Uses of engineered yeast strains and lipid compositions thereof

Abstract

Provided herein are methods and materials for using organisms (e.g., engineered yeast strain-derived) to generate lipid compositions which can modulate cellular aging, extend lifespan, and / or modulate gut homeostasis in a subject. The lipid compositions contain at least twelve of the lipids selected from a phosphatidylethanolamine (PE); a lysophosphatidylethanolamine (LPE); a diacylglycerol (DG); a monoglyceride (MG); an acyl carnitine (AcCa); a phosphatidic acid (PA); a phosphatidylglycerol (PG); a lysophosphatidylcholine (LPC); a triacylglycerol (TG); a cholesterol ester (ChE); a wax ester (WE); a phosphatidylserine (PS); a phosphatidylcholine (PC); a phosphatidylinositol (PI); a ceramide (Cer); and a lysophosphatidylserine (LPS).

Description

[0001] USES OF ENGINEERED YEAST STRAINS AND LIPID COMPOSITIONS THEREOF

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 637,565, filed on April 23, 2024, and U.S. Provisional Application Senal No. 63 / 572,647, filed on April 1, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0004] TECHNICAL FIELD

[0005] The present disclosure relates to methods and materials for using engineered yeast strain-derived lipid composition to promote longevity.

[0006] BACKGROUND

[0007] Aging manifests as the accumulation of molecular and cellular damages, resulting in significant physiological impairments across the organism over time. The gastrointestinal system experiences age-related changes that influences overall health. Gut functions as a complex barrier, responding to diverse stimuli such as metabolites, age-related changes, microbiota, and inflammation-associated processes. The dysfunction of intestine barrier has been reported to be a primary risk factor for multiple diseases, including intestinal infections, intestinal tumors, malnutrition, chronic constipation, and other age-related diseases5-9Thus, novel interventions are needed to prevent age-related decline and / or diseases.

[0008] SUMMARY

[0009] This document provides methods and materials for using engineered yeast strains and engineered yeast strain-derived lipid compositions to promote longevity. For example, this document provides methods and materials for using engineered yeast strains and engineered yeast strain-derived lipid compositions to modulate cellular aging, extend lifespan, and / or modulate gut homeostasis in a subject (e.g., a human) in need thereof. As demonstrated herein, engineered yeast strain-derived lipid compositions can incorporate into the epithelial membrane of the gut and promote gut health and lifespan. Provided herein are lipid compositions that include: a phosphatidylethanolamine (PE), a lysophosphatidylethanolamine (LPE), a diacylglycerol (DG), a monoglyceride (MG), an acyl carnitine (AcCa), a phosphatidic acid (PA), a phosphatidylglycerol (PG), a lysophosphatidylcholine (LPC), a triacylglycerol (TG), a cholesterol ester (ChE), a wax ester (WE), a phosphatidylsenne (PS), a phosphatidylcholine (PC), a phosphatidylinositol (PI), a ceramide (Cer), and a lysophosphatidylserine (LPS). Also provided herein are lipid compositions that consist of a phosphatidylethanolamine (PE), a lysophosphatidylethanolamine (LPE), a diacylglycerol (DG), a monoglyceride (MG), an acyl carnitine (AcCa), a phosphatidic acid (PA), a phosphatidylglycerol (PG), a lysophosphatidylcholine (LPC), a triacylglycerol (TG), a cholesterol ester (ChE), a wax ester (WE), a phosphatidylserine (PS), a phosphatidylcholine (PC), a phosphatidylinositol (PI), a ceramide (Cer), and a lysophosphatidylserine (LPS). A lipid composition can include at least two of the lipids selected from the group consisting of: a phosphatidylethanolamine (PE), a lysophosphatidylethanolamine (LPE), a diacylglycerol (DG), a monoglyceride (MG), an acyl carnitine (AcCa), a phosphatidic acid (PA), a phosphatidylglycerol (PG), a lysophosphatidylcholine (LPC), a triacylglycerol (TG), a cholesterol ester (ChE), a wax ester (WE), a phosphatidylsenne (PS), a phosphatidylcholine (PC), a phosphatidylinositol (PI), a ceramide (Cer), and a lysophosphatidylserine (LPS).

[0010] In some embodiments, the lipid composition includes at least three lipids. In some embodiments, the lipid composition includes at least four lipids. In some embodiments, the lipid composition includes at least five lipids. In some embodiments, the lipid composition includes at least six lipids. In some embodiments, the lipid composition includes at least seven lipids. In some embodiments, the lipid composition includes at least eight lipids. In some embodiments, the lipid composition includes at least nine lipids. In some embodiments, the lipid composition includes at least ten lipids. In some embodiments, the lipid composition includes at least eleven lipids. In some embodiments, the lipid composition includes at least twelve lipids. In some embodiments, the lipid composition includes at least thirteen lipids. In some embodiments, the lipid composition includes at least fourteen lipids. In some embodiments, the lipid composition includes at least fifteen lipids. In some embodiments, the lipid composition includes a PC and a TG. In some embodiments, the lipid composition further includes any one of the lipids selected from the group consisting of: a phosphatidylethanolamine (PE), a lysophosphatidylethanolamine (LPE), a diacylglycerol (DG), a monoglyceride (MG), an acyl carnitine (AcCa), a phosphatidic acid (PA), a phosphatidylglycerol (PG), a lysophosphatidylcholine (LPC), a cholesterol ester (ChE), a wax ester (WE), a phosphatidylserine (PS), a phosphatidylinositol (PI), a ceramide (Cer), and a lysophosphatidylserine (LPS).

[0011] In some embodiments, the PE includes from about 2% to about 22% of the lipid composition. In some embodiments, the LPE includes from about 5% to about 35% of the lipid composition. In some embodiments, the DG includes from about 0.5% to about 3% of the lipid composition. In some embodiments, the MG includes from about 0.01% to about 1% of the lipid composition. In some embodiments, the AcCa includes from about 0.01% to about 1% of the lipid composition. In some embodiments, the PA includes from about 1% to about 3% of the lipid composition. In some embodiments, the PG includes from about 0.01% to about 1% of the lipid composition. In some embodiments, the LPC includes from about 50% to about 80% of the lipid composition. In some embodiments, the TG includes from about 5% to about 25% of the lipid composition. In some embodiments, the ChE includes from about 0.01% to about 1% of the lipid composition. In some embodiments, the WE includes from about 0.01% to about 1% of the lipid composition. In some embodiments, the PS includes from about 0.01% to about 1% of the lipid composition. In some embodiments, the PC includes from about 0.5% to about 3% of the lipid composition. In some embodiments, the PI includes from about 0.5% to about 3% of the lipid composition. In some embodiments, the Cer includes from about 0.01% to about 1% of the lipid composition. In some embodiments, the LPS includes from about 0.01% to about 1% of the lipid composition.

[0012] In some embodiments, the LPC includes a plurality of subtypes of different carbon lengths. In some embodiments, the LPC includes a subtype having about 5 to about 25 carbons. In some embodiments, the subtype includes one or more double bonds. In some embodiments, the double bond occurs at position 0, 1, or 3 of the LPC subtype. In some embodiments, the LPC includes a plurality of at least eleven subtypes selected from the group consisting of LPC (10:0), LPC (14: 1), LPC (15:0), LPC (16:0), LPC (16:1), LPC (16:1), LPC (18:3), LPC (20:1), LPC (20:3), LPC (22:3), LPC (16:0), LPC (17:0), LPC (18:0), LPC (18:1), LPC (20:0), LPC (14:0), LPC (20:2), and LPC (17:0). In some embodiments, the plurality of LPC subtypes include at least twelve subtypes. In some embodiments, the plurality of LPC subtypes include at least thirteen subtypes. In some embodiments, the plurality of LPC subtypes include at least fourteen subtypes.

[0013] In some embodiments, the LPE includes a plurality of subtypes of different carbon lengths. In some embodiments, the LPE includes a subtype having about 5 to about 25 carbons. In some embodiments, the LPE subtype includes one or more double bonds. In some embodiments, the double bond occurs at position 0 or 1 of the LPE subtype. In some embodiments, the LPE includes a plurality of at least five subtypes selected from the group consisting of LPE (10:0), LPE (12:0), LPE (14:0), LPE (14:1), LPE (15:0), LPE (16:0), LPE (16:0), LPE (16:1), and LPE (18:1). In some embodiments, the plurality of LPE subtypes include at least six different subtypes. In some embodiments, the plurality of LPE subtypes include at least seven different subtypes. In some embodiments, the plurality of LPE subtypes include at least eight subtypes.

[0014] In some embodiments, the TG includes a plurality of subtypes of different carbon lengths. In some embodiments, the TG includes a subty pe having about 5 to about 30 carbons. In some embodiments, the TG subtype includes one or more double bonds. In some embodiments, the double bond occurs at position 0, 1, 2, 3, or 4 of the TG subtypes. In some embodiments, the TG includes a plurality of at least thirty' subtypes selected from the group consisting of TG (12:0_12:0_22:4), TG (15:0 14:0 14:0), TG (15:0 14:0 16:0), TG (15:0 14:0 16:1), TG (15:0 16:0 16:0), TG (15:0 16:0 16:1), TG(15:0_16:l_16:l), TG (15:0 16:1 18:1), TG(16:0_18:2_18:2), TG (18:0_ 16:0_16:0), TG (18:1 18:1 18:1), TG (18:1 18:1 18:2), TG (18: 1_18:2_18:2), TG(18:2_18:2_18:2), TG (16:0 10:0 16:0), TG(16:0_10:0_16:l), TG (16:0 14:0 16:0), TG(16:0_14:0_16:l), TG (16:0 16:0 16:1), TG (16:0_16:l_16:l), TG(16:0_16:l_16:l), TG (16:0_16:l_18: 1), TG(16:0_18:l_20:3), TG (16: l_10:0_14:0), TG(16:l_10:0_16:l), TG (16:1 10:0 18:3), TG (16: 1 12:0 16: 1), TG(16:l_14:0_14:0), TG (16:1 14:0 16:1), TG (16:1 16:1 16:1), TG (16:1 16:1 18:1), TG(16:1_16:1_18:3), TG (16:1 18:1 18:1), TG (18:0 10:0 16:0), TG(18:0_16:0_18:l), TG (18:0_16:l_16;l), TG (18:0 18:0 18:1), TG (18:0 18: 1 18: 1), TG (18:4_14:0_16: l), TG (18:4 16:1 16:1), TG(18:4_16: 1_18:1), TG (26:0 14:0 16: 1), TG (26:0_16:0_18: l), TG (26:0 16: 1 16: 1), TG (16:0 10:0 14:0), TG (16:0_18: l_18:3), TG (18:0_16:0_16:l), TG (18: 1 18: 1_18:3), TG (26:0_10:0_16:0), and TG (16:1 14:1 16:1). In some embodiments, the plurality of TG subtypes include at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 subtypes.

[0015] In some embodiments, the lipid composition is extracted from an organism. In some embodiments, the one or more of the lipids in the lipid composition is extracted from one or more types of organisms. In some embodiments, the lipids in the lipid composition is extracted from a plurality of the same organism. In some embodiments, the one or more of the lipids in the lipid composition are extracted from pluralities of two or more different organisms.

[0016] In some embodiments, the organism is a prokaryote or a eukaryote. In some embodiments, the organism is a prokaryote. In some embodiments, the prokaryote is a bacterium. In some embodiments, the organism is a eukary ote. In some embodiments, the eukaryote is an alga, or a fungus, a plant, or a mammalian cell. In some embodiments, the fungus is a yeast. In some embodiments, the mammalian cell is an animal or a human cell. In some embodiments, the non-human mammal or the human cell is a liver cell, a kidney cell, an intestine cell, a connective tissue cell, a muscle cell, a heart cell, a vessel cell, a gut bacterial cell, a stomach cell, other gut organ cells, an anus cell, a joint cell, a nerve cell, a skin cell, a nasal cavity cell, a tongue cell, an appendix cell, a diaphragm cell, a lung cell, a thyroid cell, an adrenal gland cell, an ear cell, a larynx cell, an esophagus cell, a trachea cell, a brain cell, an eye cell, a spinal cord cell, a thymus gland cell, a lymph node cell, a pancreas cell, a ureter cell, a bronchus cell, a genital cell, a pharynx cell, a salivary gland cell, a urethra bladder cell, a gallbladder cell, a placenta cell, a uterus cell, a bone marrow cell, a mouth cell, a prostate cell, a seminal vesicle cell, a hair follicle cell, a mesentery cell, a subcutaneous tissue cell, a mammary gland cell, a tooth cell, an interstitial cell, a parathyroid gland cells, a tonsil cell, a nail cell, a vestigial cell, a cancer cell.

[0017] In some embodiments, the yeast is an engineered strain overexpressing a HAP complex component, a SIR2 gene product, or both the HAP complex component and the SIR2 gene product. In some embodiments, the yeast is an engineered yeast strain overexpressing a HAP4 gene product, the SIR2 gene product, or both the HAP4 gene product and the SIR2 gene product. In some embodiments, the HAP complex component comprises a HAPl, a HAP2, a HAP3, a HAP4, and / or a HAP5 gene product. In some embodiments, the engineered yeast strain includes NH0880, NH0868, and / or NH0897. In some embodiments, the engineered yeast strain includes NH0880.

[0018] In some embodiments, the one or more of the lipids in the lipid composition is made by synthesis. In some embodiments, the synthesis is a chemical synthesis, a chemoenzymatic synthesis, or an enzyme-free synthesis.

[0019] In some embodiments, the lipid composition is formulated in an ingestible form or a topical form. In some embodiments, the ingestible form is a pharmaceutical composition or an edible. In some embodiments, the pharmaceutical composition is a tablet, a powder, a granule, a pill, a capsule, a gel, or a liquid. In some embodiments, the edible is a snack bar, a gummy, a yogurt, or a supplement. In some embodiments, the topical form is a cream, a serum, a gel, a mask, an ointment or a cleanser.

[0020] Also provided herein are methods of modulating cellular aging in a subject, the method including administering a therapeutically effective amount of the lipid composition provided herein to a subject. Also provided herein are methods of extending the lifespan of a subject, the method including administering a therapeutically effective amount of a lipid composition provided herein to the subject. Also provided herein are methods of modulating gut homeostasis in a subject, the method including administering a therapeutically effective amount of a lipid composition provided herein to the subject. In some embodiments, the lipid composition comprises at least a TG.

