closely related species (maximum likelihood method); Figure 5For the detection of structural variations in SV1 of strains HS01 and HS09, the DNA molecular weight standard used was DL2000, where 1: SV1 of strain HS01, 2: SV1 gene detection of strain HS09. Figure 6 For the detection of SV2-1 genes in strains HS01 and HS09, the DNA molecular weight standard used was DL2000, where 1: SV2-1 gene detection of strain HS01, and 2: SV2-1 gene detection of strain HS09. Figure 7 For the detection of SV2-2 genes in strains HS01 and HS09, the DNA molecular weight standard used was DL2000, where 1: SV2-2 gene detection of strain HS01, and 2: SV2-2 gene detection of strain HS09. Figure 8 The DNA molecular weight standard used was DL2000. The DNA molecular weight standard used for the detection of SV2-3 genes of strains HS01 and HS09 was DL2000. 1: SV2-3 gene detection of strain HS01, 2: SV2-3 gene detection of strain HS09. Figure 9 The mortality curves of EPA strains under different treatment times with sodium azide are shown. Figure 10 The lethality curves of EPA strain at different concentrations of tebufenozide are shown. Figure 11 The lethality curves of EPA strains under different ARTP treatment times are shown. Figure 12 The lethality curves of EPA strain at different concentrations of 2,2'-bipyridine are shown. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] The HS09 strain was obtained from the HS01 strain through mutagenesis. Based on this, the original strain HS01 and the mutagenized strain HS09 were subjected to third-generation PacBio sequencing analysis to discover and identify gene fragments related to high EPA production. The gene fragments provided by this invention have important value in high EPA production by Schizochytrium.

[0024] I. Identification and 18S sequence analysis of the mutagenic schistosomiasis strain HS09 The inventors of this application deposited the Schizochytrium strain HS01 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 13746, and obtained the Schizochytrium strain HS09 through mutagenesis.

[0025] The HS09 Schizochytrium fungus was inoculated onto a solid culture medium containing: 50 g / L glucose, 12 g / L yeast extract, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, and 0.17 g / L anhydrous calcium chloride, with 15 g / L agar powder. The medium was incubated in the dark at 28°C for 3 days. The results are shown below. Figure 1 and Figure 2 The results showed that the *Schizochytrium* colonies were light orange with irregular edges; the cell walls were thin, spherical, and transparent. The 18S rDNA sequence of the strain was consistent with... Aurantiochytrium sp. 16W-3a, Thraustochytriidae The 18S rDNA sequence similarity of sp. MBIC11070 can reach 99%. 18S rDNA amplification primers: 18S-F: CCGTAAACGATGCCGAC; 18S-R: CAATAAGATTCACCCGAGTTCTCA; Amplification was performed using Novizan 2×Taq Master Mix (Dye Plus). The PCR reaction program was: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min.

[0026] 18S partial sequence: CCGTAAACGATGCCGACTTGCGATTGCGGGTGGCTTGTATTGGGCCTCCGCAGCAGCACATGAGAAATCAAAGTCTTTGGGTTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTTACCAGGTCCAGACATAGGTAGGATTGACAGATTGAGAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTCGGCCTACTAAATAGCCGGGCGTATGGCGACATATGTGTTTGTGGCTTCTTAGAGGGACATGTTCGGTATACGAGCAGGAAGTTCGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGATGGGTTCAGCGGGTTTTTGTTGTGTTTTGCACAGCGTTGCTTTGTCGGAAGGCATGGCTAATCCTTTGAACGCCCATCGTGCTGGGGCTAGATTTTTGCAATTATTAATCTCCAACGAGGAATTCCTAGTAAACGCAAGTCATCAGCTTGCATTGAATACGTCCCTGCCCTTTGTACACACCGCCCGTCGCACCTACCGATTGAACGGTCCGATGAAACCATGGGACTACCTTTTGAGCGTTTGTTCGCGATGGAGGTGAGAACTCGGGTGAATCTTATTG。

