Archaea phosphatidylinositol production strain as well as construction method and application thereof

Abstract

The invention provides an archaea phosphatidylinositol producing strain as well as a construction method and application thereof. The molecular formula of the archaea phosphatidylinositol is C49H83O11P, and the archaea phosphatidylinositol is Saccharomyces cerevisiae which is used for over-expression of G1PDH, GGGPS, DGGGPS, CarS, AIPS and INO1. The archaea phosphatidylinositol is a saccharomyces cerevisiae which is used for over-expression of G1PDH, GGGPS, DGGGPS, CarS, AIPS and INO1. The strain has relatively high thermal stability, and the obtained archaea phosphatidylinositol uDoPhPI is also proved to be capable of forming lipidosome and has very high thermal stability.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to archaea phosphatidylinositol-producing strains, their construction methods, and applications. Background Technology

[0002] Lipids are important cell membrane components, energy storage, and signaling molecules in organisms, playing a crucial role in regulating cell membrane fluidity, energy metabolism, and signal transduction. In recent years, with the development of synthetic biology and metabolic engineering, the controlled biosynthesis of lipids with specific structures and functions using microorganisms, especially yeast strains, has become an important direction in the field of biomanufacturing. Natural yeasts (such as *Saccharomyces cerevisiae* and *Yarrowia lipolytica*) have well-defined lipid synthesis pathways, capable of synthesizing neutral lipids (mainly triacylglycerols, TAGs) and polar lipids (such as phospholipids), which have wide applications in biotechnology fields such as dietary supplements. Lipid composition and structure not only affect nutrient absorption but are also closely related to cellular aging, membrane homeostasis, and metabolic diseases. Recent studies have found that specific phospholipids and their derivatives can regulate intestinal barrier integrity, delay aging, and promote energy homeostasis. Therefore, developing engineered microbial systems capable of precisely controlling the types and structures of lipid molecules is of great significance for producing lipid compositions with specific physiological functions or nutritional value.

[0003] WO2025212524A1 discloses a unique lipid composition produced by genetically engineering yeast (specifically overexpressing the HAP4 and SIR2 genes) to reshape its lipid metabolism. This composition contains at least 12 specific lipids, among which phosphatidylcholine (PC) and triglycerides (TG), particularly TG rich in medium- and short-chain fatty acids, have been shown to be key components for synergistic effects. These lipids derived from engineered yeast can be absorbed by the intestine and integrated into the cell membrane, enhancing intestinal barrier function, regulating lipid metabolism, and delaying cellular senescence, ultimately resulting in a significant lifespan extension effect in animal models.

[0004] WO2024238964A1 discloses the production of structurally specific triglycerides (TAGs) using engineered Yarrowia yeast, aiming to mimic the structure of human milk fat for use in infant formula. The patent provides a genetically engineered Yarrowia yeast (specifically, Yarrowia lipolytica). A key feature of this yeast is the inclusion of a heterologous gene encoding a lysophosphatidyl acyltransferase (16:0 LPAAT) specific for palmitoyl-CoA (C16:0-CoA). LPAAT is a crucial enzyme in the triglyceride synthesis pathway, responsible for linking fatty acids to the sn-2 site of the glycerol backbone. By introducing this C16:0 (palmitic acid) specific LPAAT, the engineered yeast can efficiently and precisely place palmitic acid at the sn-2 position of the TAG molecule, thus producing TAGs with high sn-2 palmitic acid content, mimicking the structure of human milk fat and resulting in better absorption and utilization.

[0005] CN119331742A (A high-squalene-producing Saccharomyces cerevisiae genetically engineered strain and its application) discloses a systematic integration of cell structural biology and metabolic balance control, employing a three-dimensional linkage technology scheme of organelle compartmentalization, lipid metabolism coupling and cofactor regeneration to improve the accumulation of squalene, a squalene-producing lipid, and cell tolerance, thereby obtaining a high-squalene-producing Saccharomyces cerevisiae genetically engineered strain.

[0006] The research by Chenyang Zhang et al. disclosed the de novo synthesis 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.

[0007] The research by Jianzhi Zhang et al. disclosed the use of engineered Saccharomyces cerevisiae to achieve heterologous synthesis of archaeal polar cell membrane lipids in eukaryotic cells, and obtained a series of hybrid neutral lipids with both eukaryotic and archaeal characteristics. However, the content of archaeal cell membrane polar lipids in the yeast model 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 Lipidswith Archaeal and Eukaryotic Characteristics in Engineered Saccharomycescerevisiae. Angew Chem Int Ed Engl. 2023 Jan 23;62(4):e202214344.).

[0008] Clinical studies have confirmed that archaeal liposomes, as a safe and highly effective vaccine adjuvant, can simultaneously activate humoral and cellular immune responses, exhibiting excellent immunostimulatory capabilities. Their stability advantage primarily stems from the abundance of archaeal phosphatidylinositol in natural archaeal membranes. This component significantly enhances the structural integrity of liposomes by strengthening the hydrogen bond network and optimizing membrane structural order. Currently, the main methods for obtaining archaeal lipids include strain extraction, chemical synthesis, and enzymatic synthesis. However, existing production technologies still face multiple challenges: industrial-scale cultivation of thermophilic archaea is hampered by high engineering difficulty, long cycles, high costs, and low yields; chemical synthesis routes are complex and prone to contamination; and enzymatic methods are limited by synthesis costs and the difficulty of scaling up production. These factors collectively restrict the commercialization of archaeal liposome technology.

[0009] The production of specific functional lipids using industrial production chassis yeast strains has broad application prospects in drug delivery, vaccine adjuvants, functional foods, medical oils, liposomes, or high-energy fuels, and is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0010] To address the aforementioned technical problems, the present invention aims to provide an archaeal phosphatidylinositol production strain, its construction method, and its applications. This strain is capable of producing unsaturated di-O-phytanylphosphatidylinositol (uDoPhPI) and hybrid lipids, and has broad application prospects.

[0011] Saccharomyces cerevisiae, a recognized safe food-grade microorganism, possesses mature genetic manipulation tools, large-scale fermentation processes, and a complete industrial foundation. Utilizing it as a cell factory, efficient biosynthesis of archaic phosphatidylinositol and hybrid lipids can be achieved, providing a more feasible industrialization path for promoting the commercial application of archaic liposomes in drug delivery, vaccine adjuvants, and dietary supplements.

[0012] To achieve the above objectives, the present invention provides an archaea phosphatidylinositol producing strain, wherein the molecular formula of the archaea phosphatidylinositol is C2. 49 H 83 O 11 P, this strain is a Saccharomyces cerevisiae strain that overexpresses G1PDH, GGGPS, DGGGPS, CarS, AIPS and INO1.