[0021] Also provided herein are methods of modulating cellular aging in a subject, the method including administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strains NH0880, NH0868, and / or NH0897 to the subject. Also provided herein are methods of extending the lifespan of a subject, the method including administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strains NH0880, NH0868, and / or NH0897 to the subject. Also provided herein are methods of modulating gut homeostasis in a subject, the method including administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strains NH0880, NH0868, and / or NH0897 to the subject. Also provided herein are methods of modulating cellular aging in a subject, the method including administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strain NH0880 to the subject. Also provided herein are methods of extending the lifespan of a subject, the method including administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strain NH0880 to the subject. Also provided herein are methods of modulating gut homeostasis in a subject, the method including administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strain NH0880 to the subject.

[0022] In some embodiments, the yeast composition is formulated in an ingestible form. In some embodiments, the ingestible form is a pharmaceutical composition or an edible. In some embodiments, the pharmaceutical composition is a tablet, a powder, a granule, a pill, a capsule, a gel, a solid, or a liquid. In some embodiments, the edible is a bakery item, a beverage, a food ingredient, an animal feed, a snack bar, a gummy, a yogurt, or a supplement. In some embodiments, the beverage comprises an alcoholic drink or a non-alcoholic drink. In some embodiments, the alcoholic drink comprises a beer, a wine, a spirit, or a cider. In some embodiments, the non-alcoholic drink comprises a probiotic drink, a tea, a dairy drink, a juice, a mocktail, an alcohol- free beverage, or a sparkling drink. In some embodiments, the food ingredient comprises a yeast extract, a yeast flake, an autolyzed yeast, an active dry yeast, an instant dry yeast, a fresh yeast, a bread machine yeast, a spice, an herb, a syrup, or a cream.

[0023] In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal, a chromadorea, or an insecta. In some embodiments, the mammal is a human, a non-human primate, horse, bovine, porcine, dog, cat, or a rodent. In some embodiments, the chromadorea is a Caenorhabditis elegcms. In some embodiments, the insecta is a Drosophila.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary' skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0025] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0026] DESCRIPTION OF DRAWINGS

[0027] FIGS. 1A-1L Yeast lipid extracts affected Drosophila gut physiology and lifespan. FIG. 1A: The lifespan analysis of W1118flies fed with lipid extracts from yeast strains 270, 868, 897, and 880. n=120 flies per group. FIG. IB: SRS imaging showed metabolic changes in the guts of 45 -day old flies that were fed with lipid extract from yeast strains 270, 868, 897, and 880. FIG. 1C: The total lipids amount per gut in (FIG. IB) was quantified. FIG. ID: The lipid turnover rate in (FIG. IB) was quantified as CD / CH. FIG. IE: The gut width of 45-day old flies fed with lipid extract from yeast strains 270, 868, 897, and 880 was measured. FIG. IF: The cell number in 45-day old flies fed with lipid extract from yeast strains 270, 868, 897, and 880 was counted. FIG. 1G: The cell area of 45-day old flies fed with lipid extract from yeast strains 270, 868, 897, and 880 was measured. FIGS. 1H and II: The Esg>GFP+ cell number in 45-day old flies fed with lipid extract from yeast strains 270, 868, 897, and 880 was imaged (FIG. 1H) and quantified (FIG. II). For FIGS. 1B-1H, values are shown as mean ± SEM. n=10 flies. *, p < 0.05; **, p < 0.01; ****, p < 0.0001, ns, not significant, by one-way ANOVA with Tukey multiple comparison test.

[0028] FIGS. 2A-2F. SRS imaging detected changes in lipid metabolism in long lived yeasts. FIG. 2A: DO-SRS imaging showed the metabolic activity difference between yeast strains 270, 868, 897, and 880. The representative images are shown for n=3 independent experiments. The arrows point to the subcellular clusters in yeast cells. FIG. 2B: Quantification of total lipid level of yeasts from FIG. 2A. FIG. 2C: Quantification of lipid turnover rate of yeasts from FIG. 2A. FIGS. 2D-2F: The CD (FIG. 2D), CH (FIG. 2E), and fingerprint (FIG. 2F) region of lipid Raman spectra collected from the lipid extracts from D2O labeled yeasts 270, 868, 897, and 880. For FIGS. 2B and 2C, values are shown as mean ± SEM. n=10 ROIs per group. *, p < 0.05; **, p < 0.01; ****, p < 0.0001, ns, not significant, by one-way ANOVA with Tukey multiple comparison test. FIGS. 2D-2F: The displayed spectrum for each group was averaged from 10 spectra measured from that yeast group.

[0029] FIGS. 3A-3H. The long-lived yeast strains exhibited diverse lipidomes. FIG. 3 A: Bar graph showing relative abundance % of AcCa, Cer, DG, PA, PC, PE, PI, PS, and WE in yeast strains 270, 868, 897, and 880. FIG. 3B: Bar graph showing relative abundance % of LPC, LPE, and TG in yeast strains 270, 868, 897, and 880. FIG. 3C: Bar graph showing relative abundance % of ChE, LPS, MG, and PG in yeast strains 270, 868, 897, and 880. n=4 biological repeats. FIG. 3D: Heatmap plot of the difference in lipid species in yeast strains 270, 868, 897, and 880 were detected by lipidomics. FIGS. 3E-3G: Significantly changed lipids in LPC (FIG. 3E), LPE (FIG. 3F), and TG (FIG. 3G) in the 880 group are shown. FIG. 3H: The lipid pathway enrichment analysis of the 880 yeast strain is shown.

[0030] FIGS. 4A-4F. The 880 and 868 yeast derived lipids promoted membrane lipid homeostasis and gut integration. FIG. 4A: DO-SRS imaging showed that D-lipids derived from D2O labeled yeast strains (270, 868, 897, 880) can be incorporated into the fly guts. The zoomed-in images showed the subcellular distribution of newly synthesized lipids. The arrows indicate the lipid droplets, and the arrow heads point to the membrane structure. FIG. 4B: Smurf assay of flies fed with lipid extracts from yeasts is shown. n=80 flies from 4 independent experiments. FIGS. 4C-4D: SRS-HSI showed lipid droplet (FIG. 4C) and membrane (FIG. 4D) spectra from D-lipids fed fly guts. Averaged spectra from n=4 independent experiments. FIG. 4E: Raman spectra showed the difference in lipid component from D-lipids fed fly guts. Averaged spectra from n=4 independent experiments. FIG. 4F: The lifespan analysis of W1118flies fed with medium chain fatty acids containing TG and PC. n=120 flies per group.

[0031] FIG. 5. Lifespan analysis of the Canton S flies fed with standard food and lipid extract from the 880 yeast strain. n=120 flies per group.

[0032] FIGS. 6A-6E. SRS imaging showed age-dependent changes in gut lipids. FIG. 6A: An illustration depicting the different focus plane displayed in FIG. 6B. FIG. 6B: SRS imaging showed that the lipid abundance was different between the young and the old flies. FIG. 6C: The coregistration of Bodipy staining and SRS lipid droplet signal in fly gut are shown. FIG. 6D: Quantification of age-dependent lipid reduction in fly gut is shown. Values are mean ± SEM. n=10 flies per group. **, p < 0.01; ****, p < 0.0001, by one-way ANOVA with Tukey multiple comparison test. FIG. 6E: Raman spectrometry' showed a distinct peak in the cell silence regions of gut after feeding the flies with D2O labeled food. The CD peak intensity, which indicated newly synthesized lipid level, was reduced in the gut of the old flies. Each spectrum was averaged from n=10 flies.

[0033] FIGS. 7A-7F. DO-SRS imaging detected lipid metabolism changes in 45-day fly guts. FIG. 7 A: SRS imaging showed changes in the lipid metabolism in the guts of 25-day old flies that were fed with lipid extracts from yeast strains 270, 868, 897, and 880. FIGS. 7B and 7C: The total lipids (FIG. 7B) and lipid turnover (FIG. 7C) were quantified. FIG. 7D: The gut width of 25-day old flies fed with lipid extracts from yeast 270, 868, 897, and 880 was measured. FIG. 7E: SRS imaging showed that the fatbody lipid turnover changed from 45-day old flies fed with lipid extract from yeast strains 270, 868, 897, and 880. FIG. 7F: The lipid turnover rate was quantified. For FIGS. 7B-7D and 7F, values are shown as mean ± SEM. n=I0 flies. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001, ns, not significant, by one-way ANOVA with Tukey multiple comparison test.

[0034] FIGS. 8A-8B. Raman spectra from yeast and lipid extracts. FIG. 8A: Spontaneous Raman spectra of yeast strains 270, 868, 897, and 880 labeled with D2O is shown. There was a distinct peak at 2150 cm'1. FIG. 8B: Spontaneous Raman spectra of pure lipid extracts from yeast strains 270, 868, 897, and 880 labeled with D2O. The deuterium labeled yeast lipids showed a peak at 2176 cm'1.

[0035] FIGS. 9A-9C. PCA analysis of yeast lipids. FIG. 9A: Scatter plot showing PC A scores for Principal Component (PC) 2 vs PC 1. Data points were grouped by yeast 270, 868, 880, and 897. Positive values indicate a positive correlation between a point and the respective principal component, and a negative value indicate a negative correlation. The strength of the correlation was determined by the magnitude of the score. FIG. 9B: Bar and line plots of explained variables percentage for the first five principal components. The percentage values indicate the percentage of variation across the whole dataset that can be explained by or attributed to each component. FIG. 9C: Box plot of the PCA scores for each yeast across the first five principal components are shown. Horizontal line in box plots indicates median value, box boundaries indicate the lower and upper quartiles, and whiskers indicate maximum and minimum value (excluding outliers). FIGS. 10A-10F. Comparison between 270, 868 and 897 lipidomes. FIG. 10A- 10C: Significantly changed lipids in LPC (FIG. 10A), LPE (FIG. 10B), TG (FIG. IOC) in the 868 group are shown. FIG. 10D: The lipid pathway enrichment analysis of the yeast strain 868 is shown. FIG. 10E: Significantly changed lipids in LPC in the 897 group are shown. FIG. 10F: The lipid pathway enrichment analysis of the yeast strain 897 is shown.

[0036] DETAILED DESCRIPTION

[0037] Gut functions as a complex barrier, responding to diverse stimuli such as metabolites, age-related changes, microbiota, and inflammation-associated processes. These stimuli result in functional consequences affecting stem cell adaptation, immune responses, reinforcement of barrier function, cellular senescence, and stem cell exhaustion. The continuous interplay of functional consequences impacts epithelial homeostasis and, consequently, the barrier function of the intestine, potentially leading to epithelial barrier defects20’70'72.

[0038] Drosophila midgut is an intestinal model system, mirroring the cell types and functions found in the mammalian stomach and small intestine. The epithelium of Drosophila midgut is made up of 4 cell types: intestinal stem cells (ISCs) and their differentiating daughters called enteroblasts (EBs) (together termed progenitor cells), and 2 terminally differentiated cell types consisting of absorptive enterocytes (ECs) and secretory enteroendocrine cells (EEs)3 16. And it is highly regionalized in function, cell type and gene expression17,18. This regionalization is evident across four distinct gut regions: the proventriculus (PV), midgut region 2 (R2), and midgut regions 4-5 (R4 / R5). Spatial distribution of lipid droplets (LDs) are predominantly localized to the enterocytes (ECs) of R2 region3’19. EC is the major cell type in charge of food digestion, nutrient absorption, and energy substrate storage. ECs show the imbalance of lipid homeostasis under different settings. For instance, a decrease in the lipid droplet (LD) abundance was shown during aging9,20. Conversely, when Drosophila gut encounters pathogenic challenges, LD accumulation has been observed21.

[0039] Aging is characterized by the progressive accumulation of molecular and cellular damages, leading to significant physiological impairments throughout the organism1,2It is a complex process that involves both genetic and various environmental factors. As an important interface with the environment, the intestine must have the ability to adapt to rapidly changing conditions, such as feeding, starvation, and ingestion of toxins or pathogenic bacteria3-5. To meet these challenges, the structural integrity of intestine is a prerequisite for organismal health. The dysfunction of intestine barrier has been reported to be a primary risk factor for multiple diseases, including intestinal infections, intestinal tumors, malnutrition, chronic constipation, and other age-related diseases5-9Aging related transformation of gut microbiota includes diminishing of the cellular and / or extracellular function of intestine and alteration of cytokine expression patterns, can modulate the intestinal integrity9 1by metabolic remodeling during aging12'15.

[0040] Provided herein are methods and materials (e.g., lipid compositions and methods of making the same) for extending lifespan. For example, supplementing an animal (e.g., a fly, or a mammal) with lipids extracted from the engineered long-lived yeast strain can enhance gut health and lifespan of the animal. In some cases, the engineered long-lived yeast strain shows an increase in lipid metabolic activity and a substantial remodeling of lipid profiles. The mechanism underlying this pro-longevity effect involves integration of lipid species derived from the engineered long-lived yeast strain into the epithelial membranes of the gut, thereby bolstering the gut barrier function in animals throughout the aging process.

[0041] Thus, described herein are lipid compositions and methods of making the same, which demonstrate the pro-longevity effect.

[0042] As used herein, the term “subtype” refers to a specific lipid itself or a derivative thereof that may have distinct variations or molecular forms characterized by differences in fatty acid chain composition and / or length, the presence of specific functional groups, structural modifications, or chemical modifications, as long as it has properties of that lipid.

[0043] Engineered Lipid Composition

[0044] Provided herein are lipid compositions that can include phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), diacylglycerol (DG), monoglyceride (MG), an acyl carnitine (AcCa), phosphatidic acid (PA), phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), triacylglycerol (TG), cholesterol ester (ChE), wax ester (WE), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), ceramide (Cer), and lysophosphatidylserine (LPS). Also, provided herein are lipid compositions that consist of phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), diacylglycerol (DG), monoglyceride (MG), an acyl carnitine (AcCa), phosphatidic acid (PA), phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), triacylglycerol (TG), cholesterol ester (ChE), wax ester (WE), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), ceramide (Cer), and lysophosphatidylserine (LPS). In some embodiments, the lipid composition described herein includes at least two lipids selected from the group consisting of phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), diacylglycerol (DG), monoglyceride (MG), an acyl carnitine (AcCa), phosphatidic acid (PA), phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), triacylglycerol (TG), cholesterol ester (ChE), wax ester (WE), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), ceramide (Cer), and lysophosphatidylserine (LPS). For example, the lipid composition can include at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen of the lipids described above.

