WO2025132829A1 - Bactérie comprenant une voie de dégradation de phytate - Google Patents

Bactérie comprenant une voie de dégradation de phytate Download PDF

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WO2025132829A1
WO2025132829A1 PCT/EP2024/087460 EP2024087460W WO2025132829A1 WO 2025132829 A1 WO2025132829 A1 WO 2025132829A1 EP 2024087460 W EP2024087460 W EP 2024087460W WO 2025132829 A1 WO2025132829 A1 WO 2025132829A1
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bacterium
phytate
seq
composition
hydroxypropionate
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Max NIEUWDORP
Thi Phuong Nam BUI
Willem Meindert De Vos
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Wageningen Universiteit
Amsterdam UMC Foundation
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Wageningen Universiteit
Amsterdam UMC Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to the field of preventing and/or treating metabolic diseases, such as metabolic syndrome and insulin resistance or insulin resistance-related conditions, including dyslipidemia and type 2 diabetes mellitus as well as insulin-resistance in endocrine diseases or obesity.
  • metabolic diseases such as metabolic syndrome and insulin resistance or insulin resistance-related conditions, including dyslipidemia and type 2 diabetes mellitus as well as insulin-resistance in endocrine diseases or obesity.
  • Phytate a well-known phytochemical, is widely distributed in the plant kingdom, and especially abundant in wheat, rice and nuts. Dietary supplementation of phytate has been reported for various health benefits, including anti-diabetic activity.
  • dietary phytate supplementation reportedly promotes epithelial repair resulting in improved gut barrier function (Wu et al., Nature 2020, 586 (7827), 108-112), reduces serum levels of glycated hemoglobin HbA1c and Advanced Glycation End products (Sanchis et al., Trial. Sci. Rep. 2018, 8 91), 9619), improves glucose metabolism (Lee et al., Nutr Res 2006, 26(9), 474-479), reduced inflammation (Liu et al., Br J. Nutr 2018, 120(2), 121-130) and exerts protective effects against colon cancer (Vucenik et al., Nutr Cancer 2006, 55(2), 109-25).
  • Mitsuokella spp. are Gram-negative gut anaerobes belonging to the Negativicutes and the rumen isolate M. multacida possesses a phytase that is located in the outer membrane (d’Silva et al., Can J Microbiol 2000, 46(4), 391-5).
  • metabolic diseases in particular metabolic syndrome and insulin resistance or insulin resistance-related conditions, including dyslipidemia and type 2 diabetes mellitus as well as insulin-resistance in endocrine diseases (e.g., obese subjects with type 1 diabetes mellitus, Cushing's disease or lipodystrophy syndromes).
  • the present inventors identified Mitsuokella jalaludinii as an efficient phytate degrader in the human gut. Subsequently, a complete phytate degradation pathway in Mitsuokella jalaludinii was elucidated with NMR spectroscopy using 13C-isotope labelling and transcriptomic and genomic analysis. The genes involved in the pathway are disclosed herein and can be introduced in any suitable host bacterium to achieve phytate degradation activity.
  • the host bacterium may, for example, be Escherichia coli, Bifidobacterium spp., Lactobacillus spp., Lactococcus spp., or Lactobacillus spp.
  • a major end product of the pathway is the antimicrobial 3-hydroxypropionate.
  • 3-hydroxypropionate can be converted into propionate in a synergistic interaction with Anaerostipes rhamnosivorans both in vitro and in vivo.
  • the pathway for converting 3-hydroxypropionate into propionate has also been unravelled.
  • the present inventors envisage supplementation of a subject with a bacterium according to the present disclosure, or the synergistic combination of bacteria according to the present disclosure, to provide for a direct or synergistic effect via in situ (3-hydroxy)propionate production.
  • the present disclosure relates to a first bacterium comprising a gene set encoding a pathway for the conversion of phytate to 3-hydroxypropionate or a salt or ester thereof that allows said bacterium to convert phytate to 3-hydroxypropionate or a salt or ester thereof, for use in preventing and/or treating metabolic syndrome, insulin resistance and/or insulin resistance related condition.
  • the bacterium is capable of converting phytate into 3-hydroxypropionate or a derivative thereof via phytate metabolism and 3-hydroxypropionate synthesis.
  • the term phytate is well-known to the skilled person and can be interchangeable with the terms myo-inositol-hexakisphosphate or myo-inositol-1,2,3,4,5,6-hexakisphosphate or InsPe.
  • the present disclosure relates to a second bacterium comprising a gene set encoding a pathway for the conversion of 3-hydroxypropionate or a salt or ester thereof to propionate or a salt or ester thereof that allows said bacterium to convert 3-hydroxypropionate or a salt or ester thereof to propionate or a salt or ester thereof, for use in preventing and/or treating metabolic syndrome, insulin resistance and/or insulin resistance related condition.
  • a bacterium that comprises a gene set encoding a pathway for the conversion of phytate to 3- hydroxypropionate or a salt or ester thereof and a gene set encoding a pathway for the conversion of 3-hydroxypropionate or a salt or ester thereof to propionate or a salt or ester thereof, that allows said bacterium to convert phytate to propionate or a salt or ester thereof, for example for use in preventing and/or treating metabolic syndrome, insulin resistance and/or insulin resistance related condition.
  • the bacteria according to the present disclosure when administered to a human being or when ingested by a human being in an adequate amount, are able to survive and at least transiently colonize the gastrointestinal (Gl) tract of said human being.
  • Colonization of the first bacterium enables greater in situ production of 3- hydroxypropionate and together with colonization of the second bacterium greater in situ production of propionate is enabled.
  • Increased in situ production of 3-hydroxypropionate and/or propionate is believed to underlie beneficial effects as taught herein, e.g.
  • metabolic diseases such as metabolic syndrome and insulin resistance or insulin resistance-related complications, such as dyslipidemia and type 2 diabetes mellitus as well as insulin-resistance in endocrine diseases (e.g., obese subjects with type 1 diabetes mellitus, Cushing's disease or lipodystrophy syndromes).
  • endocrine diseases e.g., obese subjects with type 1 diabetes mellitus, Cushing's disease or lipodystrophy syndromes.
  • the present disclosure relates to a first bacterium comprising a gene set encoding a pathway for the conversion of phytate to 3-hydroxypropionate or a salt or ester thereof that allows said bacterium to convert phytate to 3-hydroxypropionate or a salt or ester thereof, in particular but not necessarily under anaerobic conditions, such as wherein the bacterium and/or its medium is not in contact with gas comprising more than 0.1 vol%, 1 vol%, 2 vol%, 3 vol%, 4 vol%, 5 vol%, or 10 vol% of oxygen.
  • the bacterial strain is preferably, but not necessarily, an isolate, e.g. a human intestinal isolate.
  • the phytate to 3-hydroxypropionate or a salt or ester thereof pathway gene set may comprise one or more (e.g., at least two or three, or all) of the genes encoding the proteins: phytase, major myo-inositol transporter lolT, myo-inositol 2-dehydrogenase, inosose dehydratase, 3D-(3,5/4)-trihydroxycyclohexane-1, 2-dione hydrolase, 5-deoxy-glucuronate isomerase, 5-keto-2-deoxygluconokinase, 5-keto-2-deoxy-D-gluconate-6 phosphate aldolase, 2-hydroxy-3-oxopropionate reductase, D-beta-hydroxypropionate permease.
  • the phytate to 3-hydroxypropionate or a salt or ester thereof pathway gene set may comprise the gene(s) encoding the proteins phosphate ABC transporter PstS, Phosphate ABC transporter PstC, Phosphate ABC transporter PstA, Na+/H+ antiporter NhaA type, phosphoglycerate mutase, triosephosphate isomerase, enolase, phosphoglycerate kinase, pyruvate kinase, NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, D-lactate dehydrogenase, pyruvate-flavodoxin oxidoreductase, acetyl-CoA hydrolase, D-lactate dehydrogenase, pyruvate carboxylase, malate dehydrogenase, NADP-dependent malic enzyme, fumarate hydratase alpha subunit, fumarate hydrat
  • the amount of phytate used to determine whether genes are upregulated or proteins are overexpressed in a bacterium as compared to when said bacterium is grown in absence of phytate may be in the range of from about 5 mM to about 100 mM, preferably from about 10 mM to about 50 mM, more preferably from about 15 mM to about 25 mM, and even more preferably about 20 mM.
  • the first bacterium according to the present disclosure may be an isolated intestinal bacterial strain, or strain derived therefrom, and/or may be an intestinal bacterium isolated from a human being, which naturally comprises an phytate to 3- hydroxypropionate or a salt or ester pathway gene set as taught herein and which is capable of converting phytate to 3-hydroxypropionate or a salt or ester (or a derivative) thereof.
  • the first bacterium according to the disclosure may be a Mitsuokella species, preferably Mitsuokella jalaludinii or relative thereof or relative thereof having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO:93.
  • the first bacterium may be Mitsuokella jalaludinii H1-1 deposited by Wageningen University on December 1, 2023 at the Westerdijk Fungal Biodiversity Institute (CBS) located at Uppsalalaan 8, 3584CT Utrecht, the Netherlands, assigned the deposit number CBS 150836.
  • CBS Westerdijk Fungal Biodiversity Institute
  • CBS 150836 also encompassed is any bacterial strain derived from the deposited bacterium.
  • the first bacterium according to the present disclosure may be a bacterium which has been transfected with the phytate to 3-hydroxypropionate or a salt or ester thereof pathway gene set as taught herein, and which is capable of converting phytate to 3-hydroxypropionate or a salt or ester (or a derivative) thereof.
  • the skilled person is well-acquainted with methods for transforming bacteria with a desired genetic construct (i.e. pathway gene set).
  • the present disclosure relates to a (second) bacterium comprising a gene set encoding a pathway for conversion of 3-hydroxypropionate or a salt or ester thereof to propionate or a salt or ester thereof that allows said second bacterium to convert 3- hydroxypropionate or a salt or ester thereof to propionate or a salt or ester thereof, in particular but not necessarily under anaerobic conditions, such as wherein the bacterium and/or its medium is not in contact with gas comprising more than 1 vol%, 2 vol%, 3 vol%, 4 vol%, 5 vol%, or 10 vol% of oxygen.
  • the bacterial strain is preferably, but not necessarily, an isolate, e.g. a human intestinal isolate.
  • the 3-hydroxypropionate or a salt or ester thereof to propionate or a salt or ester thereof pathway gene set may comprise one or more (e.g., at least two or three, or all) of the genes encoding the proteins: permease, oxoacid CoA transferase, dehydratase, electron transfer flavoprotein beta subunit, electron transfer flavoprotein alpha subunit, and/or acyl dehydrogenase.
