Methods and compositions for aging and mitochondrial health

Abstract

Provided herein, inter alia, are methods for treating, delaying and / or reducing the severity of conditions associated with age-related decline of various physiological pathways, including mitochondrial health and associated physical states, by administration of compositions that can degrade extracellular ATP (eATP), including NTPDase enzymes, nucleotidases such as apyrases and members of the GDA1_CD39 superfamily, phosphatases and ATP degrading enzyme producing yeast strains.

Description

Attorney Docket No. NB42251-WO-PCT METHODS AND COMPOSITIONS FOR AGING AND MITOCHONDRIAL HEALTH CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 590,129 filed October 13, 2023, the disclosure of which is incorporated by reference herein in its entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing submitted in an XML file, in accordance with 37 C.F.R. §§2412 is incorporated herein by reference. The xml file name is “NB42251_WO_PCT_Seq_List.xml”, the date of creation of the xml file is September 30 2024, and the size of the xml file in bytes is 150,200. FIELD OF THE INVENTION

[0003] Provided herein, inter alia, are methods for treating, delaying and / or reducing the severity of conditions associated with age-related decline of various physiological pathways, including mitochondrial health and associated physical states, by administration of compositions comprising ATP degrading enzymes including NTPDase enzymes, nucleotidases such as apyrases and members of the GDA1_CD39 superfamily, acid phosphatases and ATP degrading enzyme producing yeast strains. BACKGROUND

[0004] The biology of aging is a rapidly expanding area of biomedical research, and there is a growing appreciation that aging is a combination of various physiological changes and their complex interactions. While classical research into aging focused on mortality and longevity, there has been increasing interest in achieving a period of life free of disease and disabilities, or “healthy aging”.

[0005] Currently, twelve molecular, cellular, and systemic hallmarks of aging have been proposed, which include cellular senescence and mitochondrial dysfunction. Therefore, it is envisaged that restoring mitochondrial function will have a positive impact on health decline associated with un-healthy ageing. Although the classical role of mitochondria is oxidativeAttorney Docket No. NB42251-WO-PCT phosphorylation, which generates adenosine triphosphate (ATP) which is necessary to thermodynamically drive a multitude of biochemical reactions, extracellular adenosine triphosphate (eATP) is considered a danger-associated molecular pattern (DAMP) that is released in response to tissue damage and cellular stress and can have various effects on cellular processes.

[0006] However, the impact of eATP on healthy aging through regulation of its various hallmarks is not yet understood. In addition, aside from classical approaches to longevity, including maintenance of healthy diet and exercise, the realm of therapeutic approaches to achieve healthy aging is still vastly unexplored. Therefore, there is a need for improved methods and compositions that can extend longevity and delay the onset, as well as the incidence, of age-related diseases and disorders.

[0007] The subject matter disclosed herein addresses these needs and provides additional benefits as well. SUMMARY

[0008] Provided herein is a method of treating, delaying or reducing the severity of at least one condition selected from age-related decline in metabolic regulation, age-related alterations in immune function, age-related alterations in muscle mass and function, age- related decline in gut function, age-related cognitive decline and cell-senescence, the method comprising administering to a subject in need thereof or at risk thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof.

[0009] In some embodiments, the age-related alterations in immune function comprise a decline in immune function and an increase in inflammation; the age-related decline in gut- function comprises an age related decline in gut permeability, an age related decline in nutrient sensing and an age related decline in nutrient absorption; the age-related decline in metabolic regulation comprises a decline in insulin sensitivity, a decline in systemic glucose metabolism, a decline in lipid metabolism, and a decline in resting energy expenditure; the age-related cognitive decline comprises short-term memory loss, word-finding difficulty, alterations in sleep patterns, insomnia, slower processing speed and / or degenerative dementia; and / or the age-related alterations in muscle mass and function comprise muscle atrophy and sarcopenia.Attorney Docket No. NB42251-WO-PCT

[0010] In some embodiments, the ATP degrading enzyme comprises an apyrase. In some embodiments, the enzyme comprises a member of the GDA1_CD39 superfamily. In some embodiments, the ATP degrading enzyme comprises a member of the acid phosphatase family. In some embodiments, the enzyme is not a mammalian ATP degrading enzyme. In some embodiments, the enzyme is a bacterial ATP degrading enzyme. In some embodiments, the enzyme is derived from Gallaecimonas xiamenensis.

[0011] In some embodiments, the enzyme comprises a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-24, 57- 63, 30-32, and 90-102. In some embodiments, the enzyme further comprises an N-terminus tripeptide comprising the sequence of amino acids AGK.

[0012] In some embodiments, the ATP degrading enzyme producing yeast strain is selected from Cyberlindnera, Kluyveromyces and Saccharomyces. In some embodiments, the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii. In some embodiments, the ATP degrading enzyme producing yeast strain is Cyberlindnera jadinii and comprises the strain deposited at DSMZ under number DSM 33763 or a strain having all of the identifying characteristics of the Cyberlindnera jadinii strain deposited at DSMZ under number DSM 33763. In some embodiments, the ATP degrading enzyme producing yeast strain is Kluyveromyces lactis and comprises the strain deposited at DSMZ under number DSM 33764 or a strain having all of the identifying characteristics of the Kluyveromyces lactis strain deposited at DSMZ under number DSM 33764.

[0013] In some embodiments, the composition comprises a probiotic. In some embodiments, the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration or topical administration. In some embodiments, the composition comprises a food product, food ingredient, dietary supplement, or medicament. In some embodiments, the at least one condition is associated with mitochondrial dysfunction. In some embodiments, the subject is human. In some embodiments, the at least one condition affects one or moreAttorney Docket No. NB42251-WO-PCT cells. In some embodiments, the one or more cells comprise epithelial cells, endothelial cells, immune cells, neural cells, fibroblasts, muscle cells and / or adipose cells.

[0014] In some embodiments, the subject is a non-human animal.

[0015] Provided herein is a method of treating and / or preventing at least one physical state associated with mitochondrial health, the method comprising administering to a subject in need thereof or at risk thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof. In some embodiments, the at least one physical state is selected from the group consisting of impaired Complex I activity, impaired mitochondrial respiration, oxidative stress, DNA damage, impaired cellular function and decreased energy production.

[0016] In some embodiments, the treating and / or preventing delays the onset of at least one condition selected from age-related decline in metabolic regulation, age-related alterations in immune function, age-related alterations in muscle mass and function, age-related decline in gut function, age-related cognitive decline and cell-senescence. In some embodiments, the ATP degrading enzyme comprises an apyrase. In some embodiments, the enzyme comprises a member of the GDA1_CD39 superfamily In some embodiments, the enzyme comprises a member of theacid phosphatase family. In some embodiments, the enzyme is not a mammalian ATP degrading enzyme. In some embodiments, the enzyme is a bacterial ATP degrading enzyme. In some embodiments, the enzyme is derived from Gallaecimonas xiamenensis.

[0017] In some embodiments, the enzyme comprises a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-24, 57- 63, 30-32, and 90-102.. In some embodiments, the enzyme further comprises an N-terminus tripeptide comprising the sequence of amino acids AGK.

[0018] In some embodiments, the ATP degrading enzyme producing yeast strain is selected from Cyberlindnera, Kluyveromyces and Saccharomyces. In some embodiments, the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii. In some embodiments, the ATP degrading enzyme producing yeast strain is Cyberlindnera jadinii and comprises the strainAttorney Docket No. NB42251-WO-PCT deposited at DSMZ under number DSM 33763 or a strain having all of the identifying characteristics of the Cyberlindnera jadinii strain deposited at DSMZ under number DSM 33763. In some embodiments, wherein the ATP degrading enzyme producing yeast strain is Kluyveromyces lactis and comprises the strain deposited at DSMZ under number DSM 33764 or a strain having all of the identifying characteristics of the Kluyveromyces lactis strain deposited at DSMZ under number DSM 33764.

[0019] In some embodiments, the composition comprises a probiotic. In some embodiments, the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration or topical administration. In some embodiments, the composition comprises a food product, food ingredient, dietary supplement, or medicament. In some embodiments, the subject is human.

[0020] Provided herein is a method of treating age related decline in nutrient absorption , comprising administering to a subject in need thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain; or c) a combination thereof. In some embodiments, the treating increases the nutrient absorption in the subject relative to a subject that has not been treated with an effective amount of the composition.

[0021] In some embodiments, the ATP degrading enzyme enzyme comprises an apyrase. In some embodiments, the enzyme comprises a member of the GDA1_CD39 superfamily. In some embodiments, the enzyme comprises a member of the acid phosphatase family. In some embodiments, the enzyme is not a mammalian ATP degrading enzyme. In some embodiments, the enzyme is a bacterial ATP degrading enzyme. In some embodiments, the enzyme is derived from Gallaecimonas xiamenensis.

[0022] In some embodiments, the enzyme comprises a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-24, 57- 63, 30-32, and 90-102.. In some embodiments, the enzyme further comprises an N-terminus tripeptide comprising the sequence of amino acids AGK.Attorney Docket No. NB42251-WO-PCT

[0023] In some embodiments, the ATP degrading enzyme producing yeast strain is selected from Cyberlindnera, Kluyveromyces and Saccharomyces. In some embodiments, the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii. In some embodiments, the ATP degrading enzyme producing yeast strain is Cyberlindnera jadinii and comprises the strain deposited at DSMZ under number DSM 33763 or a strain having all of the identifying characteristics of the Cyberlindnera jadinii strain deposited at DSMZ under number DSM 33763. In some embodiments, the ATP degrading enzyme producing yeast strain is Kluyveromyces lactis and comprises the strain deposited at DSMZ under number DSM 33764 or a strain having all of the identifying characteristics of the Kluyveromyces lactis strain deposited at DSMZ under number DSM 33764.

[0024] In some embodiments, the composition comprises a probiotic. In some embodiments, the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration or topical administration. In some embodiments, the composition comprises a food product, food ingredient, dietary supplement, or medicament. In some embodiments, the subject is human.

[0025] Provided herein is a method of increasing pre-albumin / albumin levels in a subject, comprising administering to a subject in need thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof. In some embodiments, the increase is relative to a subject that has not been treated with an effective amount of the composition. In some embodiments, the pre-albumin levels are in serum. In some embodiments, the increase in pre-albumin levels reduces inflammation in a subject relative to a subject who has not been treated with an effective amount of the composition. In some embodiments, the inflammation is systemic inflammation.

[0026] In some embodiments, the ATP degrading enzyme comprises an apyrase. In some embodiments, the enzyme comprises a member of the GDA1_CD39 superfamily. In some embodiments, the enzyme comprises a member of the acid phosphatase family. In some embodiments, the enzyme is not a mammalian ATP degrading enzyme. In some embodiments, the enzyme is a bacterial ATP degrading enzyme. In some embodiments, the enzyme is derived from Gallaecimonas xiamenensis.Attorney Docket No. NB42251-WO-PCT

[0027] In some embodiments, the enzyme comprises a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-24, 57- 63, 30-32, and 90-102.. In some embodiments, the enzyme further comprises an N-terminus tripeptide comprising the sequence of amino acids AGK.

[0028] In some embodiments, the ATP degrading enzyme producing yeast strain is selected from Cyberlindnera, Kluyveromyces and Saccharomyces. In some embodiments, the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii. In some embodiments, the ATP degrading enzyme producing yeast strain is Cyberlindnera jadinii and comprises the strain deposited at DSMZ under number DSM 33763 or a strain having all of the identifying characteristics of the Cyberlindnera jadinii strain deposited at DSMZ under number DSM 33763. In some embodiments, the ATP degrading enzyme producing yeast strain is Kluyveromyces lactis and comprises the strain deposited at DSMZ under number DSM 33764 or a strain having all of the identifying characteristics of the Kluyveromyces lactis strain deposited at DSMZ under number DSM 33764.

[0029] In some embodiments, the composition comprises a probiotic. In some embodiments, the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration or topical administration. In some embodiments, the composition comprises a food product, food ingredient, dietary supplement, or medicament. In some embodiments, the subject is human.

[0030] Provided herein is a method of reducing the appearance of age-related skin changes, comprising topically applying a composition comprising: an ATP degrading enzyme enzyme; b) a at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof; to the skin of a subject, wherein the appearance of age related skin changes is reduced compared to a subject that did not apply the composition; and wherein the age related skin changes comprise fine lines, wrinkles, hyperpigmentation, melasmic skin, age spots, skin discoloration, and / or inflamed skin.Attorney Docket No. NB42251-WO-PCT

[0031] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0032] The disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure.

[0033] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Any terms defined are more fully defined by reference to the specification as a whole.

[0034] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG.1 depicts the effect of K. lactis and C. jadinii on the removal of ATP from culture as compared to controls.

[0036] FIG.2 depicts a schematic of the experimental setup for the 3D epidermal tissue model for evaluation of mitochondrial function.

[0037] FIG.3 depicts the effect of exemplary enzymes on mitochondrial Complex I activity in a 3D epidermal tissue model.

[0038] FIG.4 depicts the effect of exemplary enzymes on IL-1β expression in a 3D epidermal tissue model.

[0039] FIG.5 depicts the effect of exemplary enzymes on ATP release in a 3D epidermal tissue model.

[0040] FIG.6 depicts the effect of exemplary enzymes and yeast strains K. lactis and C. jadinii on mitochondrial Complex I activity in a murine model of colitis.Attorney Docket No. NB42251-WO-PCT

[0041] FIG.7 depicts the effect of exemplary enzymes and yeast strains K. lactis and C. jadinii on intestinal permeability measured as relative fluorescent units (RFU) in a murine model of colitis.

[0042] FIG.8 depicts the effect of exemplary enzymes and yeast strains K. lactis and C. jadinii on pre-albumin levels in a murine model of colitis.

[0043] FIG.9 depicts the effect of exemplary enzymes on mitochondrial Complex I activity in a 3D muscle tissue model.

[0044] FIG.10 depicts the effect of exemplary enzymes on mitochondrial Complex I activity in a HaCat aging cell model.

[0045] FIG.11 depicts the effect of exemplary enzymes on GDF15 levels in a HaCat aging cell model.

[0046] FIG.12 depicts the effect of exemplary enzymes and yeast strain C. jadinii on body weight in a murine model of colitis.

[0047] FIG.13 depicts the effect of exemplary enzymes and yeast strain C. jadinii on cecum weight in a murine model of colitis.

[0048] FIG.14 depicts the effect of exemplary enzymes and yeast strain C. jadinii on intestinal permeability measured as relative fluorescent units (RFU) in a murine model of colitis.

[0049] FIG.15 depicts the effect of exemplary enzymes and yeast strain C. jadinii on mitochondrial Complex I activity in a murine model of colitis. DETAILED DESCRIPTION

[0050] Aging is a gradual pathophysiological process that presents with declines in tissue and cellular functions, and significant increases in the risks of various aging-related diseases, including neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, and immune system diseases (Guo et al. Sig. Transduct Target Ther 7, 391 (2022). Although the biology of aging is not a new field of research, the growing awareness of the shift in distribution of the world’s population toward older ages has aided in promoting a surge of interest into the field of aging, particularly on the possibility ofAttorney Docket No. NB42251-WO-PCT achieving a period of life free of disease and disabilities, or “healthy aging”. (Cohen, et al. A complex systems approach to aging biology. Nat Aging 2, 580–591 (2022)). According to the World Health Organization, by 2030, 1 in 6 people in the world will be aged 60 years or over. At this time, the share of the population aged 60 years and over will increase from 1 billion in 2020 to 1.4 billion. By 2050, the world’s population of people aged 60 years and older will double (2.1 billion).

[0051] Aging is considered to be driven by a number of cellular and molecular hallmarks that are generally considered to contribute to the aging process and together determine an aging phenotype (López-Otín et al. Cell.2013 Jun 6;153(6):1194-217). As of 2023, twelve hallmarks have been proposed, which include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient- sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis (López-Otín et al. Cell. 2023 Jan 19;186(2):243-278).

[0052] Given the importance of mitochondrial dysfunction on aging, restoring mitochondrial function could potentially aid in the development of anti-aging treatments and address the health decline associated with un-healthy aging. The inventors of the present disclosure have surprisingly found that extracellular ATP (eATP) is a major driver of mitochondrial health, where eATP induces depolarization and thus impacts the Complex I activity, and that restoration of mitochondrial function can be accomplished by removal of eATP through the use of NTPDase enzymes of the GDA1_CD39 superfamily, acid phosphatases, yeast strains that express an ATP degrading enzyme or a combination thereof.

[0053] Additional markers of mitochondrial function include Growth differentiation factor 15 (GDF15), a protein that is upregulated during aging and may play a role in aging and age- related diseases. GDF15 levels are associated with a number of biological factors, including: GDF15 levels are positively correlated with age, and GDF15 is one of the most upregulated proteins during aging. GDF15 is associated with chronic inflammation, and elevated levels of GDF15 are associated with poorer recovery after acute illness. GDF15 is elevated in patients with mitochondrial diseases and is a valuable biomarker for diagnosing (Huan Liu et .al. Experimental Gerontology,Volume 146,2021,111228; Montero R et.al., PLoS One.2016 Feb 11;11(2):e0148709).

[0054] Accordingly, the present disclosure relates, at least in part, to methods for treating, delaying or reducing the severity of at least one age-related decline or alteration in the health of a subject, and treating or preventing physical states associated with mitochondrial healthAttorney Docket No. NB42251-WO-PCT through the administration of an NTPDase enzyme of the GDA1_CD39 superfamily or an ATP degrading enzyme of the acid phosphatase family, a yeast strain including Kluyveromyces lactis and Cyberlindnera jadinii, or a combination thereof. I. Definitions

[0055] The term “ATP degrading enzyme” is used to mean an enzyme capable of degrading ATP substrate. ATP degrading enzymes can be found in multiple enzyme families, included but not limited to the apyrases (EC 3.6.1.5), NTPDases (EC 3.6.1.5), acid phosphatases (EC 3.1.3.2) and alkaline phosphatases (EC 3.1.3.1).

[0056] The term "acid phosphatase" (EC 3.1.3.2) (also known as acid phosphomonoesterase, phosphomonoesterase, glycerophosphatase, acid monophosphatase, acid phosphohydrolase, acid phosphomonoester hydrolase, uteroferrin, acid nucleoside diphosphate phosphatase, acid phosphatase (class A) or orthophosphoric-monoester phosphohydrolase (acid optimum)) is used to mean an enzyme having a pH optimum for mediating hydrolysis of a phosphate ester bond in a substrate at a pH less than about pH 6.5, such as less than about pH 4.0. Acid phosphatases free attached phosphoryl groups from other molecules during digestion. It can be further classified as a phosphomonoesterase. Acid phosphatase is stored in lysosomes and functions when these fuse with endosomes, which are acidified while they function; therefore, it has an acid pH optimum. This enzyme is present in many animal and plant species. In some embodiments, the acid phosphatase used in the compositions and methods disclosed herein is not derived from Shigella. In some embodiments, the acid phosphatase used in the compositions and methods disclosed herein is not derived from a mammal.

[0057] As used herein “NTPDases” refer to enzymes also known as ectonucleotidases, Ecto- ATPases and ectonucleoside triphosphate diphosphohydrolase. NTPDases as used herein are capable of hydrolyzing extracellular nucleotides. NTPDases include four major families of ectonucleotidases: CD39 / NTPDases (ecto-nucleotide triphosphate diphosphohydrolases); ecto-nucleotide pyrophosphatase phosphodiesterases (E-NPPs); alkaline phosphatases; and ecto-5'-nucleotidases / CD73 (See Robson et al.2006 Purinergic Signalling 2:409-430). In some of any of the embodiments described herein, the NTPDase is an apyrase.

