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CN-121991819-A - Archaea phosphatidylinositol production strain, construction method and application thereof

CN121991819ACN 121991819 ACN121991819 ACN 121991819ACN-121991819-A

Abstract

The invention provides an archaea phosphatidylinositol producing strain, a construction method and application thereof. The molecular formula of the archaea phosphatidylinositol is C 49 H 83 O 11 P, and the strain is Saccharomyces cerevisiae (Saccharomyces cerevisiae) which over-expresses G1PDH, GGGPS, DGGGPS, carS, AIPS and INO 1. The strain has higher thermal stability, and the obtained archaea phosphatidylinositol uDoPhPI is also proved to be capable of forming liposome and has higher thermal stability.

Inventors

  • SI TONG
  • LI JINZE
  • ZHANG SHIZHE
  • GUO ERPENG
  • HONG ZHILAI

Assignees

  • 中国科学院深圳先进技术研究院

Dates

Publication Date
20260508
Application Date
20251231

Claims (10)

  1. 1. An archaea phosphatidylinositol producing strain, wherein the molecular formula of the archaea phosphatidylinositol is C 49 H 83 O 11 P, and the strain is saccharomyces cerevisiae (Saccharomyces cerevisiae) which over-expresses G1PDH, GGGPS, DGGGPS, carS, AIPS and INO 1.
  2. 2. The archaea phosphatidylinositol producing strain of claim 1, wherein the transcriptional units of G1PDH, GGGPS, DGGGPS, carS, AIPS and INO1 are linked into a metabolic pathway that is integrated into position 106a of the saccharomyces cerevisiae genome.
  3. 3. The method for constructing an archaea phosphatidylinositol producing strain according to claim 1 or 2, wherein the step of the construction method is to construct the archaea phosphatidylinositol producing strain expressing the above genes by introducing G1PDH, GGGPS, DGGGPS, carS, AIPS and INO1 into Saccharomyces cerevisiae (Saccharomyces cerevisiae) through a plasmid expression vector or integrating into Saccharomyces cerevisiae chromosome through genetic engineering means.
  4. 4. A method of construction according to claim 3, wherein the expressed promoter is a constitutive promoter and/or an inducible promoter.
  5. 5. The construction method according to claim 3, wherein the construction method comprises the steps of constructing transcription units of G1PDH, GGGPS, DGGGPS, carS, AIPS and INO1, assembling the multi-gene construct by the Golden Gate cloning method, connecting 6 transcription units into a metabolic pathway, and integrating the metabolic pathway into the 106a site of the yeast genome by the CRISPR/Cas method to obtain the archaea phosphatidylinositol producing strain.
  6. 6. A method for producing archaea phosphatidylinositol by using a strain, which produces archaea phosphatidylinositol by using the archaea phosphatidylinositol producing strain constructed by the method of any one of claims 3 to 5.
  7. 7. The method of claim 6, wherein the method further produces a hybrid lipid.
  8. 8. Use of the archaea phosphatidylinositol producing strain of claim 1 or 2 or the method of claim 6 or 7 for the preparation of archaea phosphatidylinositol.
  9. 9. A giant unilamellar vesicle prepared from archaea phosphatidylinositol obtained by the method of claim 6 or 7.
  10. 10. Use of the giant unilamellar vesicle of claim 9 for the preparation of a drug delivery system.