[0013] In the aforementioned archaea phosphatidylinositol producing strains, preferably, the transcriptional units of G1PDH, GGGPS, DGGGPS, CarS, AIPS, and INO1 are linked together to form a metabolic pathway, which is integrated into the 106a site of the Saccharomyces cerevisiae genome.

[0014] The present invention also provides a method for constructing the above-mentioned archaea phosphatidylinositol producing strain, wherein the steps of the construction method are as follows: by introducing G1PDH, GGGPS, DGGGPS, CarS, AIPS and INO1 into Saccharomyces cerevisiae via a plasmid expression vector or integrating them into the chromosome of Saccharomyces cerevisiae through genetic engineering, an archaea phosphatidylinositol producing strain expressing the above genes is constructed.

[0015] According to the above construction method, preferably, the expressed promoter is a constitutive promoter and / or an inducible promoter; more preferably, the promoter is an inducible promoter; and even more preferably, the inducible promoter is a P... GAL1 .

[0016] According to the above construction method, preferably, the construction method specifically includes the following steps: constructing transcription units of G1PDH, GGGPS, DGGGPS, CarS, AIPS and INO1, assembling a multi-gene construct using the Golden Gate cloning method, connecting the six transcription units into a metabolic pathway, and integrating the metabolic pathway into the 106a site of the yeast genome using the CRISPR / Cas method to obtain an archaeal phosphatidylinositol producing strain.

[0017] The present invention also provides a method for producing archaeal phosphatidylinositol using a strain, wherein the method utilizes an archaeal phosphatidylinositol producing strain constructed according to the method described above to produce archaeal phosphatidylinositol.

[0018] According to the above method, preferably, the method also produces hybrid lipids.

[0019] The present invention also provides the application of the above-mentioned archaeal phosphatidylinositol producing strain or the above-mentioned method in the preparation of archaeal phosphatidylinositol.

[0020] The present invention also provides a giant monolayer vesicle, which is prepared from archaeal phosphatidylinositol obtained by the above method.

[0021] The present invention also provides the application of the above-mentioned giant monolayer vesicles in the preparation of drug delivery systems.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention reconstructs the archaeological phosphatidylinositol (CDP-C) synthesis pathway in the eukaryotic host *Saccharomyces cerevisiae*, enabling the synthesis of archaeological membrane lipids in a eukaryotic system. This is achieved through genetic engineering of the yeast, including overexpression of G1PDH, GGGPS, DGGGPS, and CarS in yeast cells and enhanced GGPP supply to produce CDP-C ... Attached Figure Description

[0024] Figure 1 To construct the Golden Gate assembly scheme for archaeal lipid biosynthesis pathways.

[0025] Figure 2 This is a schematic diagram of the preparation and purification process of uDoPhPI. It includes fermentation culture, organic solvent extraction, rotary evaporation, silica gel column chromatography separation, and lyophilization steps.

[0026] Figure 3Production of uDoPhPI in different engineered yeast strains. The left figure illustrates the pathways present in each strain. Engineered yeast strain construction protocols used to compare uDoPhPI synthesis capabilities include 4U (MaG1PDH, MaGGGPS, MaDGGGPS, AfCarS), 4U+A (+ApAIPS), 4U+I (+ScINO1), 4U+AI (+ApAIPS +ScINO1), 4U-G+AI (+ApAIPS +ScINO1, MaG1PDH missing, replaced by Venus), and 4U-C+AI (+ApAIPS +ScINO1, AfCarS missing, replaced by Venus). The right figure shows uDoPhPI abundance determined by LC-MS / MS multiple reaction monitoring (MRM), normalized to cellular biomass. Fold change is relative to the baseline 4U strain (set as 100%). Data are presented as mean ± standard error (SEM) of three independent biological replicates.

[0027] Figure 4 The NMR spectrum of uDoPhPI is shown as a 1H NMR spectrum.

[0028] Figure 5 This figure illustrates the impact of pathway engineering on cellular lipid profiles. Different pathway engineering strategies were used to assess the lipid composition of yeast cells. Changes in lipid abundance were quantified using MRM mode of LC-MS / MS, with a 4U baseline strain as a control. Results are expressed as fold changes. Figure 5 The a represents the types of yeast endogenous phosphatidylinositol (PI) molecules, including C34:1PI, C32:0PI, and C32:1PI. Figure 5 b represents yeast triglycerides (TAG), including C45:1 TAG, C47:1 TAG, and C53:2 TAG. Figure 5 c represents the archaea neutral lipid DGGGOH. Figure 5 d represents heterozygous DGGGO-acyl esters with different fatty acid chain lengths, including C8:0, C10:0, C12:0, C14:1, C16:0, C16:1, C18:0, and C18:1. Data are expressed as mean ± standard error (SEM) from three independent biological replicates. Statistical significance was determined by one-way ANOVA, with p < 0.01 compared to 4U.

[0029] Figure 6 Flow cytometry analysis of neutral lipid accumulation in engineered yeast. Figure 6 'a' represents the fluorescence intensity histogram of strains 4U, 4U+A, 4U+I, and 4U+AI after Nile Red staining. Figure 6b represents the quantification result of the mean fluorescence intensity of neutral lipids for each strain. Data are expressed as mean ± standard error (SEM) and are from two independent biological replicates.

[0030] Figure 7 Characterization of uDoPhPI liposome formation and stability. Figure 7 Image a is a confocal microscopy image of uDoPhPI giant monolayer vesicles (GUVs) loaded with calcein, prepared by electroforming; top: single vesicle; bottom: vesicle population; green: encapsulated calcein; magenta: membrane marker DOPE-Atto647N. Figure 7 b is the average diameter of the small monolayer vesicle (SUV) measured by DLS. Figure 7 c represents the time-dependent release of calcein in the SUV during storage at 4°C. Figure 7 d represents the temperature-dependent release of calcein in the SUV under pH 7.0 conditions. Figure 7 b to Figure 7 The data in d are represented as the average of three independent preparations ± SEM. Black squares: DPPC; red dots: uDoPhPI; Figure 7 The red triangle in b: uDoPhPI after 4 weeks of storage at 4°C. Detailed Implementation

[0031] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0032] It should be noted that, unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0034] It should be understood that the terms “comprising,” “including,” and / or “containing” as used herein specify the presence of the stated features, integers, steps, components, or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, components, or combinations thereof.

[0035] The endpoints and any values ​​of the ranges disclosed in this invention 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 in this invention.