  • the 3-hydroxypropionate or a salt or ester thereof to propionate or a salt or ester thereof pathway gene set may comprise the genes encoding the proteins ATP synthase epsilon chain, ATP synthase beta chain, ATP synthase gamma chain, ATP synthase alpha chain, ATP synthase delta chain, ATP synthase FO sector subunit b, ATP synthase FO sector subunit c, ATP synthase FO sector subunit a, and/or ATP synthase protein I.
  • the first bacterium as disclosed herein is combined with the second bacterium as disclosed herein.
  • the first bacterium and/or second bacterium as disclosed herein are live bacteria.
  • the first bacterium and/or second bacterium as disclosed herein have (machinery for) ATP production and/or NADH production.
  • At least one (or at least two or three or all) of the proteins: permease, oxoacid CoA transferase, dehydratase, electron transfer flavoprotein, beta subunit, electron transfer flavoprotein, alpha subunit, and/or acyl dehydrogenase, ATP synthase epsilon chain, ATP synthase beta chain, ATP synthase gamma chain, ATP synthase alpha chain, ATP synthase delta chain, ATP synthase FO sector subunit b, ATP synthase FO sector subunit c, ATP synthase FO sector subunit a, and/or ATP synthase protein I may be overproduced (or the genes encoding therefor overexpressed) when the bacterium is grown in the presence of 3-hydroxypropionate as compared to when the (second) bacterium is grown in the absence of 3-hydroxypropionate.
  • the amount of 3-hydroxypropionate used to determine whether genes are upregulated or proteins are overexpressed in a bacterium as compared to when said bacterium is grown in absence of 3-hydroxypropionate may be in the range of from about 5 mM to about 100 mM, preferably from about 10 mM to about 50 mM, more preferably from about 15 mM to about 25 mM, and even more preferably about 20 mM.
  • the (second) bacterium according to the present disclosure may be an isolated intestinal bacterial strain, or strain derived therefrom, and/or may be an intestinal bacterium isolated from a human being, which naturally comprises a 3-hydroxypropionate or a salt or ester to propionate or salt or ester thereof pathway gene set as taught herein and which is capable of converting 3-hydroxypropionate or a salt or ester (or a derivative) thereof to propionate or a salt or ester (or derivative) thereof.
  • the (second) bacterium according to the disclosure may be an Anaerostipes species (which has ability to convert 3-hydroxypropionate to propionate), although preferably not Anaerostipes butyraticus (e.g. DSM22094T) but for example Anaerostipes caccae (e.g. DSM14662) or Anaerostipes hadrus (preferably PEL85, but preferably not DSM3319T, and/or preferably not DSM 108065).
  • the first bacterium may be Anaerostipes rhamnosivorans 1y2 deposited by Wageningen University on June 26, 2015 at the Centraalbureau voor Schimmelcultures (now named Westerdijk Fungal Biodiversity Institute) located at Uppsalalaan 8, 3584CT Utrecht, the Netherlands, assigned the deposit number CBS 140182.
  • the (second) bacterium according to the present disclosure may be a bacterium which has been transfected with the 3-hydroxypropionate or a salt or ester thereof to propionate or salt or ester thereof pathway gene set as taught herein, and which is capable of converting 3-hydroxypropionate or a salt or ester (or a derivative) thereof to propionate or a salt or ester (or derivative) thereof.
  • the skilled person is well-acquainted with methods for transforming bacteria with a desired genetic construct (i.e. pathway gene set).
  • the gene encoding phytase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:1. Additionally or alternatively, the gene encoding phytase may encode a phytase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:2. The function of the gene phytase can be seen in the conversion of phytate to (myo-)inositol and phosphate.
  • the gene encoding major myo-inositol transporter lolT may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:3. Additionally or alternatively, the gene encoding major myo-inositol transporter lolT may encode a major myo-inositol transporter lolT that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:4. The function of the gene major myo-inositol transporter lolT can be seen in the transport of inositol (across the cell membrane).
  • the gene encoding myo-inositol 2-dehydrogenase may have at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:45. Additionally or alternatively the gene encoding myo-inositol 2-dehydrogenase may encode an myo-inositol 2-dehydrogenase that has at least 70, 80, 90, 95, 99, 100% sequence identity with SEQ ID NO:46. The function of the gene myo-inositol 2-dehydrogenase can be seen in the conversion of myo-inositol to scyllo- inosose.
  • the gene encoding inosose dehydratase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:5. Additionally or alternatively, the gene encoding inosose dehydratase may encode an inosose dehydratase that has at least 70%, 80%, 90%, 95%, 99%, or 100%sequence identity with SEQ ID NO: 6.
  • the function of the gene inosose dehydratase can be seen in the conversion of scyllo-inosose to 3,5/4-trihydroxycyclohexa- 1 ,2-dione.
  • the gene encoding 3D-(3,5/4)-trihydroxycyclohexane-1, 2-dione hydrolase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:7. Additionally or alternatively, the gene encoding 3D-(3,5/4)-trihydroxycyclohexane-1, 2-dione hydrolase may encode an 3D-(3,5/4)-trihydroxycyclohexane-1 ,2-dione hydrolase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:8.
  • the gene encoding 5-deoxy-glucuronate isomerase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:9. Additionally or alternatively, the gene encoding 5-deoxy-glucuronate isomerase may encode a 5-deoxy-glucuronate isomerase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 10. The function of the gene 5-deoxy-glucuronate isomerase can be seen in the conversion of 5-deoxy-D-glucuronate to 2-deoxy-5-keto-D-gluconate.
  • the gene encoding 5-keto-2-deoxygluconokinase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:11. Additionally or alternatively, the gene encoding 5-keto-2-deoxygluconokinase may encode a 5-keto-2-deoxygluconokinase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 12. The function of the gene 5-keto-2-deoxygluconokinase can be seen in the conversion of 2- deoxy-5-keto-D-gluconate to 2-deoxy-5-keto-D-gluconate-6P.
  • the gene encoding 5-keto-2-deoxy-D-gluconate-6 phosphate aldolase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:13. Additionally or alternatively, the gene encoding 5-keto-2-deoxy-D-gluconate-6 phosphate aldolase may encode a 5-keto-2-deoxy-D-gluconate-6 phosphate aldolase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:14.
  • 5-keto-2-deoxy-D-gluconate-6 phosphate aldolase The function of the gene 5-keto-2-deoxy-D-gluconate-6 phosphate aldolase can be seen in the conversion of 5-dehydro-2-deoxy-D- Gluconate-6-phosphate to 3-oxopropionate (and/or in the conversion of 5-dehydro-2-deoxy-D- Gluconate-6-phosphate to glycerone phosphate).
  • the gene encoding 2-hydroxy-3-oxopropionate reductase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:15. Additionally or alternatively, the gene encoding 2-hydroxy-3-oxopropionate reductase may encode a 2-hydroxy-3- oxopropionate reductase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 16. The function of the gene 2-hydroxy-3-oxopropionate reductase can be seen in the conversion of 3-oxopropionate to 3-hydroxypropionate.
  • the gene encoding D-beta-hydroxypropionate permease may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:17. Additionally or alternatively, the gene encoding D-beta-hydroxypropionate permease may encode a D-beta- hydroxypropionate permease that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 18. The function of the gene D-beta-hydroxypropionate permease can be seen in 3-hydroxypropionate transport (across cell membrane).
  • the gene encoding phosphate ABC transporter PstS may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:19. Additionally or alternatively, the gene encoding phosphate ABC transporter PstS may encode a phosphate ABC transporter PstS that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NQ:20. The function of the gene phosphate ABC transporter PstS can be seen in the transport of phosphate (across the cell membrane).
  • the gene encoding Phosphate ABC transporter PstC may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:21. Additionally or alternatively, the gene encoding Phosphate ABC transporter PstC may encode a Phosphate ABC transporter PstC that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:22. The function of the gene Phosphate ABC transporter PstC can be seen in the transport of phosphate (across the cell membrane).
  • the gene encoding Phosphate ABC transporter PstA may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:23. Additionally or alternatively, the gene encoding Phosphate ABC transporter PstA may encode a Phosphate ABC transporter PstA that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:24. The function of the gene Phosphate ABC transporter PstA can be seen in the transport of phosphate (across the cell membrane).
  • the gene encoding Na+/H+ antiporter NhaA type may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:25. Additionally or alternatively, the gene encoding Na+/H+ antiporter NhaA type may encode a Na+/H+ antiporter NhaA type that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:26. The function of the gene Na+/H+ antiporter NhaA type can be seen in the transport of phosphate (across the cell membrane).
  • the gene encoding phosphoglycerate mutase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:27. Additionally or alternatively, the gene encoding phosphoglycerate mutase may encode a phosphoglycerate mutase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:28.
  • triosephosphate isomerase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:29. Additionally or alternatively, the gene encoding triosephosphate isomerase may encode a triosephosphate isomerase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NQ:30.
  • the gene encoding enolase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:31. Additionally or alternatively, the gene encoding enolase may encode an enolase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:32.
  • the gene encoding phosphoglycerate kinase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:33. Additionally or alternatively, the gene encoding Phosphoglycerate kinase may encode a phosphoglycerate kinase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:34.
  • the gene encoding pyruvate kinase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:35. Additionally or alternatively, the gene encoding pyruvate kinase may encode a pyruvate kinase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:36.
  • the gene encoding NAD-dependent glyceraldehyde-3-phosphate dehydrogenase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:37. Additionally or alternatively, the gene NAD-dependent glyceraldehyde-3-phosphate dehydrogenase may encode a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:38.
  • the function of the gene NAD-dependent glyceraldehyde-3-phosphate dehydrogenase can be seen in the conversion of glycerone phosphate to pyruvate.
  • the gene encoding D-lactate dehydrogenase_ may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:39. Additionally or alternatively, the gene D- lactate dehydrogenase_may encode a D-lactate dehydrogenase_that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NQ:40. The function of the gene D- lactate dehydrogenase_can be seen in the conversion of pyruvate to lactate.
  • the gene encoding pyruvate-flavodoxin oxidoreductase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:41. Additionally or alternatively, the gene pyruvate-flavodoxin oxidoreductase may encode an pyruvate-flavodoxin oxidoreductase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:42.
  • the function of the gene pyruvate-flavodoxin oxidoreductase can be seen in the conversion of pyruvate to acetyl-CoA.
  • the gene encoding acetyl-CoA hydrolase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:43. Additionally or alternatively, the gene acetyl- CoA hydrolase may encode an acetyl-CoA hydrolase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:44. The function of the gene acetyl-CoA hydrolase can be seen in the conversion of acetyl-CoA to acetate.
