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CN-122012535-A - Nucleotide sequence related to EPA production, engineering strain and application thereof

CN122012535ACN 122012535 ACN122012535 ACN 122012535ACN-122012535-A

Abstract

The invention relates to the field of gene engineering and microorganism technology, and in particular discloses a nucleotide sequence related to EPA production, an engineering strain and application thereof, wherein the invention reduces the consumption of irrelevant metabolic pathways by cells through mutagenesis to cause early termination or deletion of partial genes, lightens metabolic burden, is beneficial to the directional distribution of carbon sources to EPA synthetic pathways, is different from the technical route of over-expressing desaturase or elongase to improve EPA yield, introduces specific structural variation into the genome of the mutagenized schizochytrium, comprises inserting nucleotide sequences into a coding region of a target gene to cause early termination of coding proteins, and another structural variation causes deletion of genome fragments and inactivation of a plurality of genes, and the fatty acid synthesis pathway is effectively reconstructed from the global metabolism regulation level, and the second aspect provides an engineering strain which is not obviously deteriorated compared with the original strain in biomass, growth rate and culture adaptability, and simultaneously, EPA yield is obviously improved, so that the technical contradiction that the EPA yield is often accompanied with growth inhibition in the prior art is overcome, the genetic stability is high, the higher EPA yield can be still maintained in the multi-generation subculture process, and the engineering strain is suitable for continuous fermentation or batch fermentation production on an industrial scale.

Inventors

  • CHEN LIYI
  • ZHONG HUICHANG
  • CHEN SHUIRONG
  • CUI LINGLING

Assignees

  • 厦门荣盛生物有限公司

Dates

Publication Date
20260512
Application Date
20251231

Claims (14)

  1. 1. A mutated nucleotide sequence, characterized in that it has been inserted after the 360 th sequence with respect to SEQ ID No. 1 with nucleotide sequence SEQ ID No. 2, said insertion resulting in premature termination of the corresponding encoded protein.
  2. 2. The mutant nucleotide sequence according to claim 1, wherein the nucleotide sequence has a length of 2295bp relative to the insertion sequence after position 360 of SEQ ID No. 1.
  3. 3. The mutant nucleotide sequence according to claim 1 or 2, wherein the mutant nucleotide sequence comprises the sequence shown in SEQ ID No. 3.
  4. 4. A nucleotide sequence characterized in that said sequence is at least 95%, 96%, 98% or 99% identical to the nucleotide sequence set forth in SEQ ID No. 3.
  5. 5. A schizochytrium limacinum strain for high-expression EPA production, which is characterized in that the schizochytrium limacinum strain is obtained by mutagenesis treatment of an original strain HS01, and compared with the original strain, the schizochytrium limacinum strain has at least two structural variations of gene coding regions in the genome, The first structural variation, SV1, is a nucleotide sequence insertion that results in a frame shift mutation in the coding region associated with EPA synthesis; The second structural variation, SV2, is a deletion of a nucleotide sequence, which results in the deletion of at least three gene sequences.
  6. 6. The schizochytrium limacinum 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, and the length of the inserted nucleotide sequence is 2295bp.
  7. 7. The schizochytrium strain of claim 5, wherein SV1 results in partial or complete loss of function of the corresponding protein.
  8. 8. The schizochytrium limacinum strain according to claim 5, characterized in that the SV2 mutation results in a 9322bp deletion, 3 gene sequence deletions SV2-1, SV2-2, SV2-3, with nucleotide sequences shown as SEQ ID NO.4, SEQ ID NO. 5, SEQ ID NO. 6, respectively.
  9. 9. The schizochytrium strain of claim 5, wherein the structural variation is an insertion variation, a deletion variation, or a combination thereof.
  10. 10. A method of preparing a schizochytrium strain according to any of claims 5 to 9, comprising the steps of: Carrying out mutagenesis treatment on an original strain HS 01; screening the strain after mutagenesis to obtain a candidate strain with improved EPA yield; Performing genome analysis on the candidate strain to confirm that at least the structural variation SV1 and the structural variation SV2 exist in the genome of the candidate strain; thus obtaining the schizochytrium limacinum strain with high expression for producing EPA.
  11. 11. The method of claim 10, wherein the mutagenesis comprises a first chemical mutagenesis, a plasma mutagenesis.
  12. 12. The method of claim 11, wherein the first chemical mutagenesis comprises subjecting the original strain HS01 to sodium azide mutagenesis to obtain a first mutagenized population, subjecting the first mutagenized population to plasma mutagenesis to obtain a second mutagenized population, and subjecting the second mutagenized population to adaptive mutagenesis and screening under selective culture conditions comprising 2,2' -bipyridine to obtain strain HS09.
  13. 13. Use of the schizochytrium strain of any of claims 5 to 9 in the production of EPA.
  14. 14. A process for producing EPA, characterized in that a fermentation broth obtained by fermentation culture of the schizochytrium strain according to any one of claims 5 to 9 is isolated and purified to obtain EPA.