[0036] Example 1:

[0037] This embodiment provides an archaeal phosphatidylinositol production strain and its construction method, as well as a method for producing archaeal phosphatidylinositol (uDoPhPI) and preparing giant and small monolayer vesicles using this strain, as detailed below:

[0038] I. Preparation Method

[0039] (1) Construction of recombinant DNA

[0040] The plasmids constructed and used in this invention are listed in Tables 1-2 (Table 2 is a continuation of Table 1). The primers and genes were synthesized by Genewiz (China), and the primer sequences are shown in Table 3. Where "Zhang et al., 2023" refers to Zhang J, LiT, Hong Z, Ma C, Fang X, Zheng F, Teng W, Zhang C, Si T. Biosynthesis ofHybrid Neutral Lipids with Archaeal and Eukaryotic Characteristics inEngineered Saccharomyces cerevisiae. Angew Chem Int Ed Engl. 2023 Jan 23;62(4):e202214344; "Lee et al., 2015" refers to Lee, ME, DeLoache, WC, Cervantes, B. &Dueber, JE A Highly Characterized Yeast Toolkit for Modular, MultipartAssembly. ACS Synth Biol 4, 975-986 (2015).

[0041] Table 1

[0042]

[0043] Table 2

[0044]

[0045] Table 3

[0046]

[0047] The ApAIPS gene (Aeropyrum pernix, APE1526) was synthesized after codon optimization for expression in *S. cerevisiae*. The ScINO1 gene (YJL153C) of *Saccharomyces cerevisiae* was amplified from its genomic DNA. Restriction endonucleases, T4 DNA ligase, and the Gibson Assembly kit were purchased from New England Biolabs (USA). Phanta Max Master Mix was purchased from Novizan Biotechnology Co., Ltd. The QIAprep Spin Plasmid Mini-prep kit for preparing plasmid DNA from *E. coli* was purchased from Qiagen (Valencia, USA). PCR products and enzyme digestion products were purified using the QIAquick PCR purification kit and gel extraction kit, respectively. Yeast genomic DNA was extracted using the YeaStar Genomic DNA Kit (Zymo Research).

[0048] The archaeal lipid biosynthesis pathway plasmid was constructed using the Golden Gate Assembly MoClo-YTK kit (Addgene #1000000061) and modified accordingly. This system employs a two-level hierarchical assembly strategy (e.g., Figure 1 As shown, Figure 1 To construct the Golden Gate assembly scheme for the archaeal lipid biosynthesis pathway. The primary plasmid (Level 1, pYTK095 series) serves as a separate transcription unit, containing the inducible promoter P. GAL1 , Coded Sequence (CDS), Terminator T ENO1The plasmids contain a 6×His_3×Flag tag and linker elements (ConL, ConR) with BsmBI restriction sites. Primary plasmids encoding archaeal lipid synthesis-related enzymes (MaG1PDH, MaGGGPS, MaDGGGPS, and AfCarS) have been constructed in the study of Jianzhi Zhang et al. (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.). In this invention, a new primary plasmid is constructed using a two-fragment Gibson Assembly for overexpression of ApAIPS or ScINO1, or by replacing the target gene with the Venus fluorescent protein gene. The two fragments are: (1) a PCR product or synthetic gene with a 20-30bp homologous arm; and (2) the pYTK095 backbone vector obtained by PCR amplification.

[0049] Secondary plasmids (pYTK096 series) contain complete metabolic pathways composed of multiple transcriptional units and carry homologous recombination sequences for integration into the S. cerevisiae genome at site 106a. During secondary plasmid construction, six primary plasmids (pYTK095-MaG1PDH, -MaGGGPS, -MaDGGGPS, -AfCarS, -ApAIPS, and -ScINO1) were assembled with the recipient vector pYTK096-ccdB in a BsmBI system. For gene deletion variants, the corresponding primary plasmid was replaced with the corresponding Venus replacement plasmid. The total volume of the Golden Gate assembly reaction system was 5 μL, containing: 0.02 pmol of each DNA component, 1000 U of T4 DNA ligase, 0.5 μL of T4 ligase buffer, 5 U of BsmBI restriction endonuclease, and the remainder was added to sterile water. The reaction procedure was as follows: the assembly procedure consisted of 45 cycles, each cycle consisting of 1 min at 42°C and 1 min at 16°C, followed by treatment at 65°C for 20 min.

[0050] The assembled product was transformed into E. coli DH5α by chemical transformation and screened by culturing at 37°C on Luria-Bertani (LB) agar plates containing 50 μg / mL kanamycin or 100 μg / mL ampicillin. Subsequently, liquid culture was used for plasmid extraction.

[0051] (2) Construction of yeast strains

[0052] The yeast strains used in this invention are listed in Table 4. "Zhang et al., 2023" refers to Zhang J, LiT, 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; "Reider Apel et al., 2017" refers to Reider Apel, A. et al. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. NucleicAcids Res 45, 496-508 (2017).

[0053] Table 4

[0054]

[0055] Following the construction method of the previous basic strain 4U, the sequenced-validated secondary plasmid was linearized with NotI and transformed into competent Saccharomyces cerevisiae cells using the standard heat shock method. The plasmid pCUT-KanMX encoding the CRISPR / Cas system was co-transformed with the linearized donor DNA to induce a double-strand break at the 106a site, thereby achieving efficient and seamless integration of the donor DNA. Transformants were screened on YPD (yeast extract peptone glucose medium) plates containing 800 μg / mL of G418 (Beijing Wokai Biotechnology Co., Ltd., China), and correct genome integration was verified by colony PCR (diagnostic colony PCR).

[0056] (3) Production, purification and structural analysis of uDoPhPI

[0057] To prepare seed cultures, the highest-yielding strain (4U+AI) was first cultured in shake flasks containing YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 30°C and 200 rpm. To induce production, cells were then inoculated at an initial OD600 of 0.1 into 4 L of YPG medium (1% yeast extract, 2% peptone, 2% galactose) and cultured under the same conditions for 48 hours.

[0058] For the purification of uDoPhPI (e.g.) Figure 2 As shown, Figure 2 This is a schematic diagram of the preparation and purification process of uDoPhPI. It includes fermentation culture, organic solvent extraction, rotary evaporation, silica gel column chromatography, and lyophilization. Cells were collected by centrifugation, yielding 24g of stem cells. The cell pellet was ground using a mortar and pestle under liquid nitrogen conditions. The crude extract was mixed with 500mL of methanol / chloroform (2:1, v / v) and placed in a 2L separatory funnel. Phase separation was induced by sequentially adding 200mL of chloroform and 200mL of water. The lower organic phase was collected, and the remaining material was extracted 3-5 times. The combined organic phase extract was concentrated using a rotary evaporator. The concentrated extract was redissolved in 2mL of n-hexane and 2mL of chloroform and loaded onto a silica gel column (200-300 mesh). uDoPhPI was eluted using a chloroform-methanol-ammonia (75:25:0.4, v / v / v) gradient, and the result was analyzed by mass spectrometry (m / z 896.6 [M+NH4]). + The elution fractions were monitored. Fractions containing uDoPhPI were combined and lyophilized, yielding 131.4 mg of purified product. Purity was assessed by high-performance liquid chromatography (HPLC) using an Agilent 1290 HPLC system, including a G7104A pump, a G7117B diode array detector (DAD), and a G7167B autosampler.