[0027] II. Preparation of Mutagenized Schizochytrium sp. Strain HS09 1. Sodium azide mutagenesis time: Take 10 ml of the logarithmic growth phase HS01 fissuri strain suspension, add sodium azide solution to a final concentration of 10 μmol / L, gently shake and place on a shaker. Treat at 28℃ and 200 r / min for 0, 10, 20, 30, 40, 50, and 60 min respectively (0 min is the control group). Centrifuge, discard the supernatant, wash three times with 50 ml sterile water, and inoculate the bacterial suspension into a new liquid culture medium (50 g / L glucose, 12 g / L yeast extract, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, phosphate). The culture was prepared with potassium dihydrogen phosphate (1.0 g / L), ammonium sulfate (1.0 g / L), and anhydrous calcium chloride (0.17 g / L) for 24 h. After centrifugation, the supernatant was discarded, and the culture was washed three times with 50 ml of sterile water. Finally, the culture was resuspended in 500 μl of sterile water and evenly spread on solid culture medium plates (glucose 50 g / L, yeast extract 12 g / L, anhydrous sodium sulfate 15 g / L, potassium chloride 0.5 g / L, magnesium sulfate heptahydrate 4.1 g / L, potassium sulfate 0.65 g / L, potassium dihydrogen phosphate 1.0 g / L, ammonium sulfate 1.0 g / L, anhydrous calcium chloride 0.17 g / L, agar powder 15 g / L). The plates were incubated at 28℃ in the dark for 4–5 days, and the lethality rate was calculated. The lethality rate was calculated using the formula rL = (N0 – Nm) / N0 × 100% (rL: lethality rate, %); N0: colony count in the control group; Nm: colony count in the mutagenized group. The results showed a significant positive correlation between the lethality rate and the sodium azide treatment time. The cell lethality rate increased rapidly during the treatment period of 0–30 min, and then stabilized. At a treatment time of 40 min, the lethality rate reached 92.4%, and this treatment time was selected for mutagenesis.

[0028] 2. Determination of tebuconazole screening concentration: 100 μL of the HS01 fissuri strain suspension in the logarithmic growth phase was spread onto a solid medium containing tebuconazole (50 g / L glucose, 12 g / L yeast extract, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, 0.17 g / L anhydrous calcium chloride, and 15 g / L agar powder). The tebuconazole concentrations were 0, 10, 20, 30, 40, 50, and 60 mg·L⁻¹. -1 The spread solid culture medium was incubated at 28℃ in the dark for 4–5 days, and the lethality rate was calculated. The results showed that haloxyfop-R-methyl exhibited a positive correlation with the inhibitory trend of the strain at concentrations of 0–40 μg / mL; the higher the concentration of haloxyfop-R-methyl, the better the inhibitory effect and the higher the lethality rate. When the concentration of haloxyfop-R-methyl was 40–60 μg / mL, the increasing trend of the lethality rate slowed down. At a concentration of 40 mg / L, the lethality rate reached 93.7%, and the selection concentration for haloxyfop-R-methyl was 40 mg / L.

[0029] 3. Determination of ARTP mutagenesis time: Based on the statistical analysis of the lethality rate of sodium azide mutagenesis, single colonies that grew on plates containing 40 mg / L tebuconazole after 40 min of mutagenesis treatment were selected and inoculated into 250 ml shake flasks containing 50 ml of seed culture medium. After the bacterial culture reached the logarithmic growth phase (OD600 value approximately 3-4), 100 μL was evenly spread onto sterile ARTP-specific metal slides. The metal slides were then placed on the stage of the ARTP mutagenesis instrument. The ARTP mutagenesis instrument irradiation time was set to 0, 10, 20, 30, 35, 40, 50, and 60 s, with a helium flow rate of 10 L / min. -1 The power supply was 100W, and the metal slides were transferred to centrifuge tubes containing 490 μL of sterile PBS buffer. 100 μL of bacterial suspension was diluted and spread onto solid culture medium, and incubated at 28°C in the dark for 4–5 days. The lethality rate was then calculated. Based on previously reported results, mutants with better results are more likely to be obtained within a lethality range of 90–99%. Therefore, a mutagenesis treatment time of 50 s was optimal.

[0030] 4. Determination of 2,2'-bipyridine screening concentration: 100 μL of the bacterial suspension in the logarithmic growth phase was spread onto a solid culture medium containing 2,2'-bipyridine (50 g / L glucose, 12 g / L yeast extract, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, and 0.17 g / L anhydrous calcium chloride, 15 g / L agar powder). The 2,2'-bipyridine concentrations were 0, 1.5, 3, 4.5, 6, 7.5, and 9 mg·L⁻¹. -1 The spread solid culture medium was incubated at 28°C in the dark for 4–5 days, and the lethality rate was calculated. The results showed that the lethality rate was 90–95% when the concentration of 2,2'-bipyridine was 9 mg / L, and this concentration was selected as the screening concentration.