[0045] In some embodiments, the lipid composition described herein includes a PC and a TG. In some embodiments, the lipid composition containing a PC and a TG further contains one or more lipids selected from the group consisting of phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), diacylglycerol (DG), monoglyceride (MG), an acyl carnitine (AcCa), phosphatidic acid (PA), phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), cholesterol ester (ChE), wax ester (WE), phosphatidylserine (PS), phosphatidylinositol (PI), ceramide (Cer), and lysophosphatidylserine (LPS). For example, the lipid composition containing a PC and a TG can further include at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen, of the lipids described above.

[0046] In some embodiments, one or more lipids in the lipid composition can be one or more lipids of the same subtype provided herein. In some embodiments, one or more lipids in the lipid composition can be one or more lipids of different subtypes provided herein.

[0047] Lipid compositions provided herein can contain one or more than one phosphatidylethanolamines (PE). A composition provided herein can contain any appropriate amount of a phosphatidylethanolamine. In some cases, at least 2% (e.g., at least 8%, at least 12%, at least 18%, or at least 22%) of the lipid composition can be a phosphatidylethanolamine. In some embodiments, a lipid composition contains no more than 20%, no more than 18%, no more than 16%, no more than 14%, no more than 12%, no more than 10%, no more than 8%, no more than 6%, or no more than 4% of a phosphatidylethanolamine. In some cases, a lipid composition can contain from about 2% to about 22% (e.g., 2% to about 4%, about 2% to about 8%, about 2% to about 12%, about 2% to about 16%, about 2% to about 20%, about 4% to about 8%, about 4% to about 12%, about 4% to about 16%, about 4% to about 20%, about 4% to about 22%, about 8% to about 12%, about 8% to about 16%, about 8% to about 20%, about 8% to about 22%, about 12% to about 16%, about 12% to about 20%, about 12% to about 22%, about 16% to about 20%, about 16% to about 22%, or about 20% to about 22%) of a phosphatidylethanolamine. In some embodiments, a lipid composition contains about 2% to about 5% of a phosphatidylethanolamine.

[0048] Lipid compositions provided herein can contain one or more than one lysophosphatidylethanolamines (LPE). A composition provided herein can contain any appropriate amount of a lysophosphatidylethanolamine. In some cases, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35%) of the lipid composition can be a lysophosphatidylethanolamine. In some cases, a lipid composition can contain from about 5% to about 35% (e.g., about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 35%, about 25% to about 30%, or about 30% to about 35%) of a lysophosphatidylethanolamine. In some embodiments, a lipid composition contains no more than 30%, no more than 28%, no more than 26%, no more than 24%, no more than 22%, no more than 20%, no more than 18%, no more than 16%, or no more than 14% of a lysophosphatidylethanolamine. In some cases, the LPEs of the lipid composition can include subtypes of different carbon lengths. The LPEs of the lipid composition can contain subtypes of about 5 to about 25 carbons. For example, the LPEs of the lipid composition can contain LPE subtypes of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and / or 25 carbons. In some cases, the LPE subtypes of the lipid composition can contain one or more double bonds. For example, the double bonds can occur at position 0, and / or 1 of an LPE. In some cases, the LPE of the lipid composition can contain a plurality of LPE subtypes selected from the group consisting of LPE (10:0), LPE (12:0), LPE (14:0), LPE (14: 1), LPE (15:0), LPE (16:0), LPE (16:0), LPE (16:1), and LPE (18: 1). In some embodiments, the TG of the lipid composition can contain a plurality of at least two (e.g., at least three, at least four, at least five, at least six, at least seven, or at least eight) different LPE subtypes. For example, the LPEs of the lipid composition can contain a plurality of at least six different LPE subtypes. The LPEs of the lipid composition can contain a plurality of at least seven different LPE subty pes. The LPEs of the lipid composition can contain a plurality of at least eight different LPE subtypes.

[0049] Lipid compositions provided herein can contain one or more than one diacylglycerols (DG). A composition provided herein can contain any appropriate amount of a diacylglycerol. In some cases, at least 0.5% (e.g., at least 1%, at least 1.5%, at least 2%, at least 2.5%, or at least 3%) of the lipid composition can be a diacylglycerol. In some embodiments, a lipid composition contains no more than 5%, no more than 4%, no more than 3%, or no more than 2% of a diacylglycerol. In some cases, a lipid composition can contain from about 0.5% to about 3% (e.g., about 0.5% to about 1%, about 0.5% to about 1.5%, about 0.5% to about 2%, about 0.5% to about 2.5%, about 0.5% to about 3%, about 1% to about 1.5%, about 1% to about 2%, about 1% to about 2.5%, about 1% to about 3%, about 1.5% to about 2%, about 1.5% to about 2.5%, about 1.5% to about 3%, about 2% to about 2.5%, about 2% to about 3%, or about 2.5% to about 3%) of a diacylglycerol.

[0050] Lipid compositions provided herein can contain one or more than one monoglycendes (MG). A composition provided herein can contain any appropriate amount of a monoglyceride. In some cases, at least 0.01% (e.g., at least 0.02%, at least 0.04%, at least 0.06%, at least 0.08%, or at least 0.1%) of the lipid composition can be a monoglyceride. In some embodiments, a lipid composition contains no more than 0.08%, no more than 0.06%, no more than 0.04%, or no more than 0.02% of a monoglyceride. In some cases, a lipid composition can contain from about 0.01% to about 1% (e.g., about 0.01% to about 0.4%, about 0.01% to about 0.1%, about 0.01% to about 0.04%, about 0.01% to about 0.02%, about 1% to about 0.4%, about 1% to about 0.1%, about 1% to about 0.04%, about 1% to about 0.02%, about 0.4% to about 0.1%, about 0.4% to about 0.04%, about 0.4% to about 0.02%, about 0.1% to about 0.04%, about 0.1% to about 0.02%, or about 0.04% to about 0.02%) of a monoglycende.

[0051] Lipid compositions provided herein can contain one or more than one acyl carnitines (AcCa). A composition provided herein can contain any appropriate amount of an acyl carnitine. In some cases, at least 0.01% (e.g., at least 0.02%, at least 0.04%, at least 0.06%, at least 0.08%, or at least 0.1%) of the lipid composition can be an acyl carnitine. In some embodiments, a lipid composition contains no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% of an acyl carnitine. In some cases, a lipid composition can contain from about 0.01 % to about 1% (e.g., about 0.01% to about 0.4%, about 0.01% to about 0.1%, about 0.01% to about 0.04%, about 0.01% to about 0.02%, about 1% to about 0.4%, about 1% to about 0.1%, about 1% to about 0.04%, about 1% to about 0.02%, about 0.4% to about 0.1%, about 0.4% to about 0.04%, about 0.4% to about 0.02%, about 0.1% to about 0.04%, about 0.1% to about 0.02%, or about 0.04% to about 0.02%) of an acyl carnitine.

[0052] Lipid compositions provided herein can contain one or more than one phosphatidic acids (PA). A composition provided herein can contain any appropriate amount of a phosphatidic acid. In some cases, at least 1% (e.g., at least 1.5%, at least 2%, at least 2.5%, at least 3%) of the lipid composition can be a phosphatidic acid. In some embodiments, a lipid contains no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, or no more than 2% of a phosphatidic acid. In some cases, a lipid composition can contain from about 1% to about 3% (e.g., about 1% to about 2.5%, about 1% to about 2%, about 1% to about 1.5%, about 3% to about 2.5%, about 3% to about 2%, about 3% to about 1.5%, about 2.5% to about 2%, about 2.5% to about 1.5%, or about 2% to about 1.5%) of a phosphatidic acid.

[0053] Lipid compositions provided herein can contain one or more than one phosphatidylglycerols (PG). A composition provided herein can contain any appropriate amount of a phosphatidylglycerol. In some cases, at least 0.01% (e.g., at least 0.02%, at least 0.04%, at least 0.06%, at least 0.08%, or at least 0.1%) of the lipid composition can be a phosphatidylglycerol. In some embodiments, a lipid composition contains no more than 1.2%, no more than 1.0%, no more than 0.8%, no more than 0.6%, or no more than 0.4% of a phosphatidylglycerol. In some cases, a lipid composition can contain from about 0.01% to about 1% (e.g., about 0.01% to about 0.4%, about 0.01% to about 0.1%, about 0.01% to about 0.04%, about 0.01% to about 0.02%, about 1% to about 0.4%, about 1% to about 0.1%, about 1% to about 0.04%, about 1% to about 0.02%, about 0.4% to about 0.1%, about 0.4% to about 0.04%, about 0.4% to about 0.02%, about 0.1% to about 0.04%, about 0.1% to about 0.02%, or about 0.04% to about 0.02%) of a phosphatidylglycerol.

[0054] Lipid compositions provided herein can contain one or more than one lysophosphatidylcholines (LPC). A composition provided herein can contain any appropriate amount of a lysophosphatidylcholine. In some cases, at least 50% (e.g., at least 50%, at least 60%, at least 70%, or at least 80%) of the lipid composition can be a lysophosphatidylcholine. In some embodiments, a lipid composition contains no more than 75%, no more than 72%, no more than 70%, no more than 68%, no more than 66%, no more than 64%, or no more than 62% of a lysophosphatidylcholine. In some cases, a lipid composition can contain from about 50% to about 80% (e.g., about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 75% to about 70%, about 75% to about 65%, about 75% to about 60%, about 75% to about 55%, about 70% to about 65%, about 70% to about 60%, about 70% to about 55%, about 65% to about 60%, about 65% to about 55%, or about 60% to about 55%) of a lysophosphatidylcholine. In some cases, the LPC of the lipid composition can include subtypes of different carbon lengths. The LPC subtypes of the lipid composition can contain about 5 to about 25 carbons. For example, the LPCs of the lipid composition can contain LPC subtypes of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and / or 25 carbons. In some cases, the LPC subtypes of the lipid composition can contain one or more double bonds. For example, the double bonds can occur at position 0, 1, and / or 3 of the LPC. In some cases, the LPC of the lipid composition can contain a plurality of LPC subtypes of selected from the group consisting of LPC (10:0), LPC (14: 1), LPC (15:0), LPC (16:0), LPC (16: 1), LPC (16:1), LPC (18:3), LPC (20:1), LPC (20:3), LPC (22:3), LPC (16:0), LPC (17:0), LPC (18:0), LPC (18: 1), LPC (20:0), LPC (14:0), LPC (20:2), and LPC (17:0). In some embodiments, the LPCs of the lipid composition can contain a plurality of at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve) different LPC subtypes. In some embodiments, the LPCs of the lipid composition can contain a plurality of at least eleven or twelve different LPC subtypes. The LPCs of the lipid composition can contain a plurality of at least thirteen different LPC subtypes. The LPCs of the lipid composition can contain a plurality of at least fourteen different LPC subtypes.

[0055] Lipid compositions provided herein can contain one or more than one triacylglycerols (TG). A composition provided herein can contain any appropriate amount of a triacylglycerol. In some cases, at least 5% (e.g., at least 10%, at least 15%, at least 20%, or at least 25%) of the lipid composition can be a triacylglycerol. In some cases, a lipid composition can contain from about 5% to about 25% (e.g., about 5% to about 22%, about 5% to about 17%, about 5% to about 12%, about 5% to about 7%, about 22% to about 17%, about 22% to about 12%, about 22% to about 7%, about 17% to about 12%, about 17% to about 7%, or about 12% to about 7%) of a triacylglycerol. In some embodiments, a lipid composition contains no more than 20%, no more than 18%, no more than 16%, no more than 14%, no more than 12%, or no more than 10% of a triacylglycerol. In some cases, the TG can include a plurality of different carbon lengths (e.g., about 5 to about 30 carbons). The TGs subtypes of the lipid composition can contain about 5 to about 30 carbons. For example, the TGs subtypes of the lipid composition can contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and / or 30 carbons. In some cases, the TG subtypes of the lipid composition can contain one or more double bonds. For example, the double bonds can occur at position 0, 1, 2, 3, and / or 4 of the TG. In some cases, the TGs of the lipid composition can contain a plurality of TG subtypes selected from the group consisting of TG (12:0_12:0_22:4), TG (15:0_14:0_14:0), TG (15:0 14:0 16:0), TG (15:0 14:0 16:1), TG (15:0 16:0 16:0), TG (15:0_16:0_16:l), TG (15:0_16:l_16:l), TG (15:0_16: l_18:l), TG (16:0 18:2 18:2), TG (18:0_ 16:0_16:0), TG (18: 1 18: 1 18: 1), TG (18: 1 18: 1_18:2), TG (18: 1_18:2_18:2), TG (18:2_18:2_18:2), TG (16:0_10:0_16:0), TG (16:0_10:0_16: 1), TG (16:0_14:0_16:0), TG (16:0_14:0_16:l), TG (16:0_16:0_16: l), TG (16:0_16: l_16: l), TG (16:0_16: 1_16: 1) , TG (16:0_16: 1_18: 1), TG (16:0_18: l_20:3), TG (16: 1 10:0 14:0), TG (16:l_10:0_16:l), TG (16:1 10:0 18:3), TG (16: l_12:0_16: l), TG (16:1 14:0 14:0), TG (16:l_14:0_16:l), TG (16: 1_16: 1_16: 1), TG (16:1 16:1 18:1), TG (16: 1 16: 1 18:3), TG (16:1 18:1 18:1), TG (18:0 10:0 16:0), TG (18:0 16:0 18:1), TG (18:0_16:l_16;l), TG (18:0 18:0 18:1), TG (18:0 18:1 18:1), TG (18:4_14:0_16:l), TG (18:4 16:1 16:1), TG(18:4 16: 1 18: 1), TG (26:0_14:0_16:l), TG (26:0 16:0 18:1), TG (26:0_16: l_16: l) TG (16:0_10:0_14:0), TG (16:0 18: 1 18:3), TG (18:0_16:0_16: l), TG (18: 1 18: 1 18:3), TG (26 : 0 10 : 0 16 : 0), and TG (16: 1 14: 1_16: 1). In some embodiments, the TG of the lipid composition can contain a plurality of at least two (e.g., at least five, at least ten, at least fifteen, at least twenty, or at least twenty five) different TG subtypes. For example, the TGs of the lipid composition can contain a plurality of TG subtypes of at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, or at least 43 different subtypes.