[0059] For NMR analysis, purified uDoPhPI was dissolved in 500 µL of deuterated chloroform and 200 µL of deuterated methanol (70 mg / mL), and NMR data were subsequently acquired on a 400 MHz Bruker Avance III NMR spectrometer. NMR data are reported as follows (e.g., Figure 4 As shown, Figure 4 The following data are presented for the 1H NMR spectrum of uDoPhPI: chemical shift (δ, ppm), splitting pattern (s = singlet, d = doublet, t = triplet), coupling constant (J, Hz), and integral, with TMS as an internal standard. NMR spectrum acquisition and data processing were performed using Topspin 4.5.0 software (Bruker Biospin).

[0060] High-resolution mass spectrometry (HR-MS) was performed using a timsTOF Flex mass spectrometer (Bruker Daltonics) to confirm the molecular formula. Mass spectrometry was acquired in electrospray ionization (ESI) positive ion mode with the following parameters: tims scan time 100 ms, PASEF on, 100 ms, scan range m / z 100–1350; 1 / k0 0.55–1.87 Vs / cm². 2 Non-targeted analysis data were acquired in both ESI positive and negative ion modes.

[0061] (4) Yeast lipid extraction for quantitative analysis

[0062] Yeast culture and induced production were performed according to the methods described in (1)-(3). After 48 hours, approximately 2 mL of sample, corresponding to 40 OD600 units (equivalent to 12 mg of stem cells), was collected for total lipid extraction using a modified Blighand Dyer method optimized for yeast phospholipids. Cells were precipitated by centrifugation (10000×g, 5 min) and lysed by vigorous shaking with 0.1 mm glass beads added directly to a chloroform / methanol extraction solvent (1:2, v / v). The mixture was vortexed for 1 min at room temperature. Phase separation was induced by adding chloroform and water (1:1, v / v). After centrifugation (2000×g, 5 min), the lipid-containing lower organic phase was carefully collected. The upper aqueous phase and interface were extracted again using the same method, and the organic phases were combined. The combined organic extract was dried under a nitrogen stream and stored at -80°C until analysis. Before analysis, the lipids were resuspended in 500 µL of methanol / isopropanol (1:1, v / v) to obtain the lipid extract.

[0063] (5) LC-MS / MS quantitative lipid analysis

[0064] Lipid extracts were analyzed using a triple quadrupole LC-MS / MS system equipped with an Agilent 1290 HPLC and an Agilent 6470B triple quadrupole mass spectrometer, operated in electrospray ionization (ESI) positive ion mode. Quantitative analysis was performed using multiple reaction monitoring (MRM) mode.

[0065] To achieve optimal separation of different lipid classes, three different chromatographic methods were employed. For the archaea uDoPhPI and DGGGOH (Method 1), two tandemly connected C18 AR UPLC columns (2.1 × 150 mm, 2 µm; ACE) were used for chromatographic separation, with the column temperature maintained at 55 °C and the flow rate at 0.5 mL / min. The injection volume was 2 µL, and elution was performed using the following gradient: 0 min, 72% mobile phase B; 8 min, 100% mobile phase B; 11 min, 72% mobile phase B, followed by an 8 min equilibration. Mobile phase A consisted of 50% acetonitrile and 50% water containing 5 mM ammonium acetate; mobile phase B consisted of 50% isopropanol and 50% acetonitrile containing 4 mM ammonium formate in 0.1% aqueous solution. To avoid signal saturation, the sample was diluted 1000-fold when detecting DGGGOH. MRM-converted ions were monitored under fragmentation voltage of 135V and collision energy of 20V, with uDoPhPI conversion to m / z 896.6. 335 ([M+NH4]) + The conversion of DGGGOH to m / z 654.4 273.3 ([M+NH4]) + ).

[0066] For yeast endogenous phosphatidylinositol species (Method 2), a single C18 AR UPLC column (2.1 × 150 mm, 2 µm; ACE) was used, along with the same chromatographic and mass spectrometric conditions as in Method 1. The MRM-converted ions were m / z 854.6. 577.5 (C34:1 PI, [M+NH4]) + ), 828.5 551.5 (C32:0 PI, [M+NH4]) + ) and 826.5 549.6 (C32:1 PI, [M+NH4]) + ).

[0067] For yeast TAG and hybrid DGGGO-acyl esters (Method 3), these neutral lipids were separated on a single C18 AR UPLC column (2.1 × 150 mm, 2 µm; ACE) at a temperature of 55 °C and a flow rate of 0.2 mL / min. The injection volume was 2 µL, and elution was performed using the following gradient: 0 min, 0% mobile phase B; 3.3 min, 0% mobile phase B; 5 min, 24% mobile phase B; 23 min, 65% mobile phase B; 24 min, 90% mobile phase B; 28 min, 90% mobile phase B, followed by a 5 min equilibration. Mobile phase A was methanol, and mobile phase B was isopropanol, both containing formic acid and ammonium hydroxide (100:0.04:0.1, v / v / v). MRM-converted ions were monitored at a fragmentation voltage of 135 V and a collision energy of 10 V. For yeast TAG, quantitative ion m / z 549.5 was used, and the corresponding [M+NH4] was monitored. + Precursor ions included C45:1 TAG (m / z 738.6), C47:1 TAG (m / z 766.5), and C53:2 TAG (m / z 848.6). For DGGGO-acyl esters, m / z 273.3 was chosen as the quantitative ion for the corresponding [M+NH4] ions. + The precursor ions were quantified relative to each other, including DGGGO-C8:0 (m / z 780.7), DGGGO-C10:0 (m / z 808.6), DGGGO-C12:0 (m / z 836.6), DGGGO-C14:1 (m / z 862.6), DGGGO-C16:1 (m / z 890.7), DGGGO-C16:0 (m / z 892.7), DGGGO-C18:1 (m / z 918.7), and DGGGO-C18:0 (m / z 920.7).

[0068] (6) Quantitative analysis of cellular lipids by flow cytometry

[0069] Neutral lipid content was assessed using Nile Red staining combined with flow cytometry. After 48 hours of galactose induction as described above, cells were collected, washed twice with phosphate-buffered saline (PBS, pH 7.4), and stained with 100 μM Nile Red (Thermo Fisher Scientific, USA) for 30 min at room temperature in the dark. After staining, cells were washed and resuspended in PBS for analysis using a CytoFLEX S flow cytometer (Beckman Coulter, USA), with excitation at 488 nm and emission detection at 585 nm. 10,000 gating events were collected for each sample, and the mean fluorescence intensity was calculated using CytExpert software to quantify the relative changes in neutral lipid levels among different strains.