[0031] 5. Screening of sodium azide-ARTP mutagenized strains: After 40 min of sodium azide mutagenesis and screening on a medium containing 40 mg / L tebuconazole, the colonies were further mutagenized with ARTP for 50 s and then screened on a medium containing 9 mg / L 2,2'-bipyridine. The grown colonies were then fermented in 150 mL shake flasks, and their biomass, fatty acid content, EPA and DHA production were measured. Finally, strain HS09 with significantly increased EPA production was obtained. III. Comparison of shake-flask fermentation and fermenter fermentation between starting strain HS01, mutagenized strain HS09, and control strain HS08 The starting strain HS01 was compared with the mutant strain HS09 and the control strain HS08 in shake-flask fermentation. Single clones of HS01, HS09, and HS08 were inoculated into 10 ml of fermentation medium and cultured at 28°C and 180 rpm / min for 48 h to obtain primary seed culture. This primary seed culture was then inoculated at a rate of 10% (v / v) into shake flasks containing 250 ml of shake-flask medium and fermented for 120 h. The contents of EPA, DPA, and DHA were then measured. The fermentation shake-flask medium formulation was: glucose 50 g / L, yeast extract 12 g / L, anhydrous sodium sulfate 15 g / L, potassium chloride 5 g / L, magnesium sulfate heptahydrate 4.1 g / L, potassium sulfate 0.65 g / L, potassium dihydrogen phosphate 1.0 g / L, ammonium sulfate 1.0 g / L, anhydrous calcium chloride 0.17 g / L, pH 5.5-6.0.

[0032] Table 1. Comparison of shake-flask fermentation between strain HS01, HS09, and control strain HS08

[0033] Among them, the schizochymosis strain HS08 was obtained through the mutagenesis technology of patent CN 117701403 A and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No.40902.

[0034] A comparative study was conducted in an 8-ton fermenter using HS01, HS09, and control strain HS08. 1. Inoculate a single clone of Schizochytrium into a shake flask containing 2 mL of shake flask culture medium and incubate at 28°C and 180 rpm / min for 48 h to obtain a primary seed culture.

[0035] 2. Take the primary seed culture and inoculate it at a rate of 10% (v / v) into a shake flask containing 250 ml of shake flask culture medium. Incubate at 28 °C and 180 rpm / min for 24 h to obtain the secondary seed culture. The seed culture medium consists of: glucose 50 g / L, yeast extract 10 g / L, anhydrous sodium sulfate 15 g / L, potassium chloride 0.5 g / L, magnesium sulfate heptahydrate 4.1 g / L, potassium sulfate 0.65 g / L, potassium dihydrogen phosphate 1.0 g / L, ammonium sulfate 1.0 g / L, and anhydrous calcium chloride 0.17 g / L. 3. Inoculate the secondary Schizochytrium seed culture into an 8-ton fermenter at an inoculation rate of 15-35%. The fermentation medium formula is as follows: yeast extract 2.0-6.0 g / L, anhydrous sodium sulfate 20-30 g / L, anhydrous calcium chloride 0.1-0.2 g / L, potassium chloride 0.3-0.6 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, ammonium sulfate 2.0-4.0 g / L, zinc sulfate 1.0-4.0 g / L, magnesium sulfate 2.0-6.0 g / L; 4. During fermentation, the temperature was controlled at 28℃ and the pH was controlled at 5.0-6.0. Ammonia and 50% malic acid were used to adjust the pH during fermentation. After 120 hours of fermentation, the fermentation broth was obtained, and the oil content, EPA content, DPA content, and DHA content in the fermentation broth were tested.

[0036] Similar to the results of shake-flask fermentation, biomass decreased slightly, the proportion of DHA decreased significantly, and the percentages of n-3DPA and EPA increased significantly.