  • the gene encoding D-lactate dehydrogenase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:45. Additionally or alternatively, the gene D- lactate dehydrogenase may encode an D-lactate dehydrogenase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:46. The function of the gene D- lactate dehydrogenase can be seen in the conversion of pyruvate to lactate.
  • the gene encoding pyruvate carboxylase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:47. Additionally or alternatively, the gene pyruvate carboxylase may encode a pyruvate carboxylase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:48. The function of the gene pyruvate carboxylase can be seen in the conversion of pyruvate to oxaloacetate.
  • the gene encoding malate dehydrogenase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:49. Additionally or alternatively, the gene malate dehydrogenase may encode a malate dehydrogenase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NQ:50. The function of the gene malate dehydrogenase can be seen in the conversion of oxaloacetate to malate.
  • the gene encoding NADP-dependent malic enzyme may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:51. Additionally or alternatively, the gene NADP-dependent malic enzyme may encode an NADP-dependent malic enzyme that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:52. The function of the gene NADP-dependent malic enzyme can be seen in the conversion of pyruvate to malate.
  • the gene encoding fumarate hydratase alpha subunit may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:53. Additionally or alternatively, the gene fumarate hydratase alpha subunit may encode an fumarate hydratase alpha subunit that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:54. The function of the gene fumarate hydratase alpha subunit can be seen in the conversion of malate to fumarate.
  • the gene encoding fumarate hydratase beta subunit may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:55. Additionally or alternatively, the gene fumarate hydratase beta subunit may encode an fumarate hydratase beta subunit that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:56. The function of the gene fumarate hydratase beta subunit can be seen in the conversion of malate to fumarate.
  • the gene encoding succinate dehydrogenase cytochrome b558 subunit may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:57. Additionally or alternatively, the gene succinate dehydrogenase cytochrome b558 subunit may encode an succinate dehydrogenase cytochrome b558 subunit that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:58.
  • the function of the gene succinate dehydrogenase cytochrome b558 subunit can be seen in the conversion of fumarate to succinate
  • the gene encoding succinate dehydrogenase flavoprotein subunit may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:59. Additionally or alternatively, the gene succinate dehydrogenase flavoprotein subunit may encode an succinate dehydrogenase flavoprotein subunit that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:60.
  • the function of the gene succinate dehydrogenase flavoprotein subunit can be seen in the conversion of fumarate to succinate
  • the gene encoding succinate dehydrogenase iron-sulfur protein may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:61. Additionally or alternatively, the gene succinate dehydrogenase iron-sulfur protein may encode an succinate dehydrogenase iron-sulfur protein that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:62. The function of the gene succinate dehydrogenase iron-sulfur protein can be seen in the conversion of fumarate to succinate
  • the gene encoding ATP synthase epsilon chain may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:63. Additionally or alternatively, the gene ATP synthase epsilon chain may encode an ATP synthase epsilon chain that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:64.
  • the gene encoding ATP synthase beta chain may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:65. Additionally or alternatively, the gene ATP synthase beta chain may encode an ATP synthase beta chain that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:66.
  • the gene encoding ATP synthase gamma chain may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:67. Additionally or alternatively, the gene ATP synthase gamma chain may encode an ATP synthase gamma chain that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:68.
  • the gene encoding ATP synthase alpha chain may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:69. Additionally or alternatively, the gene ATP synthase alpha chain may encode an ATP synthase alpha chain that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NQ:70.
  • the gene encoding ATP synthase delta chain may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:71. Additionally or alternatively, the gene ATP synthase delta chain may encode an ATP synthase delta chain that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:72.
  • the gene encoding ATP synthase F0 sector subunit b may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:73. Additionally or alternatively, the gene ATP synthase F0 sector subunit b may encode an ATP synthase F0 sector subunit b that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:74.
  • the gene encoding ATP synthase FO sector subunit c may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:75.
  • the gene ATP synthase F0 sector subunit c may encode an ATP synthase F0 sector subunit c that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:76.
  • the gene encoding ATP synthase F0 sector subunit a may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:77. Additionally or alternatively, the gene ATP synthase F0 sector subunit a may encode an ATP synthase F0 sector subunit a that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:78. The gene encoding ATP synthase protein I may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:79. Additionally or alternatively, the gene ATP synthase protein I may encode an ATP synthase protein I that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NQ:80.
  • the gene encoding permease may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:81. Additionally or alternatively, the gene permease may encode a permease that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:82. The function of the gene permease can be seen in the transport of 3- hydroxypropionate (across the cell membrane).
  • the gene encoding oxoacid CoA transferase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:83. Additionally or alternatively, the gene oxoacid CoA transferase may encode an oxoacid CoA transferase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:84.
  • the function of the gene oxoacid CoA transferase can be seen in the conversion of 3-hydroxypropionate to 3- hydroxy propionyl-CoA.
  • the gene encoding dehydratase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:85. Additionally or alternatively, the gene dehydratase may encode an dehydratase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:86. The function of the gene dehydratase can be seen in the conversion of 3-hydroxy propionyl-CoA to Acryloyl-CoA.
  • the gene encoding electron transfer flavoprotein beta subunit may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:87. Additionally or alternatively, the gene electron transfer flavoprotein beta subunit may encode an electron transfer flavoprotein beta subunit that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:88. The function of the gene electron transfer flavoprotein beta subunit can be seen in the conversion of acryloyl-CoA to propionyl-Coa.
  • the gene encoding electron transfer flavoprotein alpha subunit may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:86.
  • the gene electron transfer flavoprotein alpha subunit may encode an electron transfer flavoprotein alpha subunit that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:90.
  • the function of the gene electron transfer flavoprotein alpha subunit can be seen in the conversion of acryloyl-CoA to propionyl-Coa.
  • the gene encoding acyl dehydrogenase may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:91. Additionally or alternatively, the gene acyl dehydrogenase may encode an acyl dehydrogenase that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:92. The function of the gene acyl dehydrogenase can be seen in the conversion of acryloyl-CoA to propionyl-Coa..
  • the gene encoding ATP synthase epsilon chain may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:63. Additionally or alternatively, the gene ATP synthase epsilon chain may encode an ATP synthase epsilon chain that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:64.
  • the gene encoding ATP synthase beta chain may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:65. Additionally or alternatively, the gene ATP synthase beta chain may encode an ATP synthase beta chain that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:66.
  • the gene encoding ATP synthase gamma chain may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:67. Additionally or alternatively, the gene ATP synthase gamma chain may encode an ATP synthase gamma chain that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:68.
  • the gene encoding ATP synthase alpha chain may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:69. Additionally or alternatively, the gene ATP synthase alpha chain may encode an ATP synthase alpha chain that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NQ:70.
  • the gene encoding ATP synthase delta chain may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:71. Additionally or alternatively, the gene ATP synthase delta chain may encode an ATP synthase delta chain that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:72.
  • the gene encoding ATP synthase F0 sector subunit b may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:73. Additionally or alternatively, the gene ATP synthase F0 sector subunit b may encode an ATP synthase F0 sector subunit b that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:74.
  • the gene encoding ATP synthase F0 sector subunit c may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:75. Additionally or alternatively, the gene ATP synthase FO sector subunit c may encode an ATP synthase FO sector subunit c that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:76.
  • the gene encoding ATP synthase F0 sector subunit a may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:77. Additionally or alternatively, the gene ATP synthase F0 sector subunit a may encode an ATP synthase F0 sector subunit a that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:78. The gene encoding ATP synthase protein I may have at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO:79. Additionally or alternatively, the gene ATP synthase protein I may encode an ATP synthase protein I that has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NQ:80.
  • the present disclosure provides a kit comprising a composition comprising the first bacterium and/or a composition comprising the second bacterium, wherein optionally the compositions comprise a physiologically acceptable carrier.
  • the present disclosure also provides a composition comprising the first bacterium and/or second bacterium according to the present disclosure and optionally a physiologically acceptable carrier.
  • the physiologically acceptable carrier may be any carrier that is suitable for keeping the bacterium/bacteria as taught herein viable until consumption by a subject (e.g. human or animal).
  • Non-limiting examples of acceptable carriers that are suitable for this purpose include any well-known physiological or pharmaceutical carriers, buffers, and excipients.
  • a suitable physiological or pharmaceutical carrier will depend upon the intended mode of administration of the composition as taught herein (e.g. oral) and the intended form of the composition (e.g. beverage, yogurt, powder, capsules, and the like). The skilled person knows how to select a physiological or pharmaceutical carrier, which is suitable for the compositions as taught herein.
  • the composition as taught herein may be in the form of a food composition, feed composition, feed supplement composition, food supplement composition or pharmaceutical composition.
  • the composition is preferably suitable for consumption by a human being.
  • the composition is a food or food supplement composition.
  • the food or food supplement composition may be selected from the group consisting of a liquid, liquid beverage (including dairy beverage and fermented beverage), yogurt, cheese, gel, gelatine, gelatine capsule, powder, paste, pressed tablet, and gel cap.
  • the composition is a liquid, preferably a liquid beverage (e.g. dairy beverage).
  • the food or food supplement composition may be a dairy product, preferably a fermented dairy product, such as yogurt or yogurt drink.
  • the composition as taught herein may be a probiotic composition.
  • Such probiotic composition may comprise the first (isolated) bacterium and/or second (isolated) bacterium as taught herein, or a strain derived therefrom.
  • the composition as taught herein further comprises one or more additional beneficial isolated intestinal bacterial strain.
  • the composition may be a symbiotic composition. It may be advantageous to add one or more prebiotic ingredients to the composition as taught herein, for example, to supplement the effects (e.g. production of 3-hydroxypropionate/propionate) of the first bacterium and/or second bacterium as taught herein.
  • the one or more prebiotic ingredients may be any prebiotic ingredients, which are suitable to enhance the activity and/or stimulate the growth of the bacterium, or a strain derived therefrom, as taught herein.
  • suitable prebiotic ingredients include fibres such as inulin, pectin, and resistant starch, as well as cellobiose, maltose, mannose, salicine, trehalose, amygdalin, arabinose, melibiose, sorbitol, rhamnose and xylose.
  • the composition as taught herein comprises a phytate-rich source and/or phytate.
  • a phytate-rich source and/or phytate may be advantageous to add to the composition as taught herein to further promote the production of 3-hydroxypropionate/propionate or a derivative thereof in the Gl tract of a mammal (e.g. human being).
  • the composition as taught herein may comprise one or more ingredients which are suitable for promoting survival and/or viability of the bacterium or strain derived therefrom as taught herein during storage and/or during exposure to bile and/or during passage through the Gl tract of a mammal (e.g. a human being).