[0058] The term “apyrase,” as used herein, refers to one or more of a calcium-activated enzyme (i.e. proteins belonging to class EC.3.6.1.5) which possesses ATP-Attorney Docket No. NB42251-WO-PCT diphosphohydrolase activity and catalyzes the hydrolysis of the gamma phosphate from ATP, and catalyzes the hydrolysis of the beta phosphate from ADP. As used herein, an apyrase can constitute an ATPase, an NTPase, or both. Apyrases are found in all eukaryotes and some prokaryotic organisms, indicating a preserved role for these enzymes across species. They possess a distinct phosphohydrolase activity, nucleotide substrate specificity, divalent cation requirement, and sensitivity to inhibitors. (See, Plesner, Int. Rev. Cytol., 158:141 (1995), and Handa & Guidotti, Biochem. Biophys. Res. Commun., 218(3):916 (1996)). In mammals, apyrase is believed to function primarily as an extracellular hydrolase specific for ATP and ADP, which function is important in the inactivation of synaptic ATP molecules following nerve stimulation. (See, Todorov et al., Nature, 387(6628):76 (1997)). Apyrase in mammals is also believed to be important in the inhibition of ADP-induced platelet aggregation. (See, Marcus et al., J. Clin. Invest., 99(6):1351 (1997)). Recombinant potato apyrase is commercially available from Sigma-Aldrich. In some embodiments, the apyrase used in the compositions and methods disclosed herein is not derived from potato. In other embodiments, the apyrase used in the compositions and methods disclosed herein is not derived from a mammal.

[0059] As used herein, the “GDA1_CD39 superfamily refers to enzymes comprised of nucleoside triphosphate diphosphohydolases with common motifs in their protein sequences. The family is named after two proteins: the yeast GDPase (GDA1) and a lymphoid cell activation antigen, CD39. These proteins, in some embodiments, are cell-surface enzymes that hydrolyze a range of NTPs, including extracellular ATP. Non-limiting examples include ecto-ATPases, apyrases, CD39s, and ecto-ATP / Dases (Knowles, 2011, Purinergic Signalling volume 7, pages 21–45; Robson et al.2006 Purinergic Signalling 2:409-430; incorporated by reference herein).

[0060] As used herein, "microorganism" or “microbe” refers to a bacterium, a fungus, a virus, a protozoan, and other microbes or microscopic organisms.

[0061] The term “sequence identity” or “sequence similarity” as used herein, means that two polynucleotide sequences, a candidate sequence and a reference sequence, are identical (i.e. 100% sequence identity) or similar (i.e. on a nucleotide-by-nucleotide basis) over the length of the candidate sequence. In comparing a candidate sequence to a reference sequence, the candidate sequence may comprise additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment ofAttorney Docket No. NB42251-WO-PCT the two sequences. Optimal alignment of sequences for determining sequence identity may be conducted using the any number of publicly available local alignment algorithms known in the art such as ALIGN or Megalign (DNASTAR), or by inspection.

[0062] The term “percent (%) sequence identity” or “percent (%) sequence similarity,” as used herein with respect to a reference sequence is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the residues in the reference polynucleotide sequence after optimal alignment of the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0063] As used herein with regard to amino acid residue positions, “corresponding to” or “corresponds to” or “correspond to” or “corresponds” refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide.

[0064] As used herein, “prevent,” “preventing,” “prevention” and grammatical variations thereof refers to a method of partially or completely delaying or precluding the onset or recurrence of a disorder or condition and / or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing an subject's risk of acquiring or reacquiring a disorder or condition or one or more of its attendant symptoms.

[0065] As used herein, the term “reducing” “or “decreasing” in relation to a particular trait, characteristic, feature, biological process, or phenomena refers to a decrease in the particular trait, characteristic, feature, biological process, or phenomena. The trait, characteristic, feature, biological process, or phenomena can be decreased by 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater than 100%.

[0066] As used herein, the term “subject” or “patient” is meant a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition including, but not limited to, a disease, disorder or condition associated with unhealthy aging or mitochondrial dysfunction. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic ofAttorney Docket No. NB42251-WO-PCT susceptibility to or risk of a disease, disorder, or condition. In some embodiments, the subject is human. In some embodiments, a subject is a patient. In some embodiments, the subject is not a non-human animal. In some embodiments, the subject is a non-human animal. In some embodiments, the non-human animal is a dog, cat, pig, hamster, rabbit, bird, mouse, or horse. In some embodiments, a subject is an subject to whom diagnosis and / or therapy is and / or has been administered. In some embodiments, any of the subjects described herein are between about 20-30 years old, 30-40 years old, 40-50 years old, 50-60 years old, or 60-70 years old, or 65 or older.

[0067] As used herein “administer” or “administering” is meant the action of introducing one or more microbial strain, an exogenous feed enzyme and / or a strain and an exogenous feed enzyme to a subject, such as by feeding or by gavage.

[0068] As used herein, “effective amount” or “therapeutically effective amount” means a quantity of exogenous enzyme(s)s to improve one or more metrics in a subject. Improvement in one or more metrics of a subject (such as, without limitation, healthy aging, mitochondrial function, improved barrier integrity, reduced mortality, or reduced pathogen infection) can be measured as described herein or by other methods known in the art. The exogenous enzymes can also be administered in one or more doses.

[0069] The term “commensal bacteria” refers to microflora (normal microflora, indigenous microbiota) consisting of those micro-organisms which are present on body surfaces covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, skin, etc.). These bacteria have been shown to impact the regulation of immunophysiological functions, including but not limited to, metabolism, ontogeny, and pathogen defense. Commensal bacteria have also been shown to promote resistance to pathogens that is mutually beneficial to the host and the commensal microbiota (Byrd et al. 2018. Nature Reviews Microbiology, 16:143-155; Kilian et al.2016, British Dental Journal 221:657-666, incorporated by reference herein).

[0070] The term “probiotic” refers to live or viable microorganisms which, when administered in effective amounts to a subject, confer a health benefit on the subject. For example, the probiotics included in the probiotic formulations described herein, which are suitable for oral or topical administration to subjects, may improve the subjects' health. The term “probiotic,” as used herein, encompasses live microorganisms as well as viable microorganisms in dormant states, including frozen microorganisms, desiccated or driedAttorney Docket No. NB42251-WO-PCT microorganisms, spores, cysts or microorganisms in various states of reduced metabolic activity, which can be reconstituted upon exposure to suitable conditions. In some embodiments, the microorganisms of the probiotic comprise or consist of one or more commensal bacteria. In other embodiments, probiotic the microorganisms of the probiotic are derived from a fecal microbial source (such as fecal microbial source obtained from an otherwise healthy subject). Probiotics are distinguished from bacterial compositions that have been killed, for example, by pasteurization or heat treatment. Administration of non- viable bacterial compositions is also contemplated in certain embodiments of the methods disclosed herein.

[0071] As used herein, the term “engraftment” refers to the colonization of one or more bacterial species (such as commensal bacterial species provided, for example, as a probiotic) or to the adherence of one or more bacterial species (such as commensal bacterial species provided, for example, as a probiotic) in a subject.

[0072] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number can be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term “about” refers to a range of - 10% to +10% of the numerical value, unless the term is otherwise specifically defined in context.

[0073] As used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

[0074] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0075] It is also noted that the term “consisting essentially of,” as used herein refers to a composition wherein the component(s) after the term is in the presence of other knownAttorney Docket No. NB42251-WO-PCT component(s) in a total amount that is less than 30% by weight of the total composition and do not contribute to or interferes with the actions or activities of the component(s).

[0076] It is further noted that the term "comprising,” as used herein, means including, but not limited to, the component(s) after the term “comprising.” The component(s) after the term “comprising” are required or mandatory, but the composition comprising the component(s) can further include other non-mandatory or optional component(s).

[0077] It is also noted that the term “consisting of,” as used herein, means including, and limited to, the component(s) after the term "consisting of.” The component(s) after the term “consisting of” are therefore required or mandatory, and no other component(s) are present in the composition.

[0078] The terms “protein” and “polypeptide” refer to compounds comprising amino acids joined via peptide bonds and may be used interchangeably. A “protein” or “polypeptide” comprises a polymeric sequence of amino acid residues. The single and 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure. The single letter X refers to any of the twenty amino acids. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. Amino acid positions in a given polypeptide sequence can be named by the one letter code for the amino acid, followed by a position number. For example, a glycine (G) at position 87 is represented as “G087” or “G87.”

[0079] As used herein, where “amino acid sequence” is recited it refers to an amino acid sequence of a protein or peptide molecule. An “amino acid sequence” can be deduced from the nucleic acid sequence encoding the protein. However, terms such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the deduced amino acid sequence but can include posttranslational modifications of the deduced amino acid sequences, such as amino acid deletions, additions, and modifications such as glycosylations and addition of lipid moieties. Also, the use of non-natural amino acids, such as D-amino acids to improve stability or pharmacokinetic behavior falls within the scope of the term “amino acid sequence”, unless indicated otherwise.

[0080] The terms “signal sequence” and “signal peptide” refer to a sequence of amino acid residues that may participate in the secretion or direct transport of the mature or precursorAttorney Docket No. NB42251-WO-PCT form of a protein. The signal sequence is typically located N-terminal to the precursor or mature protein sequence. The signal sequence may be endogenous or exogenous. A signal sequence is normally absent from the mature protein. A signal sequence is typically cleaved from the protein by a signal peptidase after the protein is secreted.

[0081] The term “mature” form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or enzyme without the signal peptide sequence and or a pro- peptide sequence.

[0082] The term “wild-type” in reference to an amino acid sequence or nucleic acid sequence indicates that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term “naturally-occurring” refers to anything (e.g, proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term “non-naturally occurring” refers to anything that is not found in nature (e.g, recombinant / engineered nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

[0083] A “biologically pure strain” means a strain containing no other strains in quantities sufficient to interfere with replication of the strain or to be detectable when assessed using techniques recognized in the field.

[0084] “Isolated” when used in connection with the organisms and cultures described herein includes not only a biologically pure strain, but also any culture of organisms which is grown or maintained other than as it is found in nature. In some embodiments, the strains are mutants, variants, or derivatives of strains of Cyberlindnera jadinii and / or Kluyveromyces lactis described herein that also provide benefits comparable to that provided by deposited strains of Cyberlindnera jadinii and / or Kluyveromyces lactis as described herein. In some embodiments, the strains are strains having all of the identifying characteristics of the deposited strains of Cyberlindnera jadinii or Kluyveromyces lactis described herein. Further, each individual strain (e.g., Cyberlindnera jadinii strain and Kluyveromyces lactis strain described herein) or any combination of these strains can also provide one or more of the benefits described herein. It will also be clear that addition of microbial strains, carriers, additives (e.g., cryoprotectants, extracts, prebiotics, postbiotics, parabiotics), enzymes, other yeasts, or the like may also provide one or more benefits or improvement of diseases, disorders, and conditions associatedAttorney Docket No. NB42251-WO-PCT with unhealthy aging or mitochondrial dysfunction, in a subject and will not constitute a substantially different yeast strain.

[0085] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0086] Unless defined otherwise herein, 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 pertains.

[0087] Other definitions of terms may appear throughout the specification. II. COMPOSITIONS A. Yeasts

[0088] Provided herein are yeast strains and compositions containing yeast strains capable of producing ATP degrading enzyme . In some of any embodiments, the ATP degrading enzyme producing yeast strains and compositions comprising ATP degrading enzyme producing yeast strains are useful for treating, delaying or reducing the severity of age-related conditions, physical states associated with mitochondrial dysfunction, treating age related decline in nutrient absorption in the gut, and increasing pre-albumin / albumin levels in a subject. In some embodiments, the ATP degrading enzyme producing yeast strains and compositions containing yeast strains are useful for the treatment and / or prevention of diseases, disorders, and conditions associated with age-related conditions. In some embodiments, the diseases, disorders, and conditions associated with an age-related condition are or include those described below. In some embodiments, the compositions disclosed herein can be used as supplements, food additives, and / or therapeutics for administration to subjects when under periods of physiologic stress or as a part of a daily nutritional regimen to prevent disease and facilitate healthy aging.Attorney Docket No. NB42251-WO-PCT

[0089] In some embodiments, the compositions provided herein contain yeast from one or more genera. For example, 2, 3, 4, 5, 6, or more different genera. In some embodiments, the compositions provided herein contain species of yeast from one or more genera. For example, one or more species from 2, 3, 4, 5, 6, or more genera. In some embodiments, the compositions provided herein contain strains of a species of yeast from one or more genera. For example, one or more strains of a species from 2, 3, 4, 5, 6, or more genera. In some embodiments, the compositions provided herein contain yeast from a single genus. In some embodiments, the compositions provided herein contain species of yeast from a single genus. For example, 2, 3, 4, 5, 6, or more species of yeast from a single genus. In some embodiments, the compositions provided herein contain one or more strains of a species of yeast from a single genus. For example, 2, 3, 4, 5, 6, or more different strains of a yeast species from a single genus. In some embodiments, the compositions provided herein contain a single strain of a yeast species, e.g., a biologically pure strain. In some embodiments, the yeast genera, species, and strains are any of those described herein.

[0090] In some embodiments, the ATP degrading enzymeproducing yeast is any yeast capable of producing an ATP degrading enzyme enzyme. In some embodiments, the ATP degrading enzyme producing yeast is of the genera Cyberlindnera, Kluyveromyces or Saccharomyces. In some embodiments, the yeast is of the genera Cyberlindnera or Kluyveromyces. In some embodiments, the yeast is of the genus Cyberlindnera. In some embodiments, the yeast is of the genus Kluyveromyces. In some embodiments, the yeast is of the genus Saccharomyces. In some embodiments, the yeast is of the species Cyberlindnera jadinii (C. jadinii), Kluyveromyces lactis (K. lactis) or Saccharomyces boulardii. In some embodiments, the yeast is of the species Cyberlindnera jadinii (C. jadinii) or Kluyveromyces lactis (K. lactis). In some embodiments, the yeast is C. jadinii. In some embodiments, the yeast is K. lactis. In some embodiments, the yeast is Saccharomyces boulardii. In some embodiments, the yeast is a strain of C. jadinii or K. lactis. In some embodiments, the yeast is a strain of C. jadinii. In some embodiments, the yeast is a strain of K. lactis. In some embodiments, the ATP degrading enzyme producing yeast strain comprises one or more of Kluyveromyces lactis, Cyberlindnera jadinii, or Saccharomyces boulardii.

[0091] In some embodiments, the compositions provided herein include ATP degrading enzyme producing yeasts of the genera Cyberlindnera, Kluyveromyces or Saccharomyces. In some embodiments, the compositions provided herein include yeasts of the generaAttorney Docket No. NB42251-WO-PCT Cyberlindnera or Kluyveromyces. In some embodiments, the compositions provided herein include yeasts of the genus Cyberlindnera. In some embodiments, the compositions provided herein include yeasts of the genus Kluyveromyces. In some embodiments, the compositions provided herein may include yeasts of the genera Cyberlindnera and Kluyveromyces. In some embodiments, the compositions provided herein may include yeasts of the species Cyberlindnera jadinii (C. jadinii) or Kluyveromyces lactis (K. lactis). In some embodiments, the composition contains C. jadinii. In some embodiments, the composition contains K. lactis. In some embodiments, the compositions provided herein include yeasts of the species C. jadinii and K. lactis. In some embodiments, the composition contains a strain of C. jadinii or K. lactis. In some embodiments, the composition contains a strain of C. jadinii. In some embodiments, the composition contains a strain of K. lactis. In some embodiments, the composition contains a strain of C. jadinii and a strain of K. lactis.

[0092] In some embodiments, the strain of C. jadinii is the strain deposited at DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D- 38124 Braunschweig - Germany] under number DSM 33763. In some embodiments, the strain of C. jadinii is a strain having all of the identifying characteristics of the C. jadinii strain deposited at DSMZ under number DSM 33763. In some embodiments, the strain of C. jadinii having all of the identifying characteristics of the C. jadinii strain deposited at DSMZ under number DSM 33763 is a live strain. In some embodiments, the strain of C. jadinii is a biologically pure strain of the strain DSM 33763. In some embodiments, the C. jadinii strain is a biologically pure strain of C. jadinii that has all of the identifying characteristics of the C. jadinii strain DSM 33763. In some embodiments, the strain of C. jadinii having all of the identifying characteristics of the C. jadinii strain deposited at DSMZ under number DSM 33763 is a live strain.

[0093] In some embodiments, the strain of K. lactis is the strain deposited at DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D- 38124 Braunschweig - Germany] under number DSM 33764. In some embodiments, the strain of K. lactis is a strain having all of the identifying characteristics of the K. lactis strain deposited at DSM under number DSM 33764. In some embodiments, the strain of K. lactis is a biologically pure strain of the strain DSM 33764. In some embodiments, the K. lactis strain is a biologically pure strain of K. lactis that has all of the identifying characteristics of the K. lactis strain DSM 33764. In some embodiments, the strain of K. lactis having all of theAttorney Docket No. NB42251-WO-PCT identifying characteristics of the K. lactis strain deposited at DSMZ under number DSM 33764 is a live strain.

[0094] An identifying characteristic, as used herein, may be a genome sequence percentage identity and / or functional behavior. Functional behavior may be defined by metabolic activity; functional pathways, e.g., signaling pathways; upregulated and downregulated genes, for example as a result of epigenetic changes; protein expression, and protein secretion. In some embodiments, the genome sequence percentage identity is at least 70, 80, 90, 95, 96, 97, 98, 99% or 100%. B. Enzymes

[0095] Provided herein are enzymes and compositions containing enzymes useful for treating, delaying or reducing the severity of age-related conditions, physical states associated with mitochondrial dysfunction, treating age related decline in nutrient absorption in the gut, and increasing pre-albumin / albumin levels in a subject. In some embodiments, the enzymes and compositions containing enzymes are useful for the treatment and / or prevention of diseases, disorders, and conditions associated with age-related conditions. In some embodiments, the diseases, disorders, and conditions associated with an age-related condition are or include those described below. In some embodiments, the compositions disclosed herein can be used as supplements, food additives, and / or therapeutics for administration to subjects when under periods of physiologic stress or as a part of a daily nutritional regimen to prevent disease and facilitate healthy aging.

[0096] Provided herein are compositions comprising ATP degrading enzymes such as NTPDase enzymes and phosphatase enzymes. In some embodiments, the phosphatase enzymes are acid phosphatases or alkaline phosphatases. NTPDases, also known as ectonucleotidases, Ecto-ATPases and ectonucleoside triphosphate diphosphohydrolase, are ectoenzymes that hydrolyze extracellular nucleotides. There are four major families of ectonucleotidases: CD39 / NTPDases (ecto-nucleotide triphosphate diphosphohydrolases); ecto-nucleotide pyrophosphatase phosphodiesterases (E-NPPs); alkaline phosphatases; and ecto-5'-nucleotidases / CD73.