[0037] Table 2 Comparison of HS01 and HS09 strains in an 8-ton fermenter

[0038] Lipid content (%) refers to the mass fraction of the cell dry weight; biomass (g / L) represents the cell dry weight. III. Genome sequencing and phylogenetic analysis of Schizochytrium Comparison of shake flask and fermenter tests between strains HS09 and HS01 showed that strain HS09 exhibited significantly increased n-3DPA and EPA content, while DHA production decreased. To further investigate the genomic changes associated with the increased n-3DPA and EPA content, genomic DNA was extracted from the original strain HS01 and the mutant strain HS09 using a fungal genome extraction kit. Whole-genome sequencing was then performed using PacBio RS II by Wuhan Zhenyue Biotechnology Co., Ltd. Assembly was performed using hifiasm software. The results showed that the genomes of HS01 and HS09 were approximately 46 Mb in size, and the genome of the mutant HS09 was highly similar to that of the original strain HS01. Figure 3 Multiple sequence alignment was performed using MAFFT software, low-information sites were removed using trimal, and a phylogenetic tree was constructed using the maximum likelihood method with IQTREE. The results showed that HS09 and Schizochytrium CCTCC M209059 (HX308) has the highest genomic similarity, and the two are highly homologous at the genomic level; at the same time, they are similar to... Hondaea They are also closely related; see the detailed phylogenetic tree for the genome. Figure 4 .

[0039] IV. Gene sequences in HS09 strain associated with high EPA production Comparative analysis of the whole genome sequences of strains HS01 and HS09 revealed 340 SNPs and Indels, and 214 genomic structural variations (SVs). Most of these variations occurred in intergenic regions, with two occurring in coding regions. One SV variation involved a 2295bp insertion in strain HS09 after nucleotide 360 ​​of the target gene (SEQ ID NO:1), relative to strain HS01. The inserted sequence fragment causing the frameshift is shown in SEQ ID NO:2. Specifically, the 2295bp insertion was integrated into the target gene through its terminal sequence (as shown in SEQ ID NO:2), forming the mutation signature sequence shown in SEQ ID NO:3. This resulted in a mutation in the related coding gene. The original coding sequence at SV1 was 834bp, with the nucleotide sequence shown in SEQ ID NO:1. After the insertion, the coding sequence became 519bp, with the nucleotide sequence shown in SEQ ID NO:3. The encoded protein terminated prematurely, and some sequences were altered. Another SV mutation resulted in a 9322bp deletion, leading to the loss of coding sequences for three genes.

[0040] (1) The first mutation SV1: a 2295bp insertion, caused a frameshift mutation encoding the related protein, resulting in premature protein termination and changes in some sequences: Further PCR identification and first-generation sequencing analysis were performed on the 2295bp insertion in strain HS09. PCR amplification primers: SV1-F: GCTCAAGTACCTCAAGCTGGCC SV1-R: GTAATGTACGGACGAAGAGGGCG Amplification was performed using Novizan 2×Taq Master Mix (Dye Plus). The PCR reaction program was: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 3.5 min, 35 cycles; 72℃ for 5 min. The nucleotide sequence encoded by the SV1 position-related gene of strain HS01 is shown in SEQ ID NO:1 The protein sequence encoded by the SV1 position-associated gene of strain HS01 is SEQ ID NO:7. The nucleotide sequence encoded by the structural variation-related gene of SV1 in strain HS09 is SEQ ID NO:3: SEQ ID NO:2 is an inserted sequence that causes changes in the coding sequence and premature termination of protein translation.

[0041] The protein sequence encoded by the SV1 structural variation-related gene SEQ ID NO:3 of strain HS09 is SEQ ID NO:8. In the protein sequence SEQ ID NO:8, KRDASDRSTLRTFFLLLSALVSACSAGFAHRGHEMHRQNRGKSKQILHFKIA* represents the altered amino acid sequence.

[0042] (2) The second SV2 mutation resulted in the deletion of three gene sequences: SV2-1, SV2-2, and SV2-3. SV2-1 gene detection primers: SV2-1-F: ATGATGGAGATTGGTGACGC SV2-1-R: TTAACGCCAGAACTTACCAAGACG Amplification was performed using Novizan 2×Taq Master Mix (Dye Plus). The PCR reaction program was: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1.5 min, 35 cycles; 72℃ for 5 min. like Figure 6 As shown, strain HS01 amplified the expected band size, while strain HS09 did not amplify the expected band. Strain HS09 lacks the SV2-1 nucleotide sequence compared to strain HS01.

[0043] SV2-1 nucleotide sequence SEQ ID NO:4: SV2-1 protein sequence SEQ ID NO:9: SV2-2 gene detection primers: SV2-2-F: ATGGAGGGACTCGGACAACG SV2-2-R: TTATAGCGCGGACTTGCTTGG Amplification was performed using Novizan 2 × Taq Master Mix (Dye Plus). The PCR reaction program was: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s, 35 cycles; 72℃ for 5 min. like Figure 7 As shown, strain HS01 amplified the expected band size, while strain HS09 did not amplify the expected band. Strain HS09 lacked the SV2-2 nucleotide sequence compared to strain HS01.