  • suitable ingredients include an enteric coating and controlled release agents allowing passage through the stomach. The skilled person knows how to select suitable ingredients for maintaining a bacterium as taught herein viable and functional, i.e. able to carry out intended function(s).
  • compositions as taught herein may further comprise one or more ingredients, which further enhance the nutritional value and/or the therapeutic value the compositions as taught herein.
  • ingredients e.g. nutritional ingredients, veterinary, medicinal agents, etc.
  • the group consisting of proteins, amino acids, enzymes, mineral salts, vitamins e.g.
  • thiamine HCI riboflavin
  • pyridoxine HCI niacin
  • inositol choline chloride
  • calcium pantothenate biotin, folic acid, ascorbic acid, vitamin B12, p-aminobenzoic acid, vitamin A acetate, vitamin K, vitamin D, vitamin E, and the like
  • sugars and complex carbohydrates e.g. water-soluble and water-insoluble monosaccharides, disaccharides, and polysaccharides
  • medicinal compounds e.g. antibiotics
  • antioxidants e.g.
  • the first bacterium and/or second bacterium as taught herein may be incorporated into the composition in lyophilized form, microencapsulated form (reviewed by, for example, Solanki et al., BioMed Res. Int. 2013, Article ID 620719), or any other form preserving the activity and/or viability of the bacterial strain.
  • the composition as taught herein may be a pharmaceutical composition.
  • the pharmaceutical composition may be for use as a supplement.
  • a pharmaceutical composition will usually comprise a pharmaceutical carrier, in addition to the first bacterium and/or second bacterium taught herein.
  • the carrier is preferably an inert carrier. The preferred form depends on the intended mode of administration and (therapeutic) application.
  • a pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver the bacterium taught herein to the Gl tract of a subject.
  • sterile water or inert solids may be used as a carrier, usually complemented with a pharmaceutically acceptable adjuvant, buffering agent, dispersing agent, and the like.
  • a pharmaceutical composition as taught herein may be in liquid form, e.g.
  • a cryoprotectant such as lactose, trehalose or glycogen can be employed.
  • the first bacterium and/or second bacterium taught herein can be administered in (separate) solid dosage forms, such as capsules, tablets, and powders, comprising lyophilized bacteria, or in (separate) liquid dosage forms, such as elixirs, syrups, and suspensions.
  • the first bacterium and/or second bacterium taught herein can be encapsulated in capsules such as gelatin capsules, together with inactive ingredients and powdered carriers, such as e.g. glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.
  • capsules such as gelatin capsules
  • inactive ingredients and powdered carriers such as e.g. glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.
  • the first bacterium and/or second bacterium as taught herein may be comprised in the composition as taught herein in an amount ranging from about 10 6 to about 10 15 colony forming units (CFU).
  • CFU colony forming units
  • the intestinal bacteria may be comprised in the composition in an amount of about 10 7 CFU to about 10 14 CFU, preferably about 10 8 CFU to about 10 13 CFU, more preferably about 10 9 CFU to about 10 12 CFU, even more preferably about 10 1 ° CFU to about 10 12 CFU, e.g. for each of the first bacterium and/or second bacterium.
  • the present disclosure also provides for a composition obtainable by: a) growing the first bacterium as taught herein and optionally a second bacterium as taught herein in an aqueous medium suitable therefor (the aqueous medium may comprise phytate); b) after step a), separating the first bacterium and optionally the second bacterium from the aqueous medium to thereby obtain the composition (i.e. the aqueous medium separated from the first bacterium and optionally the second bacterium).
  • the composition may comprise (3-hydroxy)propionate.
  • the first bacterium and/or second bacterium, or composition taught herein is not comprised in fecal matter.
  • fecal matter includes feces or a fraction thereof, such as obtained by mixing feces with aqueous medium and subsequent filtering and/or centrifugation.
  • the present disclosure is concerned with a first bacterium and/or second bacterium as taught herein or a composition as taught herein for use as a medicament, for use as a food or food supplement, or for use as a probiotic and/or symbiotic.
  • the first bacterium and/or second bacterium for use according to the present disclosure may be administered separately, sequentially or simultaneously with phytate or with a phytate source, which may be present in the same composition as the first bacterium and/or second bacterium, or in a separate composition, for example, comprising in the range of from about 5 mM to about 100 mM, preferably from about 10 mM to about 50 mM, more preferably from about 15 mM to about 25 mM, and even more preferably about 20 mM phytate, or for example comprising from about 1 mg to about 1000 mg, preferably from about 10 mg to about 500 mg, more preferably from about 15 mg to about 50 mg.
  • the amount of phytate and/or administration frequency may be chosen such that between 0.1 g and 100 grams is consumed per day, for example between 0.5 g and 10 g per day or between 0.1 g and 5 g per day.
  • the present disclosure particularly pertains to the first bacterium and/or second bacterium as taught herein or (separate) composition comprising the same as taught herein for use in maintaining, restoring and/or improving Gl health in general, and/or for preventing and/or treating conditions or diseases such as obesity, metabolic diseases, such as metabolic syndrome and insulin resistance or insulin resistance-related complications, such as (metabolic) Adenylosuccinate lyase deficiency ((M)ASLD) I (metabolic) dysfunction- associated steatohepatitis ((M)ASH), dyslipidemia and type 2 diabetes mellitus as well as insulin-resistance in endocrine diseases (e.g., obese subjects with type 1 diabetes mellitus, Cushing's disease or lipodystrophy syndromes).
  • MASLD Adenylosuccinate lyase deficiency
  • MASH metabolic) dysfunction- associated steatohepatitis
  • dyslipidemia e.g., obese subjects with type 1 diabetes mellit
  • foreseen is use for not gaining weight, such as after having lost weight e.g. following GLP1 agonist therapy.
  • foreseen is use for maintaining weight (within 1, 2, 3, 4 or 5% margin with respect to said weight), such as during and/or after GLP1 agonist therapy.
  • the first and/or second bacterium according to the present disclosure may have satiety effects since in situ propionate is provided (see Int. Dairy J. 18, 945-950 (2008)).
  • the present disclosure is also directed to a method for maintaining, restoring and/or improving Gl health in general, and/or for preventing and/or treating conditions or diseases such as obesity (i.e. a body mass index (BMI) over 25, 26, 27, 28, 29, preferably over 30), metabolic diseases, such as metabolic syndrome and insulin resistance or insulin resistance-related complications, such as dyslipidemia and type 2 diabetes mellitus as well as insulin-resistance in endocrine diseases (e.g., obese subjects with type 1 diabetes mellitus, Cushing's disease or lipodystrophy syndromes) in a subject in need thereof, said method comprising the step of increasing the level of the first bacterium and/or second bacterium as taught herein in said subject.
  • obesity i.e. a body mass index (BMI) over 25, 26, 27, 28, 29, preferably over 30
  • metabolic diseases such as metabolic syndrome and insulin resistance or insulin resistance-related complications, such as dyslipidemia and type 2 diabetes mellitus as well as insulin-resistance in endocrine diseases
  • the present disclosure may be for use in enhancing (intestinal) epithelial barrier function.
  • the present disclosure e.g. the first bacterium and/or second bacterium as taught herein, may be for use in enhancing (intestinal) epithelial barrier function.
  • the present disclosure e.g. the first bacterium and/or second bacterium as taught herein, may be for use in preventing or treating inflammatory disorders, such as rheumatoid arthritis, or inflammatory bowel disease.
  • the present disclosure e.g.
  • the first bacterium and/or second bacterium as taught herein may be for use in preventing or treating cancer such as colon cancer.
  • the present disclosure also foresees use for enhancing satiety in a subject.
  • the present disclosure shows in particular the increased propionate production in the gut that will increase satiety and as a consequence will reduce food intake and prevent weight gain (see Ruijschop et al. 2008 Chambers et al 2015).
  • the level of the first bacterium and/or second bacterium as taught herein in said subject may be increased by administering an effective amount of said first bacterium and/or second bacterium to said subject, and/or by administering an effective amount of a compound capable of increasing the level of said first bacterium and/or second bacterium in (the Gl tract of) said subject.
  • the first bacterium and/or second bacterium as taught herein may be administered concomitant with phytate or a phytate source, such as fruits, beans, grains, and nuts.
  • a phytate source such as fruits, beans, grains, and nuts.
  • the skilled person can, without undue burden, readily identify phytate-rich sources.
  • the first bacterium and/or second bacterium as taught herein may be administered to a subject following a diet which includes phytate, such as a subject following a vegetarian diet, vegan diet, or Mediterranean diet.
  • the level of the first bacterium and/or second bacterium as taught herein in the Gl tract of a subject may be increased by administering to the subject an effective amount of the first bacterium and/or second bacterium as taught herein, preferably Mitsuokella jalaludinii or relative thereof having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO:93 and/or Anaerostipes rhamnosivorans 1y2 or relative thereof having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO:94 and/or a composition as taught herein comprising said.
  • an effective amount of the first bacterium and/or second bacterium as taught herein preferably Mitsuokella jalaludinii or relative thereof having a 16S rRNA gene sequence with at least 97% sequence identity with SEQ ID NO:93 and/or Anaerostipes rhamnosivorans 1y2 or relative thereof having a 16S rRNA gene sequence
  • the subject may be selected from the group consisting of human beings, (monogastric) animals such as non-human primates, mice, rats, cats, dogs, cows, and pigs.
  • the subject is a human.
  • the disclosure also relates to a method for producing 3-hydroxypropionate and/or propionate, comprising the step of contacting the first bacterium and/or second bacterium as taught herein with a suitable energy source, e.g. phytate and/or glucose/acetate, under conditions which allow the first bacterium and/or second bacterium as taught herein to convert the energy source to 3-hydroxypropionate and/or propionate.
  • a suitable energy source e.g. phytate and/or glucose/acetate
  • the methods taught herein may be in vitro methods.
  • probiotics and ‘probiotic products’ as used herein refer to microorganisms such as intestinal bacteria, which - when administered or ingested in effective amounts - confer health benefits to the host (e.g. mammals, such as humans).
  • the host e.g. mammals, such as humans.
  • probiotics should be alive or viable when administered to a subject so as to allow the probiotics to colonize the large intestine of the host.
  • probiotics may also be dead when administered provided that substances produced by the probiotics still exert probiotic, beneficial effects on the host.
  • prebiotics and ‘prebiotic products’ as used herein generally refer to compounds that promote the growth and/or activity of Gl microorganisms that contribute to the well-being of their host.
  • Prebiotics or prebiotic products consist mainly of fermentable fibres or non- digestible carbohydrates. The fermentation of these fibres by prebiotics promotes the production of beneficial end products, such as SCFAs, particularly butyrate.