Description

Archaea phosphatidylinositol production strain, construction method and application thereof Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to an archaea phosphatidylinositol production strain, a construction method and application thereof. Background Lipids are important cell membrane components, energy storage and signaling molecules in organisms, and play a key role in regulating cell membrane fluidity, energy metabolism, signaling and the like. In recent years, with the development of synthetic biology and metabolic engineering, the realization of controllable biosynthesis of lipids of specific structure and function by microorganisms, particularly yeast strains, has become an important direction in the field of biological manufacturing. Natural yeasts (such as Saccharomyces cerevisiae, yarrowia lipolytica, etc.) have definite lipid anabolism pathways, can synthesize neutral lipids (mainly triacylglycerols, TAG) and polar lipids (such as phospholipids), and have wide application in biotechnology fields such as dietary supplements, etc. Lipid composition and structure not only affect nutrient absorption, but are also closely related to cell aging, membrane homeostasis and metabolic diseases. In recent years, it has been found that specific phospholipids and derivatives thereof can modulate intestinal barrier integrity, delay aging and promote energy homeostasis. Therefore, the development of an engineered microbial system capable of precisely regulating the type and structure of lipid molecules is of great importance for the production of lipid compositions having specific physiological functions or nutritional values. WO2025212524A1 discloses the creation of a unique lipid composition by engineering yeasts (in particular over-expressing HAP4 and SIR2 genes) to remodel their lipid metabolism. The composition comprises at least 12 specific lipids, of which Phosphatidylcholine (PC) and Triglycerides (TG), especially TG rich in medium short chain fatty acids, have proven to be key components for synergistic effects. These engineered yeast-derived lipids can be absorbed by the intestinal tract and integrated into the cell membrane, acting to enhance intestinal barrier function, regulate lipid metabolism, and delay cellular aging, ultimately achieving significant life-prolonging effects in animal models. WO2024238964A1 discloses the use of engineered Yarrowia (Yarrowia) for the production of triglycerides of specific structure (TAG) with the aim of mimicking the structure of human milk fat for infant formula. The patent provides genetically engineered yarrowia (especially yarrowia lipolytica, yarrowia lipolytica). The key feature of this yeast is the inclusion of a heterologous gene encoding a lysophosphatidic acid acyltransferase (16:0 LPAAT) specific for palmitoyl CoA (C16:0-CoA). LPAAT is a key enzyme in the triglyceride synthesis pathway responsible for linking fatty acids to the sn-2 site of the glycerol backbone. By introducing the LPAAT with specificity to C16:0 (palmitic acid), engineering yeast can efficiently and accurately place palmitic acid on the sn-2 position of TAG molecules, so that TAG with high sn-2 position palmitic acid is generated, the structure of human milk fat can be simulated, and the engineering yeast can be absorbed and utilized better. CN119331742A (high-yield squalene saccharomyces cerevisiae gene engineering strain and application thereof) discloses a three-dimensional linkage technical scheme for systematically integrating cell structure biology and metabolic balance control, and adopting organelle compartmentalization, lipid metabolism coupling and cofactor regeneration to improve accumulation amount and cell tolerance of the squalene, thereby obtaining the high-yield squalene saccharomyces cerevisiae gene engineering strain. Chenyang Zhang et al discloses the synthesis of 1, 3-oleic acid-2-palmitoyl glyceride (OPO) and 1-oleic acid-2-palmitoyl-3-linoleate from the head by multiple recombination of yeast lipid metabolism （OPL）（Zhang C, Zhou X, Wei W, Yu J, Wu Y, Liu Y, Li J, Du G, Chen J, Xu T, Lv X, Xu X, Liu L. De novo production of 1,3-olein-2-palmitin (OPO) and 1-olein-2-palmitin-3-linolein (OPL) by multiplexed reconstruction of lipid metabolism in yeasts. Metab Eng. 2025 Nov 5;94:1-14.）. Jianzhi Zhang et al, discloses a means for achieving heterologous synthesis of archaea-derived polar cell membrane lipids in eukaryotic cells by using engineered Saccharomyces cerevisiae, and obtaining a series of hybrid neutral lipids having both eukaryotic and archaeal characteristics, although the content of archaea cell membrane polar lipids in yeast models is still low （Zhang J, Li T, Hong Z, Ma C, Fang X, Zheng F, Teng W, Zhang C, Si T. Biosynthesis of Hybrid Neutral Lipids with Archaeal and Eukaryotic Characteristics in Engineered Saccharomyces cerevisiae. Angew Chem Int Ed Engl. 2023 Jan 23;62(4):e202214344.）. Clinical