[0056] Lipid compositions provided herein can contain one or more than one cholesterol esters (ChE). A composition provided herein can contain any appropriate amount of a cholesterol ester. In some cases, at least 0.01% (e.g., at least 0.02%, at least 0.04%, at least 0.06%, at least 0.08%, or at least 0.1%) of the lipid composition can be a cholesterol ester. In some embodiments, a lipid composition contains no more than 1.2%, no more than 1.0%, no more than 0.8%, no more than 0.6%, no more than 0.4%, or no more than 0.2% of a cholesterol ester. In some cases, a lipid composition can contain from about 0.01% to about 1% (e.g., about 0.01% to about 0.4%, about 0.01% to about 0.1%, about 0.01% to about 0.04%, about 0.01% to about 0.02%, about 1% to about 0.4%, about 1% to about 0.1%, about 1% to about 0.04%, about 1% to about 0.02%, about 0.4% to about 0.1%, about 0.4% to about 0.04%, about 0.4% to about 0.02%, about 0.1% to about 0.04%, about 0.1% to about 0.02%, or about 0.04% to about 0.02%) of a cholesterol ester.

[0057] Lipid compositions provided herein can contain one or more than one wax esters. A composition provided herein can contain any appropriate amount of a wax ester (WE). In some cases, at least 0.01% (e.g., at least 0.02%, at least 0.04%, at least 0.06%, at least 0.08%, or at least 0.1%) of the lipid composition can be a wax ester. In some embodiments, a lipid composition contains no more than 1.4%, no more than 1.2%, no more than 1.0%, no more than 0.8%, no more than 0.6%, or no more than 0.4% of a wax ester. In some cases, a lipid composition can contain from about 0.01% to about 1% (e.g., about 0.01% to about 0.4%, about 0.01% to about 0.1%, about 0.01% to about 0.04%, about 0.01% to about 0.02%, about 1% to about 0.4%, about 1% to about 0.1%, about 1% to about 0.04%, about 1% to about 0.02%, about 0.4% to about 0.1%, about 0.4% to about 0.04%, about 0.4% to about 0.02%, about 0.1% to about 0.04%, about 0.1% to about 0.02%, or about 0.04% to about 0.02%) of a wax ester.

[0058] Lipid compositions provided herein can contain one or more than one phosphatidylserines (PS). A composition provided herein can contain any appropriate amount of a phosphatidylserine. In some cases, at least 0.01% (e.g., at least 0.02%, at least 0.04%, at least 0.06%, at least 0.08%, or at least 0.1%) of the lipid composition can be a phosphatidylserine. In some embodiments, a lipid composition contains no more than 1.2%, no more than 1.0%, no more than 0.8%, no more than 0.6%, no more than 0.4%, or no more than 0.2% of a phosphatidylserine. In some cases, a lipid composition can contain from about 0.01% to about 1% (e.g., about 0.01% to about 0.4%, about 0.01% to about 0.1%, about 0.01% to about 0.04%, about 0.01% to about 0.02%, about 1% to about 0.4%, about 1% to about 0.1%, about 1% to about 0.04%, about 1% to about 0.02%, about 0.4% to about 0.1%, about 0.4% to about 0.04%, about 0.4% to about 0.02%, about 0.1% to about 0.04%, about 0.1% to about 0.02%, or about 0.04% to about 0.02%) of a phosphatidylserine.

[0059] Lipid compositions provided herein can contain one or more than one phosphatidylcholines (PC). A composition provided herein can contain any appropriate amount of a phosphatidylcholine. In some cases, at least 0.5% (e.g., at least 1%, at least 1.5%, at least 2%, at least 2.5%, or at least 3%) of the lipid composition can be a phosphatidylcholine. In some embodiments, a lipid composition contains no more than 3%, no more than 2.5%, no more than 2%, no more than 1.8%, no more than 1.6%, no more than 1.4%, no more than 1.2%, or no more than 1% of a phosphatidylcholine. In some cases, a lipid composition can contain from about 0.5% to about 3% (e.g., 0.5% to about 2.5%, about 0.5% to about 2%, about 0.5% to about 1.5%, about 0.5% to about 1%, about 2.5% to about 2%, about 2.5% to about 1.5%, about 2.5% to about 1%, about 2% to about 1.5%, about 2% to about 1%, or about 1.5 % to about 1%) of a phosphatidylcholine.

[0060] Lipid compositions provided herein can contain one or more than one phosphatidylinositols (PI). A composition provided herein can contain any appropriate amount of a phosphatidylinositol. In some cases, at least 0.5% (e.g., at least 1%, at least 1.5%, at least 2%, at least 2.5%, or at least 3%) of the lipid composition can be a phosphatidylinositol. In some embodiments, a lipid composition contains no more than 5%, no more than 4%, no more than 3%, no more than 2.5%, or no more than 2% of a phosphatidylinositol. In some cases, a lipid composition can contain from about 0.5% to about 3% (e.g., 0.5% to about 2.5%, about 0.5% to about 2%, about 0.5% to about 1.5%, about 0.5% to about 1%, about 2.5% to about 2%, about 2.5% to about 1.5%, about 2.5% to about 1%, about 2% to about 1.5%, about 2% to about 1%, or about 1.5 % to about 1%) of a phosphatidylinositol.

[0061] Lipid compositions provided herein can contain one or more than one ceramides (Cer). A composition provided herein can contain any appropriate amount of a ceramide. In some cases, at least 0.01% (e.g., at least 0.02%, at least 0.04%, at least 0.06%, at least 0.08%, or at least 0.1%) of the lipid composition can be a ceramide. In some embodiments, a lipid composition contains no more than no more than 1.2%, no more than 1.0%, no more than 0.8%, no more than 0.6%, no more than 0.4%, or no more than 0.2% of a ceramide. In some cases, a lipid composition can contain from about 0.01% to about 1% (e.g., about 0.01% to about 0.4%, about 0.01% to about 0.1%, about 0.01% to about 0.04%, about 0.01% to about 0.02%, about 1% to about 0.4%, about 1% to about 0.1%, about 1% to about 0.04%, about 1% to about 0.02%, about 0.4% to about 0.1%, about 0.4% to about 0.04%, about 0.4% to about 0.02%, about 0.1% to about 0.04%, about 0.1% to about 0.02%, or about 0.04% to about 0.02%) of a ceramide.

[0062] Lipid compositions provided herein can contain one or more than one lysophosphatidylserines (LPS). A composition provided herein can contain any appropriate amount of a lysophosphatidylserine. In some cases, at least 0.01% (e.g., at least 0.02%, at least 0.04%, at least 0.06%, at least 0.08%, or at least 0.1%) of the lipid composition can be a lysophosphatidylserine. In some embodiments, a lipid composition contains no more than 1.2%, no more than 1.0%, no more than 0.8%, no more than 0.6%, no more than 0.4%, or no more than 0.2% of a lysophosphatidylserine. In some cases, a lipid composition can contain from about 0.01% to about 1% (e.g., about 0.01% to about 0.4%, about 0.01% to about 0.1%, about 0.01% to about 0.04%, about 0.01% to about 0.02%, about 1% to about 0.4%, about 1% to about 0.1%, about 1% to about 0.04%, about 1% to about 0.02%, about 0.4% to about 0.1%, about 0.4% to about 0.04%, about 0.4% to about 0.02%, about 0.1% to about 0.04%, about 0.1% to about 0.02%, or about 0.04% to about 0.02%) of a lysophosphatidylserine.

[0063] In some embodiments, the lipid composition is extracted from an organism (e.g., a yeast). In some cases, one or more of the lipids in the lipid composition is extracted from one or more types of organisms (e.g., a plurality of different strains of yeast, or a plurality of yeast and a plurality of algae). In other cases, each of the lipids in the lipid composition is extracted from a plurality of the same organism (e.g., a plurality of the same strain of yeast, a plurality of the same type of animal cell). In some embodiments, all of the lipids in the lipid composition are extracted together from a plurality of the same organism (e.g., a plurality of the same strain of yeast). In some cases, one or more of the lipids in the lipid composition are extracted from two or more pluralities of different organisms (e.g., a plurality of yeast and a plurality of algae, a plurality of yeast and a plurality of algae and a plurality of bacteria. For example, the organism can be a prokaryote or a eukaryote. In some cases, the organism is a prokaryote. For example, the prokary ote can be a bacterium (e.g., a bacterium from the intestines of an animal). In some cases, the organism is a eukaryote. For example, the eukaryote can be an algae, a fungus, a plant, or a mammalian cell. The fungus can be a yeast (e.g., Saccharomyces cerevisiae). In some cases, the mammalian cell can be anon-human mammal or a human cell. For example, the non-human mammal or the human cell can be a liver cell, a kidney cell, an intestine cell (e.g., a small intestine cell, a colon cell, or a large intestine cell), a connective tissue cell (e.g., a bone cell, a tendons cell, an adipose cell, or a ligament cell), a muscle cell, a heart cell, a vessel cell (e.g., a capill ary cell, a vein cell, an artery cell, or lymphatic vessel cell), a stomach cell, other gut organ cells, an anus cell (e.g., a rectum cell), a joint cell, a nerve cell, a skin cell, a nasal cavity cell, a tongue cell, an appendix cell, a diaphragm cell, a lung cell, a thyroid cell, an adrenal gland cell, an ear cell, a larynx cell, an esophagus cell, a trachea cell, a brain cell (e.g., a cerebellum cell, a pineal gland cell, a pituitary gland cell, or hypothalamus cell), an eye cell, a spinal cord cell, a thymus gland cell, a lymph node cell, a pancreas cell, a ureter cell, a bronchus cell, a genital cell (e.g., a penis cell, a testes cell, a scrotum cell, a vas deferens cell, a vagina cell, a clitoris cell, a cervix, an ovary, a fallopian tube, or vulva bulbourethral gland cell), a pharynx cell, a salivary gland cell, a urethra bladder cell, a gallbladder cell, a placenta cell, a uterus cell, a bone marrow cell, a mouth cell, a prostate cell, a seminal vesicle cell, a hair follicle cell, a mesentery cell, a subcutaneous tissue cell, a mammary gland cell, a tooth cell, an interstitium cell, a parathyroid gland cells, a tonsil cell, a nail cell, a vestigial cell or a cancer cell. In some embodiments, the cancer cell can be derived from an non-human mammal or a human tissue. For example, the cancer cell can be derived from a liver, a kidney, an intestine (e.g., a small intestine a colon or a large intestine), a connective tissue (e.g., a bone, a tendons, an adipose or a ligament), a muscle a heart a vessel (e.g., a capillary a vein an artery or lymphatic vessel ), a stomach, other gut organ, an anus (e.g., a rectum ), a joint, a nerve, a skin, a nasal cavity, a tongue, an appendix, a diaphragm, a lung, a thy roid, an adrenal gland , an ear, a larynx, an esophagus, a trachea, a brain (e.g., a cerebellum, a pineal gland, a pituitary gland, or hypothalamus), an eye, a spinal cord, a thymus gland, a lymph node, a pancreas, a ureter, a bronchus, a genital (e.g., a penis, a testes, a scrotum, a vas deferens, a vagina, a clitoris, a cervix, an ovary, a fallopian tube, or vulva bulbourethral gland), a pharynx, a salivary gland, a urethra bladder, a gallbladder, a placenta, a uterus, a bone marrow, a mouth, a prostate, a seminal vesicle, a hair follicle, a mesentery, a subcutaneous tissue, a mammary gland, a tooth, an interstitium, a parathyroid gland, a tonsil, a nail, a vestigial organ or a body-fluid. In some embodiments, a body fluid can be blood, cerebral spinal fluid, intracellular fluid (e.g., fluid in cytosol and nucleus of a cell), an extracellular fluid intravascular fluid (e.g., blood plasma), an interstitial fluid lymphatic fluid (e.g., interstitial fluid), transcellular fluid, breast milk, synovial fluid, semen, or exosome (e.g., vesicles, extracellular vesicles).

[0064] In some embodiments, the lipid compositions provided herein can be made without extracting lipids from an algae, animal, or plant source. In some embodiments, one or more of the lipids of a lipid composition described herein can be generated using chemical synthesis techniques known in the art. In some embodiments, one or more of the lipids in the lipid compositions provided herein can be synthesized (e.g., chemical synthesis, chemoenzymatic synthesis, or enzyme-free synthesis). In some embodiments, all of the lipids in the lipid compositions described herein can be synthesized. In other embodiments, fewer than all of the lipids in the lipid compositions described herein can be synthesized.

[0065] In some embodiments, the yeast is an engineered yeast strain overexpressing a heme activator protein (HAP) gene product, a SIR2 gene product, or both a HAP gene product and a SIR2 gene product. In some embodiments, the engineered yeast strain overexpresses one or more genes that encode a HAP complex component. For example, the HAP complex component can include HAP1, HAP2, HAP3, HAP4, and / or HAP5. In some embodiments, the yeast is an engineered yeast strain overexpressing a HAP4 gene product, a SIR2 gene product, or both a HAP4 gene product and a SIR2 gene product. In some embodiments, overexpression of one or more genes encoding a HAP complex component provided herein has the same effects as the overexpression of HAP4 gene. In some cases, the engineered yeast strain is an engineered long-lived yeast strain. For example, the engineered yeast strain can be NH0880, NH0868, and / or NH0897. In some embodiments, the engineered yeast strain is NH0880. The yeast strains described herein can be any yeast species known in the art. For example, the yeast species can be a Saccharomyces (e.g., Saccharomyces cerevisiae. Saccharomyces boulardii. Saccharomyces bayanus, and Saccharomyces paradoxus). In some embodiments, any yeast species can be used to produce the engineered yeasts strains described herein.