  • SCFAs beneficial end products
  • symbiotics and ‘symbiotic products’ as used herein generally refer to compositions and/or nutritional supplements combining probiotics and one or more compounds that promote the growth and/or activity of Gl microorganisms, such as prebiotics, into one product.
  • the symbiotic beneficially affects the host by improving the survival and colonization of the probiotic in the Gl tract, by selectively stimulating the growth and/or by activating the metabolism of the probiotic, thus improving host welfare.
  • the skilled person is well-acquainted with symbiotics and knows how to select ingredients that may be combined into a symbiotic.
  • propionate and ‘propionic acid’ as used herein refer to a carboxylic acid with chemical formula CH3CH2CO2H.
  • the terms may include derivatives thereof, i.e. compounds derived from propionic acid and in particular salts of propionic acid (propionates) and esters of propionic acid ( propanoates).
  • Propionic acid can be seen as a short-chain saturated fatty acid comprising ethane attached to the carbon of a carboxy group.
  • propionates are ammonium propionate, calcium propionate, magnesium propionate, potassium propionate and sodium propionate.
  • a non-limiting example of a propanoate is ethyl propionate.
  • beneficial intestinal bacteria species refers to a bacterium species that inhabits (i.e. is innate) the mammalian (e.g. human) intestine and exerts beneficial effect(s) (e.g. protection against pathogenic bacteria species, production of butyric acid and/or butyrate and derivatives, etc.) on the Gl, metabolic and other health of a mammal in which it resides.
  • beneficial intestinal bacterial species include lactic acid bacteria from the genera Lactobacillus and Bifidobacterium.
  • beneficial intestinal bacterial species include butyrate-producing bacterial species, which use the acetyl-CoA to produce butyric acid and/or butyrate and derivative thereof, such as the bacterial strains disclosed in US 2014/0242654, WO 2014/150094 or WO 2013/032328.
  • an effective amount of the first bacterium and/or second bacterium as taught herein is an amount which is effectively useful for maintaining, restoring, and/or improving Gl heath in a human being, for converting phytate into 3- hydroxypropionate/propionate or a derivative thereof and/or for preventing and/or treating conditions or diseases described herein in a subject, preferably a human being.
  • These conditions or diseases include, without limitation, obesity, metabolic diseases, such as metabolic syndrome and insulin resistance or insulin resistance-related complications, such as dyslipidemia and type 2 diabetes mellitus as well as insulin-resistance in endocrine diseases (e.g., obese subjects with type 1 diabetes mellitus, Cushing's disease or lipodystrophy syndromes).
  • the effective amount can be readily determined without undue experimentation by a person of ordinary skill in the art.
  • a strain that derives therefrom relates to strains obtained by using the deposited strain as taught herein as starting material.
  • the strain that derives therefrom may be a mutant strain, which may be derived from a strain of the invention by means of, for instance, genetic engineering, radiation, UV light, chemical treatment.
  • such derivative or mutant strain may be a strain derived from the deposited strain as taught herein that has been subjected to growth adaptation to particular conditions resulting in an additional benefit to the derivative strain, such as more rapid growth, better survival in the gut, enhanced phytate to propionate conversion, using methods that are well- known to the skilled person. It is preferred that the derivative or mutant is functionally equivalent to the deposited strain as taught herein.
  • a preferred derivative or mutant as taught herein has substantially the same activity or function as the deposited strain as taught herein, i.e. has the ability to convert phytate to 3-hydroxypropionate/propionate and derivatives.
  • the derivative or mutant advantageously provides substantially the same benefits to a mammal (e.g. humans or other mammals) administered with said derivative or mutant as would be the case upon administration of the deposited strain.
  • the derivative or mutant strain may also be a spontaneous derivative or mutant strain having the same characteristics as described herein for the deposited strain.
  • isolated may refer to being separated from its natural environment (which may be the gut or feces for example).
  • suitable for consumption and ‘nutritionally acceptable’ refer to ingredients or substances, which are generally regarded as safe for human (as well as other mammals) consumption.
  • metabolic syndrome is well known by the skilled person. Within the present disclosure, the term encompasses all conditions diagnosed as “metabolic syndrome” by an (authorized) medical practitioner. For example, metabolic syndrome may be diagnosed if a patient has at least two or at least three of the following traits:
  • Large waist A waistline that measures at least 35 inches (89 centimeters) for women and 40 inches (102 centimeters) for men;
  • High triglyceride level 150 milligrams per deciliter (mg/dL), or 1.7 millimoles per liter (mmol/L), or higher of this type of fat found in blood;
  • HDL cholesterol Less than 40 mg/dL (1.04 mmol/L) in men or less than 50 mg/dL (1.3 mmol/L) in women of high-density lipoprotein (HDL) cholesterol;
  • Increased blood pressure 130/85 millimeters of mercury (mm Hg) or higher; Elevated fasting blood sugar — 100 mg/dL (5.6 mmol/L) or higher.
  • insulin resistance is well known by the skilled person. Within the present disclosure, the term encompasses all conditions diagnosed as “insulin resistance” by an (authorized) medical practitioner.
  • insulin resistance may be diagnosed by the gold standard for determining and quantifying insulin resistance which is the "hyperinsulinemic euglycemic clamp" (DeFronzo RA, Tobin JD, Andres R (1979). The American Journal of Physiology. 237 (3): E214-23). This method measures the amount of glucose necessary to compensate for an increased insulin level without causing hypoglycemia. The procedure may take about two hours and typically involves the following steps. Through a peripheral vein, insulin is infused at 10-120 mil per m 2 per minute.
  • glucose 20% is infused to maintain blood sugar levels between 5 mmol/L and 5.5 mmol/L.
  • the rate of glucose infusion is determined by checking the blood sugar levels every five to ten minutes (Muniyappa R, Lee S, Chen H, Quon MJ (January 2008). American Journal of Physiology. Endocrinology and Metabolism. 294 (1): E15-26). The rate of glucose infusion during the last thirty minutes of the test determines insulin sensitivity. If high levels (7.5 mg/min or higher) are required, the patient is insulinsensitive. Low levels (4.0 mg/min or lower) indicate insulin resistance. Levels between 4.0 mg/min and 7.5 mg/min are not definitive and suggest "impaired glucose tolerance", an early sign of insulin resistance.
  • the homeostatic model assessment is a method used to determine and quantify insulin resistance, as it correlates reasonably with the golden standard. See e.g. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC (1985). Diabetologia. 28 (7): 412-9. doi:10.1007/BF00280883. PMID 3899825; and/or A. S. Rudenski; D. R. Matthews; J. C. Levy; R. C. Turner (1991) Metabolism. 40 (9): 908-917.
  • a HOMA(-IR) score that deviates from a reference range can indicate insulin resistance.
  • insulin resistance refers to peripheral insulin resistance and/or hepatic insulin resistance.
  • insulin resistance-related conditions may refer to dyslipidemia. Alternatively or additionally, the term may refer to type 2 diabetes mellitus. Further, the term ‘insulin resistance-related conditions’ (or complications) may refer to insulin-resistance in endocrine disease (e.g., obese subjects with type 1 diabetes mellitus, Cushing's disease or lipodystrophy syndromes).
  • identity refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" perse has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H.
  • Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. For example NCBI Nucleotide Blast with standard settings (blastn, https://blast.ncbi.nlm.nih.gov/).
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).
  • nucleotide sequence or amino acid sequence having at least, for example, 95% "identity" to a reference sequence it is intended that the nucleotide sequence or amino acid sequence is identical to the reference sequence except that there may be up to five-point mutations per each 100 nucleotides or amino acids of the reference sequence.
  • nucleotide sequence or amino acid sequence being at least 95% identical to a reference sequence
  • up to 5% of the nucleotides or amino acids in the reference sequence may be deleted and/or substituted with another nucleotide or amino acid, and/or a number of nucleotides or amino acids up to 5% of the total nucleotides or amino acids in the reference sequence may be inserted into the reference sequence.
  • sequence identity refers to the sequence identity over the entire length of the sequence. It is further understood that, when referring to “sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids or nucleotides) are referred to.
  • the term ‘about’ as used herein indicates a range of normal tolerance in the art, for example within 2 standard deviations of the mean.
  • the term ‘about’ can be understood as encompassing values that deviate at most 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the indicated value.
  • references to an element by the indefinite article ’a’ or ‘an’ does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements.
  • the indefinite article ‘a’ or ‘an’ thus usually means ‘at least one’.
  • a level is increased or decreased when it is at least 5%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% higher or lower, respectively, than the corresponding level in a control or reference.
  • a level in a sample may be increased or decreased when it is statistically significantly increased or decreased compared to a level in a control or reference.
  • FIG. 1 Fecal phytate metabolism and identification of phytate-degrading Mitsuokella spp. a: 13 C-NMR spectra of [ 13 Ce]lnsP6 metabolism by fecal microbiome of donor A during 24h incubation. Dashed lines connect NMR signals of identified metabolites and CO2. For 3-hydroxypropionate dotted lines are used for better visibility.
  • the final metabolic products derived from [ 13 Ce]lnsP6 are [ 13 C2]acetate, [ 13 C3]propionate, and [ 13 C4]butyrate.
  • [ 13 C3]3-hydroxypropionate accumulates transiently between 4.5 - 6.5 h.
  • b Microbial composition of fecal samples, the initial time points and end time points of phytate enrichment from donor A. Top 15 abundant taxonomic groups in the samples are shown in colors and the rest is shown as other in white
  • c Changes of relative abundance of Mitsuokella jalaludinii and Mitsuokella spp. during phytate incubation and phytate consumption in fecal enrichments from donor A.
  • Figure 2 Elucidation of phytate degradation pathway in Mitsuokella jalaludinii DSM13811 T .
  • a Faster growth of M. jalaludinii in 10mM and 40mM phytate as compared to growth in 10mM and 40mM myo-inositol while similar metabolite production was observed
  • b 13 C-NMR analysis shows rapid [ 13 Ce]phytate degradation with [ 13 Ce]lns(2)P and [ 13 Ce]lnositol as intermediates and [ 13 C3]3-hydroxypropionate, [ 13 C3]lactate, [ 13 C4]succinate, and [ 13 C2]acetate as end metabolites
  • c BIRD- ⁇ 1 H, 13 C ⁇ HMQC spectrum of the M.
  • A. rhamnosivorans in phytate degradation, a-b Metabolite production and substrate consumption by monoculture of M. jalaludinii (a) and coculture of M. jalaludinii and
  • [ 13 C4]succinate is again formed as a mixture with partially or non- 13 C-labeled succinate.
  • the same InsP intermediates can be observed between 2 - 7.5 h as for the monoculture (Fig. 3b).