[0097] In some embodiments, any of the compositions provided herein are capable of removing phosphatases from ATP. In some embodiments, any of the compositions provided herein are capable of removing phosphatases form NTP. In some embodiments, theAttorney Docket No. NB42251-WO-PCT composition is an apyrase and is capable of removing phosphates from ATP and / or NTP. In some embodiments, the composition is an NTPDase and is capable of removing phosphates from ATP and / or NTP. In some embodiments, the composition is an acid phosphatase and is capable of removing phosphates from ATP and / or NTP. In some embodiments, the composition is an alkaline phosphatase and is capable of removing phosphates from ATP and / or NTP.

[0098] In some embodiments, any of the compositions provided herein are capable of breaking the bond between a sugar and a purine in ATP. In some embodiments, the composition is a purine nucleoside phosphorylase (PNP) and is capable of breaking the bond between a sugar and a purine in ATP.

[0099] NTPDases are constitutively expressed in many tissues and can be differentiated on the basis of cellular location. In some embodiments, the NTPDases provided herein comprise NTPDases 1, 2, 3 and 8, located on the cell surface; In some embodiments, the NTPDases provided herein comprise 5 and 6 and are localized within the cell and undergo secretion after heterologous expression. In some embodiments, the NTPDases provided herein comprise 4 and 7 and are entirely intracellularly located and face the lumen of cytoplasmic organelles. NTPDases located at the cell surface require Ca2+ or Mg2+ ions for activity. These subtypes contribute to the recycling of nucleosides from salvage pathways and extracellular nucleoside phosphates.

[0100] Due to their role in nucleotide metabolism, NTPDases help control the availability of extracellular nucleotide agonists at P2 receptors and modulate P2 receptor function as a result. The distribution of P2 receptors throughout the body, and the wide-spread nature of purinergic signaling, suggests an important role for NTPDases in a range of cellular processes. Location, substrate preference and hydrolysis rates vary between subtypes; however, all NTPDases contain five highly conserved sequence domains, known as apyrase conserved regions (APCR1 to APCR5).

[0101] In some of any of the embodiments provided herein, the enzyme has phosphatase activity. A phosphatase is an enzyme that dephosphorylates its substrates. Enzymes exhibiting phosphatase activity include: apyrases (also known as NTPDases, ATP- diphosphohydrolases (EC 3.6.1.5)), inorganic pyrophosphatases (EC 3.6.1.1,) acid phosphatase (EC 3.1.3.2), alkaline phosphatases (EC 3.1.3.1), trimetaphosphatases (EC 3.6.1.2) and exopolyphosphatases (EC 3.6.1.11). This phosphatase activity action is directlyAttorney Docket No. NB42251-WO-PCT opposite to that of phosphorylases and kinases, which attach phosphate groups to their substrates by using energetic molecules like ATP. In some of any embodiments, any of the NTPDase enzymes are capable of hydrolyzing ATP, ADP, CTP, CDP, UTP, UDP, GTP, and / or GDP. In some embodiments, the NTPDase enzyme is capable of hydrolyzing ATP. In some of any embodiments, the NTPDase enzyme is soluble. In some of any embodiments, the NTPDase enzyme is secreted.

[0102] In some embodiments, the ATP degrading enzyme is not an endogenous ATP degrading enzyme. In some embodiments, the ATP degradingenzyme is a mammalian, bacterial, plant or fungal ATP degrading enzyme. In some embodiments, the ATP degrading enzyme is not a mammalian ATP degrading enzyme. In some embodiments, the ATP degrading enzyme is a bacterial ATP degrading enzyme. In some embodiments, the ATP degrading enzyme is a GDA1_CD39 superfamily apyrase. In some embodiments, the ATP degrading enzyme is an acid phosphatase superfamily enzyme. In some embodiments, the ATP degrading enzyme is derived from Legionella sp., Beggiatoa sp., Gallaecimonas sp., Betaproteobacteria sp., Klebsiella sp., Serratia sp., Herbaspirillum sp., Acinetobacter sp., Paraburkholderia sp., or Pantoea beijingensis sp.

[0103] In some embodiments, the ATP degrading enzyme enzyme is derived from Legionella pneumophila, Legionella fallonii ATCC 700992, Gallaecimonas xiamenensis, Legionella santicrucis DSM23075, Legionella quateirensis, Betaproteobacteria bacterium, Klebsiella aerogenes, Serratia rubidaea, Herbaspirillum autotrophicum, Acinetobacter apis, Acinetobacter boissieri, Paraburkholderia bonniea or Pantoea beijingensis.

[0104] The enzymes for use in the compositions and methods disclosed herein are active at least from about pH 3 to pH 9 (such as any of about pH 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8 or 9) or from pH 3.5 to 5.5 or from pH 3.5 to 7 or from pH 5 to 7. The enzyme can be a member of the GDA1_CD39 superfamily or the acid phosphatase family. In some embodiments, the enzyme can be a member of the GDA1_CD39 superfamily including apyrases derived from potato. In some embodiments, the enzyme can be a member of the GDA1_CD39 superfamily excluding apyrases derived from potato. In some embodiments, the enzyme has activity from pH 3 to pH 9.Attorney Docket No. NB42251-WO-PCT

[0105] Other enzymes for use in the compositions and methods disclosed herein are active at least from about pH 3 to pH 9 (such as any of about pH 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8 or 9) or from pH 3.5 to 5.5 or from pH 3.5 to 7 or from pH 5 to 7 and are a member of the GDA1_CD39 superfamily or the acid phosphatase family. In some embodiments, the enzyme comprises an amino acid sequence having at least 35% or at least 60% sequence identity (such as any of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with the full length amino acid sequence of one or more of SEQ ID NO: 9-16, 50-56 or 77-89. In other embodiments, the enzyme comprises an amino acid sequence having at least 35% sequence identity (such as any of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with the full length mature amino acid sequence of any one of SEQ ID NO: 17-24, 57-63, 30-32, and 90-102. In some embodiments, the full-length amino acid sequence further comprises a signal sequence. In some embodiments, the amino acid sequence further contains a N-terminus AGK tripeptide.

[0106] In some embodiments, the enzyme comprises an amino acid sequence having at least 35% sequence identity (such as any of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with the full length amino acid sequence of SEQ ID NO: 12 (CRC22110). In other embodiments, the enzyme comprises an amino acid sequence having at least 35% sequence identity (such as any of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,Attorney Docket No. NB42251-WO-PCT 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with the mature amino acid sequence of SEQ ID NO: 20.

[0107] In some embodiments, the enzyme comprises an amino acid sequence having at least 35% sequence identity (such as any of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with the amino acid sequence of SEQ ID NO: 30, 31 or 32.

[0108] In other embodiments, the enzyme comprises an amino acid sequence having at least 35% sequence identity (such as any of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with the mature amino acid sequence of SEQ ID NO: 63. In other embodiments, the enzyme comprises an amino acid sequence having at least 35% sequence identity (such as any of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with the full length amino acid sequence of SEQ ID NO: 56.

[0109] In some embodiments, the full length amino acid sequence further comprises a signal sequence. In some embodiments, the amino acid sequence further contains a N-terminus AGK tripeptide.

[0110] Provided herein are compositions comprising ATP degrading enzyme enzymes containing one or more modifications. In some embodiments, the modification comprises a deletion, insertion or substitution. In some embodiments, the modification comprises a truncation. In some embodiments, the truncation is of the N-terminus. In some embodiments,Attorney Docket No. NB42251-WO-PCT the truncation is of the C-terminus. In some embodiments, the modification comprises an insertion. In some embodiments, the insertion is on the N-terminus. In some embodiments, the insertion is on the C-terminus.

[0111] In some of any embodiments, any of the enzymes provided herein comprise a truncation in the C-terminus of an amino acid having the full length mature amino acid sequence of one or more of SEQ ID NOs: 17-24, 57-63 or 90-102.

[0112] In some embodiments, any of the enzymes provided herein comprises an amino acid sequence having at least 35% (such as any of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with one or more of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

[0113] In some embodiments, any of the enzymes described herein can be of mammalian, fungal or bacterial origin. In some embodiments, any of the enzymes described herein is not a mammalian enzyme. In some embodiments, any of the enzymes described herein is a bacterial enzyme. In some embodiments, any of the enzymes described herein is an ATP degrading enzyme that can be of mammalian, fungal or bacterial origin. In some embodiments, any of the enzymes described herein is not a mammalian ATP degrading enzyme enzyme. In some embodiments, any of the enzymes described herein is a bacterial ATP degrading enzyme enzyme. B. Vectors

[0114] The terms “nucleic acid,” “ polynucleotide”, and “nucleic acid fragment” are used interchangeably herein and refer to a polymer of RNA or DNA that is single- or double- stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

[0115] A DNA construct comprising a nucleic acid encoding a phosphatase polypeptide disclosed herein (such as a nucleic acid comprising any of SEQ ID NOs:1-8 or 43-49) can be constructed such that it is suitable to be expressed in a host cell. Because of the knownAttorney Docket No. NB42251-WO-PCT degeneracy in the genetic code, different polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also known that, depending on the desired host cells, codon optimization may be required prior to attempting expression.

[0116] A polynucleotide encoding a phosphatase polypeptide of the present disclosure can be incorporated into a vector. Vectors can be transferred to a host cell using known transformation techniques, such as those disclosed below.

[0117] A suitable vector may be one that can be transformed into and / or replicated within a host cell. For example, a vector comprising a nucleic acid encoding an phosphatase polypeptide disclosed herein can be transformed and / or replicated in a bacterial host, fungal, or mammalian cell as a means of propagating and amplifying the vector. The vector may also be suitably transformed into an expression host, such that the encoding polynucleotide is expressed as a functional phosphatase enzyme.

[0118] A non-limiting representative useful vector is pTrex3gM (see, Published US Patent Application 20130323798), pTTT (see, Published US Patent Application 20110020899), and p2JM103BBI (see Vogtentanz, Protein Expr Purif, 55:40-52, 2007), which can be inserted into genome of host. The vectors pTrex3gM, pTTT, and p2JM103BBI can be modified with routine skill such that they comprise and express a polynucleotide encoding a phosphatase polypeptide of the invention.

[0119] An expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the phosphatase polypeptide to a host cell organelle such as a peroxisome, or to a particular host cell compartment. For expression under the direction of control sequences, the nucleic acid sequence of the phosphatase polypeptide is operably linked to the control sequences in proper manner with respect to expression.

[0120] For expression and production of a protein of interest (e.g., an ATP degrading enzyme described herein) in a cell, one or more expression vector or expression cassette comprising one or more copy of a polynucleotide encoding one or more ATP degrading enzyme described herein, and in some instances comprising multiple copies, is transformed into the cell under conditions suitable for expression of the variant. In some embodiments, aAttorney Docket No. NB42251-WO-PCT polynucleotide sequence encoding one or more ATP degrading enzyme described herein (as well as other sequences included in the vector) is integrated into the genome of the host cell, while in other embodiments, a plasmid vector comprising a polynucleotide sequence encoding one or more ATP degrading enzyme described herein remains as autonomous extra- chromosomal element within the cell. Some embodiments provide both extrachromosomal nucleic acid elements as well as incoming nucleotide sequences that are integrated into the host cell genome. The vectors described herein are useful for production of the one or more ATP degrading enzyme described herein. In some embodiments, a polynucleotide construct encoding one or more subtilisin variant described herein is present on an integrating vector that enables the integration and optionally the amplification of the polynucleotide encoding the variant into the host chromosome. Examples of sites for integration are well known to those skilled in the art. In some embodiments, transcription of a polynucleotide encoding one or more ATP degrading enzyme described herein is effectuated by a promoter that is the wild-type promoter for the parent ATP degrading enzyme. In some other embodiments, the promoter is heterologous to the one or more ATP degrading enzyme described herein, but is functional in the host cell. Exemplary promoters for use in bacterial host cells include, but are not limited to the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoters; the promoter of the B. stearothermophilus maltogenic amylase gene; the B. amyloliquefaciens (BAN) amylase gene; the B. subtilis alkaline protease gene; the B. clausii alkaline protease gene; the B. pumilis xylosidase gene; the B. thuringiensis cryIIIA; and the B. licheniformis alpha-amylase gene. Additional promoters include, but are not limited to the A4 promoter, as well as phage Lambda PR or PL promoters and the E. coli lac, trp or tac promoters, and the Bacillus rrn promoters such as rrnI, rrnB, rrnL and rrnE ribosomal RNA promoters and variants thereof.

[0121] A polynucleotide encoding a phosphatase polypeptide disclosed herein can be operably linked to a promoter, which allows transcription in the host cell. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of promoters for directing the transcription of the DNA sequence encoding an phosphatase, for example in a bacterial, fungal, or mammalian host, include the aprE promoter (SEQ ID NO: 46), the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA or celA promoters, the promoters of the Bacillus licheniformis amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylaseAttorney Docket No. NB42251-WO-PCT gene (amyM), the promoters of the Bacillus amyloliquefaciens amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes, and the like.

[0122] For transcription in a fungal host, examples of useful promoters include those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid stable α-amylase, Aspergillus niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase and the like. When a gene encoding a phosphatase polypeptide is expressed in a bacterial species such as an E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Along these lines, examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters. Expression in filamentous fungal host cells often involves cbh1, which is an endogenous, inducible promoter from T. reesei. See Liu et al. (2008) Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0123] The coding sequence can be operably linked to a signal sequence. The DNA encoding the signal sequence may be a DNA sequence naturally associated with the phosphatase polypeptide gene of interest to be expressed or may be from a different genus or species from which a portion of the phosphatase is derived. A signal sequence and a promoter sequence comprising a DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence may be the Trichoderma reesei cbh1 signal sequence, which is operably linked to a cbh1 promoter.

[0124] An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding a phosphatase. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

[0125] The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xxsC, a marker giving rise toAttorney Docket No. NB42251-WO-PCT hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., Published International PCT Application WO 91 / 17243.

[0126] Synthetic genes coding for the protein sequences corresponding to SEQ ID NOs.: 9- 16,50-56 and 77-89 can be generated using molecular biology methods known in the art. Synthetic genes include SEQ ID Nos: 1-8, 43-49 and 64-76. These genes can further be cloned into a suitable expression vector resulting in expression plasmids containing one or more of: a promoter (for example, the aprE promoter (SEQ ID NO: 25)); a signal sequence (for example, the aprE signal sequence (SEQ ID NO.26) encoding a peptide of SEQ ID NO: 27); an oligonucleotide to facilitate the secretion of the target protein (for example, an oligonucleotide encoding the peptide Ala-Gly-Lys); a synthetic nucleotide sequence encoding the mature region of the gene of interest (for example, a synthetic oligonucleotide having at least 35% (such as any of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with the synthetic oligonucleotide of any of SEQ ID NOs: 1-8, 43-49 or 64-76); and / or a terminator (such as the lat terminator (SEQ ID NO.28)). In some embodiments, any of the oligonucleotides or encoded proteins provided herein can contain a C-terminal His tag. The expression plasmid can further be transformed into a suitable expression host cell (such as a mammalian or bacterial or fungal expression host cell). C. Host Cells

[0127] An isolated cell, either comprising a DNA construct (such as any of the DNA constructs disclosed herein) or an expression vector (such as any of the expression vectors disclosed herein, for example, an expression vector comprising any of the polynucleotides of SEQ ID NOs:1-8, 43-49 and 64-76 that encodes a polypeptide of any one of SEQ ID NOs:17- 24, 57-6330-32, and 90-102), is advantageously used as a host cell in the recombinant production of a phosphatase polypeptide. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according toAttorney Docket No. NB42251-WO-PCT conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector in connection with the different types of host cells.

[0128] Examples of suitable bacterial host cells are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species such as Streptomyces murinus; lactic acid bacterial species including Lactococcus sp. such as Lactococcus lactis; Lactobacillus sp. including Lactobacillus reuteri; Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism.

[0129] A suitable yeast host cell can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, S. pombe species. A strain of the methylotrophic yeast species, Pichia pastoris, can be used as the host organism. Alternatively, the host organism can be a Hansenula species.

[0130] Suitable host cells among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma sp. such as Trichoderma ressei can be used as a host. A phosphatase polypeptide expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different as present in the wild-type phosphatase. The type and / or degree of glycosylation may impart changes in enzymatic and / or biochemical properties.

[0131] Suitable mammalian host cells include, without limitation, Chinese hamster ovary (CHO) cells or human embryonic kidney HEK) cells. Additional host cells can include insect sells, such as S2 cells.Attorney Docket No. NB42251-WO-PCT

[0132] It is advantageous to delete genes from expression hosts, where the gene deficiency can be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell having one or more inactivated genes. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbh1, cbh2, egl1, and egl2 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.

[0133] General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001), supra. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No.6,022,725. Reference is also made to Cao et al. (2000) Science 9:991-1001 for transformation of Aspergillus strains. Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding a phosphatase is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques.

[0134] A method of producing any of the enzymes disclosed herein may comprise cultivating a host cell under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and / or culture medium.

[0135] The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell and obtaining expression of a polypeptide. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

[0136] Any of the fermentation methods well known in the art can suitably be used to ferment the transformed or the derivative fungal strain as described above. In some embodiments, fungal cells are grown under batch or continuous fermentation conditions.

[0137] Separation and concentration techniques are known in the art and conventional methods can be used to prepare a concentrated solution or broth comprising a polypeptide of the invention.

[0138] After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain a solution. Filtration, centrifugation, microfiltration,Attorney Docket No. NB42251-WO-PCT rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra-filtration, extraction, or chromatography, or the like, are generally used.

[0139] It may at times be desirable to concentrate a solution or broth containing the polypeptide to optimize recovery. Use of un-concentrated solutions or broth would typically increase incubation time in order to collect the enriched or purified enzyme precipitate. D. Enzyme Compositions

[0140] Any of the enzymes for use in the methods disclosed herein can be formulated in compositions (for example, pharmaceutical or nutritional compositions). As already mentioned, any of the enzyme compositions described herein can be useful in treating and / or preventing diseases associated with unhealthy aging and / or mitochondrial dysfunction. In one embodiment, the composition is preferably a pharmaceutical composition, comprising an enzyme according to the disclosure. Said pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the composition is not a pharmaceutical composition, and is sold to reduce the risk of disease.

[0141] In some embodiments, the composition includes between 1nM to 100 nM of any of the enzymes described herein, or any ranges therebetween. In some embodiments, the composition includes between 1 pM to 1nM of any of the enzymes described herein or any ranges therebetween. In some embodiments, the composition includes between 100nM to 1 µM of any of the enzymes described herein or any ranges therebetween. In some embodiments, any of the compositions described herein contain about 1nM, 5nM, 10nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM or 100nM of any of the enzymes described herein or any ranges therebetween.

[0142] In some embodiments, the composition is in a form such as liquids, solids, semisolids or a combination thereof. The composition can be a complete oral nutritional supplement. The composition can also be in a form such as lozenges, lollipops, sachets, dissolvable films or a combination thereof.

[0143] In some embodiments, the composition is in a form such as a food, a beverage and combinations thereof. The composition can include an ingredient such as a thickener. In an alternative embodiment, the composition is a topical compound.

[0144] The composition can be presented in any form, for example as a tablet, as anAttorney Docket No. NB42251-WO-PCT injectable fluid or as an infusion fluid etc. Moreover, the composition, protein, nucleotide and / or vector according to the invention can be administered via different routes, for example bronchially, topically, or orally.

[0145] In some embodiments, the skin care component comprises any “dermatologically acceptable” composition. In some embodiments, the dermatologically acceptable composition is suitable for use in contact with human skin tissue without undue toxicity, incompatibility, instability, allergic response, and the like.