[0066] In some embodiments, the engineered yeast strains (e.g., NH0880, NH0868, and / or NH0897) include one or more additional genetic modifications to improve the therapeutic efficacy of the lipids extracted from the engineered yeasts. For example, the genetic modification (e.g. mutations in the gene PRT1 or overexpression of the gene GCN4) can target a protein synthesis process (e.g., global protein synthesis machinery) in the yeast strains described herein. In some embodiments, the yeast strains described herein can both produce a lipid profile described herein and inhibit / reduce the protein synthesis in the yeast strains. Such genetic modifications targeting a protein synthesis machinery can be performed using homologous recombination, RNA interference (e.g., small interfering RNAs or short hairpin RNAs), microRNA, morpholino, Transcription Activator-Like Effector Nucleases (TALENS), Zinc Finger Nucleases (ZFN), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas9 system, Proteolysis Targeting Chimeras (PROTAC) system, or antisense oligonucleotides. Pharmaceutical Composition The lipid composition disclosed herein can be included in and / or formulated as a pharmaceutical composition. Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include oral, parenteral, e.g., intravenous, intradermal, subcutaneous, intratumoral, intramuscular or subcutaneous administration.

[0067] The lipid composition provided herein can be formulated in an ingestible form or a topical form. In some embodiments, the ingestible form is a pharmaceutical composition or an edible. For example, the pharmaceutical composition can be in the form of a liquid, solution, suspension, tablet, powder, granule, pill, capsule, gel, cream, mist, atomized vapor, aerosol, soft gelatin capsule, or hard gelatin capsule. In some embodiments, for oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets can be coated by methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspension, or they can be presented as a dry product for constitution with saline or other suitable liquid vehicle before use. Liquid preparations also can contain pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles, preservatives, buffer salts, flavoring agents, coloring agents, and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the compound. In some cases, the lipid composition is an edible (e.g., a snack bar, a gummy, a yogurt, or a supplement).

[0068] A lipid composition provided herein can be administered by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. In some cases, the lipid composition in a topical form can be a cream, a serum, a gel, a mask, an ointment, a cleanser, salves, a gel, or as generally known in the art.

[0069] In some embodiments, methods provided herein includes a yeast composition comprising an engineered yeast strain NH0880, NH0868, and / or NH0897. In some embodiments, the yeast composition can be formulated in an ingestible form. The ingestible form can be a pharmaceutical composition or an edible. For example, the pharmaceutical composition can be a tablet, a powder, a granule, a pill, a capsule, a gel, a solid, or a liquid. In some embodiments, the edible is a bakery item, a beverage, a food ingredient, an animal feed, a snack bar, a gummy, a yogurt, or a supplement. A bakery item can be a bread, a pastry, a dough, a bagel, a pretzel, a roll, and / or a bun. In some embodiments, the beverage can be an alcoholic drink or a non-alcoholic drink. For example, an alcoholic dnnk can be a beer, a wine, a spirit, or a cider. Additionally, a non-alcoholic drink can be a probiotic dnnk, a tea, a dairy drink, a juice, a mocktail, an alcohol-free beverage, or a sparkling drink. In some embodiments, the food ingredient is a yeast extract, a yeast flake, an autolyzed yeast, an active dry yeast, an instant dry yeast, a fresh yeast, a bread machine yeast, a spice, an herb, a syrup, or a cream. The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0070] Methods of Use

[0071] Provided herein are methods of modulating cellular aging in a subject. In some cases, methods provided herein can include administering a therapeutically effective amount of a lipid composition described herein to the subject. In some embodiments, the lipid composition described herein for modulating cellular aging in a subject comprises at least a TG. For example, the lipid composition for modulating cellular aging in a subject can comprise at least a TG and a PC. In some embodiments, the lipid composition described herein for modulating cellular aging in a subject comprises one or more lipids extracted from a yeast strain (e.g., NH0880, NH0868, and / or NH0897) comprising one or more generic modifications targeting a protein synthesis machinery (e.g., global protein synthesis). In some embodiments, the methods provided herein for modulating cellular aging in a subject can include administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strains NH0880, NH0868, and / or NH0897 described herein to the subject. In some embodiments, the engineered yeast strains (e.g., NH0880, NH0868, and / or NH0897) further comprise one or more genetic modifications targeting a protein synthesis machinery. In some embodiments, methods provided herein for modulating cellular aging in a subject can include administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strain NH0880 described herein to the subject. In some cases, the yeast strain NH0880 can comprise one or more genetic modifications targeting a protein synthesis machinery.

[0072] In some cases, the methods provided herein can slow age-related decline in a subject. In some embodiments, the lipid composition described herein for slowing age-related decline in a subject comprises at least a TG. For example, the lipid composition for slowing age-related decline in a subject can comprise at least a TG and a PC. For example, age-related decline can include cellular dysfunction and / or deterioration, or decline in intestinal barrier function. In some embodiments, age- related decline can include cognitive symptoms / cognitive decline. For example, cognitive symptoms / cognitive decline can include difficulties with multitasking, memory (e.g., holding information in mind), finding words, concentration (e.g., sustaining attention), impaired reasoning, and / or cognitive impairment (e.g., slow thinking and / or processing information). In some embodiments, age-related decline can include physical symptoms / physical decline. For example, physical symptoms / physical decline can include reduced muscle mass and strength, reduced bone densify and size, hearing impairment, weakening vision, increased risk of arthritis, hypertension, heart disease, diabetes, reduced physical fitness (e.g., strength, endurance, flexibility, agility), and / or reduced level of daily activities.

[0073] Also, provided herein are methods of extending the lifespan of a subject. The method can include administering a therapeutically effective amount of a lipid composition disclosed herein to the subject. In some embodiments, the lipid composition described herein for extending the lifespan of a subject comprises at least a TG. For example, the lipid composition for extending the lifespan of a subject can comprise at least a TG and a PC. In some embodiments, the lipid composition described herein for extending the lifespan of a subject comprises one or more lipids extracted from a yeast strain (e.g., NH0880, NH0868, and / or NH0897) comprising one or more genetic modifications targeting a protein synthesis machinery (e.g., global protein synthesis). In some embodiments, the methods provided herein for extending the lifespan of a subject can include administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strains NH0880, NH0868, and / or NH0897 described herein to the subject. In some embodiments, the engineered yeast strains (e.g., NH0880, NH0868, and / or NH0897) further comprise one or more genetic modifications targeting a protein synthesis machinery. In some embodiments, the methods provided herein for extending the lifespan of a subject can include administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strain NH0880 described herein to the subject. In some cases, the yeast strain NH0880 can comprise one or more genetic modifications targeting a protein synthesis machinery.

[0074] In some cases, methods provided herein can extend the life of a subject by at least 3% (e.g., at least 5%, at least 7%, at least 12%, at least 17%, at least 20% or at least 25%). In some embodiments, the methods provided herein can extend the life of a subject by about 3% (e.g., about 5%, about 7%, about 12%, about 17%, about 20% or about 25%). In some embodiments, the methods provided herein can extend the healthspan of a subject by at least 3% (e.g., at least 5%, at least 7%, at least 12%, at least 17%, at least 20% or at least 25%). In some embodiments, the methods provided herein can extend the healthspan of a subject by about 3% (e.g., about 5%, about 7%, about 12%, about 17%, about 20% or about 25%). Extending healthspan can include delaying the onset of age-related diseases and conditions from what it otherwise would have been if such administration was not performed.

[0075] Also, provided herein are methods of modulating gut homeostasis in a subject. The method can include administering a therapeutically effective amount of a lipid composition disclosed herein to the subject. In some embodiments, the lipid composition described herein for modulating gut homeostasis in a subject comprises at least a TG. For example, the lipid composition for modulating gut homeostasis in a subject can comprise at least a TG and a PC. In some embodiments, the lipid composition described herein for modulating gut homeostasis in a subject comprises one or more lipids extracted from ayeast strain (e.g., NH0880, NH0868, and / or NH0897) comprising one or more genetic modifications targeting a protein synthesis machinery (e.g., global protein synthesis). In some embodiments, the methods provided herein for modulating gut homeostasis in a subject can include administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strains NH0880, NH0868, and / or NH0897 described herein to the subject. In some embodiments, the engineered yeast strains (e.g., NH0880, NH0868, and / or NH0897) further comprise one or more genetic modifications targeting a protein synthesis machinery . In some embodiments, the methods provided herein for modulating gut homeostasis in a subject can include administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strain NH0880 described herein to the subject. In some cases, the yeast strain NH0880 can comprise one or more genetic modifications targeting a protein synthesis machinery.

[0076] The modulating gut homeostasis can include promoting gut integration by incorporating in the membrane and lipid droplet biosynthesis. In some cases, the methods provided herein can modulate lipid metabolic pathways, lipid signaling pathways, lipid membrane order, and / or expression of diverse lipid classes. In some cases, methods provided herein can mitigate loss of lipids from the intestine. In some embodiments, the methods provided herein can improve gut health. For example, the methods provided herein can strengthen the gut barrier function (e.g., making the gut membrane more ordered and compact resulting in a membrane that is resistant to age- related damage).

[0077] In some embodiments, the subject is an animal. The animal can be a mammal, a chromadorea, or an insecta. Non- limitation examples of mammals include, humans, non-human primates (e.g., monkeys), horses, bovine species, porcine species, dogs, cats, mice, or rats. A chromadorea can be a Caenorhabditis . An insecta can be a Drosophila.

[0078] A “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. For example, an effective amount is one that achieves a desired therapeutic effect, e.g., an amount necessary to treat a disease, or to reduce risk of development of disease or disease symptoms (also referred to as a prophylactically effective amount). An effective amount can be administered in one or more administrations, applications, or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. For example, a therapeutically effective amount of a lipid composition as provided herein can be effective to modulate the cellular aging, extend the lifespan, and / or modulate the gut homeostasis in a subject (e.g., a mammal). The lipid composition provided herein can be administered one or more times per day (e.g., one time per day, two times per day, three times per day, four times per day, or five times per day) to one or more times per week, including once every other day, once every three days, or twice a week. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. Various factors can influence the actual amount used for a particular application. For example, the frequency of administration, duration of treatment, combination of other agents, site of administration, stage of disease (if present), and the anatomical configuration of the treated area may require an increase or decrease in the actual amount administered.

[0079] The frequency of administration of lipid compositions provided herein can be any frequency. For example, the frequency of administration can be from about three times a day to about once a week, or more specifically, from about twice a day to about once a week. In addition, the frequency of administration can remain constant or can be variable during the duration of treatment. As with the amount administered, various factors can influence the actual frequency of administration used for a particular application. For example, the amount (dose), duration of treatment, combination of agents, site of administration, stage of disease (if present), and the anatomical configuration of the treated area may require an increase or decrease in administration frequency. For example, the methods can include administering a first dose, followed by one or more additional doses at a later time (e.g., a “booster” dose), e.g., 1, 2, 3, 4, 5, 6, or 7 days from a previously administered dose. For example, administration can include administering two, three, four, or five doses, administered 1, 2, 3, 4, 5, 6, or 7 days apart. The length of time between multiple doses can be the same (e.g., 2 days between each dose, 3 days between each dose, 4 days between each dose, etc.), or the length of time between multiple doses can be different between each dose (e.g., 4 days between the first and second dose, 3 days between the second and third dose, and 2 days between the third and fourth dose).

[0080] An effective duration for administering a lipid composition provided herein can be any duration that reduces age-related decline, modulates cellular aging, extends lifespan, extends healthspan, and / or modulates gut homeostasis in the subject without producing significant toxicity to the mammal. In some cases, the effective duration can vary from several days to several weeks, to several months, or longer. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the effective amount, frequency of administration, use of route of administration, and severity of the subject’s condition.

[0081] Dosage, toxicity and therapeutic efficacy of the lipid compositions disclosed herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compositions that exhibit high therapeutic indices are preferred. While compositions that exhibit toxic side effects may be used, care should be taken to minimize and reduce side effects. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compositions used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models. Such information can be used to more accurately determine useful doses in humans.

[0082] EXAMPLES

[0083] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0084] Example 1: Long-lived yeast-derived lipids promoted longevity in Drosophila

[0085] Methods and materials

[0086] Stimulated Raman scattering microscopy

[0087] An upright laser-scanning microscope (DIY multiphoton, Olympus) equipped with a 25 x water objective (XLPLN, WMP2, 1.05 NA, Olympus) was utilized for near-IR throughput. The experimental setup involved a synchronized pulsed pump beam (tunable 720-990 nm wavelength, 5-6 ps pulse width, and 80 MHz repetition rate) and a Stokes beam (wavelength at 1032 nm, 6 ps pulse width, and 80 MHz repetition rate) provided by a picoEmerald system (Applied Physics & Electronics) coupled to the microscope. The pump and Stokes beams were collected in transmission using a high NA oil condenser (1.4 NA).

[0088] To selectively detect the stimulated Raman loss signal, a high optical density (O.D.) shortpass filter (950 nm, Thorlabs) was employed, which blocked the Stokes beam and allowed the transmission of the pump beam only. The pump beam was directed onto a Si photodiode for signal detection. The output current from the photodiode was terminated, filtered, and demodulated using a lock-in amplifier operating at 20 MHz. The demodulated signal was then fed into the FV-OSR software module (Olympus) integrated with the FV3000 microscope system to generate images during laser scanning. All acquired images were of size 512 x 512 pixels, with a dwell time of 80 ps and an imaging speed of approximately 23 seconds per image. To minimize background interference, a background image was obtained at 1900 cm-1and subtracted from all stimulated Raman scattering (SRS) images using Fiji (ImageJ) software.