  • d M. jalaludinii (MJ): Log2fold change of differential expression of genes involved in phytate degradation; inositol and phosphate uptake and inositol metabolic genes comparing between monoculture and coculture. Functions of these genes are indicated on the right.
  • e A. rhamnosivorans-.
  • Figure 4 In vivo phytate conversion by M. jalaludinii and A. rhamnosivorans in mice
  • a experimental design of in vivo microbial phytate conversion.
  • Three groups of 8 mice were treated with either phytate (0.1 mg/g body weight) alone, or phytate (0.1 mg/g body weight) together with M. jalaludinii (10 A 9 cells per dose) or phytate (0.1 mg/g body weight) with M. jalaludinii (MJ) and A. rhamnosivorans (A.rham) (10 A 9 cells per strain per dose) every second day for two weeks. After two weeks, a [ 13 Ce]phytate oral challenge of the same bacterial dosage were performed for the respective group.
  • mice A group of four mice was sacrificed after 3 h or 6 h. Cecum and plasma samples were harvested for inositol phosphate extraction and measurement while colon samples were used for quantification of M. jalaludinii and A. rhamnosivorans via qPCR.
  • Significant lower levels of cecal [ 13 Ce] phytate in microbial treatment groups compared to control groups at 3 h (b) and 6 h (c) after oral challenge.
  • Figure 5 Propionate and phytate-derived bacterial metabolites improved epithelial barrier integrity via activating tight junction genes
  • a 20mM propionate co-incubation increased TEER values as compared to the control
  • b Increased activity of tight junction genes Claudin-1, Claudin-2 and E-cadherin upon propionate exposure.
  • d Increased activity of tight junction genes Claudin-1 and E-cadherin upon bacterial supernatant exposure compared to the medium control.
  • Figure 6 Concentration independent synergy between M. jalajudinii and A. rhamnosivorans for phytate breakdown
  • a Growth curves of co-cultures (blue) of M. jalajudinii and A. rhamnosivorans and monocultures (pink) of M. jalajudinii in 10 mM, 20 mM, and 40 mM phytate during 24 h incubation
  • FIG. 7 3-hydroxypropionate metabolism by A. rhamnosivorans.
  • A. rhamnosivorans Metabolite production and substrate consumption of phytate (a) and myo-inositol (b) by monocultures and co-cultures of M. jalaludinii and A. rhamnosivorans. Cell numbers of M. jalaludinii and A. rhamnosivorans quantified by qPCR in the coculture on myo-inositol (c) and phytate (d).
  • e Principal coordination plot of the transcriptional profiles obtained by RNA-seq of M.
  • A. rhamnosivorans. 3-hydroxypropionate is imported via a permease (AR1Y2_1114) and further converted to 3-hydroxy propionyl-CoA, Acryloyl_CoA, propionyl-CoA and eventually propionate via oxoacid CoA transferase (AR1Y2_1115), dehydratase (AR1 Y2_1116), acad-etf complex (AR1Y2_1112, 1113 and 1117).
  • Acyl-CoA dehydrogenase/Etf complex (AR1 Y2_1112, 1113 and 1117) is predicted to be involved in energy conservation via by coupling the electrochemical gradient generated via a membrane bound Rnf complex with ATP synthesis.
  • Figure 11 Production of bacterial supernatant of the co-culture for epithelial barrier function test using a Caco-2 cell model. The experiment was performed in YCFA medium supplemented with phytate. Around 20 mM phytate was repeatedly added after adjusting the pH to neutral ( ⁇ 7). Bacterial supernatants was collected before and after substrate addition for 5 days. In the end of the growth, propionate, acetate and succinate were found as major end metabolites with nearly 90 mM propionate. Red arrows indicate the time of phytate addition.
  • ATP synthase protein l MJ 0860 - may be used in addition or alternatively to 0860 above
  • sequences disclosed in the description are preferred.
  • sequences of the sequence listing may be used.
  • EXAMPLE 1 Identification of microbial phytate metabolism in the human gut and its relation with host health
  • This Example shows that Misuokella jalaludinii or another bacterium comprising the identified phytate degradation pathway of Misuokella jalaludinii is able to convert phytate into the antimicrobial 3-hydroxypropionate.
  • the Example also shows the synergy of M. jalaludinii with A. rhamnosivorans in phytate breakdown to beneficial SCFAs (i.e. propionate, acetate, succinate). This also supports that the beneficial effects of dietary phytate on host health may derive from the modulation of the microbiome.
  • Stool of donor A was used to extract gDNA and inositol phosphates to further determine the microbial composition by 16S rRNA sequencing analysis and inositol phosphate profiles by 1 D- and 2D-NMR analyses (see below for detailed protocol). 1D- and 2D-NMR measurements. All chemicals were used without further purification unless specified.
  • Deuterated solutions (deuterium oxide (D2O), sodium deuteroxide (NaOD) and deuterium chloride (DCI)) were obtained from Eurisotop, perchloric acid (HCIO4) from Supelco, potassium hydroxide (KOH) from Carl Roth, ammonia solution from Sigma Aldrich, tetramethyl phosphonium bromide (TMPBr) from Alfa Aesar, titanium oxide particles (Titanosphere 5 pm) from GL Sciences.
  • D2O deuterium oxide
  • NaOD sodium deuteroxide
  • DCI deuterium chloride
  • HMQC measurements of cecal and plasma extracts were recorded with 128 scans, and P1 pulses determined for every single sample, and otherwise identical parameters.
  • BIRD- ⁇ 1 H, 13 C ⁇ HMQC-NMR spectra were processed without digital water suppression with manual phasing and automatic baseline correction.
  • capped NMR tubes were roughly sterilized by rubbing with 70 % ethanol and radiation with UV light for 20 min in an aseptic laminar flow hood.
  • Thawn aliquots of bacterial culture samples were centrifuged (5 min, 5000 g, 4 °C) and 500 pL of the supernatant were transferred under aseptic conditions into a sterilized NMR tube and 55 pL of D2O (from a previously unopened bottle which was kept under aseptic conditions) were added for locking during NMR measurements. Samples were kept at 4 °C until the measurement of 13 C-NMR spectra on the same day.
  • NMR samples which contained multiple InsP species according to the 13 C-NMR were lyophilized, redissolved in D2O, and lyophilized again. Finally, the samples are taken up in 500 pL of D2O and pH adjusted to 6.0 with NaOD and DCI and submitted for recording HMQC and HMQC-C LIP-COSY spectra.
  • Microbiome profiling analysis DNA from using from 1ml bacterial culture of fecal phytate enrichments using a repeated bead-beating protocol.
  • 16S V3V4 amplicon sequences were parsed using a vsearch (v2.15.2) based pipeline. Paired end reads were merged, with max differences set to 100 and allowing for staggered overlap.
  • ASVs were inferred from reads with lower than 1 .5 expected error rate using the cluster unoise with centroids algorithm with a minsize of 4, after which chimeras were removed using the uchime3 denovo method.
  • ASV abundances were determined by mapping the merged reads against the ASV sequences with identical matches sequence set using the usearch_global algorithm with a 0.97 distance cut off. Taxonomy was assigned using R (V4.0.5) and the dada2 assign taxonomy function using the silva (v132) reference database.
  • the HELI US study is a population-based multi-ethnic prospective cohort study, based in Amsterdam, The Netherlands. Baseline data collection took place between 2011 and 2015 among the six largest ethnic groups in Amsterdam (those of T urkish,ixie, African Surinamese, South-Asian Surinamese Ghanaian and Dutch origin). People in these groups who were aged 18-70 were randomly, stratified by ethnicity, recruited from the municipal registry. Data collection consisted of a questionnaire and a physical examination including the collection of biological samples. Fecal samples were collected from participants. Microbiome data from the HELIUS study was processed. ASV variants with an average abundance of more than 0.1 % were selected as representatives.
  • Colonies surrounded with clear zones as indication of phytate degradation activities were picked up and streaked on new agar medium to confirm the degradation activity. This streaking was repeated a several times before being inoculated in a liquid medium containing phytate as a sole energy and carbon source.
  • the identity of the isolate was determined via 16S rRNA sequencing at Macrogen, and a draft genome of the Mitsuokella isolate was sequenced by Illumina NovaSeq 6000 S4 PE150 XP and assembled using Genome de novo assembly pipeline at Eurofins.
  • a phytase phylogenetic tree was constructed from amino acid sequences of phytases and phosphatases from all hosts ranging from mammals to bacterial species that were retrieved from the NCBI database. This included previously reported phytase and phosphatase amino acid sequences from all hosts, E. coli phytase, Akkermansia phytase and Mitsuokella phytase. All amino acid sequences were aligned using the Clustal_X programme.
  • a phylogenetic tree was constructed using the neighbour-joining algorithm by the MEGA 7 with 1000 bootstraps to obtain confidence levels for the branches.
  • Mitsuokella jalaludinii DSM13811 T was obtained from DSMZ culture collection and Anaerostipes rhamnosivorans 1y-2 T was isolated previously and is available as DSM26241 T . These two bacteria were routinely maintained in a modified YCFA medium supplemented with myo-inositol (SIGMA) for A. rhamnosivorans or phytate (SIGMA) for M. jalaludinii.
  • the growth experiments were performed in duplicate in 20 ml bicarbonate-buffered medium supplemented with 20 mM phytate filled with N2/CO2 (80:20, v/v) gas in the head phase.
  • Phytate and inositol were filter sterilized as stock solutions of 0.2 M and 0.5 M. respectively.
  • the condition in which the bacteria were added to 20 ml bicarbonate-buffered medium without substrate was used as control.
  • the growth was monitored via metabolite formation by HPLC and optical density measurement by a spectrophotometer at a wavelength of 600 nm.
  • jalaludinii DSM13811 T and A. rhamnosivorans 1y-2 T were pre-cultured in YCFA medium supplemented with 20 mM phytate or 20 mM myo-inositol respectively. These precultures were added with
  • the bacteria were grown in monoculture and coculture in a medium containing 10 mM, 20 mM and 40 mM phytate or 10 mM, 20 mM or 40 mM myo-inositol in 96 well plate incubated in an anaerobic tent for growth monitoring for 48 h. At the end of the growth, the bacterial supernatants were collected to analyze the substrate consumption and metabolite production. The experiment was performed in biological triplicate.
  • rhamnosivorans was grown in 20mM myo-inositol supplemented with either 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM of 3-hydroxypropionate, and the growth was monitored by OD600 measurement every 30 minutes in a plate reader placed in an anaerobic tent. The experiment was performed in biological triplicate.