[0146] In some embodiments, any of the skin care components or compositions herein are configured for topical application to keratinous tissue. In some embodiments, any of the skin care components or compositions may include dimethicone and a particulate material as well as a water-soluble UV-blocking active at specific amounts, and such compositions may provide the desired feels including non-greasy, non-sticky (indicated by Mean Break Time), non-shiny, refreshing and easy to spread (indicated by dynamic viscosity). In some embodiments, any of the skin care components or compositions provided herein are UV- blocking compositions or sunscreen compositions.

[0147] In some embodiments, the composition may include at least a portion of or be an oral care composition. Illustrative oral care compositions may include, but are not limited to, a toothpaste (dentifrice), a prophylactic paste, a tooth powder, a tooth polish, a tooth gel (e.g., whitening gel), a chewing gum, a lozenge, a mouthwash, mouth rinse, a whitening strip, patch, suspension, emulsion, hydrogel, paste, multiphase solution, a paint-on gel, varnish, veneer, and tube, syringe or dental tray comprising a gel or paste, liquid, powder, or a gel or paste coated on an application support such as dental floss or a toothbrush (e.g., a manual, electric, sound, a combination thereof or ultrasound toothbrush). In some embodiments, the oral care composition comprises a combination of one or more oral care compositions.

[0148] The present composition may include a variety of optional ingredients that are known for use in personal care composition, as long as the optional ingredient(s) do not unduly alter product stability, aesthetics, or performance. The optional ingredients, when incorporated into the composition, should be suitable for contact with human keratinous tissue without undue toxicity, incompatibility, instability, allergic response, and the like within the scope of sound judgment. The compositions herein may comprise from about 0.0001% to about 50%; from about 0.001% to about 20%; or, alternately, from about 0.01% to about 10%, by weight of the composition, of optional ingredients. Some non-limitingAttorney Docket No. NB42251-WO-PCT examples of optional ingredients include abrasives, absorbents, opacifying agents, colorings / colorants (e.g., pigments, dyes, and lakes), particles, essential oils, anti-caking agents, foaming agents, anti-foaming agents, oil control agents, binders, biological additives, vitamins, minerals, peptides, sugar amines, flavonoid compounds, anti-oxidants, preservatives, plant extract, phytosterols, protease inhibitors, tyrosinase inhibitors, exfoliating agents, skin lightening agents, sunless tanning agents, anti-acne actives, anti-cellulite actives, anti-wrinkle actives, phytosterols and / or plant hormones, N-acyl amino acid compounds, antimicrobials, antifungals, moisturizers, emollients, humectants, lubricating agents, fragrances, anti-dandruff agents, buffering agents, bulking agents, chelating agents, biocides, denaturants, astringents, external analgesics, anti-inflammatory agents, sunscreen agents, film formers and / or polymers for aiding film-forming properties and substantivity of the composition, propellants, reducing agents, sequestrants, conditioning agents, and combinations of these.

[0149] In some embodiments, any of the compositions provided herein can optionally comprise pharmaceutically acceptable excipients, stabilizers, activators, carriers, permeators, propellants, disinfectants, diluents and preservatives. Suitable excipients are commonly known in the art of pharmaceutical formulation and may be readily found and applied by the skilled artisan, references for instance Remmington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia Pa., 17th ed.1985.

[0150] For oral administration, the protein can, for example, be administered in solid dosage forms, such as capsules, tablets (e.g., with an enteric coating), and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. A In some embodiments, any of the enzymes provided herein (e.g. apyrases) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulphate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. LiquidAttorney Docket No. NB42251-WO-PCT dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[0151] Enteric coatings arrest the release of the active compound from orally ingestible dosage forms. Depending upon the composition and / or thickness, the enteric coatings are resistant to stomach acid for required periods of time before they begin to disintegrate and permit slow release of ATP degrading enzymes in the lower stomach, small intestines, or large intestine. Examples of some enteric coatings are disclosed in U.S. Pat. No.5,225,202 (incorporated by reference). Examples of enteric coatings comprise beeswax and glyceryl monostearate; beeswax, shellac and cellulose, optionally with neutral copolymer of polymethacrylicacid esters; copolymers of methacrylic acid and methacrylic acid methylesters or neutral copolymer of polymethacrylic acid esters containing metallic stearates (for references enteric coatings see: U.S. Pat. Nos.4,728,512, 4,794,001, 3,835,221, 2,809,918, 5,225,202, 5,026,560, 4,524,060, 5,536,507). Most enteric coating polymers begin to become soluble at pH 5.5 and above, with a maximum solubility rates at pH above 6.5. Enteric coatings may also comprise subcoating and outer coating steps, for instance for pharmaceutical compositions intended for specific delivery in the lower GI tract, i.e. in the colon (pH 6.4 to 7.0, ileum pH 6.6), as opposed to a pH in the upper intestines, in the duodenum of the small intestines the pH ranges 7.7-8 (after pancreatic juices and bile addition). The pH differences in the intestines may be exploited to target the enteric-coated ATP degrading enzyme composition to a specific area in the gut. It also allows the selection of a specific ATP degrading enzyme that is most active at a particular pH in the intestine.

[0152] Besides the fact that a protein according to the invention can be incorporated in a pharmaceutical composition such an ATP degrading enzyme can also be part of a nutritional composition or a nutraceutical.

[0153] A protein according to the invention can be added to a nutrient (such as milk) but can also be produced within said nutrient (for instance by molecular engineering). Moreover, tablets and / or capsules can be prepared which are subsequently added to a nutrient or which can be taken directly by a human being.

[0154] In some embodiments, the compositions provided herein can be formulated for consumption of a non-human animal. In some embodiments, the compositions provided herein can be formulated for consumption a dog, cat, pig, hamster, rabbit, bird, mouse, or horse.Attorney Docket No. NB42251-WO-PCT

[0155] In yet another aspect, the invention features beverage and food products comprising an ATP degrading enzyme effective to address unhealthy aging or mitochondrial dysfunction in a subject in need thereof. The beverage products can contain from 1 to 10,000, e.g., 1 to 200, 200 to 500, 500 to 1,000, 1,000 to 5,000, or 5,000 to 10,000, units per milliliter. The food products can contain from 1 to 10,000, e.g., 1 to 200, 200 to 500, 500 to 1,000, 1,000 to 5,000, or 5,000 to 10,000, units per gram.

[0156] In some embodiments, any of the compositions provided herein may be prepared in various forms and may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intraperitoneal, subcutaneous, enteral, sublingual, or rectal routes. Preferably, the composition may be prepared for oral administration, e.g. as tablets, capsules and the like, or as injectable, e.g. as liquid solutions or suspensions. In some embodiments, the pharmaceutical composition is an injectable. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection are also encompassed, for example the pharmaceutical composition may be in lyophilized form.

[0157] In some embodiments, any of the compositions provided herein are suitable for parenteral administration, e.g., by injection or infusion, for example by bolus injection or continuous infusion. In some embodiments, the composition is a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and / or dispersing agents. Alternatively, any of the compositions provided herein may be in dry form, for reconstitution before use with an appropriate sterile liquid. In some embodiments, the ATP degrading enzyme is lyophilized. In some embodiments, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, e.g. for reconstitution with sterile water containing a preservative). For injection, e.g. intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer’s injection, lactated Ringer’s injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, as required. For injection, the pharmaceutical composition may be provided, for example, in a pre-filled syringe. E. Yeast FormulationsAttorney Docket No. NB42251-WO-PCT

[0158] The compositions disclosed herein, e.g., probiotics, generally contain at least one yeast able to treat or prevent or reduce the severity of diseases, disorders, and conditions associated with age or mitochondrial dysfunction as described herein. In some embodiments, the composition is formulated in freeze-dried (lyophilized) form. For example, the compositions containing yeast can comprise granules or gelatin capsules, for example hard gelatin capsules, including a yeast, e.g., yeast strain, disclosed herein.

[0159] In some embodiments, the yeast of the composition described herein are dried. In some embodiments, the compositions disclosed herein contain lyophilized yeast.

[0160] Lyophilization of yeast is a well-established procedure in the art. In some embodiments, the compositions disclosed herein contain spray dried yeast. Spray drying of yeast is a well-established procedure in the art. In some embodiments, the compositions disclosed herein contain drum dried yeast. Drum drying, in some cases referred to as roll drying, of yeast is a well-established procedure in the art. Alternatively, the compositions may contain a live, active yeast culture.

[0161] In some embodiments, the compositions provided herein contain yeast in frozen, dried, freeze-dried, liquid or solid format, in the form of pellets or frozen pellets, or in a powder or dried powder. In some embodiments, the compositions provided herein contain yeast described herein in a frozen format or in the form of pellets or frozen pellets. In some embodiments, the compositions provided herein contain yeast described herein in a dried or freeze-dried format. In some embodiments, the compositions provided herein contain yeast described herein in a powder or dried powder. In some embodiments, the yeast is lyophilized.

[0162] In some embodiments, any of the compositions disclosed herein, e.g., probiotics, are encapsulated to enable delivery of the yeast, such as a yeast disclosed herein, to the intestine. Encapsulation protects the composition from degradation until delivery at the target location through, for example, rupturing with chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier surfaces, self- aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule.

[0163] The compositions disclosed herein can be administered orally and may be in the form of a tablet, capsule, powder, chewable gum, or granule. In some embodiments, the tabletAttorney Docket No. NB42251-WO-PCT is an effervescent tablet, a chewable tablet, or a lyophilized tablet. In some embodiments, the tablet, capsule, powder, or granule is formulated for prolonged release, e.g., prolonged- release tablet, prolonged-release capsule, or prolonged-release granule. For example, prolonged-release formulations may release the dose over a period of time, such as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. Other ingredients (such as vitamin C or minerals, for example), may be included as oxygen scavengers and prebiotic substrates to improve the delivery and / or partial or total colonization and survival in vivo. Alternatively, the compositions (such as probiotic compositions) disclosed herein may be administered orally as a food or nutritional product or as a pharmaceutical product.

[0164] In some embodiments, the compositions disclosed herein are formulated as a probiotic. Alternatively, in some embodiments, the compositions disclosed herein are formulated as a non-viable composition, such as a pasteurized or heat-treated yeast composition.

[0165] In some embodiments, the composition described herein is administered in the form of a sachet, or suspension.

[0166] The composition, e.g., probiotic, disclosed herein may include a therapeutically effective amount of a yeast disclosed herein. A therapeutically effective amount of a yeast, e.g., yeast strain, is sufficient to exert a beneficial effect upon a subject, e.g., a subject having a disease, disorder, or condition described herein. In some embodiments, the composition contains an effective amount of Cyberlindnera jadinii and / or Kluyveromyces lactis to treat and / or prevent any of the age-related conditions described herein. In some embodiments, the composition contains an effective amount of Cyberlindnera jadinii and / or Kluyveromyces lactis to treat and / or prevent a disease, disorder, or conditions associated with age as described herein. A therapeutically effective amount of yeast, e.g., yeast strain, may be sufficient to result in delivery to and / or partial or total colonization of the subject’s intestine. In some embodiments, therapeutically effective amount of yeast, e.g., yeast strain, is sufficient to survive for a specific duration in a subject’s intestine. In some embodiments, the duration is, is at least, is about 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, one month or longer. In some embodiments, the duration is, is about, or is at least 12 days.

[0167] In some embodiments, the composition containing yeast described herein may be administered topically. For example, in some cases, treatment of inflammation, e.g., arisingAttorney Docket No. NB42251-WO-PCT from age and / or age-related disease, disorders, or conditions, may be accomplished by topical administration. In some embodiments, the inflammation may be treated or prevented by oral administration of compositions containing yeast described herein.

[0168] Provided herein is a method of visibly reducing the signs of skin aging, comprising applying any one the compositions described herein. In some embodiments, the method reduces a visible appearance of wrinkles when compared to skin to which the composition was not applied. ( Li, X., et. al. The Skin and Inflamm-Aging. Biology (Basel). 2023 Nov 2;12(11):1396).

[0169] For topical administration, the compositions containing yeast described herein may be in any form suitable for application to the body surface, such as a cream, lotion, sprays, solution, gel, ointment, paste, patch, plaster, paint, bioadhesive, suspensions or the like, and / or may be prepared so as to contain liposomes, micelles, and / or microspheres. Such a formulation may be used in combination with an occlusive overlayer so that moisture evaporating from the body surface is maintained within the formulation upon application to the body surface and thereafter.

[0170] Topical formulations include those in which the active ingredient(s), e.g., yeast, is (are) dissolved or dispersed in a dermatological vehicle known in the art (e.g. aqueous or non-aqueous gels, ointments, water-in-oil or oil-in-water emulsions). Constituents of such vehicles may comprise water, aqueous buffer solutions, non-aqueous solvents (such as ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycofurol or glycerol), oils (e.g. a mineral oil such as a liquid paraffin, natural or synthetic triglycerides such as Miglyol™, or silicone oils such as dimethicone). In some embodiments, the topical compositions include pH buffering agent(s) that when dissolved in a water component of the composition provide a pH in the range of 5 to 7 (e.g., about pH 5.5).

[0171] Methods of producing topical compositions, e,g., topical pharmaceutical compositions, such as creams, ointments, lotions, sprays and sterile aqueous solutions or suspensions are well known in the art. Suitable methods of preparing topical pharmaceutical compositions are described, for example in WO 95 / 10999, U.S. Pat. No.6,974,585, WO 2006 / 048747 as well as in documents cited in any of these references.

[0172] In some embodiments, the yeast strain may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial,Attorney Docket No. NB42251-WO-PCT intraperitoneal, subcutaneous, enteral, sublingual, or rectal routes. Preferably, the composition may be prepared for oral administration, e.g. as tablets, capsules and the like, or as injectable, e.g. as liquid solutions or suspensions. In some embodiments, the composition is an injectable. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection are also encompassed, for example the composition may be in lyophilized form.

[0173] In some embodiments, the yeast strain is suitable for parenteral administration, e.g., by injection or infusion, for example by bolus injection or continuous infusion. In some embodiments, the yeast strain is in a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and / or dispersing agents. Alternatively, any of the yeast strain may be in dry form, for reconstitution before use with an appropriate sterile liquid. In some embodiments, the yeast strain is lyophilized. In some embodiments, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, e.g. for reconstitution with sterile water containing a preservative). For injection, e.g. intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer’s injection, lactated Ringer’s injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, as required. For injection, the pharmaceutical composition may be provided, for example, in a pre-filled syringe.

[0174] In some embodiments, the composition contains one or more yeasts as described herein at an amount, independently, of at least or at least about 105, 106, 107, 108, 109, 1010, or 1011colony forming units (CFU). In some embodiments, the composition contains one or more yeasts as described herein at an amount, independently, of at least or at least about 109, 1010, or 1011CFU. In some embodiments, the composition contains one or more yeasts as described herein at an amount, independently, of at least or at least about 1010CFU. In some In some embodiments, the composition contains one or more yeasts as described herein at an amount, independently, of about 1 x 105to about 1 x 1011CFU; for example, from about 1 x 106to about 1 x 1011CFU, from about 1 x 107to about 1 x 1011CFU, from about 1 x 108to about 1 x 1011CFU, or from about 1 x 109to about 1 x 1011CFU, or from about 1 x 1010to about 1 x 1011CFU. In some embodiments, the compositions contain one or more yeasts asAttorney Docket No. NB42251-WO-PCT described herein at an amount, independently, of from or from about 1 x 108to about 1 x 1011CFU, 1 x 109to about 1 x 1011CFU, 1 x 109to about 1 x 1010CFU. In some embodiments, the compositions contain one or more yeasts as described herein at an amount, independently, from or from about 0.5 x 1010to about 5 x 1010CFU, from or from about 1 x 1010to about 5 x 1010CFU, from or from about 1.5 x 1010to about 5 x 1010CFU, from or from about 2 x 1010to about 5 x 1010CFU, from or from about 2.5 x 1010to about 5 x 1010CFU, from or from about 3 x 1010to about 5 x 1010CFU, from or from about 3.5 x 1010to about 5 x 1010CFU, from or from about 4 x 1010to about 5 x 1010CFU, or from or from about 4.5 x 1010to about 5 x 1010CFU. In some embodiments, the compositions contain one or more yeasts as described herein at an amount, independently, of or of about 1 x 1010, 1.5 x 1010, 2 x 1010, or 2.5 x 1010CFU. In some embodiments, the compositions contain one or more yeasts as described herein at an amount, independently, of or of about 2.5 x 1010CFU. In some embodiments, the CFU may be a CFU per weight, e.g., gram, of composition. Thus, in some of any of the embodiments, the CFU is a CFU per weight of the composition. In some embodiments, the CFU described herein is a CFU per gram of the composition (CFU / g). In some embodiments, the CFU described herein is a CFU per kilogram of the composition (CFU / kg). In some embodiments, the compositions contain one or more yeasts as described herein at an amount, independently, of or of about 2.5 x 1013CFU / kg.

[0175] In some embodiments, the weight of the composition administered to a subject, e.g., human, is between 50mg and 3000mg, between 100mg and 2500mg, between 150mg and 2000mg, between 200mg and 1500mg, between 250mg and 1000mg, between 300mg and 950mg, between 400mg and 900mg, between 450mg and 850mg, between 500mg and 800mg, between 550mg and 750mg, or between 600mg and 700mg. In some embodiments, the yeast in any of the compositions disclosed herein is administered at a dose of between 200mg and 3000mg, between 200mg and 2500mg, between 200mg and 2000mg, between 200mg and 1500mg or between 200mg and l000mg. In some embodiments, the yeast in any of the compositions disclosed herein is administered at a dose of or of about 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, 1500mg, 2000mg, 2500mg, or 3000mg. In some embodiments, the yeast in any of the compositions disclosed herein is administered at a dose of or of about 500mg, 1000mg, 1500mg, 2000mg, 2500mg, or 3000mg. In some embodiments, the yeast in any of the compositions disclosed herein is administered at a dose of or of about 100mg, 200mg, 300mg, 400mg, or 500mg.

[0176] In some embodiments, the composition contains a therapeutically effectiveAttorney Docket No. NB42251-WO-PCT amount of Cyberlindnera jadinii and / or Kluyveromyces lactis, e.g., as described herein, to treat, delay or reduce diseases, disorders, and conditions associated therewith as described herein. For example, the composition may contain a therapeutically effective amount of a strain of Cyberlindnera jadinii and / or Kluyveromyces lactis as described herein, effective to treat, delay or reduce the severity of at least one condition associated with aging or mitochondrial health. In some embodiments, the therapeutically effective amount is at least about 1 x 1010CFU / g to at least about 5 x 1010CFU / g of composition. In some embodiments, the therapeutically effective amount is or is about 2.5 x 1010CFU / g composition.

[0177] In some embodiments, the amount of yeast, e.g., as described herein, of the composition is a suitable daily dose for a subject, e.g., human subject. In some embodiments, the subject is a human. In some embodiments, the subject is an adult human. In some embodiments, the subject is a human child. The compositions provided herein may be repeatedly administered to a subject.

[0178] In some embodiments, the amount of yeast, e.g., as described herein, of the composition is a suitable daily dose for a subject, e.g., a non-human animal.Typically, a probiotic is optionally combined with at least one suitable prebiotic compound. A prebiotic compound is usually a non-digestible carbohydrate such as an oligo- or polysaccharide, or a sugar alcohol, which is not degraded or absorbed in the upper digestive tract. Known prebiotics include commercial products such as inulin and transgalacto- oligosaccharides.