[0089] Spontaneous Raman spectroscopy

[0090] Raman spectra of all samples were acquired using a confocal Raman microscope (XploRA PLUS, Horiba) connected to a Raman spectrometer. A diode line focus laser with a wavelength of 532 nm (~40 mW at the sample) was utilized, and the laser beam was focused onto the cells using a 100x objective (MPLN100X, Olympus). The laser power was optimized to ensure cell integrity and prevent damage. Detection of the Raman signal was performed using a cooled charge coupled device (CCD) detector fitted to a spectrometer with a grating of 2400 grooves per mm. The acquisition time for each Raman spectrum was 60 seconds. The instrument calibration was validated using the silicon line at 520 cm '. Background spectra were recorded for each sample point at the same focus plane and immediately subtracted from the original spectrum. All ratio calculations were conducted on the raw data before applying any normalization or baseline correction procedures. Data analysis and processing were performed using Originlab software (Origin Lab Corporation, Northampton, MA). Yeast strains and culture

[0091] The Saccharomyces cerevisiae strains used in this study were derived from the BY4741 background (BY4741 MATa hisS l leu2 \() m,ell5 \() uraSAO). The engineered yeast strains (Strains 868, 880, and 898) were constructed as described elsewhere (Li et al., 2020). Detailed information about the strains tested can be found in Table 1. Glycerol stocks of frozen S. cerevisiae cells were initially cultured on yeast extract peptone dextrose (YPD) plates. Incubated at 30 °C for 2 days, these plates were then stored at 4 °C. Every four weeks, the strains were re-streaked onto fresh YPD plates. Subsequently, a single colony was chosen and introduced into 5 mL synthetic defined (SD, prepared from CSM powder from Sunrise Science, #1001-100, with 2% glucose) medium. The culture was incubated at 30 °C with shaking at 250 rpm for 24 hours. Afterward, the cells were transferred to either a 20 mL SD liquid culture or SD solid agar plates for experimental preparations. Cultures were grown until reaching an optical densify at 600 nm (OD600) of 0.6-0.8.

[0092] Table 1 : Yeast strains

[0093] Total yeast lipid extraction

[0094] Lipid extraction was performed using the chloroform-methanol method. Initially, yeast cells were cultured, and after harvesting by centrifugation at 1000g for 5 minutes at room temperature, the supernatant was completely removed and discarded. The cells were then frozen at -80°C overnight. Subsequently, the dry samples were mixed with 0.5 mL methanol, 0.25 mL chloroform, and 0.5 mL water. The mixture underw ent ultrasonication in ice water for 20 minutes. An additional 0.25 mL chloroform was added, and the sample was sonicated for an additional 10 minutes. The resulting mixture was centrifuged at 4000g at 4 °C for 5 minutes. The lower layer, containing the extracted lipids, was then evaporated in a fume hood overnight. Following evaporation, membrane-like substances were observed at the bottom of the tubes.

[0095] Fly husbandry

[0096] Wild-type flies (wl 118 stock #5905) were initially acquired from the Bloomington Stock Center and have been maintained in the laboratory for multiple generations. The flies were kept under standard conditions, including a temperature of 25 °C, a humidity level of 60%, and a 12-hour dark / light cycle. They were fed with com meal-based fly food (Nutri-Fly, Cat. No. 66-113; Genesee Scientific Corporation), following established protocols.

[0097] For live yeast supplementation, circular squares with a diameter of 0.7 cm were cut from solid yeast agar medium cultured with yeast. These yeast squares were positioned on top of the fly food to act as a feeding source for the flies. To ensure a fresh and nutn ent-rich food source, replace these yeast squares every three days.

[0098] To feed flies with yeast lipids, the lipids were mixed with 500 LLL of 0.2 M NaOH and incubated at 75°C for 5 minutes. Following incubation, the lipid mixture was added to 2 mL of com meal and thoroughly mixed before being provided as a food source to the flies. In the short- and medium-chain fatty acid-contaming phosphatidylcholine (PC) feeding experiments, 4:0 PC, 6:0 PC, 10:0 PC, and 12:0 PC (Cat. No. 850303, 850305, 850325, 850335; Avanti Research) were mixed in equal proportions and added to cornmeal at a final concentration of 5 mM. For the short- and medium-chain fatty acid-containing triglyceride (TG) feeding experiments, 5 mM 1,2,3-trioctanoylglycerol (Cat. No. 870111; Avanti Research) was supplemented into cornmeal. In the co-feeding experiment, PC and TG were equally mixed and added to cornmeal at a final concentration of 5 mM.

[0099] Drosophila lifespan assay

[0100] For lifespan experiments, 2-day-old W1118or Canton S flies were collected and sorted by gender under CO2 anesthesia into separate experimental groups, with 20 flies per vial. The flies were transferred to fresh food every 2 days to maintain a consistent nutrient supply. The number of deceased flies was recorded daily to track their lifespan.

[0101] Lipid extraction

[0102] Lipid extractions were performed according to the methodology of Bligh and Dyer (Bligh EG, Dyer WJ, 1959). In brief, the cell pellets were resuspended in 200 pL water, transferred to a glass vial, and 750 pL 1 :2 (v / v) CHC13: MeOH was added and vortexed well. Then 250 pL CHC13 was added and vortexed well. Finally, 250 pL ddH2O was added and vortexed well. The samples were centrifuged at 3000 RPM for 5 min at 4 °C. The lower phase was transferred to a new glass vial and dried under nitrogen stored at -20 °C until subsequent lipid analysis.

[0103] LC-MS / MS

[0104] Separation of lipids was performed on an Accucore C30 column (2.6 pm, 2.1 mm x 150 mm, Thermo Scientific). The column oven temperature was 45 °C. A binary gradient elution was carried out using different ratios of eluents A (acetonitrile: water, 60:40, v / v) and B (isopropanol: acetonitrile, 90:10, v / v), both containing 10 mM ammonium formate and 0.1% formic acid and the following gradient: -3 to 0 min isocratic elution with 30% B for the equilibration of the column; 0-2 mm, 30%-43% B; 2-2.1 mm, 43-55%; 2.1-12 min, 55-65% B; 12-18 mm, 65%-85% B; 18-20 mm, 85%-100% B; 20-25 mm, 100% B; 25-25.1 mm, 100 - 30%; 25. 1-28 min, 30% B for column washing and equilibration. The flow rate was set to 260 pL min-1and the temperature of the sample tray at 10 °C. The Q Exactive MS was operated in a full MS scan mode (resolution 70,000 at m / z 200) followed by ddMS2 (17,500 resolution) in both positive and negative mode. The AGC target value was set at 1E6 and 1E5 for the MS and MS / MS scans, respectively. The maximum injection time was 200 ms for MS and 50 ms for MS / MS. HCD was performed with a stepped collision energy' of 30 ± 10% for negative and 25%, 30% for positive ion mode with an isolation window of 1.5 Da.

[0105] Data analysis and post-processing

[0106] Data were analyzed with LipidSearch 4.2.21 software. To facilitate comparison, lipid profiles were normalized to the total intensity of WT (designated as 270) control. Only peaks with molecular identification grade: A or B were accepted (A: lipid class and fatty acid completely identified or B: lipid class and some fatty acid identified). SRplot (http: / / www.bioinformatics.com.cn / en), a free web platform, was used to generate cluster plot. Significantly changed lipid species (FO1.5. P- value<0.05) were submitted to Lipid Ontology (LION) for lipid ontology analysis. Data visualization was performed on Prism 7 software (GraphPad Software, Inc.).

[0107] Yeast lYO-laheling experiments

[0108] To investigate the changes in metabolic activity of yeast, the yeast cells were transferred to 50% D2O culture media and incubated for 12 hours. After incubation, 5 mL yeast cells were harvested by centrifuging the culture at 3,000 rpm for 5 mins at 4°C. The supernatant was carefully removed without disturbing the cell pellet. The yeast cells were then fixed using a 3.8% paraformaldehyde (PFA) solution for 30 minutes. Following fixation, the cells were immobilized onto glass slides coated with poly-lysine for 10 minutes. Raman measurements and stimulated Raman scattering (SRS) imaging were subsequently performed on the immobilized yeast cells.

[0109] Fly DiO-labeling experiments

[0110] To invest gate the changes in metabolic activity of flies at different ages, 20- day (middle age) and 40-day (old age) adult flies were transferred to the 20% D2O fly food for 5 days. Then five flies at ages 25 days and 45 days were randomly selected, sacrificed, and their guts were dissected in PBS and fixed in 4% PFA for 15 minutes. After fixation, the tissues were washed three times with PBS in glass wells. The tissues were then sandwiched between a cover slide and the bottom slide with PBS solution. Then, nail polish was used to seal the surrounding of cover slides.

[0111] Data Analysis

[0112] Scripts and functions used for processing the Raman spectra were self-written using built-in functions provided by MATLAB. The spectral pre-processing consists of several steps that include background removal, baseline correction, and vector normalization. MATLAB software was used to import the raw spectra data. Background was subtracted, and the files were converted into an array where the spectra has been interpolated at every cm'1. The raw data was then graphed for verification, and the baseline correction was performed. The resulting spectra were vector normalized and averaged for each group to reduce the amount of noise on the graph of the biomolecular signals. Each spectral peak was assigned to the vibrations of a particular chemical bond or function group.

[0113] Lipidomics data were analyzed with LipidSearch 4.2.21 software. Only peaks with molecular identification grade: A or B were accepted (A: lipid class and fattyacid completely identified or B: lipid class and some fatty acid identified). Relative abundances are expressed as ratios of a peak intensity normalized to total peak intensities. Data visualization was performed on Prism 9 software (GraphPad Software, Inc.).

[0114] Statistical analysis

[0115] The survival data were analyzed using the Kaplan-Meier Log-rank test in MATLAB. Other data sets were analyzed using appropriate statistical tests, including the Student's t-test, ANOVA with post hoc Tukey's multiple comparison, or ANOVA with post hoc Dunnetf s comparison test, performed using GraphPad Prism software. For ratio data, analysis was conducted using raw intensity values without baseline correction or normalization. Statistical significance was indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, while nonsignificant differences were denoted as “ns”.

[0116] Results

[0117] Lipids from a long-lived yeast strain promoted Drosophila gut health and lifespan

[0118] As both sirtuins and the mitochondria (regulated by Hap4) are related to lipid metabolism (Horvath and Daum, 2013; Ye X, 2017), the effects of lipids from the engineered long-lived yeast strains 868 (yeast strain overexpressing Hap4). 897 (yeast strain overexpressing Sir 2), and 880 (yeast strain overexpressing both Hap4 and Sir 2) on lipid metabolism and longevity of flies were tested.

[0119] The lifespans of W1118flies fed with lipid extracts from three long-lived yeast strains (880, 868, 897) and the WT strain (270), as well as flies fed on standard food without extra lipids were compared. It was demonstrated that the average median (surviving 50% of the population) and maximum (the longest surviving 10% of the population) lifespans significantly increased in 880-fed flies, whereas only a modest change was observed in 868-fed files. There was no significant difference among 897- fed flies, 270-fed flies and flies fed on the standard food (FIG. 1A). These results were verified by another wild-type Drosophila line. As shown in FIG. 5, Canton S (CS) flies (Iliadi et al., 2009) fed on 880-lipids exhibited a much more extended lifespan compared to the fl77775flies.

[0120] To determine the effects of yeast lipids on the health of fly guts, SRS microscopy was used to visualize lipid metabolism in both the basal and lateral sides of gut epithelial cells of young (7-day) and old (45-day) flies, which were subjected to diets containing different lipid extract (FIGS. 6A and IB). Under normal conditions, the lipid signal, detected by SRS at wavenumber 2850 cm'1, predominantly localized to lipid droplets (LDs) within the gut epithelial cells. The specificity of the LD signal was further verified using conventional lipid dye staining (Bodipy 493 / 503) (FIG. 6C). With a small extent, this signal was observed in the non-LD regions of the cytoplasm (FIG. 6B). The abundance of membrane organelles in gut cells, know n to participate in metabolic processes, underscores the prevalence of phospholipids within these membranes. Consequently, the non-LD lipid signal detected in this study may originate from the membrane structures associated with these organelles. Corroborated with findings from other studies (Regan et al., 2016; Song et al., 2014), SRS imaging revealed that LDs were evenly distributed in both the basal and lateral sides of the polygonal cells in the R2 region of the young fly gut. However, in old flies the abundance of LDs was markedly reduced and the membrane lipids in the guts were also significantly diminished (FIGS. 6B and 6D). These findings revealed the dynamic changes in lipid distribution, both in terms of LDs and membrane-like structures, as the flies underwent the aging process.

[0121] Compared to the flies fed with 270 lipids and standard food (control), middleage (25-day) and old-age (45-day) files fed with 880, 868, 897 lipids show ed increased LD abundance in the guts (FIGS. IB, 1C, 7A, and 7B). To elucidate if the lipid content in these LDs was newly synthesized lipids or translocated from other cellular compartments or tissues, the flies were treated with D2O-labeled standard foods supplemented with or without yeast lipids, respectively. Deuterium (D) from D2O can be incorporated into lipids, replacing the hydrogen (H) ions in the carbonhydrogen (CH) bonds, and forming carbon-deuterium (CD) bonds by enzy matic reaction (Li et al., 2022; Shi et al., 2018). The CD bonds generated a peak at 2140 cm'1in the cell silence region of Raman spectrum, which served as a biomarker for the metabolic activity of new-synthesized lipids (FIG. 7E) (Li et al., 2022). A higher CD signal was observed in the guts of flies fed with lipids derived from long-lived yeasts (880, 868, 897) (presented as CD / CH2 ratio, also known as K2140 / 2850), suggesting enhanced de novo lipid synthetic activity in the gut (FIGS. ID and 7C). However, lipid synthesis in the fat body, the main fat storing organ in Drosophila, was not affected by these lipid supplements (FIGS. 7E and 7F). Moreover, it was found that the gut width increased in 868 and 880 lipids treated groups, while it decreased in 897 lipid treated group (FIGS. IE and 7D).

[0122] The dimension of the gut depends on both cell size and cell number(Jasper, 2020). Despite minimal changes in cell size, a notable increase in EC (polygonal cell) number was observed in 868 and 880 lipid-treated groups (FIGS. IF and 1G). In the adult Drosophila midgut, intestinal stem cells (ISCs) give rise to enterocytes (ECs). Therefore, the stem cell activity was tested by counting intestinal stem cells (labeled with esgGal4 > UAS-GFP) (Biteau et al., 2010). Significant decreases in stem cell counts (relative to total cell number) were observed in 880 and 868 lipid-treated groups, but an insignificant increase was detected in 897 group (FIGS. 1H and II). The discrepancy in stem cell and enterocyte number could likely be attributed to EC apoptosis within the gut. It is known that over-proliferation of ISCs and apoptosis of ECs concur in the aged gut, leading to dysplasia and disruption of intestinal integrity (Gan et al., 2021; Jasper, 2020).