  • Phytate, myo-inositol, succinate, 3-hydroxypropionate and other short-chain fatty acids and alcohols were quantified on a Shimadzu HPLC system equipped with a Shodex sugar SH1821 6 pm, 8.0 x 300 mm column. The column was kept at 45 °C while running with 0.005 M H2SO4 as eluent under a flow of 1 ml/min. The detector was a refractive index detector. Chemicals at HPLC quality were used to prepare the standard curves. All analyses were performed in duplicate.
  • Pellets were washed with 20 ml, 20 mM TE-buffer (pH 7) at 4700 g for 30 min at 4 °C and re-suspended in 150 pl TE buffer.
  • Cell suspension was incubated with Lysozyme at 37 °C for 10 min.
  • Cell lysis and RNase inactivation was performed by addition of a mix containing 4 pl p-mercaptoethanol, 1 pl proteinase-K and 150 pl of Gram-positive lysis solution (Gram positive DNA extraction kit, Masterpure). Lysis was done at 65 °C for 15 min while vortexing every 5 min.
  • M. jalaludinii DSM13811 T and A. rhamnosivorans were grown either together or alone in a bicarbonate buffer medium supplemented with 20 mM phytate or 20 mM inositol. The experiments were performed in triplicate. The bacteria were harvested after 17 h incubation. 1 mL bacterial supernatants were collected to perform HPLC measurement for substrate consumption and metabolite production while the cell pellets were used for quantification of M. jalaludinii and A. rhamnosivorans. qPCR primers for M.
  • jalaludinii were designed to quantify M. jalaludinii in the cocultures and murine microbiome using Primer3 and Blast based on 16S rRNA sequence of M. jalaludinii DSM13811 T .
  • the primers were validated using gDNA of M. jalaludinii, which resulted in an 83 bp amplicon.
  • the 16S rRNA gene of M. jalaludinii was used to optimize temperature and make standard curves.
  • the qPCR programme was 95 °C for 5 min and 35 cycles consisting of 95 °C for 30 s, 60 °C for 10 s and 72 °C for 40 s; 95 °C for 1 min and 60 °C for 1 min.
  • DNA copies were calculated based on standard curves.
  • qPCR primers for A. rhamnosivorans were used as previously designed and validated. 100 ml bacterial cultures were used for RNA extraction and sequencing as described above. Obtained reads were aligned against reference genomes (BioProject PRJNA223472) of M. jalaludinii and A. rhamnosivorans using bowtie2 (V2.5.1). Gene counts were gathered in R (V4.0.5) using feature counts function of the R subread package (V4.3). Differential expression analysis was performed using DESeq2 (V 1.38.3). For analysis were re-annotated using an available genome from GenBank (BioProject PRJNA223472) and re-annotating the genome in Rapid Annotation using Subsystem Technology (RAST).
  • RAST Subsystem Technology
  • mice were received at the age of 4 weeks and randomized and divided in 2 mice per cage. Acclimatization time was 2 weeks. To reduce the interference of endogenous murine microbiome, mice were treated with antibiotic cocktail of 1 mg ml -1 ampicillin, 5 mg ml -1 streptomycin and 1 mg ml -1 colistin via oral gavage one time in 1 week prior to the intervention as described previously.
  • antibiotic cocktail 1 mg ml -1 ampicillin, 5 mg ml -1 streptomycin and 1 mg ml -1 colistin via oral gavage one time in 1 week prior to the intervention as described previously.
  • 0.2 ml oral administration of 0.1 mg/g body weight phytate or 0.1 mg/g body weight phytate and 10 9 CFU of MJ or 0.1 mg/g body weight phytate and 10 9 CFU of MJ and 10 9 CFU of AR were given to a group of 8 mice every second day for 2 weeks.
  • Bacterial suspension was prepared in 10 % trehalose.
  • [ 13 Ce]phytate-AR-MJ, [ 13 Ce]phytate-MJ or [ 13 Ce]phytate challenge were given to a group of 8 mice in corresponding group.
  • the sample is then centrifuged (10 min, 21 000 g, 4 °C) in 2 mL tubes and the supernatant transferred into a separate 5 mL or 15 mL tube containing TiO2 beads (Titanosphere 5 pm, GL Sciences, 0.5 mg of TiO2 per 1 mg original cecum content material), which were already washed with 1 mL Milli-Q® water and 1 mL 1 M perchloric acid.
  • the extract and TiC>2 beads were mixed by briefly vortexing and on a rotary shaker for 20 min at 4 °C.
  • lyophilized eluates were redissolved in 500 pL D2O and lyophilized again.
  • the lyophilized samples were taken up in 500 pL D2O and defined volumes of NaOD and DCI were used to adjust the pH to 7.0. Afterwards, 2.86 pL of a TMPBr stock solution (20 mM in D2O) were added for quantification and submitted for NMR measurement.
  • InsPs The extractions of InsPs from plasma samples were performed based on the procedures of TiO 2 purification method. Briefly, 60-100 pL of plasma was thawed on ice and an equal amount of perchloric aid (2 M, 4 °C) was added. The sample was rotated for 30 min at 4 °C and then centrifuged (15.000 g, 10 min, 4 °C). Transfer the supernatant to pre-washed TiC>2 beads (5 mg per sample, 5020-75000 GL Sciences) and incubate at 4 °C with rotation for 20 minutes. After that, the suspension was centrifuged (3.500 g, 1 min, 4 °C) and the supernatant was discarded.
  • perchloric aid 2 M, 4 °C
  • CE-ESI-QQQ analysis of ⁇ Ce] InsPs in plasma samples Known amounts of isotopic standards (2 pM [ 18 O2] 5-lnsP?) were spiked into samples for quantitation of [ 13 Ce] PP-lnsPs and [ 18 0i2] lnsP 6 (2 pM) were spiked into samples for quantitation of [ 13 Ce] InsPs.
  • 2 pM [ 18 O2] 5-lnsP? and 2 pM [ 1s Oi2] InsPe were spiked.
  • the samples were diluted 20-fold, then 4 pM [ 18 C>2] 5-lnsP 7 and 20 pM [ 18 0i2] InsPe were spiked.
  • a CE-ESI-QQQ system is used for the measurement. Set MS source parameters and MRM transitions as shown in table. The injection for each sample is 30 nL.
  • the elution started with 13 % mobile phase A, increased to 17 % mobile phase A between 0 min and 16 min then to 19 % mobile phase A between 16 min to 19 min. Between 19 min to 21 min, mobile phase A was increased to 30 % then held for 2 min.
  • Stock solution of myo-inositol (2 mg/mL) was prepared in ultrapure water, then was diluted in 87 % acetonitrile (starting mobile phase condition for analysis) to obtain five calibration levels (0.1 pg/mL, 0.5 pg/mL, 1 pg/mL, 5 pg/mL, 10 pg/mL) for quantification of myo-inositol in plasma. Each calibration levels have three replicates.
  • the mass isotopologue spectra of ([M-57] + ) fragment of the derivatives of acetate (m/z 117-1119, mo-m2), propionate (m/z 131-134, mo-ms) and butyrate (m/z 173-149, mo- u) were monitored.
  • the standard curve were prepared in a range of 10 pM to 10 mM for acetate, 3 pM to 3 mM for propionate and butyrate. These ranges covered all measured points.
  • Caco-2 cell testing To investigate the effects of microbial metabolites on barrier integrity, TEER (Transepithelial electrical resistance) experiments in Caco-2 cells were performed. As propionate is the major metabolite from phytate break-down by the coculture, effect of propionate on barrier integrity was accessed in Caco-2 cell with transwell setting. To produce bacterial supernatants for Caco-2 cell work, a coculture of M. jalaludinii and A. rhamnosivorans was grown in YCFA containing 20mM phytate. The pH of the cultures was monitored every day and adjusted to neutral pH after addition of substrates. The pH adjustment and substrate addition were conducted every day for 5 days. Bacterial supernatants were collected at all time points for HPLC measurement.
  • TEER Transepithelial electrical resistance
  • TEER analyses were performed at day 7, 14, and 21 in culture. After 21 days, the medium in the apical compartment was replaced with medium containing either 20mM propionate; 10 % M. jalaludinii and A. rhamnosivorans supernatant or 10 % YCFA medium as control. TEER measurement was performed at TO; 4 h; 7 h; 24 h and 30 h. Cells with fresh medium was used as control condition. At the end of the experiment, the supernatant in the apical compartment was collected for HPLC analysis while Caco-2 cells were washed with 300 pL of PBS and collected in 300 pL of Trizol/Tripure®. These cells were then stored at -80°C freezer for RNA extraction and qPCR to quantify the activity of tight junction genes. The experiment was performed in biological triplicate.
  • Human tight junction genes (Claudin 1 , Occludin, E-cadherin, Claudin 2 and ZO1) were amplified using specific primers, with expression being normalized to 18S and 36B4 genes.
  • the qPCR programme was 10 minutes at 95°C, 39 cycles consisting of 15 seconds at 95 °C and 30 seconds at 60 °C and melting curves were obtained at between 65 °C to 95 °C with an increment of 0.5 °C every 5 seconds. Fold changes were calculated using 2' ACq .
  • the qPCR was performed with technical duplicates.
  • Fecal phytate metabolism identifies human Mitsuokella spp. as efficient phytate degraders.
  • the inventors incubated fresh fecal samples from a healthy donor (A) in a medium supplemented with [ 13 Ce]phytate as the sole carbon and energy source. The supernatants from [ 13 Ce]phytate enrichments were collected over time and used for the analysis of 13 C-labelled components by 13 C-NMR. In parallel, non-labelled phytate fecal enrichments were repeatedly transferred to fresh phytate media to further enrich for phytate degrading microbes.
  • the inventors observed that the fecal microbiome metabolized [ 13 Ce]phytate within a few hours to [ 13 C2]acetate and [ 13 C3]3-hydroxypropionate, which was subsequently converted to [ 13 C3]propionate after 24h (Fig. 1a).
  • genomic DNA was isolated from phytate enrichments after two transfers during which bacteria involved in phytate metabolism were highly enriched and subjected to 16S rRNA gene amplicon sequencing.
  • the inventors observed a microbial community enriched from fecal microbiomes with Ruminococcaceae, Mitsuokella and Butyricicoccus as the most abundant taxa (Fig. 1 b).
  • Fig. 1 b the relative abundance of most species decreased, whereas that of Mitsuokella spp. increased in both enrichments up to 10 % of total microbiome, with M. jalaludinii as the most dominant species (Fig. 1b-c).
  • Mitsuokella as prevalent taxon in the human Gl tract and its health relation. T o further investigate the prevalence of Mitsuokella in the general population and determine its ecological niche, the inventors analyzed the microbiome of 6039 Amsterdam-located subjects in HELIUS cohort with different ethnicities. The inventors found three amplicon sequence variants (ASV) of Mitsuokella which represented 89 % of all Mitsuokella counts. These sequence variants were highly similar to those belonging to Mitsuokella multacida or Mitsuokella jalaludinii. A total of 1542 out of 6039 subjects were positive for one of these clades, indicating the high prevalence of Mitsuokella in the general population.