[0070] (7) Heat resistance test

[0071] Engineered yeast strains were cultured under galactose-induced conditions for 48 hours as described above. The final culture was diluted in YPD medium to an OD600 of 0.001 to ensure a suitable cell density for colony-forming unit (CFU) counting. Equal volumes (20 µL) of the diluted culture were treated at 50 °C for different times (0 min, 10 min, 20 min, 30 min, and 60 min), while untreated samples stored at 30 °C served as controls. Immediately after heat treatment, the samples were plated onto YPD agar and incubated at 30 °C for 48 hours. Colonies were counted, and the survival rate at each time point was calculated as the percentage of surviving cells relative to the untreated control. Data are presented as mean ± standard error (SEM) of four independent biological replicates.

[0072] (8) Preparation of giant monolayer vesicles (GUVs)

[0073] Reference lipids included DOPE-Atto647N (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, labeled Atto647N) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), purchased from Avanti Polar Lipids (Alabaster, USA) and stored in chloroform solution at -20°C until use.

[0074] GUVs were prepared using an electrodeposition method on a VesiclePrepPro device (Nanion Technologies GmbH, Munich, Germany). Purified uDoPhPI and DOPE-Atto647N were mixed in chloroform at a molar ratio of 1000:1. 15 μL of the lipid mixture (1 mg / mL) was evenly spread onto the conductive surface of a glass cover slide coated with indium tin oxide (ITO) (Visiontek Systems Ltd, UK). The lipid-coated ITO slide was placed under vacuum for at least 15 min to ensure complete solvent evaporation. A rubber O-ring (18 mm inner diameter) was placed on the lipid-coated surface, and 280 μL of a 200 mM sucrose solution containing 20 μM calcein was added. A second ITO-coated slide was then placed on top to seal the reaction chamber. The assembled electrodeposition chamber was connected to the VesiclePrepPro electrode, and an alternating electric field was applied according to the multi-step procedure described in Table 5, which outlines the GUV electrodeposition preparation process. After electrodeposition was completed, GUV was collected by gentle centrifugation and immediately observed using a Nikon AX confocal laser scanning microscope with a CFI Plan Apochromat Lambda D60× oil immersion lens (Nikon, Japan) and excitation wavelengths of 488 nm (calcein) and 647 nm (DOPE-Atto647N).

[0075] Table 5

[0076]

[0077] (9) Preparation of small monolayer vesicles (SUVs)

[0078] Calcein-loaded SUVs were prepared using a membrane hydration and extrusion method. Purified uDoPhPI (5 mg) dissolved in chloroform was transferred to a brown glass vial, and the solvent was evaporated under a nitrogen stream. The vial was then dried under vacuum for 30 min to remove residual solvent. The dried lipid membrane was hydrated with 500 μL of a 106 mM calcein solution, vigorously vortexed, and sonicated in an ultrasonic water bath at 45 °C for 30 min. The resulting multilayer vesicle suspension was extruded 21 times through a 100 nm pore size polycarbonate membrane using a small extruder (AvantiPolar Lipids) to generate SUVs. Unencapsulated calcein was removed by gel filtration onto a Sephadex G-50 column (Sigma-Aldrich, USA) pre-equilibrated with calcium- and magnesium-free PBS (pH 7.0) (Cytiva, USA). DPPC liposomes were prepared using the same procedure. Particle size distribution and polydispersity index were determined at 25°C using a DynaPro NanoStar dynamic light scattering instrument (Wyatt Technology, USA).

[0079] (10) Temperature-dependent calcein release experiment

[0080] The temperature-dependent release of calcein from uDoPhPI and DPPC vesicles was evaluated within the temperature range of 25–95 °C at pH 7.0. Liposome suspensions were diluted in PBS to a lipid concentration of 0.1 mg / mL. Samples were incubated for 5 min at each test temperature, and 200 μL aliquots were added in triplicate to Corning black 96-well microplates (Catalog #3916, Corning, USA). Fluorescence intensity was measured using an Agilent BioTek Synergy H1 microplate reader (Winooski, USA) at excitation and emission wavelengths of 488 nm and 528 nm, respectively. The percentage of calcein release was calculated as F / Fmax × 100%, where F represents the fluorescence intensity measured after incubation at the test temperature, and Fmax represents the maximum fluorescence intensity obtained after complete vesicle lysis with 2 μL of 10% (v / v) Triton X-100 at the test temperature.

[0081] II. Data Results

[0082] (1) Engineering and structural characterization of uDoPhPI production in Saccharomyces cerevisiae

[0083] In their study (Zhang J, Li T, Hong Z, Ma C, Fang X, Zheng F, Teng W, Zhang C, Si T. Biosynthesis of Hybrid Neutral Lipids with Archaealand Eukaryotic Characteristics in Engineered Saccharomyces cerevisiae. AngewChem Int Ed Engl. 2023 Jan 23;62(4):e202214344.), Jianzhi Zhang et al. constructed a yeast strain named 4U. This strain was derived from a Saccharomyces cerevisiae chassis strain with enhanced GGPP supply capacity and expressed archaeal lipid biosynthesis genes MaG1PDH, MaGGGPS, and MaDGGGPS from Methanosarcina acetivorans, as well as AfCarS from Archaeeoglobus fulgidus. This strain can generate CDP-archaeol intermediates and, due to metabolic competition with the DGGGP precursor, mainly accumulates neutral archaeal lipids DGGGOH and heterozygous DGGGO-fatty acid esters (DGGGO-FA). Based on fragmentation pattern analysis using tandem mass spectrometry (MS / MS), basal levels of uDoPhPI were detected despite the lack of specific AIPS, suggesting that yeast endogenous phosphatidylinositol synthase (Pis1p) may possess some substrate-nonspecific activity towards CDP-archaeol. However, the yield of uDoPhPI in the 4U strain was too low for purification and structural characterization.

[0084] To increase the yield of uDoPhPI, this invention considers two candidate genes: one is archaetidylinositol phosphate synthase (ApAIPS) from Aeropyrumpernix, which has been shown in vitro to catalyze the condensation reaction of CDP-archaetidylinositol with inositol-3-phosphate; the other is the yeast INO1 gene, which encodes ScIno1p, the rate-limiting enzyme in inositol-3-phosphate biosynthesis. The modular Golden Gate assembly method (constructing the Golden Gate assembly scheme for the archaeological lipid biosynthesis pathway, as shown in the figure) is employed. Figure 1 As shown in the figure, this invention constructs strains that overexpress ApAIPS and ScIno1p pathways individually or in combination.