[0123] Taken together, these results demonstrated that lipid extracts from the long- lived yeast strain overexpressing Sir2, HAP, or both influenced lipid metabolism and the gut morphology in flies, while lipid extracts from the strain (880) with both Sir2 and Hap overexpression significantly promoted the longevity of the animal.

[0124] SRS imaging revealed lipid metabolism changes in the engineered yeast strains

[0125] To understand how lipid products from engineered yeasts affect fly gut pathophysiology, metabolic activity and lipid composition changes in the engineered yeast strains were analyzed by spontaneous Raman microscopy. Yeast cultures were incubated in D2O labeled media, and Raman spectra were collected. The whole spectra were characterized by dominant peaks at 2935 cm'1in the CH region (CH3 vibrational modes from proteins) and 2150 cm'1(newly synthesized biomolecules) in the cell silence region (FIG. 8A). This indicated that the yeasts were enriched with protein, and new macromolecules were synthesized during D2O culture.

[0126] To clearly dissect the transformation of lipid profiles in the engineered yeasts, total lipids were extracted from deuterium (D)-labeled yeasts, and collected Raman spectra of the pure lipid. D-labeled lipids from the engineered yeast strains displayed a distinct peak at 2176 cm'1, distinct from the CD peak (2150 cm'1) found in the Raman spectra of the whole yeast (FIG. 8B). This confirmed that the CD peak at 2150 cm'1represented a mixture of newly synthesized macromolecules while lipids took a small portion of it. Moreover, shape differences were observed in the CD and CH stretching regions among these three yeast strains, suggesting variations in the lipid components (FIGS. 2D and 2E). Especially, lipids from strain 880 displayed a distinct Raman shape with a unique peak at 2885 cm1(FIG. 2E), corresponding to CH2 asymmetric stretching. The band around 2885 cm1was markedly enhanced by Fermi resonance in ordered lipid packing, whereas the band around 2850 cm1was not affected by structural changes of the lipid hydrocarbon chains (Yue et al., 2012). Thus, the degree of lipid ordering could be represented by the ratio between the areas under Raman bands at 2885 cm ’ and 2850 cm ’ (Y ue et al., 2012). In this study, a higher ratio of 2885 / 2850 indicated the higher degree of lipid ordering in 880-derived lipids. Consistently, a narrow peak at 1295 cm'1(The -(CH2)n-in-phase twisting mode) in 880 lipids while a broad peak at 1302 cm'1in other lipid extracts (FIG. 2F) were found. This Raman peak shape remodeling also indicated the highly ordered (CH2)n chain (Larkin, 2011), which aligned with what was found in the CH region. All these data indicated that the lipids underwent a transition to a more ordered phase within the 880 yeast strain, in contrast to other strains where lipids might mainly be in an amorphous state. Additionally, the remaining fingerprint region (400-1650 cm'1), corresponding to various types of C-C stretching and vibration models, also exhibited significant peak differences between strains 880 and other strains (FIG. 2F).

[0127] Next, subcellular lipid metabolic activity in single yeast cells was quantitatively examined by DO-SRS imaging. The 868, 897, and 880 yeast strains exhibited increased lipid-to-protein ratio (CH2 / CH3, 2850 / 2935) and newly synthesized lipid ratio (CD / CH, 2176 / 2850), indicating an upregulation of the lipid synthetic pathway in these strains (FIG. 2A-C). Of note, the lipids detected by SRS imaging were not evenly distributed but clustered to certain subcellular structures in both label-free and deuterium labeled yeast cells (FIG. 2A).

[0128] These findings collectively showed the transformation of lipid composition and metabolic pathways within the engineered long-lived yeast strains, particularly showcasing the emergence of more ordered (saturated) short-chain lipids in the 880 yeast strain.

[0129] The engineered yeast strains exhibited significant remodeling in lipid classes

[0130] To elucidate the lipid compositions in the engineered yeast strains, untargeted lipidomics was conducted using reversed-phase liquid chromatography-mass spectrometer (RPLC-MS) on lipid extracts from the four engineered yeast strains. A systematic annotation of 195 lipid species from 16 major classes was achieved, including triacylglycerols (TG), diacylglycerols (DG), monoglycerides (MG), cholesterol esters (ChE), phosphatidylethanolamines (PE), phosphatidylcholines (PC), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylglycerols (PG), phosphatidic acids (PA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), ceramides (Cer), acyl carnitine (AcCa), and wax esters (WE) (FIGS. 3A-D).

[0131] The comprehensive comparison of lipid classes between the groups showed that a predominant remodeling process occurred in engineered yeasts. Using principal component analysis (PCA), a clear separation was found between different yeast stains based on the changes of each lipid class (FIG. 9A). The first component (PCI) showed a clear separation between 880, 897, and the other two strains: 270, and 868, accounting for almost 48.26% of the differences. The second principal component (PC2), accounting for 27.85% of the differences, demonstrated differences between the 880 / 868 and 270 / 897 strains (FIG. 9B). The top varied lipid classes were manifested in PE, LPC, LPE, and TG (FIGS. 9A-9C). In the 880 and 868 strains, there was a significant reduction in major nonpolar lipids of yeast, including TGs, ChEs, and WEs, but showed an increase in polar lipids such as PE, PA, and LPE. Pathway enrichment analysis revealed that membrane components and lipid-mediated signaling pathways ranked among the most significantly altered pathways across all yeast strains (FIGS. 3H, 10D, and 10F). These data collectively demonstrated that genetic manipulation in yeasts significantly impacted the lipid signaling pathway, storage lipid metabolism, and / or membrane components of yeasts. To explore the potential lipid species contributing to the extended lifespan in the 880-fed flies, the lipid subtypes in the 880 strain was subsequently compared with other yeast stains, particularly focusing on the chain length and unsaturation of fatty acids (FIG. 3D). The 880 yeast exhibited higher levels of phospholipids, characterized by saturated short- to medium-chain fatty acids (<12C), such as PC (4:0 16:0), PC (6:0 18: 1), PC (4:0_18:l), PC (16: 1 12:0), and PC (16:0_10:0) (FIG. 3D). Conversely, TG having long-chain fatty acids (C12-C18), with or without a carboncarbon double bond, were consistently reduced in the 880 yeasts. Lipidomics detected shortening of average acyl chain length, coupled with an increase in lipid saturation in TGs and phospholipids, was consistent with the Raman spectra analysis shown in FIGS. 2E and 2F. Although the 868 yeast strain posted similar changes of these lipid species, a group of uniquely changed lipid species was detected in the 880 yeast strain including TG (8:0_12:0_12:0), TG (8: 0 12: 0 14: 0), and TG (8:0_8:0_8:0) (FIGS. 3D and 10E).

[0132] The untargeted lipidomic analysis offered a comprehensive profile of the intricate lipid composition and metabolic disparities between the long-lived yeast strains and the wild type, especially, the preference of fatty acid usage leaning towards the short- and medium-chain in the 880 and 868 yeast strains.

[0133] Long-lived yeast-derived lipids promoted membrane lipid homeostasis and gut integration in flies

[0134] The maintenance of barrier function is intricately tied to the composition and level of membrane lipids (Braun et al., 2011). Previous studies have been widely conducted from a microbiota and signaling pathway perspectives on the complex regulation of lipid metabolism in Drosophila gut (Becher et al., 2012; Fanson and Taylor, 2012; Hamby and Becher, 2016; Hoang et al., 2015). However, little is known about the role of yeast derived lipid / metabolites in association with the fly. It was tested if the consolidation of barrier function and maintenance of lipid homeostasis by administering lipids from the long-lived yeast strains is linked to the lifespan extension in Drosophila.

[0135] SRS imaging of lateral guts showed that aging flies fed with 880 and 868 lipids maintained a single-layer gut columnar epithelium with well-aligned nuclei (normally found in young fly gut, as shown in FIG. 4A), in contrast to the tumor-like, disorganized epithelial structures with unpolarized clustering cells featured in the old control guts (FIGS. 6B and 4A). Contrary to the 880 and 868 lipid-fed flies, the 897 lipid-fed flies exhibited a significant LD accumulation on the apical side and irregular epithelial structures (FIG. 4A). The morphological changes in the gut were reflected in barrier function. A blue dye, typically impervious to the gut (Rera et al., 2012), was fed to aged (45-day) flies. A low consistent proportion of Smurfs (dye leaking to body cavities) was shown in control flies, with no Smurfs observed in the 880 and 868 fed flies, and a slightly more smurfs (nonsignificant) in the 897 fed flies (FIG. 4B).

[0136] To track yeast lipids within fly gut cells, flies were fed with equal amounts of lipid extracts from D2O-labeled yeasts and performed SRS imaging at the peak 2176 cm'1, which represents D-labeled lipids from yeasts (D-lipids). It was found that D- lipids were mainly accumulated to LD-like structures in the guts of the 897-fed flies, while evenly distributed to the cytoplasm of gut cells in the 868- and 880-fed flies (FIG. 4A). Nonetheless, much less CD signal was detected in WT (270) lipids-fed fly guts compared to other groups. This may be due to the low lipid synthesis or high lipid turnover in 270 yeasts, resulting in fewer incorporation of D-lipids in fly guts. Notably, there were no LD-like lipid clusters detected in the 868- and 880-fed flies, suggesting underlying mechanisms of lipid homeostatic regulation were distinct from that in the 897-fed flies. Hyperspectral imaging (HSI) revealed no significant difference in the shape of LD spectra from different strains (FIG. 4C), indicating that the main composition of storage lipids were not changed among the groups. However, the lipid signal, indicated as the Raman peak at 2850 cm'1, on the membrane structures of cytoplasm and cell membranes in gut cells largely increased in the 880- and 868-lipid feeding groups (FIG. 4D). These results suggested that lipids from the 880 and 868 yeast strains might regulate gut integration by participating in membrane biosynthesis and maintaining LD metabolic homeostasis.

[0137] As shown in FIG. 1 A, the 880-derived lipids can extend the lifespan to a greater extent than 868 although they shared a similar lipid profile. Since a group of TGs enriched with medium-chain fatty acids was upregulated in 880 specifically, it was tested if this lipid group plays a role in regulating the lifespan. To test the role of this lipid group in regulating the lifespan, flies were fed with medium-chain fatty acid containing TGs and compared their lifespans with flies fed with short- or mediumchain fatty acids containing PC (which was shared by 880 and 868) or both. It was found that medium-chain fatty acid containing TGs could extend the lifespan, whereas PC cannot. Intriguingly, feeding with both PC and TG, which resembled the specific lipid combination enriched in 880, can increase the lifespan synergistically (FIG. 4E). These results indicated that a healthier lifespan could be achieved by controlling the phospholipid and fatty acid levels through the dietary intake of specific lipid groups.

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[0204] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A lipid composition comprising:(a) a phosphatidylethanolamine (PE);(b) a lysophosphatidylethanolamine (LPE);(c) a diacylglycerol (DG);(d) a monoglyceride (MG);(e) an acyl carnitine (AcCa);(f) a phosphatidic acid (PA);(g) a phosphatidy lglycerol (PG);(h) a lysophosphatidylcholine (LPC);(i) a triacylglycerol (TG);(j) a cholesterol ester (ChE);(k) a wax ester (WE);(l) a phosphatidy lserine (PS);(m) a phosphatidylcholine (PC);(n) a phosphatidylinositol (PI);(o) a ceramide (Cer); and(p) a lysophosphatidylserine (LPS).

2. A lipid composition consisting of:(a) a phosphatidy lethanolamine (PE);(b) a lysophosphatidylethanolamine (LPE);(c) a diacylglycerol (DG);(d) a monoglyceride (MG);(e) an acyl carnitine (AcCa);(f) a phosphatidic acid (PA);(g) a phosphatidylglycerol (PG);(h) a lysophosphatidylcholine (LPC);(i) a triacylglycerol (TG);(j) a cholesterol ester (ChE);(k) a wax ester (WE);(l) a phosphatidy lserine (PS);(m)a phosphatidylcholine (PC);(n) a phosphatidylinositol (PI);(o) a ceramide (Cer); and(p) a lysophosphatidylserine (LPS).

3. A lipid composition comprising at least two of the lipids selected from the group consisting of:(a) a phosphatidy lethanolamine (PE);(b) a lysophosphatidylethanolamine (LPE);(c) a diacylglycerol (DG);(d) a monoglyceride (MG);(e) an acyl carnitine (AcCa);(f) a phosphatidic acid (PA);(g) a phosphatidy lglycerol (PG);(h) a lysophosphatidylcholine (LPC);(i) a triacylglycerol (TG);(j) a cholesterol ester (ChE);(k) a wax ester (WE);(l) a phosphatidy lserine (PS);(m)a phosphatidylcholine (PC);(n) a phosphatidylinositol (PI);(o) a ceramide (Cer); and(p) a lysophosphatidylserine (LPS).

4. The lipid composition of claim 3, comprising at least three lipids.

5. The lipid composition of claim 4, comprising at least four lipids.

6. The lipid composition of claim 5, comprising at least five lipids.

7. The lipid composition of claim 6, comprising at least six lipids.

8. The lipid composition of claim 7, comprising at least seven lipids.

9. The lipid composition of claim 8, comprising at least eight lipids.

10. The lipid composition of claim 9, comprising at least nine lipids.

11. The lipid composition of claim 10, comprising at least ten lipids.

12. The lipid composition of claim 11, comprising at least eleven lipids.

13. The lipid composition of claim 12, comprising at least twelve lipids.

14. The lipid composition of claim 13, comprising at least thirteen lipids.

15. The lipid composition of claim 14, comprising at least fourteen lipids.

16. The lipid composition of claim 15, comprising at least fifteen lipids.

17. The lipid composition of any of the above claims, comprising a PC and a TG.

18. The lipid composition of claim 17, further comprising any one of the lipids selected from the group consisting of:(a) a phosphatidylethanolamine (PE);(b) a lysophosphatidylethanolamine (LPE);(c) a diacylglycerol (DG);(d) a monoglyceride (MG);(e) an acyl carnitine (AcCa);(f) a phosphatidic acid (PA);(g) a phosphatidy lglycerol (PG);(h) a lysophosphatidylcholine (LPC);(i) a cholesterol ester (ChE);(j) a wax ester (WE);(k) a phosphatidylserine (PS);(l) a phosphatidylinositol (PI);(m)a ceramide (Cer); and(n) a lysophosphatidylserine (LPS).