  • ASV amplicon sequence variants
  • Mitsuokella is closely related to known fiber-degrading species, including ruminal Selenomonas spp., one of which has been reported to have an active periplasmic phytase. Mitsuokella spp. were also strongly associated with the Prevotella Enterotype, which has been linked with high fiber intake and health status. The prevalence of different clades of Mitsuokella spp. varied slightly among the ethnicities but most subjects harbored M. jalaludinii.
  • M. jalaludinii is highly prevalent in human.
  • M. jalaludinii remained as the most prevalent Mitsuokella species in both genders with higher prevalence in male. It was recently reported that the abundance of fecal Mitsuokella spp. was significantly lower in cardiometabolic patients compared to the healthy population. This prompted the inventors to further study the role of M. jalaludinii in phytate degradation and its contribution to metabolic health.
  • M. jalaludinii was grown in bicarbonate buffered medium containing either [ 13 Ce]phytate or [ 13 C6]myo-inositol as the sole energy and carbon source.
  • M. jalaludinii converted [ 13 Ce]phytate rapidly to various metabolites with [ 13 C3]3-hydroxypropionate, [ 13 C3]lactate and [ 13 C4]succinate as major end metabolites (Fig. 2b).
  • the inventors identified the accumulation of myo-inositol-2-monophosphate (lns(2)P) and myo-inositol during the first 7.5 h via 2D-NMR (Fig. 2c), suggesting that these are intermediates of phytate degradation.
  • the capacity to use myo-inositol was confirmed by the growth of M. jalaludinii and conversion of [ 13 C6]myo-inositol to [ 13 C3]3-hydroxypropionate, [ 13 C3]lactate and [ 13 C4]succinate.
  • the production of 3-hydroxypropionate from phytate assured our previous hypothesis that Mitsuokella spp. were responsible for the efficient fecal phytate degradation in which 3-hydroxypropionate was detected as intermediate (Fig. 1a).
  • A. rhamnosivorans in phytate It has been shown that supplementation of A. rhamnosivorans in fecal phytate enrichment increased propionate formation.
  • A. rhamnosivorans was indeed able to convert 3-hydroxypropionate to propionate particularly in presence of glucose as carbon source, indicating a co-metabolic conversion (Fig. 7a-c).
  • glucose consumption was reduced in the presence of 3-hydroxypropionate, suggesting inhibition of the glucose metabolism.
  • This was further supported by a slower growth of A. rhamnosivorans on myoinositol in the presence of increased 3-hydroxypropionate levels (Fig. 7d).
  • the inventors assessed the differential expression of phytate degradation pathway genes of M. jalaludinii and myo-inositol degradation pathway genes of A. rhamnosivorans during growth in monocultures compared with cocultures on phytate and myo-inositol using transcriptomic analyses.
  • the results of the metabolic time-course measurements showed high concentrations of 3-hydroxypropionate only in monocultures of M. jalaludinii at the expense of high propionate concentration in the cocultures in both phytate and myo-inositol (Fig. 8a-b).
  • rhamnosivorans were 3-fold higher than that of M. jalaludinii with myo-inositol as a carbon source (Fig. 8c-d).
  • A. rhamnosivorans had small effects on the overall transcription profiles of M. jalaludinii grown in phytate in contrast to strong influence on M. jalaludinii transcriptomic profile grown in myo-inositol (Fig. 8e).
  • the expression of genes involved in phytate dephosphorylation, inositol uptake, inositol fermentation was highly similar between monoculture and coculture in phytate (Fig.
  • the inventors administered non-labeled phytate with either no microbes, M. jalaludinii alone or M. jalaludinii together with A. rhamnosivorans for two weeks before [ 13 Ce]phytate oral challenge (Fig. 4a).
  • the inventors quantified 13 C-inositol phosphates by NMR and CE-MS analyses in mouse cecum and plasma as well as the bacteria in mouse colon after 3 h and 6 h oral gavage.
  • the inventors observed that the [ 13 Ce]phytate level was significantly lower after 3 h and 6 h oral challenge in groups receiving bacteria (Fig. 4b-c), indicating the active conversion of phytate by M. jalaludinii or M. jalaludinii plus A. rhamnosivorans in the murine digestive tract.
  • Mice that received the bacteria both M. jalaludinii with and without
  • A. rhamnosivorans had less than 0.5 nmol of [ 13 Ce]phytate per mg cecal content as opposed to 4 nmol of [ 13 Ce]lnsP6 per mg cecal content from mice that did not receive the bacteria after 3 h oral challenge. This is reflected by high levels of M. jalaludinii in mouse colon samples of bacterial treatment groups compared with the phytate-only group (Fig. 4d). Similarly, the level of A. rhamnosivorans was high in mouse colon samples after treatment while completely absent in M. jalaludinii treatment and only phytate groups (Fig. 4e).
  • the inventors detected large enrichment of 13 C-propionate but not 13 C-acetate or minor 13 C-butyrate in mouse cecal samples, indicating the major production of propionate from oral phytate (Fig. 10).
  • Fig. 10 the major production of propionate from oral phytate
  • Microbial phytate derived metabolites improved barrier integrity via activating tight junction genes in Caco-2 cell model.
  • the inventors examined effects of the SCFA propionate as a major end metabolite from phytate degradation on barrier integrity using a Caco-2 cell model. The inventors incubated 20 mM propionate in 3-week seeded Caco-2 cells on transwell and measured the barrier integrity by transepithelial electrical resistance (TEER) analysis over the course of 30 h and quantified the expression of genes involved in tight junction by qPCR.
  • TEER transepithelial electrical resistance
  • the inventors observed a significant increase of TEER upon propionate incubation as compared to control (Fig. 5a).
  • the inventors found that tight junction genes Claudin-1 , Claudin-2 and E-cadherin were upregulated in propionate condition which is in line with the TEER measurement (Fig. 5b).
  • the inventors added phytate to a coculture of M. jalajudinii and A. rhamnosivorans and adjusted pH back to neutral in the end of fermentation of each addition (Fig. 11).
  • the inventors observed that 10 % supplementation of the bacterial supernatant to the apical compartment improved epithelial barrier integrity (Fig.
  • Treatment 2 6 weeks, twice daily supplement with 10 8 living cells of a bacterium according to the present disclosure, i.e. Mitsuokella jalaludinii.
  • Treatment 3 6 weeks, twice daily supplement with 10 8 living cells of a bacterium according to the present disclosure, i.e. Mitsuokella jalaludinii and Anaerostipes rhamnosivorans.
  • Treatment 3 6 weeks, twice daily supplement with 10 8 living cells of a bacterium according to the present disclosure, i.e. Mitsuokella jalaludinii and Anaerostipes rhamnosivorans in combination with 25 gram nuts.
  • Escherichia coli is transformed according to standard methods with a gene set according to the present invention and comprising (PTP-like) phytase, major myo-inositol transporter lolT, myo-inositol 2-dehydrogenase, inosose dehydratase, 3D-(3,5/4)-trihydroxycyclohexane-1, 2-dione hydrolase, 5-deoxy-glucuronate isomerase, 5-keto-2-deoxygluconokinase, 5-keto-2-deoxy-D-gluconate-6 phosphate aldolase, 2-hydroxy- 3-oxopropionate reductase, and D-beta-hydroxypropionate permease.
  • PTP-like phytase phytase
  • major myo-inositol transporter lolT myo-inositol 2-dehydrogenase
  • inosose dehydratase 3D-(3,5
  • Competent Cells A single colony of E. coli is transferred into 5 mL of LB broth and incubated overnight at 37°C with shaking. Then, 50 mL of fresh LB broth is inoculated with 1 mL of the overnight culture. The culture is incubated at 37°C with shaking until it reaches an optical density at 600 nm (OD600) of 0.4-0.6. The culture is then transferred to a pre-chilled centrifuge tube and centrifuged at 4°C for 10 minutes at 3000 x g. The supernatant is discarded and the cell pellet is resuspended in 20 mL of ice-cold molecular biology-grade water. The, the cell pellet is centrifuged again and resuspended in 2.5 mL of ice-cold 10% glycerol, and transferred to small tubes.
  • OD600 optical density at 600 nm
  • Transformation 1-5 pL of each plasmid DNA (containing the target genes) is transferred to 50 pL of competent cells on ice, and gently mixed, and then incubated on ice for 30 minutes. The mixture is then transferred to a pre-chilled electroporation cuvette. The cells are electroporated using appropriate settings for E. coli. Immediately, 1 mL of SOC medium is added to the electroporated cells and the mixture is transferred to a sterile tube. The cells are incubated at 37°C with shaking for 1-2 hours.
  • the transformed cells are plated onto LB agar plates containing the appropriate antibiotics for selection of successfully transformed cells. The plates are incubated at 37°C overnight.
  • the presence of the target genes is verified by PCR or DNA sequencing, and protein expression is confirmed by performing enzyme assays or Western blotting. It is found that the host bacterium is capable of converting phytate to 3-hydroxypropionate under anaerobic conditions (UHPLC-MS/MS as disclosed herein).
  • EXAMPLE 4 Use of other host bacteria (2) Additionally, Escherichia coli is transformed according to methods as described in Example 3 with a gene set according to the present invention and comprising permease, oxoacid CoA transferase, dehydratase, electron transfer flavoprotein, beta subunit, Electron transfer flavoprotein, alpha subunit, Acyl dehydrogenase, and oxoacid CoA transferase It is found that the host bacterium is capable of converting 3-hydroxypropionate to propionate under anaerobic conditions.

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Abstract

L'invention concerne une bactérie comprenant un ensemble de gènes de la voie du phytate au 3-hydroxoypropionate qui permet à cette bactérie de convertir le phytate en 3-hydroxypropionate ou en un sel ou un ester de celui-ci. La bactérie peut être utilisée en tant que probiotique ou supplément pour favoriser la production de propionate dans le tractus gastro-intestinal, prévenant et/ou traitant ainsi les affections ou maladies qui bénéficient de la production de propionate, par exemple les maladies métaboliques, les syndromes métaboliques, l'obésité, la résistance à l'insuline ou les affections liées à la résistance à l'insuline, en particulier la dyslipidémie, la (M)ASLD-(M)ASH, le diabète sucré de type 2 et la résistance à l'insuline dans les maladies endocriniennes.
PCT/EP2024/087460 2023-12-20 2024-12-19 Bactérie comprenant une voie de dégradation de phytate Pending WO2025132829A1 (fr)

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