[0179] In some embodiments, a probiotic composition described herein is formulated to include a prebiotic compound in an amount of from about 1 to about 30% by weight respect to the total weight composition, (e.g., from 5 to 20% by weight). Carbohydrates may be selected from the group consisting of: fructo- oligosaccharides (or FOS), short-chain fructo-ol igosaccharides, inulin, isomalt- oligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), human milk oligosaccharides, beta- glucans, gum arabic modified and resistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers. In one aspect, the prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible carbohydrates, generally obtained by the conversion of the beet sugar and including a saccharose molecule to which three glucose molecules are bonded.

[0180] The composition containing yeast described herein may further containAttorney Docket No. NB42251-WO-PCT pharmaceutically acceptable excipients or carriers. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art. Examples of suitable carriers include, without limitation, lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include, without limitation, ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binders, lubricants, suspending agents, coating agents, or solubilizing agents. Examples of suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include, without limitation, sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

[0181] In some embodiments, for example when the composition described herein is formulated as a probiotic, the composition contains one or more excipients. In some embodiments, the one or more excipient is fructose, lactose monohydrate, and / or colloidal anhydrous silica.

[0182] The composition disclosed herein may be formulated as a food product. For example, a food product may provide nutritional benefit in addition to the therapeutic effect of the invention, such as in a nutritional supplement. Similarly, a food product may be formulated to enhance the taste of the composition of the invention or to make the composition more attractive to consume by being more similar to a common food item, rather than to a pharmaceutical composition. In some embodiments, the food product is a fruit juice; bar; cheese; fresh fermented product; pickle; kimchi; miso; kombucha; kefir or other fermented milks; tempeh; indigenous fermented food; sauerkraut and other fermented vegetables; coffee; cocoa and other fermented food containing yeast; sourdough bread; beer; cereal; milk; powder milk; infant formula; a composition for sportsmen like energy drinks, protein solutions / powders; specialized nutrition, e.g., for elderly or infants; hospital nutrition; medical foods.Attorney Docket No. NB42251-WO-PCT

[0183] In certain embodiments, the compositions disclosed herein contain a single yeast species or strain and do not contain any other yeast species or strains. Such compositions may comprise only de minimis or biologically irrelevant amounts of other yeast or bacterial strains or species. Such compositions may be a culture that is substantially free from other species of organism.

[0184] The composition for use in accordance with the methods disclosed herein may or may not require marketing approval.

[0185] In some cases, the lyophilized yeast, e.g. yeast strain, is reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.

[0186] Any of the compositions containing yeast disclosed herein may contain pharmaceutically acceptable excipients, diluents or carriers.

[0187] In some embodiments, provided herein is a pharmaceutical composition comprising: one or more yeasts or yeast strains as disclosed herein; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the yeast is present at an effective amount to treat, delay or reduce the severity of at least on diseases, disorders, or conditions associated with age or mitochondrial health as described herein, including symptoms thereof.

[0188] In some embodiments, the invention provides the above pharmaceutical composition, containing a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol and sorbitol.

[0189] In some embodiments, the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol and water.

[0190] In some embodiments, the invention provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.

[0191] In some embodiments, the invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer.

[0192] In some embodiments, the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodiumAttorney Docket No. NB42251-WO-PCT benzoate, sorbic acid and esters of p- hydroxybenzoic acid.

[0193] In some embodiments, the invention provides the above pharmaceutical composition, wherein said yeast, as described herein, is lyophilized.

[0194] In some embodiments, the above pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphere having 50% relative humidity, at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the yeast as measured in colony forming units, remains after a period of at least about 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.

[0195] The yeast species and strains disclosed herein can be cultured using standard microbiology techniques such as those described. III. Methods

[0196] Provided herein are enzymes, yeasts and compositions containing enzymes and / or yeasts useful for treating, delaying or reducing the severity of age-related conditions, physical states associated with mitochondrial dysfunction, treating age related nutrient absorption in the gut, and increasing pre-albumin / albumin levels in a subject. In some embodiments, the enzymes, yeasts and compositions containing enzymes and / or yeasts are useful for the treatment and / or prevention of diseases, disorders, and conditions associated with age-related conditions. In some embodiments, the diseases, disorders, and conditions associated with an age-related condition are or include those described below. In some embodiments, the compositions disclosed herein can be used as supplements, food additives, and / or therapeutics for administration to subjects when under periods of physiologic stress or as a part of a daily nutritional regimen to prevent disease and facilitate healthy aging.

[0197] In some of any embodiments, the enzymes, yeasts and compositions containing enzymes and / or yeasts provided herein are capable of reducing the severity of age-related conditions. In some embodiments, the enzymes, yeasts and compositions containing enzymes and / or yeasts provided herein are capable of reducing the severity of age-related conditions by at least 1%, at least 5%, 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a reference. In some of any embodiments, the reference is a subject that has not been treated with an effective amount of the enzymes, yeasts and compositions containing enzymes and / or yeasts provided herein.

[0198] Aging, or the time dependent accumulation of cellular damage, as well asAttorney Docket No. NB42251-WO-PCT progressive loss of tissue and organ function over time (Childs et al. Nat. Med.2015 Dec; 21(12):1424-1435) is a process that occurs over multiple decades of life. Without wishing to be bound by theory, provided herein are methods capable of treating signs and symptoms associated with aging by reversing said signs and symptoms associated with aging. In some embodiments, any of the compositions provided herein can be usedin a method to reverse cellular damage in a cell to a level commensurate with one, two, three or four or more decades less than the chronological age of the cell. In some embodiments, the method provided herein is a method of decreasing or reversing the biological age of a cell relative to the chronological age of a cell. In some embodiments, the decreasing or reversing is 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more relative to the biological age of a cell. In some embodiments, the decreasing is one, two, three or four or more decades relative to the biological age of a cell.Metabolic changes influence all cellular activities and can also contribute to the onset and progression of many neurodegenerative disorders, which become more prevalent with aging (Trigo et al. FEBS Letters 596, 2022, 1095-1110). Metabolic changes can include reductions in insulin sensitivity, systemic glucose metabolism, lipid metabolism, and changes in resting energy expenditure, among others (Palmer, et. Al. J. Clin Invest 2022 Aug 15; 132(16):e158451). In addition, the increase in age increases the risk for developing age-related diseases, including Alzheimer’s. Cellular senescence, with major effects on the maintenance of normal tissue homeostasis, as well as pathological conditions, is a principle causative factor in the organismal aging process and facilitates aging and aging-associated diseases. Cellular senescence is a gradual decline of the proliferation and differentiation ability as well as the physiological function of cells over time. In 1956, Denham Harman proposed the free radical theory of aging, arguing that degenerative changes during aging are mediated by the harmful effects of free radicals generated during normal cellular metabolism. (Guo et al. Sig. Transduct Target Ther 7, 391 (2022). In addition, aging is accompanied by a marked decline in muscle mass known as sarcopenia, as well as a significant decline in muscle power referred to as dynapenia (Law et al., 2016; Annu Rev Gerontol Geriatr.36(1):205-228).

[0199] During the aging process, mitochondrial function, especially mitochondrial membrane potential (MMP) and respiratory capacity per mitochondrion, decline, which is often accompanied by increased production of reactive oxygen species (ROS). ROS are by- products of oxidative phosphorylation and are important signaling molecules. Mitochondrial dysfunction and reduced antioxidant detoxification result in oxidative stress, lipid oxidation,Attorney Docket No. NB42251-WO-PCT and mtDNA damage. In addition, constant loss in MMP during aging might reflect reduced electron transport chain capacity, with diminished Complex I activity in the brain, liver, skeletal muscle, as well as the immune system. Decline in Complex I activity is a driver for mitochondrial dysfunction, and has been shown to be associated with a number of health- related conditions, including health decline with aging (Enders et al. Int. J. Mol. Sci 2023, 24, 10818). Mitochondrial function also plays a role in the gastrointestinal epithelium by maintaining intestinal health. Emerging studies implicate the involvement of mitochondrial dysfunction in inflammatory bowel disease (IBD) and may serve as a starting point for investigation into mitochondrial-targeted interventions for IBD therapy. (Ho et al. Annu. Rev. Physiol 202284:435-59).

[0200] During aging, a decline in metabolic health can result in mitochondrial dysfunction, which promotes metabolic inflammation. This metabolic inflammation, in turn, can further impair mitochondrial function (Yuan et al. Oxidative Medicine and Cellular Longevity Vol 2022, Article ID 8803404). The aging process also has additional repercussions on the immune system, including the ability to maintain healthy commensal microbiota. Normally, the immune system identifies microbes through the detection of evolutionarily conserved structures, referred to as microbe-associated molecular patterns (MAMPs), by utilizing specialized pattern recognition receptors (PRRs). The activation of these PRRs instigates the production of pro-inflammatory cytokines, orchestrating a vigorous immune responses aimed at eradicating the pathogen. However, this process can concurrently lead to collateral damage to the host’s own tissues, potentially resulting in life-threatening outcomes. Cell wall glycoconjugates of commensal bacteria such as Firmicutes can act as a signaling molecule that is widely distributed within the body, and the sequestration of cell wall glycoconjugates by albumin within the plasma, can aid in inhibiting PRR activation. Several studies have indicated that older adults display an altered composition of the gut microbiota, and if the ability of albumin to sequester the glycoconjugates is impaired, it could result in a rise in systemic inflammation (Tang and Wu, 2023 Cell Host & Microbe 31:1422-1425).

[0201] Age-related changes in nutrient intake may also contribute to age related gut dysbiosis. Diet is one of the primary contributors to gut health, and advanced age is associated with deterioration in various aspects of nutrient intake and absorption including dentition, salivary function, digestion, and intestinal transit time. In addition, gut dysbiosis— commonly defined as a disturbance of or change in the density and / or composition of gut microbiota—has been linked to a wide variety of diseases and health conditions, including cystic fibrosis, inflammatory bowel conditions (irritable bowel syndrome, Crohn’s disease,Attorney Docket No. NB42251-WO-PCT and colon cancer, neurological diseases (Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis)), and musculoskeletal conditions including frailty, osteoporosis, rheumatoid arthritis, and gout (Buford, Microbiome 2017; 5:80).

[0202] Without being bound to theory, it is believed that administration of a composition comprising an ATP degrading enzyme, such as apyrases, including apyrases of the GDA1_CD39 superfamily and acid phosphatases, including at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence of any of SEQ ID NOs: 17-24, 57-63, and 30-32.; or at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 66%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence of SEQ ID Nos: 17-24, 57-63, 30-32, and 90-102) can a) treat, delay or reduce the severity of at least one age related condition by removal of eATP b) treat and / or prevent at least one physical state associated with mitochondrial health by removal of eATP; c) increase age related nutrient absorption in the gut by removal of eATP, and / or d) increase pre-albumin / albumin levels in a subject by removal of eATP.

[0203] Without being bound to theory, it is believed that administration of composition comprising at least one of Kluyveromyces lactis and Cyberlindnera jadinii can a) treat, delay or reduce the severity of at least one age related condition by removal of eATP b) treat and / or prevent at least one physical state associated with mitochondrial health by removal of eATP; c) increase age related nutrient absorption in the gut by removal of eATP, and / or d) increase pre-albumin / albumin levels in a subject by removal of eATP.

[0204] Without being bound to theory, it is believed that administration of a composition comprising an at least one of Kluyveromyces lactis and Cyberlindnera jadinii and an ATP degrading enzyme, such as apyrases, including apyrases of the GDA1_CD39 superfamily or ATP degrading enzyme of the acid phosphatase family, including at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least aboutAttorney Docket No. NB42251-WO-PCT 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence of any of SEQ ID NOs: 17-24, 57- 63, 30-32, and 90-102; can a) treat, delay or reduce the severity of at least one age related condition by removal of eATP b) treat and / or prevent at least one physical state associated with mitochondrial health by removal of eATP; c) increase age related nutrient absorption in the gut by removal of eATP, and / or d) increase pre-albumin / albumin levels in a subject by removal of eATP.

[0205] A source of ATP degrading enzyme can be any ATP degrading enzyme enzyme, or any composition comprising the ATP degrading enzyme and any means which is capable of producing a functional ATP degrading enzyme in the context of the current invention, such as DNA or RNA nucleic acids encoding an ATP degrading enzyme. The nucleic acid encoding the ATP degrading enzyme may be embedded in suitable vectors such as plasmids, phagemids, phages, (retro)viruses, transposons, gene therapy vectors and other vectors capable of inducing or conferring production of ATP degrading enzyme. Also, native or recombinant microorganisms, such as bacteria, fungi, protozoa and yeast may be applied as a source of ATP degrading enzyme in the context of the current invention. In some embodiments, the source of ATP degrading enzyme in the context of the current invention is not a mammalian ATP degrading enzyme. In some embodiments, the source of ATP degrading enzyme in the context of the current invention is a bacterial ATP degrading enzyme.

[0206] The polynucleotide encoding the ATP degrading enzyme may be optimized for expression of the ATP degrading enzyme. For example, codon optimization of the nucleotide sequence may be used to improve the efficiency of translation in expression systems for the production of the ATP degrading enzyme. Accordingly, the polynucleotide encoding of the ATP degrading enzyme may be codon-optimized. The skilled artisan is aware of various tools for codon optimization, such as those described in: Ju Xin Chin, Bevan Kai-Sheng Chung, Dong-Yup Lee, Codon Optimization OnLine (COOL): a web-based multi-objective optimization platform for synthetic gene design, Bioinformatics, Volume 30, Issue 15, 1 Aug. 2014, Pages 2210-2212; or in: Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D, JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res.2005 Jul 1;33(Web Server issue):W526-31). The nucleic acid molecule may comprise heterologous elements (i.e., elements, which in nature do notAttorney Docket No. NB42251-WO-PCT occur on the same nucleic acid molecule as the coding sequence for the ATP degrading enzyme), e.g. for expression (such as heterologous expression) of the ATP degrading enzyme. For example, a nucleic acid molecule may comprise a heterologous promoter, a heterologous enhancer, a heterologous UTR (e.g., for optimal translation / expression), a heterologous Poly-A-tail, and the like. In some embodiments the nucleic acid molecule may comprise an element conferring resistance against an antibiotic. In other embodiments, the nucleic acid molecule does not comprise an element conferring resistance against an antibiotic.

[0207] In some embodiments, the polynucleotide encoding the ATP degrading enzyme may be manipulated to insert, delete or alter certain nucleic acid sequences. In some embodiments, the polynucleotide encoding the ATP degrading enzyme is manipulated for changes to introduce restriction sites, to amend codon usage, to add or optimize transcription and / or translation regulatory sequences, etc. In some embodiments, the polynucleotide encoding the ATP degrading enzyme is altered to introduce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) substitutions, deletions and / or insertions into the amino acid sequence of the ATP degrading enzyme. In some embodiments, the polynucleotide encoding the ATP degrading enzyme can contain mutations to modify stability, post-translational modifications, or mutations to increase expression yield. In some embodiments, the polynucleotide encoding the ATP degrading enzyme can contain amino acids for the attachment of covalent groups or to introduce tags, including Histidine (His) tags. In some embodiments, the mutation may be “silent”, i.e. not reflected in the amino acid sequence due to the redundancy of the genetic code as described above. In some embodiments, the nucleic acid encoding the ATP degrading enzyme can be randomly or directionally mutated to introduce different properties in the encoded amino acids.

[0208] In some embodiments, the ATP degrading enzyme is encoded by a nucleic acid that is at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleic acid sequence of any of SEQ ID Nos: 1-8, 43-49 and 64-76; or at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 66%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at leastAttorney Docket No. NB42251-WO-PCT about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleic acid sequence of SEQ ID Nos: 1-8, 43-49, and 64-76.

[0209] In some embodiments, the composition comprising an ATP degrading enzyme contains a nucleic acid that is at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence of any of SEQ ID NOs: 1-8, 43-49, and 64-76; or at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 66%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence of SEQ ID Nos: 1-8, 43-49 and 64-76.

[0210] In some embodiments, the age-related condition is selected from age-related decline in metabolic regulation, age-related alterations in immune function, age-related alterations in muscle mass and function, age-related decline in gut function, age-related cognitive decline, and cell-senescence. In some embodiments, the age-related condition is age-related decline in metabolic regulation. In some embodiments, the age-related condition is age-related alterations in immune function. In some embodiments, the age-related condition is age-related alterations in muscle mass and function. In some embodiments, the age-related condition is age-related decline in gut function. In some embodiments, the age- related condition is age-related cognitive decline. In some embodiments, the age-related condition is cell-senescence. In some embodiments, the age-related condition is associated with mitochondrial dysfunction.

[0211] In some embodiments, the age-related decline in metabolic regulation includes a decline in insulin sensitivity, a decline in systemic glucose metabolism, a decline in lipid metabolism, and a decline in resting energy expenditure. In some embodiments, the age- related decline in metabolic regulation includes conditions associated with a decline in insulin sensitivity, a decline in systemic glucose metabolism, a decline in lipid metabolism, and a decline in resting energy expenditure.Attorney Docket No. NB42251-WO-PCT

[0212] In some embodiments, the age-related condition is age-related alterations in immune function. In some embodiments, the age-related alteration in immune function includes a decline in immune function and an increase in inflammation. In some embodiments, the inflammation does not include inflammation of the skin or oral mucosa. In some embodiments, the age-related alteration in immune function does not include an increase in inflammation. In some embodiments, the age-related alteration in immune function includes conditions associated with a decline in immune function and an increase in inflammation.

[0213] In some embodiments, the age-related condition is age-related alterations in muscle mass and function. In some embodiments, the age-related alterations in muscle mass and function include muscle atrophy and sarcopenia. In some embodiments, the age-related alterations in muscle mass and function include conditions associated with muscle atrophy and sarcopenia.

[0214] In some embodiments, the age-related condition is age-related decline in gut function. In some embodiments, the age-related condition is age-related decline in gut function includes a decline in gut permeability, an age related decline in nutrient sensing and an age related decline in absorption. In some embodiments, the age-related condition is age- related decline in gut function includes conditions associated with a decline in gut permeability, an age related decline in nutrient sensing and absorption.

[0215] In some embodiments, the age-related condition is age-related cognitive decline. In some embodiments, the age-related cognitive decline includes changes in the structure and function of synapses. In some embodiments, the age-related cognitive decline includes short- term memory loss, word-finding difficulty, alterations in sleep patterns, insomnia, slower processing speed and / or degenerative dementia. In some embodiments, the degenerative dementia is Alzheimer’s disease, vascular dementia, or dementia with Lewy bodies. In some embodiments, the age-related condition is cell-senescence.

[0216] In some embodiments, provided herein is a method for treating, delaying or reducing the severity of at least one condition selected from age-related decline in metabolic regulation, age-related alterations in immune function, age-related alterations in muscle mass and function, age-related decline in gut function, age-related cognitive decline, and cell- senescence, where the method includes administering to an individual in need thereof or at risk thereof an effective amount of a) a composition comprising an ATP degrading enzyme;Attorney Docket No. NB42251-WO-PCT b) a composition comprising at least one of Kluyveromyces lactis and Cyberlindnera jadinii, or c) a combination thereof.

[0217] In some embodiments, a subject is suffering from a relevant disease, disorder or condition including, but not limited to, a disease, disorder or condition associated with unhealthy aging or mitochondrial dysfunction. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, the subject is human. In some embodiments, the subject is not a non-human animal. In some embodiments, a subject is a patient. In some embodiments, a subject is a subject to whom diagnosis and / or therapy is and / or has been administered. In some embodiments, any of the subjects described herein are between about 20-30 years old, 30-40 years old, 40-50 years old, 50-60 years old, or 60-70 years old, or 65 or older.