19. The lipid composition of any of the above claims, wherein the PE comprises from about 2% to about 22% of the lipid composition.

20. The lipid composition of any of the above claims, wherein the LPE comprises from about 5% to about 35% of the lipid composition.

21. The lipid composition of any of the above claims, wherein the DG comprises from about 0.5% to about 3% of the lipid composition.

22. The lipid composition of any of the above claims, wherein the MG comprises from about 0.01% to about 1 %the lipid composition.

23. The lipid composition of any of the above claims, wherein the AcCa comprises from about 0.01% to about 1% of the lipid composition.

24. The lipid composition of any of the above claims, wherein the PA comprises from about 1% to about 3% of the lipid composition.

25. The lipid composition of any of the above claims, wherein the PG comprises from about 0.01% to about 1% of the lipid composition.

26. The lipid composition of any of the above claims, wherein the LPC comprises from about 50% to about 80% of the lipid composition.

27. The lipid composition of any of the above claims, wherein the TG comprises from about 5% to about 25% of the lipid composition.

28. The lipid composition of any of the above claims, wherein the ChE comprises from about 0.01% to about 1% of the lipid composition.

29. The lipid composition of any of the above claims, wherein the WE comprises from about 0.01% to about 1% of the lipid composition.

30. The lipid composition of any of the above claims, wherein the PS comprises contain from about 0.01% to about 1% of the lipid composition.

31. The lipid composition of any of the above claims, wherein the PC comprises from about 0.5% to about 3% of the lipid composition.

32. The lipid composition of any of the above claims, wherein the PI comprises from about 0.5% to about 3% of the lipid composition.

33. The lipid composition of any of the above claims, wherein the Cer comprises from about 0.01% to about 1% of the lipid composition.

34. The lipid composition of any of the above claims, wherein the LPS comprises from about 0.01% to about 1% of the lipid composition.

35. The lipid composition of any of the above claims, wherein the LPC comprises a plurality of subtypes of different carbon lengths.

36. The lipid composition of any of the above claims, wherein the LPC comprises a subtype having about 5 to about 25 carbons.

37. The lipid composition of any of the above claims, wherein the LPC subtype comprises one or more double bonds.

38. The lipid composition of any of the above claims, wherein a double bond occurs at position 0, 1, or 3 of the LPC subtype.

39. The composition of any of the above claims, wherein the LPC comprises a plurality of at least eleven subtypes selected from the group consisting of LPC (10:0), LPC (14: 1), LPC (15:0), LPC (16:0), LPC (16: 1), LPC (16:1), LPC (18:3), LPC (20: 1), LPC (20:3), LPC (22:3), LPC (16:0), LPC (17:0), LPC (18:0), LPC (18: 1), LPC (20:0), LPC (14:0), LPC (20:2), and LPC (17:0).

40. The lipid composition of any of the above claims, wherein the plurality of LPC subtypes comprise at least twelve subtypes.

41. The lipid composition of any of the above claims, wherein the plurality of LPC subtypes comprise at least thirteen subtypes.

42. The lipid composition of any of the above claims, wherein the plurality of LPC subtypes comprise at least fourteen subtypes.

43. The lipid composition of any of the above claims, wherein the LPE comprises a plurality of subtypes of different carbon lengths.

44. The lipid composition of any of the above claims, wherein the LPE comprises a subtype having about 5 to about 25 carbons.

45. The lipid composition of any of the above claims, wherein the LPE subtype comprises one or more double bonds.

46. The lipid composition of any of the above claims, wherein a double bond occurs at position 0 or 1 of the LPE subtype.

47. The lipid composition of any of any of the above claims, wherein the LPE comprises a plurality of at least five subty pes selected from the group consisting of LPE (10:0), LPE (12:0), LPE (14:0), LPE (14: 1), LPE (15:0), LPE (16:0), LPE (16:0), LPE (16:1), and LPE (18: 1).

48. The lipid composition of any of the above claims, wherein the plurality of LPE subtypes comprise at least six different subtypes.

49. The lipid composition of any of the above claims, wherein the plurality of LPE subtypes comprise at least seven different subtypes.

50. The lipid composition of any of the above claims, wherein the plurality of LPE subtypes comprise at least eight subtypes.

51. The lipid composition of any of the above claims, wherein the TG comprises a plurality of subtypes of different carbon lengths.

52. The lipid composition of any of the above claims, wherein the TG comprises a subtype having about 5 to about 30 carbons.

53. The lipid composition of any of the above claims, wherein the TG subtype comprises one or more double bonds.

54. The lipid composition of any of the above claims, wherein a double bond occurs at position 0, 1,2, 3, or 4 of the TG subtype.

55. The lipid composition of any of any of the above claims, wherein the TG comprises a plurality of at least thirty subtypes selected from the group consisting of TG (12:0_12:0_22:4), TG(15:0_14:0_14:0), TG(15:0_14:016:0), TG (15 : 0 14: 0 16: 1), TG (15:0 16:0 16:0), TG (15 :0_l 6:0 16: 1), TG (15:0_16: 1_16: 1), TG (15:0_16: 1_18: 1), TG(16:0_18:2_18:2), TG(18:0_ 16:0 16:0), TG (18: 1 18:1 18:1), TG (18: 1 18: 1 18:2), TG(18: 1 18:2 18:2), TG(18:2_18:2_18:2), TG(16:0_10:0_16:0), TG (16:0 10:0 16:1), TG(16:0_14:0_16:0), TG(16:0_14:0_16:l), TG (16:0 16:0 16:1), TG(16:0_16:l_16:l), TG(16:0_16:l_16:l), TG (16:0 16: 1 18: 1), TG(16:0_18:l_20:3), TG(16:l_10:0_14:0), TG (16: 1 10:0 16: 1), TG(16:l_10:0_18:3), TG(16:l_12:0_16:l), TG (16: l_14:0_14:0), TG(16:l_14:0_16:l), TG (16:1 16:1 16:1), TG (16: 1 16: 1_18: 1), TG(16:1_16:1_18:3), TG (16:1 18:1 18:1), TG (18:0 10:0 16:0), TG(18:0_16:0_18:l), TG(18:0_16:l_16;l), TG (18:0 18:0 18: 1), TG (18:0_l 8: 1 18: 1), TG(18:4_14:0_16:l), TG (18:4_16:1_16:1), TG(18:4_16:1_18:1), TG (26:0 14:0 16:1), TG (26:0 16:0 18: 1), TG(26:0_16:l_16:l), TG(16:0_10:0_14:0), TG (16:0 18:1 18:3), TG(18:0_16:0_16:l), TG (18:1 18:1 18:3), TG (26:0 10:0 16:0), and TG (16: 1 14: 1 16: 1).

56. The lipid composition of any of the above claims, wherein the plurality of TG subtypes comprise at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 subtypes.

57. The lipid composition of any of the above claims, wherein the lipid composition is extracted from an organism.

58. The lipid composition of any of the above claims, wherein one or more of the lipids in the lipid composition is extracted from one or more types of organisms.

59. The lipid composition of any of the above claims, wherein each of the lipids in the lipid composition is extracted from a plurality of the same organism.

60. The lipid composition of any of the above claims, wherein one or more of the lipids in the lipid composition are extracted from pluralities of two or more different organisms.

61. The lipid composition of any of the above claims, wherein the organism is a prokaryote or a eukaryote.

62. The lipid composition of any of the above claims, wherein the organism is a prokaryote.

63. The lipid composition of any of the above claims, wherein the prokaryote is a bacterium.

64. The lipid composition of any of the above claims, wherein the organism is a eukaryote.

65. The lipid composition of any of the above claims, wherein the eukaryote is an alga, a fungus, a plant, or a mammalian cell.

66. The lipid composition of any of the above claims, wherein the fungus is a yeast.

67. The lipid composition of any of the above claims, wherein the mammalian cell is a non-human mammal or a human cell.

68. The lipid composition of any of the above claims, wherein the non-human mammal or the human cell is a liver cell, a kidney cell, an intestine cell, a connective tissue cell, a muscle cell, a heart cell, a vessel cell, a gut bacterial cell, a stomach cell, other gut organ cells, an anus cell, a joint cell, a nerve cell, a skin cell, a nasal cavity cell, a tongue cell, an appendix cell, a diaphragm cell, a lung cell, a thyroid cell, an adrenal gland cell, an ear cell, a larynx cell, an esophagus cell, a trachea cell, a brain cell, an eye cell, a spinal cord cell, a thymus gland cell, a lymph node cell, a pancreas cell, a ureter cell, a bronchus cell, a genital cell, a pharynx cell, a salivary gland cell, a urethra bladder cell, a gallbladder cell, a placenta cell, a uterus cell, a bone marrow cell, a mouth cell, a prostate cell, a seminal vesicle cell, a hair follicle cell, a mesentery cell, a subcutaneous tissue cell, a mammary gland cell, a tooth cell, an interstitial cell, a parathyroid gland cells, a tonsil cell, a nail cell, a vestigial cell, or a cancer cell.

69. The lipid composition of any of the above claims, wherein the yeast is an engineered yeast strain overexpressing a HAP complex component, a SIR2 gene product, or both the HAP complex component and the SIR2 gene product.

70. The lipid composition of any of the above claims, wherein the yeast is an engineered yeast strain overexpressing a HAP4 gene product, the SIR2 gene product, or both the HAP4 gene product and the SIR2 gene product.

71. The lipid composition of claim 69, wherein the HAP complex component comprises a HAP1, a HAP2, a HAP3, a HAP4, and / or a HAP5 gene product.

72. The lipid composition of any of the above claims, wherein the engineered yeast strain comprises NH0880, NH0868, and / or NH0897.

73. The lipid composition of any of the above claims, wherein the engineered yeast strain comprises NH0880.

74. The lipid composition of any of the above claims, wherein one or more of the lipids in the lipid composition is made by synthesis.

75. The lipid composition of any of the above claims, wherein the synthesis is a chemical synthesis, a chemoenzymatic synthesis, or an enzyme-free synthesis.

76. The lipid composition of any of the above claims, wherein the lipid composition is formulated in an ingestible form or a topical form.

77. The lipid composition of any of the above claims, wherein the ingestible form is a pharmaceutical composition or an edible.

78. The lipid composition of claim 77, wherein the pharmaceutical composition is a tablet, a powder, a granule, a pill, a capsule, a gel, or a liquid.

79. The lipid composition of claim 77, wherein the edible is a snack bar, a gummy, a yogurt, or a supplement.

80. The lipid composition of claim 76, wherein the topical form is a cream, a serum, a gel, a mask, an ointment, or a cleanser.

81. A method of modulating cellular aging in a subject, the method comprising administering a therapeutically effective amount of the lipid composition of any of the above claims to the subject.

82. A method of extending the lifespan of a subject, the method comprising administering a therapeutically effective amount of the lipid composition of any of the above claims to the subject.

83. A method of modulating gut homeostasis in a subject, the method comprising administering a therapeutically effective amount of the lipid composition of any of the above claims to the subject.

84. The method of any one of claims 81-83, wherein the lipid composition comprises at least a TG.

85. The method of any one of claims 81-84, wherein the lipid composition comprises at least a TG and a PC.

86. A method of modulating cellular aging in a subject, the method comprising administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strains NH0880, NH0868, and / or NH0897 to the subject.

87. A method of extending the lifespan of a subject, the method comprising administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strains NH0880, NH0868, and / or NH0897 to the subject.

88. A method of modulating gut homeostasis in a subject, the method comprising administering a therapeutically effect ve amount of a yeast composition comprising the engineered yeast strains NH0880, NH0868, and / or NH0897 to the subject.

89. A method of modulating cellular aging in a subject, the method comprising administering a therapeutically effect ve amount of a yeast composition comprising the engineered yeast strain NH0880 to the subject.

90. A method of extending the lifespan of a subject, the method comprising administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strain NH0880 to the subject.

91. A method of modulating gut homeostasis in a subject, the method comprising administering a therapeutically effective amount of a yeast composition comprising the engineered yeast strain NH0880 to the subject.

92. The method of any one of claims 86-91, wherein the yeast composition is formulated in an ingestible form.

93. The method of claim 92, wherein the ingestible form is a pharmaceutical composition or an edible.

94. The method of claim 93, wherein the pharmaceutical composition is a tablet, a powder, a granule, a pill, a capsule, a gel, a solid, or a liquid.

95. The method of claim 93, wherein the edible is a bakery item, a beverage, a food ingredient, an animal feed, a snack bar, a gummy, a yogurt, or a supplement.

96. The method of claim 95, wherein the bakery item comprises a bread, a pastry, a dough, a bagel, a pretzel, a roll, and / or a bun.

97. The method of claim 95, wherein the beverage comprises an alcoholic drink or a non-alcoholic drink.

98. The method of claim 97, wherein the alcoholic drink comprises a beer, a wine, a spirit, or a cider.

99. The method of claim 97, wherein the non-alcoholic drink comprises a probiotic drink, a tea, a dairy drink, a juice, a mocktail, an alcohol-free beverage, or a sparkling drink.

100. The method of claim 95 wherein the food ingredient comprises a yeast extract, a yeast flake, an autolyzed yeast, an active dry yeast, an instant dry yeast, a fresh yeast, a bread machine yeast, a spice, an herb, a syrup, or a cream.

101. The method of any of claims 81-100, wherein the subject is an animal.

102. The method of claim 101, wherein the animal is a mammal, a chromadorea, or an insecta.

103. The method of claim 102, wherein the mammal is a human, a non-human primate, horses, bovine species, porcine species, dogs, cats, or a rodent.

104. The method of claim 102, wherein the chromadorea is a Caenorhabditis elegans.

105. The method of claim 102, wherein the insecta is a Drosophila.

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