[0044] SV2-2 nucleotide sequence SEQ ID NO:5: SV2-2 protein sequence SEQ ID NO:10: SV2-3 gene detection primers: SV2-3-F: ATGGAAGGGGAAAACTCGTGTGG SV2-3-R: TCATCGCAATGTGCAATTTGTCT Amplification was performed using Novizan 2 × Taq Master Mix (Dye Plus). The PCR reaction program was: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min. like Figure 8 As shown, strain HS01 amplified the expected band size, while strain HS09 did not amplify the expected band. Strain HS09 lacks the SV2-3 nucleotide sequence compared to strain HS01.

[0045] SV2-3 nucleotide sequence SEQ ID NO:6: SV2-3 protein sequence SEQ ID NO:11: This invention obtains a high-EPA-content mutant strain HS09 by mutagenesis of the starting strain HS01 with low EPA content. By comparing the genomes of the starting strain HS01 and the high-EPA-content mutant strain HS09, mutations were found in four coding genes. These mutations were verified by PCR and first-generation sequencing. The changes in these four coding genes are of great value for improving EPA yield, and the related sequences can serve as important markers for identifying the HS09 strain.

Claims

1. A mutant nucleotide sequence, characterized in that, The nucleotide sequence has an insertion of nucleotide sequence SEQ ID NO:2 after SEQ ID NO:1 at position 360, and this insertion causes the corresponding encoded protein to terminate prematurely.

2. The mutant nucleotide sequence according to claim 1, characterized in that, The nucleotide sequence is the insertion sequence after position 360 relative to SEQ ID NO:1, and the length of the insertion sequence is 2295 bp.

3. The mutant nucleotide sequence according to claim 1 or 2, characterized in that, The mutant nucleotide sequence includes the sequence shown in SEQ ID NO:

3.

4. A nucleotide sequence, characterized in that, The sequence is at least 95%, 96%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO:

3.

5. A schistocytic strain for high expression of EPA production, characterized in that, Obtained from the original strain HS01 through mutagenesis, and compared to the original strain, its genome contains at least two structural variations in gene coding regions. The first structural variation, SV1, is a nucleotide sequence insertion that results in a frameshift mutation in the coding region associated with EPA synthesis. The second structural variant, SV2, is a deletion of a nucleotide sequence, which results in the deletion of at least three gene sequences.

6. The schistocytic strain according to claim 5, characterized in that, The nucleotide sequence encoded by the SV1 position-related gene is shown in SEQ ID NO:1, followed by a nucleotide sequence of 2295 bp in length.

7. The schistocytic strain according to claim 5, characterized in that, The SV1 causes partial or complete loss of function of the corresponding protein.

8. The schistocytic strain according to claim 5, characterized in that, The SV2 mutation resulted in a 9322bp deletion, with three gene sequences deleted: SV2-1, SV2-2, and SV2-3, whose nucleotide sequences are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

9. The schistocytic strain according to claim 5, characterized in that, The structural variation is an insertion variation, a deletion variation, or a combination thereof.

10. A method for preparing the schistocytic chyme strain according to any one of claims 5 to 9, characterized in that, Includes the following steps: The original strain HS01 was subjected to mutagenesis. The mutagenized strains were screened to obtain candidate strains with increased EPA production; Genome analysis of the candidate strains confirmed the presence of at least the structural variants SV1 and SV2 in their genomes. This resulted in obtaining a schistocytic strain that highly expresses EPA production.

11. The preparation method according to claim 10, characterized in that, The mutagenesis process includes first chemical mutagenesis and plasma mutagenesis.

12. The preparation method according to claim 11, characterized in that, The first chemical mutagenesis includes the original strain HS01 being mutated by sodium azide to obtain a first mutagenic bacterial group, the first mutagenic bacterial group being subjected to plasma mutagenesis to obtain a second mutagenic bacterial group, and the second mutagenic bacterial group being subjected to adaptive mutagenesis and screening under selective culture conditions containing 2,2′-bipyridine to obtain strain HS09.

13. The use of the fissuri strain according to any one of claims 5 to 9 in the production of EPA.

14. A method for producing EPA, characterized in that, Fermentation broth was obtained by fermenting the schistocytic strain according to any one of claims 5 to 9, and the fermentation broth was separated and purified to obtain EPA.

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