[0218] Provided herein is a method of administering an effective amount of any of the compositions provided herein to a subject, thereby reducing the severity of the conditions so as to resemble an individual without the disease.

[0219] Provided herein is a method of administering an effective amount of any of the compositions provided herein to a subject, thereby reducing the severity of the conditions so as to resemble an individual at least about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7years, 8 years, 9 years or 10 years younger.

[0220] In some embodiments, provided herein is a method of administering an effective amount of any of the compositions provided herein to a subject, thereby reducing the severity of the conditions so as to resemble an individual at least between about 1-10 years, 10-20 years, 30-40 years, 40-50 years or 50-60 years younger than the age of the subject.

[0221] In some embodiments, provided herein is a method of administering an effective amount any of the compositions provided herein to a subject, thereby reducing the severity of the conditions so as to resemble an individual at least between about 1-10 years, 10-20 years, 30-40 years, 40-50 years or 50-60 years younger than the age of the subject.

[0222] In some embodiments, provided herein is a method of administering an effective amount any of the compositions provided herein to a subject, thereby reducing the age of theAttorney Docket No. NB42251-WO-PCT cells of the subject so as to resemble the cells of an individual at least between about 1-10 years, 10-20 years, 30-40 years, 40-50 years or 50-60 years younger than the age of the subject.

[0223] In some embodiments, the individual is healthy. In some embodiments, the individual does not display any of the conditions described herein. In some embodiments, the individual does not display any of the diseases or disorders described herein. In some embodiments, the individual is a healthy young adult. In some embodiments, the individual is between 10-20 years old. In some embodiments, the individual is at least between about 1-10 years, 10-20 years, 30-40 years, 40-50 years or 50-60 years younger than the age of the subject. In some embodiments, the individual is between 20-30 years old. In some embodiments, the individual is between 30-40 years old. In some embodiments, the individual is between 40-50 years old. In some embodiments, the individual is between 50-60 years old.

[0224] In some embodiments, the individual is a similar age as the subject. In some embodiments, the individual is healthy and is a similar age as the subject. In some embodiments, the individual does not display any of the conditions described herein and is a similar age as the subject. In some embodiments, the individual does not display any of the diseases or disorders described herein and is a similar age as the subject.In some embodiments, any of the compositions provided herein can be substantially purified or enriched for the NTPDase enzyme, yeast strain or combination of interest such that the sample is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or greater of the desired ATP degrading enzyme, yeast strain or combination. In some embodiments, about 40%, 30%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the composition comprises another yeast strain, enzyme, bacterial strain, probiotic, prebiotic or secondary component. In some embodiments, the secondary component is active. In some embodiments, the secondary component is inactive.

[0225] In some embodiments, any of the compositions provided herein can be administered for at least 1 week. In some embodiments, any of the compositions provided herein can be administered for between 1 week and 2 weeks. In some embodiments, any of the compositions provided herein can be administered for between 1 week and 3 weeks. In some embodiments, any of the compositions provided herein can be administered for between 1 week and 4 weeks. In some embodiments, any of the compositions provided herein can be administered for between 1 week and 5 weeks. In some embodiments, any of theAttorney Docket No. NB42251-WO-PCT compositions provided herein can be administered for between 1 week and 6 weeks. In some embodiments, any of the compositions provided herein can be administered for between 1 week and 7 weeks. In some embodiments, any of the compositions provided herein can be administered for between 1 week and 8 weeks. In some embodiments, any of the compositions provided herein can be administered for more than 8 weeks.

[0226] In some embodiments, any of the compositions provided herein can be administered for at least one month. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 2 months. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 3 months. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 4 months. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 5 months. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 6 months. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 7 months. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 8 months. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 9 months. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 10 months. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 11 months. In some embodiments, any of the compositions provided herein can be administered for between 1 month and 12 months. In some embodiments, any of the compositions provided herein can be administered for more than 12 months.

[0227] In some embodiments, any of the conditions described herein is associated or can be associated with mitochondrial dysfunction. In some embodiments, any of the conditions described herein can affect one or more cells. In some embodiments, the one or more cells include epithelial cells, endothelial cells, immune cells, neural cells, fibroblasts, muscle cells and / or adipose cells. Cells to be treated according to the present disclosure include epithelial cells, endothelial cells, immune cells, neural cells, fibroblasts, muscle cells and / or adipose cells.

[0228] In some embodiments, provided herein is a method of treating and / or preventing at least one physical state associated with mitochondrial health. In some embodiments, the at least one physical state associated with mitochondrial health is selected from impairedAttorney Docket No. NB42251-WO-PCT Complex I activity, impaired mitochondrial respiration, oxidative stress, DNA damage, impaired cellular function and decreased energy production. In some embodiments, the at least one physical state associated with mitochondrial health is impaired Complex I activity. In some embodiments, the at least one physical state associated with mitochondrial health is impaired mitochondrial respiration. In some embodiments, the at least one physical state associated with mitochondrial health is oxidative stress. In some embodiments, the at least one physical state associated with mitochondrial health is DNA damage. In some embodiments, the at least one physical state associated with mitochondrial health is impaired cellular function. In some embodiments, the at least one physical state associated with mitochondrial health is decreased energy production.

[0229] In some embodiments, the treating and / or preventing at least one physical state associated with mitochondrial health delays the onset of at least one condition selected from age-related decline in metabolic regulation, age-related alterations in immune function, age- related alterations in muscle mass and function, age-related decline in gut function, age- related cognitive decline and cell-senescence.

[0230] In some embodiments, provided herein is a method of treating and / or preventing at least one physical state associated with mitochondrial health, the method comprising administering to a subject in need thereof or at risk thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one of Kluyveromyces lactis and Cyberlindnera jadinii, or c) a combination thereof.

[0231] Also provided herein are methods for treating age related nutrient absorption in the gut. In some embodiments, any of the methods provided herein can increase age related nutrient absorption through changes in dentition, salivary function, digestion, and intestinal transit time. In some embodiments, the changes include an increase in salivary function, digestion, and intestinal transit time.

[0232] Provided herein is a method of treating age related decline in nutrient absorption in the gut, comprising administering to a subject in need thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one of Kluyveromyces lactis and Cyberlindnera jadinii; or c) a combination thereof. In some embodiments, the increase is relative to a subject that has not been treated with an effective amount of the composition.

[0233] Also provided herein are methods of increasing pre-albumin / albumin levels in aAttorney Docket No. NB42251-WO-PCT subject. In some embodiments, the method includes an increase in pre-albumin / albumin that increases the sequestration of glycoconjugates by any of the compositions provided herein. Any of the glycoconjugates described herein can be or Eukaryotic origin. Any of the glycoconjugates described herein can be or Prokaryotic origin. In In some embodiments, the glycoconjugate can be of mammalian, non-mammalian, fungal, bacterial or plant origin. In some embodiments, the glycoconjugate is of bacterial origin. In some embodiments, the bacterial origin is of commensal bacterial origin.

[0234] Provided herein is method of increasing pre-albumin / albumin levels in a subject, comprising administering to a subject in need thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one of Kluyveromyces lactis and Cyberlindnera jadinii, or c) a combination thereof. In some embodiments, the increase is relative to a subject that has not been treated with an effective amount of the composition. In some embodiments, the pre-albumin levels are present in serum. In some embodiments, the increase in pre-albumin levels reduces inflammation in a subject relative to a subject who has not been treated with an effective amount of the composition. In some embodiments, the inflammation is systemic inflammation.

[0235] The current disclosure also provides compositions comprising a source of ATP degrading enzyme, amongst which are pharmaceutical and nutraceutical compositions comprising a source of ATP degrading enzyme. The compositions may optionally comprise pharmaceutically acceptable excipients, stabilizers, activators, carriers, permeators, propellants, disinfectants, diluents and preservatives. Suitable excipients are commonly known in the art of pharmaceutical formulation and may be readily found and applied by the skilled artisan, references for instance Remmington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia Pa., 17th ed.1985.

[0236] In another embodiment, the compositions comprising a source of ATP degrading enzyme or a composition comprising Kluyveromyces lactis and / or Cyberlindnera jadinii are suitable for oral administration and comprise an enteric coating to protect the ATP degrading enzyme or yeast strain from the adverse effects of gastric juices and low pH. Enteric coating and controlled release formulations are well known in the art (references as described above). Enteric coating compositions in the art may comprise of a solution of a water-soluble enteric coating polymer mixed with the active ingredient(s) such as ATP degrading enzyme or yeast strain and other excipients, which are dispersed in an aqueous solution and which may subsequently be dried and / or pelleted. The enteric coating formed offers resistance to attackAttorney Docket No. NB42251-WO-PCT of ATP degrading enzyme or yeast strain by atmospheric moisture and oxygen during storage and by gastric fluids and low pH after ingestion, while being readily broken down under the alkaline conditions which exist in the lower intestinal tract.

[0237] Also provided herein is a method of reducing the appearance of age-related skin changes, comprising topically applying a composition comprising an ATP degrading enzyme; b) a at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof; to the skin of a subject, wherein the appearance of age related skin changes is reduced compared to a subject that did not apply the composition; and wherein the age related skin changes comprise fine lines, wrinkles, hyperpigmentation, melasmic skin, age spots, skin discoloration, and / or inflamed skin. EXEMPLARY EMBODIMENTS 1. A method of treating, delaying or reducing the severity of at least one condition selected from age-related decline in metabolic regulation, age-related alterations in immune function, age-related alterations in muscle mass and function, age-related decline in gut function, age-related cognitive decline and cell-senescence, the method comprising administering to a subject in need thereof or at risk thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading producing yeast strain, or c) a combination thereof. 2. The method of embodiment 1, wherein the age-related alterations in immune function comprise a decline in immune function and an increase in inflammation; the age- related decline in gut-function comprises an age related decline in gut permeability, an age related decline in nutrient sensing and an age related decline in nutrient absorption; the age-related decline in metabolic regulation comprises a decline in insulin sensitivity, a decline in systemic glucose metabolism, a decline in lipid metabolism, and a decline in resting energy expenditure; the age-related cognitive decline comprises short-term memory loss, word-finding difficulty, alterations in sleep patterns, insomnia, slower processing speed and / or degenerative dementia; and / or the age-related alterations in muscle mass and function comprise muscle atrophy and sarcopenia.Attorney Docket No. NB42251-WO-PCT The method of embodiment 1 or embodiment 2, wherein the ATP degrading enzyme comprises an apyrase, a member of the GDA1_CD39 superfamily or a member of the acid phosphatase family. The method of any one of embodiments 1-3, wherein the enzyme is not a mammalian ATP degrading enzyme. The method of any one of embodiments 1-4, wherein the enzyme is a bacterial ATP degrading enzyme. The method of any one of embodiments 1-5, wherein the enzyme is derived from Gallaecimonas xiamenensis. The method of any one of embodiments 1-6, wherein the enzyme comprises a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-24, 57- 63, 30-32, and 90-102. The method of embodiment 7, wherein the enzyme further comprises an N-terminus tripeptide comprising the sequence of amino acids AGK. The method of any one of embodiments 1-8, wherein the ATP degrading enzyme producing yeast strain is selected from Cyberlindnera, Kluyveromyces and Saccharomyces. The method of any one of embodiments 1-9, wherein the ATP degrading producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii. The method of any one of embodiments 1-10, wherein the enzyme producing yeast strain is Cyberlindnera jadinii and comprises the strain deposited at DSMZ under number DSM 33763 or a strain having all of the identifying characteristics of the Cyberlindnera jadinii strain deposited at DSMZ under number DSM 33763. The method of any one of embodiments 1-11, wherein the ATP degrading enzyme producing yeast strain is Kluyveromyces lactis and comprises the strain deposited at DSMZ under number DSM 33764 or a strain having all of the identifying characteristics of the Kluyveromyces lactis strain deposited at DSMZ under number DSM 33764.Attorney Docket No. NB42251-WO-PCT The method of any one of embodiments 1-12, wherein the composition comprises a probiotic. The method of any one of embodiments 1-13, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. The method of any one of embodiments 1-14, wherein the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration or topical administration. The method of any one of embodiments 1-15, wherein the composition comprises a food product, food ingredient, dietary supplement, or medicament. The method of any one of embodiments 1-16, wherein the at least one condition is associated with mitochondrial dysfunction. The method of any one of embodiments 1-17, wherein the subject is human. The method of any one of embodiments 1-17, wherein the subject is a non-human animal. The method of any one of embodiments 1-19, wherein the at least one condition affects one or more cells. The method of embodiment 20, wherein the one or more cells comprise epithelial cells, endothelial cells, immune cells, neural cells, fibroblasts, muscle cells and / or adipose cells. A method of treating and / or preventing at least one physical state associated with mitochondrial health, the method comprising administering to a subject in need thereof or at risk thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof. The method of embodiment 22, wherein the at least one physical state is selected from the group consisting of impaired Complex I activity, impaired mitochondrial respiration, oxidative stress, DNA damage, impaired cellular function and decreased energy production. The method of embodiment 22 or embodiment 23, wherein the treating and / or preventing delays the onset of at least one condition selected from age-related decline in metabolic regulation, age-related alterations in immune function, age-related alterations in muscle mass and function, age-related decline in gut function, age- related cognitive decline and cell-senescence.Attorney Docket No. NB42251-WO-PCT The method of any one of embodiments 22-25, wherein the enzyme comprises an apyrase, a member of the GDA1_CD39 superfamily or a member of the acid phosphatase family. The method of any one of embodiments 22-25, wherein the enzyme is not a mammalian ATP degrading enzyme. The method of any one of embodiments 22-26, wherein the enzyme is a bacterial ATP degrading enzyme. The method of any one of embodiments 22-27, wherein the enzyme is derived from Gallaecimonas xiamenensis. The method of any one of embodiments 22-28, wherein the enzyme comprises a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-24, 57- 63, 30-32, and 90-102. The method of embodiment 29, wherein the enzyme further comprises an N-terminus tripeptide comprising the sequence of amino acids AGK. The method of any one of embodiments 22-30, wherein the ATP degrading enzyme producing yeast strain is selected from Cyberlindnera, Kluyveromyces and Saccharomyces. The method of any one of embodiments 22-31, wherein the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii. The method of any one of embodiments 22-32, wherein the ATP degrading enzyme producing yeast strain is Cyberlindnera jadinii and comprises the strain deposited at DSMZ under number DSM 33763 or a strain having all of the identifying characteristics of the Cyberlindnera jadinii strain deposited at DSMZ under number DSM 33763. The method of any one of embodiments 22-33, wherein the ATP degrading enzyme producing yeast strain is Kluyveromyces lactis and comprises the strain deposited at DSMZ under number DSM 33764 or a strain having all of the identifyingAttorney Docket No. NB42251-WO-PCT characteristics of the Kluyveromyces lactis strain deposited at DSMZ under number DSM 33764. The method of any one of embodiments 22-34, wherein the composition comprises a probiotic. The method of any one of embodiments 22-35, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. The method of any one of embodiments 22-36, wherein the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration or topical administration. The method of any one of embodiments 22-37, wherein the composition comprises a food product, food ingredient, dietary supplement, or medicament. The method of any one of embodiments 22-38, wherein the subject is human. The method of any one of embodiments 22-38, wherein the subject is a non-human animal. A method of treating age related decline in nutrient absorption, comprising administering to a subject in need thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain; or c) a combination thereof. The method of embodiment 41, wherein the treating increases the nutrient absorption in the subject relative to a subject that has not been treated with an effective amount of the composition. The method of embodiment 41 or embodiment 42, wherein the enzyme comprises an apyrase, a member of the GDA1_CD39 superfamily or a member of the acid phosphatase family. The method of any one of embodiments 41-43, wherein the enzyme is not a mammalian ATP degrading enzyme. The method of any one of embodiments 41-44, wherein the enzyme is a bacterial ATP degrading enzyme. The method of any one of embodiments 41-45, wherein the enzyme is derived from Gallaecimonas xiamenensis. The method of any one of embodiments 41-46, wherein the enzyme comprises a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,Attorney Docket No. NB42251-WO-PCT 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-24, 57- 63, 30-32, and 90-102. The method of embodiment 47, wherein the enzyme further comprises an N-terminus tripeptide comprising the sequence of amino acids AGK. The method of any one of embodiments 41-48, wherein the ATP degrading enzyme producing yeast strain is selected from Cyberlindnera, Kluyveromyces and Saccharomyces. The method of any one of embodiments 41-49, wherein the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii. The method of any one of embodiments 41-50, wherein the ATP degrading enzyme producing yeast strain is Cyberlindnera jadinii and comprises the strain deposited at DSMZ under number DSM 33763 or a strain having all of the identifying characteristics of the Cyberlindnera jadinii strain deposited at DSMZ under number DSM 33763. The method of any one of embodiments 41-51, wherein the ATP degrading enzyme producing yeast strain is Kluyveromyces lactis and comprises the strain deposited at DSMZ under number DSM 33764 or a strain having all of the identifying characteristics of the Kluyveromyces lactis strain deposited at DSMZ under number DSM 33764. The method of any one of embodiments 41-52, wherein the composition comprises a probiotic. The method of any one of embodiments 41-53, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. The method of any one of embodiments 41-54, wherein the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration or topical administration. The method of any one of embodiments 41-55, wherein the composition comprises a food product, food ingredient, dietary supplement, or medicament. The method of any one of embodiments 41-56, wherein the subject is human.Attorney Docket No. NB42251-WO-PCT The method of any one of embodiments 41-56, wherein the subject is a non-human animal. A method of increasing pre-albumin / albumin levels in a subject, comprising administering to a subject in need thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof. The method of embodiment 59, wherein the increase is relative to a subject that has not been treated with an effective amount of the composition. The method of embodiment 59 or embodiment 60, wherein the pre-albumin levels are in serum. The method of any one of embodiments 59-61, wherein the increase in pre-albumin levels reduces inflammation in a subject relative to a subject who has not been treated with an effective amount of the composition. The method of embodiment 62, wherein the inflammation is systemic inflammation. The method of any one of embodiments 59-63, wherein the enzyme comprises an apyrase, amember of the GDA1_CD39 superfamily or a member of the acid phosphatase family. The method of any one of embodiments 59-64, wherein the enzyme is not a mammalian ATP degrading enzyme. The method of any one of embodiments 59-65, wherein the enzyme is a bacterial ATP degrading enzyme. The method of any one of embodiments 59-66, wherein the enzyme is derived from Gallaecimonas xiamenensis. The method of any one of embodiments 59-67, wherein the enzyme comprises a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-24, 57- 63, 30-32, and 90-102. The method of embodiment 68, wherein the enzyme further comprises an N-terminus tripeptide comprising the sequence of amino acids AGK.Attorney Docket No. NB42251-WO-PCT The method of any one of embodiments 59-69, wherein the ATP degrading enzyme producing yeast strain is selected from Cyberlindnera, Kluyveromyces and Saccharomyces. The method of any one of embodiments 59-70, wherein the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii. The method of any one of embodiments 59-71, wherein the ATP degrading enzyme producing yeast strain is Cyberlindnera jadinii and comprises the strain deposited at DSMZ under number DSM 33763 or a strain having all of the identifying characteristics of the Cyberlindnera jadinii strain deposited at DSMZ under number DSM 33763. The method of any one of embodiments 59-72, wherein the ATP degrading enzyme producing yeast strain is Kluyveromyces lactis and comprises the strain deposited at DSMZ under number DSM 33764 or a strain having all of the identifying characteristics of the Kluyveromyces lactis strain deposited at DSMZ under number DSM 33764. The method of any one of embodiments 59-73, wherein the composition comprises a probiotic. The method of any one of embodiments 59-74, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. The method of any one of embodiments 59-75, wherein the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration or topical administration. The method of any one of embodiments 59-76, wherein the composition comprises a food product, food ingredient, dietary supplement, or medicament. The method of any one of embodiments 1-77, wherein the subject is human. The method of any one of embodiments 1-77, wherein the subject is a non-human animal. A method of reducing the appearance of age-related skin changes, comprising topically applying a composition comprising: an ATP degrading enzyme; b) a at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof; to the skin of a subject,Attorney Docket No. NB42251-WO-PCT wherein the appearance of age-related skin changes is reduced compared to a subject that did not apply the composition; and wherein the age related skin changes comprise fine lines, wrinkles, hyperpigmentation, melasmic skin, age spots, skin discoloration, and / or inflamed skin. The method of embodiment 80, wherein the ATP degrading enzyme comprises an apyrase, a member of the GDA1_CD39 superfamily or a member of the acid phosphatase family. The method of any one of embodiments 80-81, wherein the enzyme is not a mammalian ATP degrading enzyme. The method of any one of embodiments 80-82, wherein the enzyme is a bacterial ATP degrading enzyme. The method of any one of embodiments 80-83, wherein the enzyme is derived from Gallaecimonas xiamenensis. The method of any one of embodiments 80-84, wherein the enzyme comprises a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-24, 57- 63, 30-32, and 90-102. The method of embodiment 85, wherein the enzyme further comprises an N-terminus tripeptide comprising the sequence of amino acids AGK. The method of any one of embodiments 80-86, wherein the ATP degrading enzyme producing yeast strain is selected from Cyberlindnera, Kluyveromyces and Saccharomyces. The method of any one of embodiments 80-87, wherein the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii. The method of any one of embodiments 80-88, wherein the ATP degrading enzyme producing yeast strain is Cyberlindnera jadinii and comprises the strain deposited at DSMZ under number DSM 33763 or a strain having all of the identifyingAttorney Docket No. NB42251-WO-PCT characteristics of the Cyberlindnera jadinii strain deposited at DSMZ under number DSM 33763. The method of any one of embodiments 80-89, wherein the ATP degrading enzyme producing yeast strain is Kluyveromyces lactis and comprises the strain deposited at DSMZ under number DSM 33764 or a strain having all of the identifying characteristics of the Kluyveromyces lactis strain deposited at DSMZ under number DSM 33764. The method of any one of embodiments 80-91, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient. The method of any one of embodiments 80-91, wherein the composition is formulated for topical administration. The method of any one of embodiments 80-92, wherein the subject is human. The method of any one of embodiments 80-93, wherein the subject is a non-human animal.Attorney Docket No. NB42251-WO-PCT EXAMPLES EXAMPLE 1. PREPARATION AND CHARACTERIZATION OF ENZYME CANDIDATES