[0085] Selected ion monitoring mode (m / z 896.6) was used for mass spectrometry (MS). 335.0, [M+NH4] + Semi-quantitative analysis showed that the yield of uDoPhPI increased sequentially, in the order: 4U < 4U+A < 4U+I < 4U+AI (e.g., ...). Figure 3 As shown, Figure 3 Production of uDoPhPI in different engineered yeast strains. The left figure illustrates the pathways present in each strain. Engineered yeast strain construction protocols used to compare uDoPhPI synthesis capabilities include 4U (MaG1PDH, MaGGGPS, MaDGGGPS, AfCarS), 4U+A (+ApAIPS), 4U+I (+ScINO1), 4U+AI (+ApAIPS +ScINO1), 4U-G+AI (+ApAIPS +ScINO1, MaG1PDH missing, replaced by Venus), and 4U-C+AI (+ApAIPS +ScINO1, AfCarS missing, replaced by Venus). The right figure shows uDoPhPI abundance determined by LC-MS / MS multiple reaction monitoring (MRM), normalized to cellular biomass. Fold change is relative to the baseline 4U strain (set as 100%). Data are presented as mean ± standard error (SEM) of three independent biological replicates. Among them, the 4U+AI strain was identified as the optimal strain for large-scale purification (e.g., Figure 2 (As shown).

[0086] Under galactose-induced conditions, the 4U+AI strain was fermented in 4L culture and harvested after 48 hours, yielding 24.0 g of stem cell weight (DCW). A modified Bligh-Dyer extraction method was used to obtain 2.02 g of total lipids, corresponding to 84.2 mg lipid / g DCW. The crude extract was separated by silica gel column chromatography with a chloroform-methanol-ammonia gradient (e.g., ...). Figure 2 (As shown).

[0087] Mass spectrometry ion monitoring revealed that uDoPhPI was enriched in the elution fraction with a volume fraction of 70-80% chloroform. These fractions were combined and lyophilized, yielding 131.4 mg of product, corresponding to 6.5% of total yeast lipids, with a yield of 5.5 mg uDoPhPI / g DCW. High-resolution mass spectrometry analysis confirmed its molecular formula as C2. 49 H 83 O 11 P, detected [M+NH4] +The m / z value was 896.6010, consistent with the calculated mass of 896.6011 (Δ = -0.11 ppm). Furthermore, the fragmentation patterns of uDoPhPI in both positive and negative ion MS / MS spectra were consistent with existing data. Based on the A210 absorption peak area, the purity of the separated uDoPhPI was determined to be >96.8%.

[0088] Nuclear magnetic resonance (NMR) spectroscopy further confirmed the structure of uDoPhPI (e.g. Figure 4 As shown, Figure 4 (NMR hydrogen spectrum of uDoPhPI). 1 1H NMR spectra (400 MHz, CDCl3 / CD3OD=5:2) showed characteristic signals of unsaturated geranyl-geranyl chains: δ 5.33 (d, 2H), δ 5.11 (d, 6H), δ 1.90–2.15 (m, 24H), δ 1.67 (s, 12H), and δ 1.60 (s, 18H). The inositol head group exhibited complex multiplets in the δ 3.25–4.23 range, while the glycerol signal partially overlapped with it in the δ 3.45–4.17 range. 13 C10 NMR further confirmed the structure, with quaternary carbon signals at δ 140.5–131.0 and olefinic carbon signals at δ 124.2–120.3. Glyceryl skeletal carbons were located at δ 76.8, 69.4, and 65.2, and inositol ring carbons at δ 76.4–71.2. These NMR data are consistent with those of similar compounds reported in the prior art. Two-dimensional NMR analysis of uDoPhPI confirmed the presence of the inositol head group in the COSY spectrum and the connection between the geraniol-geraniol chain and the glycerol skeleton in the HMBC correlation spectrum, further confirming its structure. Combined with high-resolution mass spectrometry (HR-MS), these spectroscopic data collectively established the structure of this compound as follows: having C10⁻¹⁴ ... 20 An unsaturated di-O-phytylphosphatidylinositol with an isoprene chain linked to a glycerophosphate backbone via an ether bond.

[0089] (2) The metabolic effects of uDoPhPI production on yeast lipid metabolism

[0090] This invention characterizes the effects of uDoPhPI overproduction on cellular lipid metabolism, focusing on endogenous phospholipids, archaea neutral lipids, and hybrid lipid species. This invention employs a multiple reaction monitoring (MRM) LC-MS / MS method for quantitative analysis. For uDoPhPI, a calibration curve was established using the purified substance to achieve absolute quantification. Commercially available C34:1 PI standards were used to quantify yeast endogenous PI species. For neutral lipids, including yeast triacylglycerols (TAG), archaea DGGGOH, and hybrid DGGGO-acyl esters, relative quantification was performed using MRM ion counting, normalized to a 4U baseline strain (e.g., ...). Figure 5 As shown, Figure 5 The impact of pathway engineering on cellular lipid profiles. Data are presented as mean ± standard error (SEM) from three independent biological replicates. Statistical significance was determined by one-way ANOVA (p < 0.01 compared to 4U).

[0091] Using these quantitative methods, this invention first analyzed the inositol phospholipid library ( Figure 3 Despite the absence of ApAIPS, the baseline 4U strain still produced 11.5 ± 1.2 mg uDoPhPI / g stem cell weight (DCW), demonstrating the substrate-nonspecific activity of the endogenous enzyme. Expression of ApAIPS in 4U+A increased uDoPhPI levels by 2.58 ± 0.19-fold, indicating the functional activity of the archaea enzyme in yeast. Overexpression of ScINO1 in 4U+I increased uDoPhPI levels by 3.06 ± 0.34-fold, confirming the limiting effect of the inositol precursor. The co-engineered 4U+AI strain achieved the highest yield, with uDoPhPI levels increasing by 3.95 ± 0.54-fold compared to baseline.

[0092] This increase in uDoPhPI production comes at the cost of sacrificing endogenous PI (e.g., Figure 5 As shown in a, Figure 5'a' represents the yeast endogenous phosphatidylinositol (PI) molecule species, including C34:1 PI, C32:0 PI, and C32:1 PI. Data are expressed as mean ± standard error (SEM) from three independent biological replicates. For the more abundant PI species C34:1 PI and C32:1 PI in yeast, overexpression of ScINO1 alone increased their levels by 1.27 ± 0.11 fold and 1.25 ± 0.24 fold, respectively, thus confirming the limiting role of inositol precursors in endogenous PI biosynthesis. However, expression of ApAIPS alone (0.98 ± 0.05 fold and 1.14 ± 0.04 fold, respectively) or in combination with ScINO1 overexpression (0.82 ± 0.12 fold and 1.07 ± 0.24 fold, respectively) had limited effects on C34:1 PI and C32:1 PI relative to the 4U baseline strain. Comparison between 4U+AI and 4U+I revealed that, in the presence of ApAIPS, the enhanced inositol supply resulting from ScIno1p overexpression was allocated between the endogenous PI pathway and the archaea PI pathway. A different pattern of change was observed for C32:0 PI (a minor PI molecule in yeast). All engineered strains (4U+A, 4U+I, and 4U+AI) showed a significant increase in C32:0 PI relative to 4U (7.36±2.16-fold, 6.44±0.75-fold, and 7.91±1.06-fold, respectively), but due to the low basal abundance of C32:0 PI, its impact on overall endogenous PI levels remained limited.