Description

Nucleotide sequence related to EPA production, engineering strain and application thereof Technical Field The invention relates to the technical field of nucleotides, and particularly discloses a nucleotide sequence related to EPA production, an engineering strain and application thereof. Background EPA (eicosapentaenoic acid) is a long-chain omega-3 series polyunsaturated fatty acid, is a key precursor for biosynthesis of eicosanoids, and is an important signal molecule involved in inflammation, so that EPA has multiple functions in regulating physiological health of organisms, such as anti-inflammation, antioxidation, preventing cardiovascular diseases, anticancer, treating depression, regulating blood pressure, blood fat, blood sugar and the like, has potential beneficial effects on atherosclerosis plaque factors and losing weight, plays positive roles in preventing Alzheimer's disease, relieving retinopathy and the like, but cannot synthesize EPA by human bodies per se, and can only be taken from food. Deep sea fish oil is a main source of EPA industrial production, but the fatty acid component in fish oil is complex, and the content of various chain lengths and saturation can directly influence the efficacy of the fish oil. Moreover, fish oil has heavy smell and high purification cost, which leads to the shortage of supply. EPA in fish oils accumulates through the food chain, so microorganisms such as algae, lower fungi, and marine bacteria are the primary sources of EPA. The DHA-producing bacteria commonly used at present are schizochytrium, and EPA-producing bacteria are Shewanella sp. Photobacteriumprofundum respectively. The fermentation production of EPA has not been realized in large scale, and the biomass of several EPA producing bacteria commonly used is far lower than that of schizochytrium. Schizochytrium is a marine microalgae with high DHA yield, but the EPA content in the schizochytrium is relatively low, the effect of changing the proportion of unsaturated fatty acid in the schizochytrium is limited by means of culture medium optimization, fermentation regulation and control and the like, and researchers improve EPA strains by means of mutation breeding, laboratory adaptive evolution, genetic engineering and the like, so that EPA production strains with excellent characters and high efficiency are obtained. In schizochytrium, biosynthesis of EPA and DHA is realized by catalysis of a series of enzymes in related anabolic pathways, and current research results show that the synthetic pathways of the schizochytrium EPA and DHA mainly comprise 2 (1) fatty acid synthase (FATTY ACID SYNTHASE, FAS) pathways, which take acetyl-CoA and malonyl-CoA as substrates, synthesize palmitic acid (C16:0) through multiple condensation, reduction, dehydration, reduction and other steps, and form final products of a FAS pathway stearic acid (SAC 18:0) through catalysis of a series of desaturases and desaturases, and then synthesize EPA, DPA, DHA and the like under the action of the series of desaturases and the elongases. (2) The polyketide synthase pathway (Polyketide SYNTHASE PATHWAY, PKS pathway) synthesizes EPA or DHA mainly from acetyl CoA and malonyl CoA as precursors under the action of polyketide synthase, but specific synthetic pathways and mechanisms are currently under debate. Meanwhile, organisms are a complex whole, and a wide gene regulation network exists inside the organisms. Any mutation of other genes related to metabolism can also indirectly influence the EPA and DHA production, and analysis of the genome of the high-yield EPA strain is helpful for digging related genes of EPA and DHA biosynthesis pathways, and provides favorable conditions for further improving the EPA production of schizochytrium. Disclosure of Invention In view of the problems of the prior art, the first aspect of the present invention proposes a mutated nucleotide sequence, which has been inserted after the 360 th step with respect to SEQ ID NO. 1 with the nucleotide sequence SEQ ID NO. 2, which insertion results in premature termination of the corresponding encoded protein. In some specific embodiments of the mutant nucleotide sequence of the first aspect, the nucleotide sequence is an insert sequence after position 360, relative to SEQ ID NO. 1, the insert sequence having a length of 2295 bp. In some specific embodiments of the mutant nucleotide sequences of the first aspect, the mutant nucleotide sequences comprise SEQ ID NO. 3. In a second aspect the invention provides a nucleotide sequence which is at least 95%, 96%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO. 3. In a third aspect, the present invention provides a schizochytrium limacinum strain for highly expressing EPA, obtained by mutagenesis treatment of an original strain HS01, having at least two structural variations in the coding region of the gene in the genome as compared to the original strain, The first structural variation, SV1,