[0238] Phylogenetically diverse sequences from enzyme families GDA1 / CD39 and acid phosphatase which are known to have ATP hydrolysis activity were identified from the NCBI or internal databases. Gene sequences of interest were codon-optimized based on codon preference of Bacillus subtilis and synthetic genes encoding the sequences for various enzymes were generated (by Generay Biotech (Shanghai) Co., Ltd) using techniques standard techniques. The sequences of codon optimized genes (SEQ ID NO: 1-8, 43-49), the native full length polypeptide sequences (SEQ ID NO: 9-16, 50-56) and the predicted mature enzymes (SEQ ID NO: 17-24, 57-63) are listed on Table 1. Genes were cloned into expression vector p2JM103BBI (Vogtentanz, Protein Expr Purif.55:40-52, 2007). The expression of the codon-optimized nucleotide sequence encodes the predicted mature polypeptide driven by aprE promoter (SEQ ID NO: 25, using the nucleotide sequence SEQ ID NO: 26 encoding the aprE signal peptide (SEQ ID NO: 27) with an additional 3 amino acids (Ala-Gly-Lys) between the signal sequence and the predicted mature sequence of the genes of interest (to facilitate the secretion of the target protein in B. subtilis), and is stopped by lat terminator (SEQ ID NO: 28). Competent B. subtilis cells were transformed with each corresponding expression vector and plated on Luria Agar plates supplemented with 5 ppm chloramphenicol. The colony was inoculated in a flask containing LB medium and grew at 37°C with 200 rpm shaking for 5-6 h. Seed culture was inoculated into a production medium containing minerals (e.g., potassium sulfate, magnesium sulfate, ferrous sulfate, calcium chloride, citric acid, etc.), one or more carbon sources (e.g., glucose), a complex nitrogen source (e.g. hydrolyzed soy peptone, yeast extract) and a buffering component (e.g. MOPS, Tris, etc.). Cultures were grown at 32°C with 250 rpm shaking for 24-40 h.

[0239] General purification procedure was as follows. Culture broth was collected by centrifugation, concentrated and supplemented with ammonium sulfate to a final concentration of 1M, then applied to a hydrophobic interaction column (e.g. phenyl FF, butyl FF) which was pre-equilibrated with 50 mM Tris pH 7.5 containing 1M ammonium sulfate. The column was eluted with a stepwise mode (0.75, 0.5, 0.25, 0.02 M ammonium sulfate, H2O, 20% ethanol) and fractions containing active enzymes of interest were pooled and buffer-exchanged to 20 mM Tris pH7.5. Pooled sample was loaded on an anion-exchangeAttorney Docket No. NB42251-WO-PCT chromatography (e.g. Q FF) and eluted with a gradient mode (0-0.5 M NaCl). Fractions containing purified enzymes were identified based on SDS-PAGE analysis and activity tests, and they were pooled. Purified samples were stored at -20°C in buffer containing 20 mM Tris pH7.5, 150 mM NaCl, and 40% glycerol until use. s ID re)Attorney Docket No. NB42251-WO-PCT

[0240] In a separate Experiment, samples were assayed to determine their ability to hydrolyze ATP or ADP at a pH of 3 to 9 using a standard malachite green phosphate detection kit (Sigma Aldrich cat no. MAK307). Reference enzymes were purchased from Sigma Aldrich, including Potato apyrase (catalog # A6535), recombinant human CD39 His- tag (catalog # SRP0623), and calf intestinal alkaline phosphatase (CIAP, catalog #P0114).

[0241] The enzyme reactions were carried out in 96-well plates with a total volume of 150 µL and contained 250 µM of a substrate, 50 mM of a buffer, 5 mM CaCl2, and a variable concentration of enzyme. The substrate was ATP or ADP. The buffer was glycine (pH 3), acetate (pH 4, 4.5, 5), bis-Tris (pH 5.5, 6, 6.5), or Tris (pH 7, 8, 9). The concentration of enzyme varied from about 1 µg / mL to 1 ng / mL and was adjusted based on the activity of the enzyme so that the amount of phosphate released during the assay was with the dynamic range of the assay (5 – 100 µM phosphate). The enzyme concentration was determined by the absorbance at 280 nm and using the predicted molecular weight and molar extinction coefficient to calculate the concentration. The enzyme reaction was initiated by the addition of 15 µL enzyme with 135 µL of the other reaction components such that the final reaction composition was as previously described. The reaction was stopped after 5 min by mixing 80 µL of the reaction mixture with 20 µL of the malachite green working reagent. The malachite green working reagent was prepared according to the manufacturer’s protocol by mixing 100 parts reagent A with 1 part reagent B. Phosphate standard solutions (0 – 80 µM) and a negative control reaction without enzyme were also mixed with the malachite green working reagent. All samples were tested in duplicate. The malachite green reaction mixtures were allowed to rest at room temperature for 45-60 min and the absorbance at 620 nm was measured. Lastly, the absorbance of the phosphate standards were used to convert the absorbance readings of each sample into phosphate concentrations and the negative control was subtracted from each reaction. The amount of phosphate in micromoles released per milligram of protein per minute of reaction time (µmol / mg / min) with ATP or ADP as a substrate is displayed in Table 2 and Table 3. Table 2. Specific activity (µmol / mg / min) of enzymes on ATP at different pH valuesAttorney Docket No. NB42251-WO-PCT

[0242] As shown in Table 2 and Table 3, CRC22110 was the most active enzyme, demonstrating activity along a wide pH range including from pH 3 to pH 9. The tested enzymes had pH optima near neutral pH (pH 6-8) and have a broad activity range between pH 5 – 7, with some maintaining activity even to pH 9. EXAMPLE 2. EXPRESSION AND CHARACTERIZATION OF TRUNCATED FORMS OF ENZYMES OF INTEREST

[0243] The CRC22110 G. xiamenensis apyrase molecule, expressed in B subtilis with N- terminal AGK peptide described in Example 1 was selected for further evaluation, includingAttorney Docket No. NB42251-WO-PCT truncations of the predicted sequence (SEQ ID NO: 29). Various C-terminal truncations of the CRC22110 apyrase were designed to remove residues C-terminal to the final cysteine which is involved in 1 of the 7 disulfide bonds in the mature enzyme: CRC22110-V1 (SED IQ NO: 30), CRC22110-V2 (SEQ ID NO: 31), and CRC22110-V3 (SEQ ID NO: 32). The methods of expression of truncation forms of CRC22110 are described below, wherein certain instances an additional 3 residues, AGK, are included at the predicted N-terminus of the mature polypeptide sequence. A first DNA fragment comprising a (5’) skfA gene flanking region (5’ skfA gene FR, SEQ ID NO:33) was operably linked to a polynucleotide construct (eg. expression cassette) comprising an upstream (5’) B. subtilis rrnI-p2 promoter region DNA sequence (SEQ ID NO:34) operably linked to DNA sequence of the B. subtilis aprE 5’ untranslated region (5’ UTR) (SEQ ID NO:35) operably linked to DNA encoding the B. subtilis aprE signal sequence (SEQ ID NO:26) operably linked to a DNA sequence encoding the desired C-terminal truncated G. xiamenensis apyrases with the addition of the nucleotide sequences encoding the tripeptide AGK at the N-termini (polypeptide sequences SEQ ID NO: 36, 37 or 38), operably linked to the Bacillus amyloliquefaciens BPN terminator (SEQ ID NO:39) operably linked to a (3’) skfA gene flanking region (3’ skfA gene FR) (SEQ ID NO:40). A second DNA fragment comprising a (5’) amyE gene flanking region (5’ amyE gene FR) (SEQ ID NO:41) was operably linked to a polynucleotide construct (eg. expression cassette) comprising an upstream (5’) B. subtilis rrnI-p2 ( SEQ ID NO:34) promoter region DNA sequence operably linked to DNA sequence of the B. subtilis aprE 5’ untranslated region (5’ UTR) (SEQ ID NO:35) operably linked to DNA encoding the B. subtilis aprE signal sequence (SEQ ID NO:26) operably linked to a DNA sequence encoding the desired C-terminal truncated G. xiamenensis apyrases with the addition of the nucleotide sequences encoding the tripeptide AGK at the N-termini (polypeptide sequences SEQ ID NO: 36, 37 or 38), and operably linked to Bacillus amyloliquefaciens BPN terminator (SEQ ID NO:39) operably linked to a (3’) amyE gene flanking region (3’ amyE gene FR) (SEQ ID NO:42). More particularly, these DNA fragments were assembled using standard molecular biology techniques and were used as templates to develop linear DNA expression cassettes for generation of a 2 copy strain. A suitable Bacillus subtilis strain, comprising nine protease deletion was used to integrate the first and second linear DNA expression cassette fragment described above in the genome using standard molecule biology techniques.

[0244] The relative enzyme activity on ATP substrate was then determined for truncated forms of the CRC22110 apyrase as described below.Attorney Docket No. NB42251-WO-PCT

[0245] Culture supernatants from B subtilis fermentations were obtained by filtration and ammonium sulfate and 1M Tris pH8 was added to a final concentration of 1M ammonium sulfate and 20mM Tris pH8. The sample was centrifuged and filtered. The filtrate was loaded onto 300ml phenyl Sepharose column on equilibrated in 20mM Tris and 1M ammonium sulfate pH8. A linear gradient from 0% to 100% 20mM Tris pH8 was run at 10ml / min over 300 minutes. Elution fractions were collected and ATPase activity was monitored using ATPase activity assay. The active fractions were pooled, concentrated and buffer exchanged into 20mM Tris pH8. This pool was loaded onto 25ml Q Sepharose column that was equilibrated in 20mM Tris pH8. They were then eluted with step gradient from 50mM NaCl in 20mM Tris, followed by 100mM NaCl, 200mM NaCl, 400mM and 500mM NaCl in 20mM Tris pH8. The active and pure fractions (>95% by inspection on SDS-PAGE) were pool as purified protein, they were then quantitated.

[0246] Protein quantitation by UPLC: Protein quantitation methods: Protein concentration was determined by UPLC (Ultra Performance Liquid Chromatography) and by OD280 densitometry). For UPLC determination, the purified enzyme was diluted in 20mM Tris pH8 and concentration was determined by separation of protein components using a Zorbax 300 SB-C3 column (Agilent) and running a linear gradient of 0.1% Trifluoroacetic acid in water (Buffer A) and 0.1% Trifluoroacetic acid in Acetonitrile (Buffer B) with detection at 220nm column on UHPLC. A 10ul sample was loaded onto the column and the peak area of the diluted sample was determined. The enzyme concentration of the samples was calculated using a standard curve of the purified reference enzyme for example full length CRC22110. The protein concentration was also determined by OD280 measurement. The purified enzyme was diluted in 20mM Tris pH8, their OD280 was measured in a quartz cuvette. The protein concentration was calculated based on their respective extinction coefficients. The final concentration was calculated based on the average of UPLC method and OD280 determination.

[0247] ATPase activity comparison: ATPase activity assay was performed using samples diluted to 0.003ppm or 0.0015ppm in assay buffer (50mM Tris pH8, 5mM CaCl2, 0.01% Tween 80) using the ATPase assay described previously. Samples of 10ul of diluted enzyme were added into 0.25mM ATP in assay buffer to start the reaction. The reaction mix was incubated at 25℃ for 10mins, then 50ul of reaction mix was added into 100ul of QuantiChrom™ Malachite Green reagent (VWR cat # 75878) and reaction mixtures were then incubated at 25℃ for 20mins, then the OD620 was measured. ATPase activity wasAttorney Docket No. NB42251-WO-PCT compared after subtracting the blank (no enzyme control). The relative ATPase activity was calculated based on the background subtracted OD relative to full length CRC22110. The results are shown on Table 4 below.

[0248] As shown on Table 4, the relative ATPase activity of CRC22110 and the C-terminal truncation is highly comparable, thus confirming the ability to truncate this region of the protein sequence without affecting enzyme function.EXAMPLE 3. PREPARATION AND CHARACTERIZATION OF YEAST CANDIDATES

[0249] This Example describes the evaluation of various yeast strains for their ability to remove ATP, when ATP was added to the growth media.

[0250] The following deposit were previously made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

[0251] Cyberlindnera jadinii strain (DGCC3540) deposited under accession number DSM 33763 on January 19, 2021, at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig - Germany].

[0252] Kluyveromyces lactis strain (DGCC3550) deposited under accession number DSM 33764 on January 19, 2021, at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig - Germany].

[0253] Pichia membranifaciens (P. membranifaciens), Kluyveromyces lactis (K. Lactis (DGCC3550)) previously deposited under Budapest treaty on the internaitonal recognition of the deposit of microorganisms under accession number DSM 33764, and Cyberlindnera jadinii (C. jadinii (DGCC3540)) previously deposited under Budapest treaty on the internaitonal recognition of the deposit of microorganisms under accession number DSMAttorney Docket No. NB42251-WO-PCT 33763, were grown overnight at 27֯C in YPD media. The culture was used as an inoculum and added to fresh media containing 1mM ATP at 1% v / v. Cells were grown at 37 ֯C anaerobically to mimic colon conditions for temperature and oxygen availability.

[0254] Supernatants were collected at 0 and 4 hr time points, and ATP was measured by using ATP determination kit (ThermoFisher Cat No. A22066) as per manufacturer’s instructions. Briefly, supernatants were collected at 0 and 4 hrs timepoints and filtered via 0.2 micron filter to remove the cells. ATP levels were measured by using A22066 kit. Results were normalized to media only control.

[0255] As shown in FIG.1, K. lactis and C. jadinii were capable of removing ATP as compared to the controls. These results suggest that both the yeast strains have the capability of degrading eATP. EXAMPLE 4. EVALUATION OF MITOCHONDRIAL FUNCTION IN AN EPIDERMAL TISSUE MODEL

[0256] This Example describes the use of exemplary enzymes in a 3D EpiDerm tissue model.

[0257] Briefly, a 3D Epiderm tissue model (Cat No EPI 200) was obtained from MatTek Corporation, MA, USA. Tissues were maintained and used in the experiment according to the manufactures protocol (https: / / www.mattek.com / mattekproduct / epiderm / ).

[0258] To determine the role of ATP on mitochondrial function, tissues were incubated with 10 mM ATP or GTP or AMP or CRC22110-V1 enzyme (100nM) or ATP+ CRC22110- V1 enzyme for 3 hrs at 37C. Test reagents were added at the top of the tissue exposed to air and as shown in the experimental set up (FIG.2). Tissues and supernatants were collected at 3 hrs, and tissues were subjected to Complex I measurement to evaluate the viability of the mitochondria under different conditions according to the manufactures protocol. Briefly, tissues were homogenized in PBS to prepare a protein extract, and 500 ug of total protein was used to measure Complex I activity by using the colorimetric Complex I activity Assay kit (Abcam, catalog #ab109721). Samples were normalized to cells only control. As shown in FIG.3, the presence of ATP decreases the activity of Complex I relative to cells only control, while the addition of AMP showed minimal effect on Complex 1 activity. The addition of exemplary enzyme CRC22110-V1 restored Complex 1 activity relative to cells only control.

[0259] In a separate experiment, the collected supernatants from the 3 hr time points were used to measure cytokines by LEGENDplex Multi-Analyte Flow Assay (BioLegend Cat NoAttorney Docket No. NB42251-WO-PCT 740808). Briefly, 100 ul of the supernatant was used to measure cytokines by using manufacturer’s instructions. Cytokine levels were represented as Mean Fluorescent Units (MFI). As shown in FIG.4, ATP is capable of inducing IL-1β expression in 3D epidermal tissues, while addition of exemplary enzyme CRC22110-V1 was able to reduce the impact of ATP on tissues.

[0260] The collected supernatants were further evaluated for ATP release using an ATP Determination Kit (Cat No A22066) similar to as described in Example 3. As shown in FIG. 5, ATP accelerates the release of ATP by tissues, while the addition of exemplary enzyme CRC22110-V1 reduced the effect of ATP release.