[0093] Notably, the accumulation of neutral lipids and hybrid lipids increased in all engineered strains (e.g., Figure 5 b, Figure 5 c Figure 5 As shown in d, Figure 5 b represents yeast triglycerides (TAG), including C45:1 TAG, C47:1 TAG, and C53:2 TAG. Figure 5 c represents the archaea neutral lipid DGGGOH. Figure 5 d represents heterozygous DGGGO-acyl esters with different fatty acid chain lengths, including C8:0, C10:0, C12:0, C14:1, C16:0, C16:1, C18:0, and C18:1. Data are expressed as mean ± standard error (SEM) from three independent biological replicates. Statistical significance was determined by one-way ANOVA, p < 0.01 compared to 4U. DGGGOH levels were increased by 5.36 ± 1.30-fold with ApAIPS expression, by 7.09 ± 2.15-fold with ScIno1p overexpression, and by 5.95 ± 1.19-fold with both modifications present simultaneously. Figure 5c). For heterozygous DGGGO-acyl esters (such as... Figure 5 As shown in d, Figure 5 d represents heterozygous DGGGO-acyl esters with different fatty acid chain lengths, including C8:0, C10:0, C12:0, C14:1, C16:0, C16:1, C18:0, and C18:1. The fold changes of the more abundant DGGGO-C16:1 and DGGGO-C16:0 are comparable to those of DGGGOH, while species with lower abundance show a more significant increase, similar to the case of the minor C32:0 PI. TAG content was also increased in all engineered strains ( Figure 5 (b) Among the 4U+A cells, C45:1 TAG showed the most significant increase (8.90 ± 0.73 fold). Flow cytometry analysis of Nile Red stained cells confirmed the increased accumulation of neutral lipids (e.g., ...). Figure 6 As shown, Figure 6 Flow cytometry analysis of neutral lipid accumulation in engineered yeast. Figure 6 'a' represents the fluorescence intensity histogram of strains 4U, 4U+A, 4U+I, and 4U+AI after Nile Red staining. Figure 6 b represents the quantification of the mean fluorescence intensity of neutral lipids for each strain. Data are expressed as mean ± standard error (SEM), from two independent biological replicates. In 4U+A, the mean fluorescence intensity was increased by 2.57 ± 0.51 times relative to 4U; in 4U+I, it was increased by 2.92 ± 0.60 times; and in 4U+AI, it was increased by 4.73 ± 0.94 times. These pleiotropic effects suggest that regulation of inositol metabolism affects a broader range of lipid homeostasis beyond the target phospholipid pathway, consistent with previous state-of-the-art findings regarding crosstalk between storage lipid metabolism and PI biosynthesis.

[0094] (3) Analysis of substrate nonspecificity of enzymes in the inositol lipid pathway

[0095] Following the observation of substrate-nonspecific activity of yeast Pis1p for CDP-archaeolide in 4U strains, this invention further investigated whether other endogenous enzymes also exhibit similar cross-domain substrate flexibility. This invention utilizes GoldenGate assembly for modular gene substitution (…). Figure 1 In a 4U+AI context, each archaic gene was replaced with Venus fluorescent protein to assess the contribution of each enzyme and identify potential metabolic crosstalk between archaic and eukaryotic pathways.

[0096] This invention first examines MaG1PDH ( Figure 3This enzyme generates a glycerol-1-phosphate backbone that is unique to archaea lipids and is stereochemically distinct. Replacing MaG1PDH in the 4U-G+AI strain resulted in a significant decrease in uDoPhPI production (>77% compared to 4U+AI), but a certain level of uDoPhPI was still detectable (0.89±0.03 times the baseline of 4U). Based on the stereochemical analysis results of previous technical reports (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.), even without the expression of exogenous G1PDH, the DGGGOH produced by yeast still only contains the G1P backbone. Therefore, it is inferred that the residual uDoPhPI is also likely to contain an archaeal G1P backbone. However, to definitively establish this inference, direct stereochemical confirmation of the chirality of uDoPhPI is still needed, because current NMR data cannot determine the chiral configuration of glycerol. This endogenous G1P synthesis reveals a hidden metabolic capacity in S. cerevisiae, echoing observations in E. coli; however, the significant decrease in uDoPhPI levels after G1PDH deletion also confirms that archaea G1PDH remains essential for efficient production.

[0097] Even after removing AfCarS in 4U-C+AI, the generation of uDoPhPI remained at a high level, 2.32 ± 0.18 times relative to the 4U baseline. Figure 3 This represents 58% of the 4U+AI complete pathway level. This result indicates that yeast endogenous CDP-diacylglycerol synthase Cds1p can accept archaea DGGGP substrates in a substrate-nonspecific manner to generate CDP-archaeolyl, echoing the flexibility shown by Pis1p in accepting CDP-archaeolyl for PI synthesis. These differential effects reveal different metabolic limiting factors: although G1P supply requires specific archaea enzymes due to limited endogenous activity, both the CDP activation and PI synthesis steps exhibit a considerable degree of enzyme substrate nonspecificity. This suggests that the lipid evolutionary boundaries between different biological domains may be maintained more by differences in precursor availability than by absolute enzyme specificity, with key transferases retaining ancestral flexibility towards structurally diverse substrates.

[0098] Since previous technical reports (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 ChemInt Ed Engl. 2023 Jan 23;62(4):e202214344.) have shown that MaGGGPS and MaDGGGPS are essential for the generation of DGGGOH in yeast, and that their generation is completely lost after deletion of these genes, their role in uDoPhPI production was not tested in this invention.

[0099] (4) The heat resistance of uDoPhPI-producing yeast strains is enhanced.

[0100] To investigate whether the production of uDoPhPI confers similar stress resistance to that observed in engineered yeast strains and E. coli strains containing archaea ether lipids, this invention placed engineered yeast strains under a heat shock condition of 50°C and monitored their survival by colony-forming unit counting. The 4U+AI strain (which produced the highest level of uDoPhPI) was [identified as an example]. Figure 3 Compared to the control strain, this strain exhibited significantly enhanced heat resistance. After treatment at 50°C for 10 minutes, it maintained a survival rate of 76.54±6.36%, while the survival rates of its parental *Saccharomyces cerevisiae* strain and the 4U strain were only 37.81±16.38% and 4.24±1.90%, respectively. This protective effect persisted throughout the entire treatment time: at 30 minutes, the 4U+AI strain maintained a survival rate of 43.85±3.88%, and at 60 minutes, it still maintained a survival rate of 5.75±1.63%; at these times, all other strains almost completely lost their viability. Conversely, the 4U-G+AI strain, which showed a significant reduction in uDoPhPI production... Figure 3 The archaea exhibited the worst heat resistance, with a survival rate of only 1.92 ± 1.92% after 10 minutes and complete loss of viability after 20 minutes. This inverse correlation between uDoPhPI levels and heat sensitivity suggests that archaeal phospholipid content directly affects the thermal stability of yeast cell membranes.