[0261] In summary, the results demonstrate that exogenous ATP (eATP) is capable of inducing a decline in Complex I activity in skin epithelial cells, and that the addition of exemplary ATP degrading enzymes can remove the exogenous ATP pool and restore mitochondrial Complex I activity, which is associated with mitochondrial heath and has been implicated in the aging process. EXAMPLE 5. EVALUATION OF EXEMPLARY ENZYMES AND YEAST STRAINS ON MITOCHONDRIAL ACTIVITY AND ASSOCIATED INTESTINAL HEALTH

[0262] This Example describes the use of exemplary enzymes and yeast strains outlined in the Examples above, for the assessment of mitochondrial function and impact on intestinal health in a mouse model of antibiotic induced colitis.

[0263] Apc / minJ mice, which possess a mutated adenomatous polyposis coli gene and are more prone to develop colitis and tumors, that were 6-7 weeks old were subjected to an antibiotics cocktail of vancomycin, ampicillin, and neomycin (1mg / mL) for 10 days in drinking water. Wild type mice and Apc / minJ mice that were not subjected to the antibiotic cocktail were used as controls. After the 10 days, mice were split into 6 groups, as outlined in Table 5, and as follows: Group 1 (WT mice) and Group 2 (Mutant Apc / MinJ) mice that had not received antibiotic treatments were used as control groups and drank only distilled water, Group 3 (Mutant Apc / MinJ) received vehicle control, Groups 4 and 5 (Mutant Apc / MinJ) received yeasts strains K. lactis and C. jadinii, respectively, by oral gavage (PO) once per day from day 1 to day 13, and Group 6 received exemplary enzyme CRC22110-V3 by gavage (PO) at 6 hour intervals for a total of twice per day from day 1 to day 13.Attorney Docket No. NB42251-WO-PCT

[0264] Animals were given antibiotics via drinking water for 10 days except for groups 1 and 2. Animals were gavaged with vehicle (group 3), ATP degrading enzyme or yeasts daily. On day 13 animals were sacrificed by exsanguination from the abdominal aorta after isoflurane anesthesia. Blood and colon tissues were collected. Colon tissues were stored at - 80°C and blood was processed and serum was stored at -20°C for further analysis.

[0265] In a first experiment, treatment groups were assessed for their effect on mitochondrial Complex I activity. Colon tissues collected from mice were washed in cold PBS and then tissues were homogenized. Protein concentration was determined by nanodrop. 500 ug of total protein was used for the determination of Complex I activity by using ab287839 kit as per manufacturer’s instructions. As shown in FIG.6, Mutant Apc / MinJ mice treated with antibiotics (MT+Abx) demonstrated decreased Complex I activity in colon tissues as compared to non-antibiotic treated Mutant Apc / MinJ mice controls (MT). The addition of C. jadinii and CRC22110-V3 to Apc / MinJ mice treated with antibiotics (MT+Abx+C. jadinii and MT+Abx+CRC22110-V3, respectively) demonstrated a restoration of Complex I activity as compared to MT control.

[0266] In a second experiment, treatment groups were assessed for their effect on gut permeability. On day 13, mice were fasted for 4 hrs. and then mice were gavaged with 150 μl of 80 mg / ml FITC dextran (4kDa). After 4 hours, mice were sacrificed and blood was collected and processed. Fluorescence was measured at 530 nm with excitation at 485 nm. As shown in FIG.7, Mutant Apc / MinJ mice treated with antibiotics (MT+Abx) showed an increase in intestinal permeability measured by relative fluorescent units (RFU). Mutant Apc / MinJ mice that were not treated with antibiotics (MT), and those treated with antibioticsAttorney Docket No. NB42251-WO-PCT and K. lactis showed a smaller increase in RFUs compared to WT non -antibiotic treated control (WT), while mice treated with antibiotics and C. jadinii (MT+Abx+ C. jadinii) or CRC22110-V3 (MT+Abx+CRC22110-V3) did not demonstrate an increase in RFUs, and showed similar and / or improved levels relative to WT control.

[0267] In a third experiment, since albumin levels have been shown to be positively correlated with decreases in inflammation, treatment groups were assessed for their effect on pre-albumin levels. Pre-albumin was measured in the serum by using Pre-albumin ELISA kit (Cat No. KA2070). Serum samples (100 ul / well) were added to ELISA plate and incubated for 30 mins at room temperature. After incubation wells were washed and bound pre-albumin were quantified by using enzyme-antibody conjugate and TMB as a substrate. Measurements were done at OD450 nm and values are reported in FIG.7 after subtracting the background. As shown in FIG.8, Mutant Apc / MinJ mice treated with antibiotics (MT+Abx) showed a decrease in pre-albumin levels, while the addition of C. jadinii (MT+Abx+ C. jadinii) AND CRC22110-V3 (MT+Abx+CRC22110-V3) were capable of restoring pre-albumin to levels similar to wild type (WT) and Mutant Apc / MinJ(MT) non-antibiotic treated controls.

[0268] In summary, antibiotics treatment induces gut inflammation, with eATP playing a critical role in the manifestation of inflammation. These results suggest that removal of eATP by using an ATPase or yeast is capable of negating the impact of inflammation by improving the gut barrier and albumin levels, both of which are associated with the aging process. EXAMPLE 6. BIOCHEMICAL CHARACTERIZATION OF ADDITIONAL ATP DEGRADING ENZYME CANDIDATES

[0269] Additional samples of the phylogenetically diverse enzymes described in Example 1 were assayed to determine their ability to hydrolyze ATP. Reference enzymes were purchased from Sigma Aldrich, including Potato apyrase (catalog # A6535), recombinant human CD39 His-tag (catalog # SRP0623), and calf intestinal alkaline phosphatase (CIAP, catalog #P0114).

[0270] The ability of candidate enzymes to degrade ATP was measured similar to as described in Example 1.

[0271] The amount of phosphate in micromoles released per milligram of protein per minute of reaction time (µmol / mg / min) with ATP as a substrate is displayed in Table 6 and Table 7.Attorney Docket No. NB42251-WO-PCT

[0272] As shown in Table 6 and Table 7, CRC34822 was the most active enzyme at pH 5 of the enzymes tested in this experiment. EXAMPLE 7. EVALUATION OF MITOCHONDRIAL FUNCTION IN A 3D MUSCLE TISSUE MODEL

[0273] This Example describes the use of exemplary enzymes in a 3D Bovine muscle tissue model.

[0274] Briefly, bovine muscle cells (Cat No. B354-05) were obtained from Sigma. Cells were grown in a 3D Matrigel (Corning Cat No.356237) according to manufacturer’s protocols to provide a 3D tissue model.

[0275] To determine the role of ATP on mitochondrial function, muscle tissues grown inAttorney Docket No. NB42251-WO-PCT Matrigel were incubated with exemplary enzymes 10 mM ATP or exemplary enzymes CRC22110-V1 (10 nM) or CRC34822 (10nM and 100nM) or CRC33930 (10 nM) or CRC33849 (10nM) or CRC22105 (10 nM) for 3 days at 37C.

[0276] Tissues were collected at 3 hrs and subjected to complex I measurement to evaluate the functionality of the mitochondria under different conditions according to the manufactures protocol, and similar to as described in Example 4. Briefly, tissues were homogenized in PBS to prepare a protein extract, and 500 ug of total protein was used to measure Complex I activity by using Complex I activity Assay kit (ab109721). Samples were normalized to cells only control. As shown in FIG.9, the presence of ATP decreases the activity of Complex I relative to cells only control, while the addition of the exemplary enzymes restored Complex 1 activity. EXAMPLE 8. EVALUATION OF MITOCHONDRIAL FUNCTION IN AN AGING CELL MODEL

[0277] This Example describes the use of exemplary enzymes in an aging HaCat cell model.

[0278] To test the role of ATP on mitochondrial health with aging, HaCat cells were obtained from Acellerate and were grown for 20 generations. In this experiment cells were passaged every third day and ATP or ATP degrading enzymes were added on the cells after 24hrs of passaging. Cells were incubated with 5 mM ATP + LPS (1ug / mL) or CRC22110- V1 (10 nM) or CRC34822 (100nM) or alkaline phophatase (10 nM ) or potato apyrase (10 nM).

[0279] Cells were collected at passage 1, 10 and 20 and subjected to Complex I measurement to evaluate the viability of the mitochondria under different conditions according to the manufacture’s protocol. Briefly, cells were homogenized in PBS to prepare a protein extract, and 500 ug of total protein was used to measure complex I activity by using Complex I activity Assay kit (Abcam ab109721) similar to as described in Example 4 and Example 7.

[0280] As shown in FIG.10, Complex I activity decreased in cells that were passaged from generation 1 to 20, and this decline was accelerated with the addition of extracellular ATP. The exemplary ATP degrading enzymes tested were able to reverse the complex I activity decline suggesting their role in improving cell health.

[0281] In an additional experiment, levels of Growth differentiation factor 15 (GDF15), aAttorney Docket No. NB42251-WO-PCT protein that is upregulated during aging and may play a role in aging and age-related diseases, was assessed under similar conditions.

[0282] To test the levels of GDF15, HaCat cells were incubated with LPS (1ug / mL) or LPS+CRC22110-V1 (10 nM) or CRC34822 (100nM) or potato apyrase (10nM) for 24 hrs. After 24 hrs supernatants were collected and GDF15 levels were measured by using Human GDF15 ELISA kit (Cat No. Cat #BMS2258, Thermo Fisher Scientific)

[0283] As shown in FIG.11, LPS induces stress on the cells and thus increase in GDF15 levels, while the exemplary ATP degrading enzymes tested were able to decrease the levels of GDF15 as compared to stressed cells suggesting their positive impact on cell health and functionality. EXAMPLE 9. EVALUATION OF ADDITIONAL EXEMPLARY ENZYMES AND YEAST STRAINS ON MITOCHONDRIAL ACTIVITY AND ASSOCIATED INTESTINAL HEALTH

[0284] This Example describes the use of additional exemplary enzymes and yeast strains outlined in the Examples above, for the assessment of mitochondrial function and impact on intestinal health in a mouse model of antibiotic induced inflammation.

[0285] 6-7 weeks old AJ mice were subjected to an antibiotics cocktail of vancomycin, ampicillin, and neomycin (1mg / mL) for 10 days in drinking water similar to as described in Example 5 and sacrificed after three days of stopping the antibiotics. AJ mice that were not subjected to the antibiotic cocktail were used as controls. Groups 2 to 6 received yeast strain C. jadinii (live or dead) by oral gavage (PO) once per day from day 1 to day 13, and Group 7 received exemplary enzyme CRC34822 by gavage (PO) once per day from day 1 to day 13.Attorney Docket No. NB42251-WO-PCT

[0286] Animals were weighed at different time points including at the beginning of antibiotic administration, throughout antibiotic administration and after antibiotic administration was ceased. As shown in FIG.12, mice with antibiotic administration showed loss in body weight as compared to control mice (Naïve; No antibiotics), whereas C. jadinii yeast and CRC34822 enzyme intervention was able to negate the weight loss associated with antibiotic administration.

[0287] On day 13 animals were sacrificed by exsanguination from the abdominal aorta after isoflurane anesthesia. Blood and colon tissues were collected. Colon tissues were stored at -80°C and blood was processed and serum was stored at -20°C for further analysis.

[0288] In a first experiment, treatment groups were assessed for their effect on cecum weight. Colon tissues collected from mice were weighed. As shown in FIG.13, mice with antibiotics administration showed significant increase in cecum weight as compared to control mice (No antibiotics), whereas C. jadinii and CRC34822 intervention was able to negate the negative impact of antibiotics on cecum weight.

[0289] In a second experiment, treatment groups were assessed for their effect on gut permeability. On day 13, mice were fasted for 4 hrs. and then mice were gavaged with 150 μl of 80 mg / ml FITC dextran (4kDa). After 4 hours, mice were sacrificed, and blood was collected and processed. Fluorescence was measured at 530 nm with excitation at 485 nm. As shown in FIG.14, antibiotics administration impacts gut permeability as shown as high FITC Dextran signature in the serum of mice treated with antibiotics (Abx), while C. jadinii and CRC34822 intervention was able to negate the negative impact of antibiotics on gut permeability.

[0290] In a third experiment, treatment groups were assessed for their effect on mitochondrial Complex I activity. Ileum was collected from the animals and used to prepare a protein lysate by using a lysis buffer (Invitrogen™ Cell Extraction Buffer (Cat. No.Attorney Docket No. NB42251-WO-PCT FNN0011) supplemented with 0.5M EDTA and Thermo Scientific™ Halt™ Protease Inhibitor Cocktail (100X) (Cat.No.78430).

[0291] 500 ug of total protein was used for the determination of Complex I activity by using ab109721 kit as per manufacturer’s instructions. As shown in FIG.15, mice treated with antibiotics (Abx) demonstrated decreased Complex I activity in ileum tissues as compared to non-antibiotic treated controls (Control). The addition of C. jadinii and CRC34822 to mice treated with antibiotics demonstrated a restoration of Complex I activity as compared to MT control.

[0292] In summary, antibiotics treatment induces gut inflammation, with eATP playing a critical role in the manifestation of inflammation. These results suggest that removal of eATP by using an ATP degrading enzyme or yeast that produces an ATP degrading enzyme is capable of negating the impact of inflammation by improving the gut barrier and mitochondrial activity, both of which are associated with the aging process.

Claims

Attorney Docket No. NB42251-WO-PCT CLAIMS We claim:

1. A method of treating, delaying or reducing the severity of at least one condition selected from age-related decline in metabolic regulation, age-related alterations in immune function, age-related alterations in muscle mass and function, age-related decline in gut function, age-related cognitive decline and cell-senescence, the method comprising administering to a subject in need thereof or at risk thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof.

2. The method of claim 1, wherein the age-related alterations in immune function comprise a decline in immune function and an increase in inflammation; the age- related decline in gut-function comprises an age related decline in gut permeability, an age related decline in nutrient sensing and an age related decline in nutrient absorption; the age-related decline in metabolic regulation comprises a decline in insulin sensitivity, a decline in systemic glucose metabolism, a decline in lipid metabolism, and a decline in resting energy expenditure; the age-related cognitive decline comprises short-term memory loss, word-finding difficulty, alterations in sleep patterns, insomnia, slower processing speed and / or degenerative dementia; and / or the age-related alterations in muscle mass and function comprise muscle atrophy and sarcopenia.

3. The method of claim 1or claim 2, wherein the ATP degrading enzyme comprises an apyrase, a member of the GDA1_CD39 superfamily or a member of the acid phosphatase family.

4. The method of any one of claims 1-3, wherein the enzyme is not a mammalian ATP degrading enzyme.

5. The method of any one of claims 1-4, wherein the enzyme is a bacterial ATP degrading enzyme.Attorney Docket No. NB42251-WO-PCT 6. The method of any one of claims 1-5, wherein the enzyme comprises a polypeptide that is at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-24, 57-63, 30-32, and 90-102.

7. The method of any one of claims 1-6, wherein the ATP degrading enzyme producing yeast strain is selected from Cyberlindnera, Kluyveromyces and Saccharomyces.

8. The method of any one of claims 1-7, wherein the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii.

9. The method of any one of claims 1-8, wherein the ATP degrading enzyme producing yeast strain is Cyberlindnera jadinii and comprises the strain deposited at DSMZ under number DSM 33763 or a strain having all of the identifying characteristics of the Cyberlindnera jadinii strain deposited at DSMZ under number DSM 33763.

10. The method of any one of claims 1-9, wherein the ATP degrading enzyme producing yeast strain is Kluyveromyces lactis and comprises the strain deposited at DSMZ under number DSM 33764 or a strain having all of the identifying characteristics of the Kluyveromyces lactis strain deposited at DSMZ under number DSM 33764.

11. The method of any one of claims 1-10, wherein the composition comprises a probiotic.

12. The method of any one of claims 1-11, wherein the composition comprises a pharmaceutical composition and at least one pharmaceutically acceptable carrier and / or excipient.

13. The method of any one of claims 1-12, wherein the composition is formulated for oral administration, intravenous administration, intramuscular administration, parenteral administration or topical administration.Attorney Docket No. NB42251-WO-PCT 14. The method of any one of claims 1-13, wherein the composition comprises a food product, food ingredient, dietary supplement, or medicament.

15. The method of any one of claims 1-14, wherein the at least one condition is associated with mitochondrial dysfunction.

16. The method of any one of claims 1-15, wherein the subject is human.

17. The method of any one of claims 1-16, wherein the subject is a non-human animal.

18. The method of any one of claims 1-17, wherein the at least one condition affects one or more cells.

19. The method of claim 18, wherein the one or more cells comprise epithelial cells, endothelial cells, immune cells, neural cells, fibroblasts, muscle cells and / or adipose cells.

20. A method of treating and / or preventing at least one physical state associated with mitochondrial health, the method comprising administering to a subject in need thereof or at risk thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof.

21. The method of claim 20, wherein the at least one physical state is selected from the group consisting of impaired Complex I activity, impaired mitochondrial respiration, oxidative stress, DNA damage, impaired cellular function and decreased energy production.

22. The method of claim 20 or claim 21, wherein the treating and / or preventing delays the onset of at least one condition selected from age-related decline in metabolic regulation, age-related alterations in immune function, age-related alterations in muscle mass and function, age-related decline in gut function, age-related cognitive decline and cell-senescence.

23. The method of any one of claims 20-22, wherein the ATP degrading enzyme comprises an apyrase, a member of the GDA1_CD39 superfamily or a member of the acid phosphatase family.Attorney Docket No. NB42251-WO-PCT 24. The method of any one of claims 20-23, wherein the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii.

25. A method of treating age related decline in nutrient absorption, comprising administering to a subject in need thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain; or c) a combination thereof.

26. The method of claim 25, wherein the treating increases the nutrient absorption in the subject relative to a subject that has not been treated with an effective amount of the composition.

27. The method of claim 25 or claim 26, wherein the ATP degrading enzyme comprises an apyrase, a member of the GDA1_CD39 superfamily or a member of the acid phosphatase family.

28. The method of any one of claims 25-27, wherein the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii.

29. A method of increasing pre-albumin / albumin levels in a subject, comprising administering to a subject in need thereof an effective amount of a) a composition comprising an ATP degrading enzyme; b) a composition comprising at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof.

30. The method of claim 29, wherein the increase is relative to a subject that has not been treated with an effective amount of the composition.

31. The method of claim 29 or claim 30, wherein the pre-albumin levels are in serum.

32. The method of any one of claims 29-31, wherein the increase in pre-albumin levels reduces inflammation in a subject relative to a subject who has not been treated with an effective amount of the composition.

33. The method of claim 32, wherein the inflammation is systemic inflammation.Attorney Docket No. NB42251-WO-PCT 34. The method of any one of claims 29-33, wherein the ATP degrading enzyme comprises an apyrase, a member of the GDA1_CD39 superfamily or a member of the acid phosphatase family.

35. The method of any one of claims 29-34, wherein the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii.

36. A method of reducing the appearance of age-related skin changes, comprising topically applying a composition comprising: an ATP degrading enzyme; b) a at least one ATP degrading enzyme producing yeast strain, or c) a combination thereof; to the skin of a subject, wherein the appearance of age-related skin changes is reduced compared to a subject that did not apply the composition; and wherein the age-related skin changes comprise fine lines, wrinkles, hyperpigmentation, melasmic skin, age spots, skin discoloration, and / or inflamed skin.

37. The method of claim 36, wherein the ATP degrading enzyme comprises an apyrase, a member of the GDA1_CD39 superfamily or a member of the acid phosphatase family.

38. The method of claim 36 or claim 37, wherein the ATP degrading enzyme producing yeast strain is selected from Kluyveromyces lactis, Cyberlindnera jadinii, and Saccharomyces boulardii.

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