[0101] (5) Formation and characterization of uDoPhPI liposomes

[0102] Archaeosome lipids can form stable archaeosomes, which have been explored for use as drug delivery carriers and vaccine adjuvants. Previous technologies for archaeosome-based phosphatidylinositol liposomes relied on chemically synthesized saturated archaeosome phosphatidylinositol (di-O-phytylphosphatidylinositol, DoPhPI) or natural lipid extracts derived from thermophilic archaea, primarily containing glycosylated forms (approximately 90% glucosyl-DoPhPI and approximately 10% DoPhPI). However, whether non-glycosylated, unsaturated DoPhPI obtained through heterologous yeast biosynthesis can form stable vesicle structures remains unknown.

[0103] Therefore, this invention investigated the film-forming ability and stability of uDoPhPI derived from yeast. First, using an electroforming method, giant unilamellarvesicles (GUVs) were successfully prepared from uDoPhPI / DOPE-Atto647 (1000:1). These GUVs exhibited a uniform spherical morphology and effectively encapsulated calcein, indicating the formation of a complete lipid bilayer structure (e.g., ...). Figure 7 As shown in a, Figure 7 Characterization of uDoPhPI liposome formation and stability. Figure 7 Image a is a confocal microscope image of uDoPhPI giant monolayer vesicles (GUVs) loaded with calcein, prepared by electroforming; top: single vesicle; bottom: vesicle population; green: encapsulated calcein; magenta: membrane marker DOPE-Atto647N).

[0104] In characterizing small unilamellar vesicles (SUVs), purified uDoPhPI was hydrated in calcein solution and extruded through a 100 nm polycarbonate membrane. Dynamic light scattering (DLS) analysis showed that the sample was a monodisperse population with an average particle size of 111.0 ± 4.1 nm. DPPC liposomes prepared using the same method showed a larger average particle size of 210.1 ± 6.2 nm, which may reflect the difference in membrane curvature stress between ester-bonded and ether-bonded lipids. During storage at 4 °C for 4 weeks, uDoPhPI archaeal liposomes exhibited good colloidal stability with minimal particle size variation (117.2 ± 0.2 nm). Figure 7 As shown in b, Figure 7 b is the average diameter of the small monolayer vesicle (SUV) measured by DLS. Figure 7 b to Figure 7The data in d are represented as the average of three independent preparations ± SEM. Black squares: DPPC; red dots: uDoPhPI; Figure 7 The red triangle in b represents uDoPhPI after 4 weeks of storage at 4°C, while DPPC liposomes rapidly aggregated, rendering accurate DLS analysis impossible within hours. Membrane integrity assessment results showed significant calcein leakage from DPPC liposomes, with the leakage rate increasing from 13.5% to 53.6% within 4 weeks (e.g., ...). Figure 7 As shown in c, Figure 7 c represents the time-dependent release of calcein in the SUV during storage at 4°C. Figure 7 b to Figure 7 Data in d are expressed as the average of three independent preparations ± SEM. Black squares: DPPC; red dots: uDoPhPI). In contrast, uDoPhPI archaeal liposomes effectively retained their encapsulated contents, with the leakage rate increasing only slightly from 8.3% to 13.4%. Figure 7 (c) shows superior long-term stability.

[0105] To evaluate the thermal stability of SUVs, this invention monitored the temperature-dependent release of calcein within a temperature range of 25-95°C (e.g., Figure 7 As shown in d, Figure 7 d represents the temperature-dependent release of calcein in SUVs under pH 7.0 conditions. Figure 7 b to Figure 7 Data in d are expressed as the average of three independent preparations ± SEM. Black squares: DPPC; red dots: uDoPhPI). DPPC liposomes showed almost no leakage below their phase transition temperature (42°C), but exhibited rapid release at 50°C, reaching approximately 90%. In contrast, uDoPhPI liposomes maintained almost complete encapsulation of calcein throughout the temperature range, with no release detected even at 95°C. Figure 7 (d). The results of this invention show that unsaturated DoPhPI, despite lacking the saturation and glycosylation modifications typical of natural archaeal membranes, still retains the ability to form vesicles and significant thermal stability, making it suitable for biotechnological applications.

[0106] The above embodiments illustrate and describe the main features and advantages of the present invention in detail. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of this application.

Claims

1. An archaea strain for producing phosphatidylinositol, wherein, The molecular formula of the archaeal phosphatidylinositol is C 49 H 83 O 11 P, this strain is a Saccharomyces cerevisiae strain that overexpresses G1PDH, GGGPS, DGGGPS, CarS, AIPS and INO1.

2. The archaea phosphatidylinositol-producing strain according to claim 1, wherein, The transcriptional units of G1PDH, GGGPS, DGGGPS, CarS, AIPS, and INO1 are linked together to form a metabolic pathway, which is integrated into the 106a site of the Saccharomyces cerevisiae genome.

3. The method for constructing the archaea phosphatidylinositol-producing strain according to claim 1 or 2, wherein, The construction method involves the following steps: introducing G1PDH, GGGPS, DGGGPS, CarS, AIPS, and INO1 into Saccharomyces cerevisiae via plasmid expression vectors or integrating them into the chromosome of Saccharomyces cerevisiae through genetic engineering to construct an archaea phosphatidylinositol-producing strain expressing the above genes.

4. The construction method according to claim 3, wherein, The expressed promoter is a constitutive promoter and / or an inductive promoter.

5. The construction method according to claim 3, wherein, The specific steps of the construction method are as follows: construct transcription units of G1PDH, GGGPS, DGGGPS, CarS, AIPS and INO1, assemble a multi-gene construct using the Golden Gate cloning method, connect the six transcription units into a metabolic pathway, and integrate the metabolic pathway into the 106a site of the yeast genome using the CRISPR / Cas method to obtain an archaeal phosphatidylinositol producing strain.

6. A method for producing archaeal phosphatidylinositol using a strain, wherein the method utilizes an archaeal phosphatidylinositol producing strain constructed according to any one of claims 3-5 to produce archaeal phosphatidylinositol.

7. The method according to claim 6, wherein, The method also produces hybrid lipids.

8. The use of the archaea phosphatidylinositol producing strain according to claim 1 or 2, or the method according to claim 6 or 7, in the preparation of archaea phosphatidylinositol.

9. A giant monolayer vesicle obtained from archaeal phosphatidylinositol obtained by the method of claim 6 or 7.

10. The application of the giant monolayer vesicles of claim 9 in the preparation of drug delivery systems.

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